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PD-1 increases PTEN phosphatase activity while decreasing PTEN protein stability by inhibiting CK2.

Publication Date: 2013 Jun 3 PMID: 23732914
Authors: Patsoukis, N. - Li, L. - Sari, D. - Petkova, V. - Boussiotis, V. A.
Journal: Mol Cell Biol

Programmed death (PD)-1 is a potent inhibitor of T cell responses. PD-1 abrogates activation of PI3K/Akt pathway but the mechanism remains unclear. We determined that during TCR/CD3 and CD28-mediated stimulation, PTEN is phosphorylated by CK2 in the Ser380/Thr382/Thr383 cluster within the C-terminus regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380/Thr382/Thr383 cluster within the C-terminus domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3 and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism via which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis.

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ADAM17 controls endochondral ossification by regulating terminal differentiation of chondrocytes.

Publication Date: 2013 Jun 3 PMID: 23732913
Authors: Hall, K. C. - Hill, D. - Otero, M. - Plumb, D. A. - Froemel, D. - Dragomir, C. L. - Maretzky, T. - Boskey, A. - Crawford, H. - Selleri, L. - Goldring, M. B. - Blobel, C. P.
Journal: Mol Cell Biol

Endochondral ossification is a highly regulated process that relies on properly orchestrated cell-cell interactions in the developing growth plate. This study is focused on understanding the role of a crucial regulator of cell-cell interactions, the membrane-anchored metalloproteinase ADAM17, in endochondral ossification. ADAM17 releases growth factors, cytokines and other membrane proteins from cells and is essential for EGF-receptor signaling and for processing TNFalpha. Here we report that mice lacking ADAM17 in chondrocytes (A17DeltaCh) have a significantly expanded zone of hypertrophic chondrocytes in the growth plate and retarded growth of long bones. This abnormality is caused by an accumulation of the most terminally differentiated type of chondrocytes that produce a calcified matrix. Inactivation of ADAM17 in osteoclasts or endothelial cells does not affect the zone of hypertrophic chondrocytes, suggesting that the main role of ADAM17 in the growth plate is in chondrocytes. This notion is further supported by in vitro experiments showing enhanced hypertrophic differentiation of primary chondrocytes lacking Adam17. The enlarged zone of hypertrophic chondrocytes in A17DeltaCh mice resembles that described in mice with mutant EGFR signaling or lack of its ligand TGFalpha, suggesting that ADAM17 regulates terminal differentiation of chondrocytes during endochondral ossification by activating the TGFalpha/EGFR signaling axis.

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Single quantum dot tracking reveals that an individual multivalent HIV-1 Tat-protein transduction domain can activate machinery for lateral transport and endocytosis.

Publication Date: 2013 Jun 3 PMID: 23732912
Authors: Suzuki, Y. - Roy, C. N. - Promjunyakul, W. - Hatakeyama, H. - Gonda, K. - Imamura, J. - Pillai, B. V. - Ohuchi, N. - Kanzaki, M. - Higuchi, H. - Kaku, M.
Journal: Mol Cell Biol

The mechanisms underlying the cellular entry of the HIV-1-Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent-TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon crosslinking of heparan-sulfate proteoglycans (HSPGs). The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic-internalization. However, bivalent-TatP did not sufficiently promote either cell-surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell-entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo-delivery nanomachines.

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A stress-activated, p38 MAPK-ATF/CREB pathway regulates post-transcriptional, sequence-dependent decay of target RNAs.

Publication Date: 2013 Jun 3 PMID: 23732911
Authors: Gao, J. - Wagnon, J. L. - Protacio, R. M. - Glazko, G. V. - Beggs, M. - Raj, V. - Davidson, M. K. - Wahls, W. P.
Journal: Mol Cell Biol

Broadly conserved, mitogen-activated/stress-activated protein kinases (MAPK/SAPK) of the p38 family regulate multiple cellular processes. They transduce signals via dimeric, basic leucine zipper (bZIP) transcription factors of the ATF/CREB family (such as Atf2, Fos and Jun) to regulate the transcription of target genes. We report additional mechanisms for gene regulation by such pathways exerted through RNA stability controls. The Spc1 (Sty1/Phh1) kinase-regulated Atf1-Pcr1 (Mts1-Mts2) heterodimer of the fission yeast Schizosaccharomyces pombe controls the stress-induced, post-transcriptional stability and decay of sets of target RNAs. Whole transcriptome RNA-seq data revealed that decay is associated nonrandomly with transcripts that contain an M26 sequence motif. Moreover, the ablation of an M26 sequence motif in a target mRNA is sufficient to block its stress-induced decay. Conversely, engineered M26 motifs can render a stable mRNA into one that is targeted for decay. This Stress-Activated RNA Decay (SARD) provides a mechanism to reduce the expression of target genes without shutting off transcription itself. Thus, a single p38-ATF/CREB signal transduction pathway can coordinately induce (promote transcription and RNA stability) and repress (promote RNA decay) transcript levels for distinct sets of genes, as is required for developmental decisions in response to stress and other stimuli.

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DIRECT PROTEIN INTERACTIONS ARE RESPONSIBLE FOR IKAROS-GATA AND IKAROS-CDK9 COOPERATIVITY IN HEMATOPOIETIC CELLS.

Publication Date: 2013 Jun 3 PMID: 23732910
Authors: Bottardi, S. - Mavoungou, L. - Bourgoin, V. - Mashtalir, N. - Affar, E. B. - Milot, E.
Journal: Mol Cell Biol

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here we establish that Ik interaction with GATA transcription factors and cyclin dependent kinase 9 (Cdk9), a component of the Positive Transcription Elongation Factor b (P-TEFb), is required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicates that the C-terminal zinc-finger domain of Ik interacts directly with the C-terminal zinc-finger of GATA1, GATA2 and GATA3 whereas the N-terminal zinc-finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions is demonstrated in vivo in COS-7 and primary hematopoietic cells, where Ik facilitates Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 does not efficiently interact with Cdk9 or GATA proteins in vivo, perturbs Cdk9/P-TEFb recruitment to Ik-target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform reveals that Ik exon 6 is dispensable for interaction with Mi2 and GATA proteins but is essential for Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.

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EGLN3 Inhibition of NF-kappaB Is Mediated by Prolyl Hydroxylase-independent Inhibition of IKKgamma Ubiquitination.

Publication Date: 2013 Jun 3 PMID: 23732909
Authors: Fu, J. - Taubman, M. B.
Journal: Mol Cell Biol

NF-kappaB transcription factors are crucial regulators of inflammation, immunity, stress responses and cell differentiation. Many studies have demonstrated that ubiquitination of IKKgamma, a regulatory subunit of the IKK (I-kappaB kinase), is instrumental in the activation of IKK and NF-kappaB. We and others previously identified EGLN3, a member of a family of prolyl hydroxylases, as a negative regulator of the NF-kappaB pathway. Here we report that EGLN3, but not EGLN1 or 2, interacts with and inhibits K63-linked ubiquitination of IKKgamma. The effect appears to be related to inhibition of IKKgamma ubiquitination mediated by cIAP1, rather than to stimulation of IKKgamma deubiquitination by the deubiquitinases A20 and CYLD. EGLN3 does not affect the protein levels of cIAP1 or its E2 ubiquitin-conjugating enzymes UbcH5 and Ubc13. EGLN3 hydroxylase activity is not responsible for its effect on IKKgamma ubiquitination and NF-kappaB signaling. Instead, interaction with IKKgamma is required for the ability of EGLN3 to inhibit IKKgamma ubiquitination and IKK-NF-kappaB signaling. EGLN3 competes with cIAP1 for IKKgamma binding, leading to inhibition of cIAP1-IKKgamma interaction, IKKgamma ubiquitination and IKK-NF-kappaB signaling. This study provides novel insights into EGLN3 function and sheds new light on the regulation of IKKgamma ubiquitination and NF-kappaB.

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Pin1 Regulates the Dynamics of c-Myc DNA Binding to Facilitate Target Gene Regulation and Oncogenesis.

Publication Date: 2013 May 28 PMID: 23716601
Authors: Farrell, A. S. - Pelz, C. - Wang, X. - Daniel, C. J. - Wang, Z. - Su, Y. - Janghorban, M. - Zhang, X. - Morgan, C. - Impey, S. - Sears, R. C.
Journal: Mol Cell Biol

The Myc oncoprotein is considered a master regulator of gene transcription by virtue of its ability to modulate expression of a large percentage of all genes. However, mechanisms that direct Myc's recruitment to DNA and target gene selection to elicit specific cellular functions have not been well elucidated. Here, we report that the Pin1 prolyl-isomerase enhances recruitment of Serine62-phosphorylated Myc and its co-activators to select promoters during gene activation, followed by promoting Myc's release associated with its degradation. This facilitates Myc's activation of genes involved in cell growth and metabolism, resulting in enhanced pro-proliferative activity, even while controlling Myc levels. In cancer cells with impaired Myc degradation, Pin1 still enhances Myc DNA binding, although it no longer facilitates Myc degradation. Thus, we find that Pin1 and Myc are co-overexpressed in cancer, and this drives a gene expression pattern that we show is enriched in poor outcome breast cancer subtypes. This study provides new insight into mechanisms regulating Myc DNA binding and oncogenic activity, it reveals a novel role for Pin1 in the regulation of transcription factors, and it elucidates a mechanism that can contribute to oncogenic cooperation between Pin1 and Myc.

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Generation of mice deficient in both KLF3/BKLF and KLF8 reveals a genetic interaction and a role for these factors in embryonic globin gene silencing.

Publication Date: 2013 May 28 PMID: 23716600
Authors: Funnell, A. P. - Mak, K. S. - Twine, N. A. - Pelka, G. J. - Norton, L. J. - Radziewic, T. - Power, M. - Wilkins, M. R. - Bell-Anderson, K. S. - Fraser, S. T. - Perkins, A. C. - Tam, P. P. - Pearson, R. C. - Crossley, M.
Journal: Mol Cell Biol

Kruppel-like factors 3 and 8 (KLF3 and KLF8) are highly related transcriptional regulators that bind to similar sequences of DNA. We have previously shown that in erythroid cells there is a regulatory hierarchy within the KLF family, whereby KLF1 drives the expression of both the Klf3 and Klf8 genes and KLF3 in turn represses Klf8 expression. While the erythroid roles of KLF1 and KLF3 have been explored, the contribution of KLF8 to this regulatory network has been unknown. To investigate this, we have generated a mouse model with disrupted KLF8 expression. Whilst these mice are viable, albeit with a reduced lifespan, mice lacking both KLF3 and KLF8 die at around E14.5, indicative of a genetic interaction between these two factors. In the fetal liver, Klf3 Klf8 double mutant embryos exhibit greater dysregulation of gene expression than either of the two single mutants. In particular, we observe derepression of embryonic, but not adult, globin expression. Taken together, these results suggest that KLF3 and KLF8 have overlapping roles in vivo and participate in the silencing of embryonic globin expression during development.

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SWI/SNF Chromatin-remodeling Factor Smarcd3/Baf60c Controls EMT by Inducing Wnt5a Signaling.

Publication Date: 2013 May 28 PMID: 23716599
Authors: Vincent Jordan, N. - Prat, A. - Abell, A. N. - Zawistowski, J. S. - Sciaky, N. - Karginova, O. A. - Zhou, B. - Golitz, B. T. - Perou, C. M. - Johnson, G. L.
Journal: Mol Cell Biol

We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNAi screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal EpCAM(-) breast cancer cells to an epithelial EpCAM+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these ten genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM(-) breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of Claudin-low triple negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.

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Lack of tRNA modification isopentenyl-A37 alters mRNA decoding and causes metabolic deficiencies in fission yeast.

Publication Date: 2013 May 28 PMID: 23716598
Authors: Lamichhane, T. N. - Blewett, N. H. - Crawford, A. K. - Cherkasova, V. A. - Iben, J. R. - Begley, T. J. - Farabaugh, P. J. - Maraia, R. J.
Journal: Mol Cell Biol

Transfer-RNA isopentenyltransferases (Tit1) modify tRNA position 37, adjacent to the anticodon, to N6-isopentenyladenosine (i6A37) in all cells yet the tRNA subsets selected for modification vary among species and relevance to phenotypes are unknown. We examined i6A37 function in Schizosaccharomyces pombe tit1+ and tit1-Delta cells using a beta-galactosidase codon-swap reporter whose catalytic activity is sensitive to accurate decoding of codon 503. I6A37 increased activity of tRNACys at a cognate codon and tRNATyr at a near-cognate codon, suggesting that i6A37 promotes decoding activity generally and increases fidelity at cognate codons while decreasing fidelity at non-cognate codons. S. pombe cells lacking tit1+ exhibit slow growth in glycerol or rapamycin. While existing data link U34 wobble base modifications to translation of functionally related mRNAs, whether this might extend to the anticodon-adjacent position 37, was unknown. Indeed we found a biased presence of i6A37-cognate codons in high abundance mRNAs for ribosome subunits and energy metabolism, congruent with the observed phenotypes and the idea that i6A37 promotes translational efficiency. Polysome profiles confirm decreased translational efficiency of mRNAs in tit1-Delta cells. As subsets of i6A37-tRNAs differ among species as do their cognate codon-sensitive mRNAs, these genomic variables may underlie associated phenotypic differences.

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AKAP-LBC COORDINATES A p38 ACTIVATING SIGNALING COMPLEX CONTROLLING COMPENSATORY CARDIAC HYPERTROPHY.

Publication Date: 2013 May 28 PMID: 23716597
Authors: Perez Lopez, I. - Cariolato, L. - Maric, D. - Gillet, L. - Abriel, H. - Diviani, D.
Journal: Mol Cell Biol

In response to stress, the heart undergoes a remodeling process associated with cardiac hypertrophy that eventually leads to heart failure. A-kinase anchoring proteins (AKAPs) have been shown to coordinate numerous pro-hypertrophic signaling pathways in cultured cardiomyocytes. It remains however to be established whether AKAP-based signaling complexes control cardiac hypertrophy and remodeling in vivo. In the current study, we show that AKAP-Lbc assembles a signaling complex composed of the kinases PKN, MLTK, MKK3 and p38alpha that mediates the activation of p38 in cardiomyocytes in response to stress signals. To address the role of this complex in cardiac remodeling, we generated transgenic mice displaying cardiomyocyte-specific overexpression of a molecular inhibitor of the interaction between AKAP-Lbc and the p38-activating module. Our results indicate that disruption of the AKAP-Lbc/p38 signaling complex inhibits compensatory cardiomyocyte hypertrophy in response to aortic banding-induced pressure overload and promotes early cardiac dysfunction associated with increased myocardial apoptosis, stress gene activation, and ventricular dilation. Attenuation of hypertrophy results from a reduced protein synthesis capacity, as indicated by a decreased phosphorylation of 4E-binding protein 1 and ribosomal protein S6. In conclusion, these results indicate that AKAP-Lbc enhances p38-mediated hypertrophic signaling in the heart in response to abrupt increases in the afterload.

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The Keap1-Nrf2 System Prevents Onset of Diabetes Mellitus.

Publication Date: 2013 May 28 PMID: 23716596
Authors: Uruno, A. - Furusawa, Y. - Yagishita, Y. - Fukutomi, T. - Muramatsu, H. - Negishi, T. - Sugawara, A. - Kensler, T. W. - Yamamoto, M.
Journal: Mol Cell Biol

Transcription factor Nrf2 (NF-E2-related factor 2) regulates a broad cytoprotective response to environmental stresses. Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein for Cullin3-based ubiquitin E3 ligase, and negatively regulates Nrf2. Whereas the Keap1-Nrf2 system plays important roles in oxidative stress response and metabolism, the roles Nrf2 plays in the prevention of diabetes mellitus remains elusive. Here we show that genetic activation of Nrf2 signaling by Keap1 gene hypomorphic knockdown (Keap1flox/--) markedly suppresses the onset of diabetes. When Keap1flox/-- were crossed with diabetic db/db mice, blood glucose levels became lower through improvement of both insulin secretion and insulin resistance. Keap1flox/-- also prevented high-calorie diet-induced diabetes. Oral administration of the Nrf2 inducer CDDO-Im also attenuated diabetes in db/db mice. Nrf2 induction altered antioxidant-, energy consumption-, and gluconeogenesis-related gene expressions in metabolic tissues. Thus, the Keap1-Nrf2 system is a critical target for preventing the onset of diabetes mellitus.

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STAT5 outcompetes STAT3 to regulate the expression of the oncogenic transcriptional modulator BCL6.

Publication Date: 2013 May 28 PMID: 23716595
Authors: Walker, S. R. - Nelson, E. A. - Yeh, J. E. - Pinello, L. - Yuan, G. C. - Frank, D. A.
Journal: Mol Cell Biol

Inappropriate activation of the transcription factors STAT3 and STAT5 has been shown to drive cancer pathogenesis through dysregulation of genes involved in cell survival, growth, and differentiation. Although STAT3 and STAT5 are structurally related, they can have opposite effects on key genes, including BCL6. BCL6, a transcriptional repressor, has been shown to be oncogenic in diffuse large B cell lymphoma. BCL6 also plays an important role in breast cancer pathogenesis, a disease in which STAT3 and STAT5 can be activated individually or concomitantly. To determine the mechanism by which these oncogenic transcription factors regulate BCL6 transcription, we analyzed their effects at the level of chromatin and gene expression. We found that STAT3 increases expression of BCL6, and enhances recruitment of RNA polymerase II phosphorylated at a site associated with transcriptional initiation. STAT5, by contrast, represses BCL6 expression below basal levels, and decreases the association of RNA polymerase II at this gene. Furthermore, the repression mediated by STAT5 is dominant over STAT3-mediated induction. STAT5 exerts this effect by displacing STAT3 from one of the two regulatory regions to which it binds. These findings may underlie the divergent biology of breast cancers containing activated STAT3 alone or in conjunction with activated STAT5.

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ARF6-regulated endocytosis of growth factor receptors links cadherin-based adhesion to canonical Wnt signaling in epithelia.

Publication Date: 2013 May 28 PMID: 23716594
Authors: Pellon-Cardenas, O. - Clancy, J. - Uwimpuhwe, H. - D'Souza-Schorey, C.
Journal: Mol Cell Biol

Wnt signaling has an essential role in embryonic development as well as stem/progenitor cell renewal, and its aberrant activation is implicated in many diseases including several cancers. beta-catenin is a critical component of Wnt-mediated transcriptional activation. Here we show that ARF6 activation during canonical Wnt signaling promotes the intracellular accumulation of beta-catenin via a mechanism that involves the endocytosis of growth factor receptors and robust activation of extracellular signal-regulated kinase (ERK). ERK promotes casein kinase 2-mediated phosphorylation of alpha-catenin leading to destabilization of the adherens junctions and subsequent increase in cytoplasmic pools of active beta-catenin and E-cadherin. ERK also phosphorylates LRP6 to amplify the Wnt transduction pathway. The aforementioned Wnt-ERK signaling pathway initiates lumen filling of epithelial cysts by promoting cell proliferation in three-dimensional cell cultures. This study uncovers a mechanism responsible for the switch in beta-catenin functions in cell adhesion at the adherens junctions and Wnt-induced nuclear signaling.

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Telomere length influences cancer cell differentiation in vivo.

Publication Date: 2013 May 28 PMID: 23716593
Authors: Hirashima, K. - Migita, T. - Sato, S. - Muramatsu, Y. - Ishikawa, Y. - Seimiya, H.
Journal: Mol Cell Biol

Limitless reproductive potential is one of the hallmarks of cancer cells. This ability is due to the maintenance of telomeres, erosion of which causes cellular senescence or death. While most cancer cells activate telomerase, a telomere-elongating enzyme, it remains elusive as to why cancer cells often maintain shorter telomeres than the cells in the surrounding normal tissues. Here, we show that forced telomere elongation in cancer cells promotes their differentiation in vivo. We elongated the telomeres of human prostate cancer cells that possess short telomeres by enhancing their telomerase activity. The resulting cells had long telomeres and retained the ability to form tumors in nude mice. Strikingly, these tumors exhibited many duct-like structures and reduced N-cadherin expression, reminiscent of well-differentiated adenocarcinoma. These changes were caused by telomere elongation and not by enhanced telomerase activity. Gene expression profiling revealed that tumor formation was accompanied by the expression of innate immune system-related genes, which have been implicated in maintaining tumor cells in an undifferentiated state and poor prognosis cancers. In tumors derived from the telomere-elongated cells, upregulation of such gene sets is not observed. Our observations suggest a functional contribution of short telomeres to tumor malignancy by regulation of cancer cell differentiation.

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Upregulation of the Rab27a-dependent trafficking and secretory mechanisms improves lysosomal transport, alleviates endoplasmic reticulum stress and reduces lysosome overload in cystinosis.

Publication Date: 2013 May 28 PMID: 23716592
Authors: Johnson, J. L. - Napolitano, G. - Monfregola, J. - Rocca, C. J. - Cherqui, S. - Catz, S. D.
Journal: Mol Cell Biol

Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes for cystinosin, the lysosomal cystine transporter. Although many cellular dysfunctions have been described in cystinosis, the mechanisms leading to these defects are not well understood. Here, we show that increased lysosomal overload induced by accumulated cystine leads to cellular abnormalities including vesicular transport defects and increased endoplasmic reticulum (ER) stress, and that correction of lysosomal transport improves cellular function in cystinosis. We found that Rab27a was expressed in proximal tubular cells (PTCs) and partially colocalized with the lysosomal marker LAMP-1. The expression of Rab27a but not other small GTPases including Rab3 and Rab7 was downregulated in kidneys from Ctns-/- mice and in human PTCs from cystinotic patients. Using Total Internal Reflection Fluorescence microscopy, we found that lysosomal transport is impaired in Ctns-/- cells. Ctns-/- cells showed significant ER expansion and a marked increase in the unfolded protein response-induced chaperones Grp78 and Grp94. Upregulation of the Rab27a-dependent vesicular trafficking mechanisms rescued the defective lysosomal transport phenotype and reduced ER stress in cystinotic cells. Importantly, reconstitution of lysosomal transport mediated by Rab27a led to decreased lysosomal overload manifested as reduced cystine cellular content. Our data suggest that upregulation of the Rab27a-dependent lysosomal trafficking and secretory pathways contribute to the correction of some of the cellular defects induced by lysosomal overload in cystinosis, including ER stress.

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MiR-33 regulates glucose metabolism.

Publication Date: 2013 May 28 PMID: 23716591
Authors: Ramirez, C. M. - Goedeke, L. - Rotllan, N. - Yoon, J. H. - Cirera-Salinas, D. - Mattison, J. A. - Suarez, Y. - de Cabo, R. - Gorospe, M. - Fernandez-Hernando, C.
Journal: Mol Cell Biol

Metabolic diseases are characterized by the failure of regulatory genes or proteins to effectively orchestrate specific pathways involved in the control of many biological processes. In addition to the classical regulators, recent discoveries have shown the remarkable role of small non-coding RNAs (microRNAs) in the post-transcriptional regulation of gene expression. In this regard, we have recently demonstrated that miR-33a/b, intronic microRNAs (miRNA) located within the sterol regulatory element-binding protein (SREBP) genes, regulate lipid metabolism in concert with their host genes. Here, we show that miR-33b also cooperates with SREBP1 in regulating glucose metabolism by targeting phosphoenolpyruvate carboxykynase (PCK1) and glucose-6-phosphatase (G6PC), key regulatory enzymes of hepatic gluconeogenesis. Overexpression of miR-33b in human hepatic cells inhibits PCK1 and G6PC expression leading to a significant reduction of glucose production. Importantly, hepatic SREBP1c/miR-33b levels correlate inversely with the expression PCK1 and G6PC upon glucose infusion in rhesus monkeys. Taken together, these results suggest that miR-33b works in concert with its host gene to ensure a fine-tuned regulation of lipid and glucose homeostasis, highlighting the clinical potential of miR-33a/b as novel therapeutic targets for a range of metabolic diseases.

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Mextli is a Novel eIF4E-Binding Protein that Promotes Translation in Drosophila.

Publication Date: 2013 May 28 PMID: 23716590
Authors: Hernandez, G. - Miron, M. - Han, H. - Liu, N. - Magescas, J. - Tettweiler, G. - Frank, F. - Siddiqui, N. - Sonenberg, N. - Lasko, P.
Journal: Mol Cell Biol

Translation is a fundamental step in gene expression and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5' cap structure of the mRNA by eIF4E. eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs) which, by competing with eIF4G for eIF4E binding, act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3 and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germline stem cells (GSCs) and early stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germline stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.

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The B55 alpha regulatory subunit of Protein Phosphatase 2A mediates FGF-induced p107 dephosphorylation and growth arrest in chondrocytes.

Publication Date: 2013 May 28 PMID: 23716589
Authors: Kolupaeva, V. - Daempfling, L. - Basilico, C.
Journal: Mol Cell Biol

FGF-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types, and underlies several genetic skeletal disorders caused by activating FGFR mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the Rb protein family member p107 by Protein Phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report we show that the PP2A-B55alpha holoenzyme is responsible for this phenomenon. Only the B55alpha regulatory subunit of PP2A was able to bind p107 and this interaction was induced by FGF in chondrocytes but not in other cell types. siRNA mediated knockdown of B55alpha prevented p107 dephosphorylation and FGF-induced growth arrest of RCS chondrocytes. Importantly, the B55alpha subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55alpha is also the site of cyclin-dependent kinase (CDK) binding, B55alpha association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55alpha subunit itself on several Serine residues that drastically increases the affinity of B55alpha for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55alpha dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals.

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HBV X protein-induced aberrant epigenetic modifications contributing to human hepatocellular carcinoma pathogenesis.

Publication Date: 2013 May 28 PMID: 23716588
Authors: Tian, Y. - Yang, W. - Song, J. - Wu, Y. - Ni, B.
Journal: Mol Cell Biol

Hepatocellular carcinoma (HCC) remains one of the most prevalent malignant diseases worldwide, and the majority of cases are related to hepatitis B virus (HBV) infection. Interactions between the HBV-encoded X protein (HBx) and host factors are known to play major roles in the onset and progression of HBV-related HCC. These dynamic molecular mechanisms are extremely complex and lead to prominent changes in the host genetic and epigenetic architecture. This review summarizes the current knowledge about HBx-induced epigenetic changes, including aberrations in DNA methylation, histone modifications and microRNAs expression, and their roles in HBV-infected liver cells and HBV-related HCC. Moreover, the HBx-mediated epigenetic control of HBV covalently-closed circular (ccc) DNA is also discussed. Although this field of study is relatively new, the accumulated evidence has indicated that the epigenetic events induced by HBx play important roles in the development of HBV-related HCC. On-going research will help to identify practical applications of the HBV-related epigenetic signatures as biomarkers for early HCC detection or as potential targets to prevent and treat HBV-related HCC.

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Editorial board.

Publication Date: 2013 Jun PMID: 23698642
Authors:
Journal: Mol Cell Biol

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Publisher's Expression of Concern.

Publication Date: 2013 Jun PMID: 23698641
Authors:
Journal: Mol Cell Biol

The American Society for Microbiology (ASM) and Molecular and Cellular Biology (MCB) are issuing this Expression of Concern to alert readers to doubts about the integrity of the data in papers coauthored by Dr. Shigeaki Kato. In 2012, ASM was notified that the University of Tokyo Institute of Molecular and Cellular Biosciences is conducting an investigation of possible scientific misconduct by Dr. Kato. Among the publications under investigation for possible data manipulation are these five MCB articles. The Expression of Concern is only for papers coauthored by Dr. Kato that have been published since 2007, in accordance with the DHHS/ORI six-year limitation on research misconduct (http://ori.dhhs.gov/sites/default/files/42_cfr_parts_50_and_93_2005.pdf). MCB has contacted the Research Promotion Department of the University of Tokyo and asked to be informed of the findings of their ongoing investigation. Once ASM has been notified of the outcome of the investigation by the University of Tokyo, MCB will take appropriate action regarding these publications.

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Articles of significant interest selected from this issue by the editors.

Publication Date: 2013 Jun PMID: 23698640
Authors:
Journal: Mol Cell Biol

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Immune mediators regulate CFTR expression through a bifunctional airway-selective enhancer.

Publication Date: 2013 May 20 PMID: 23689137
Authors: Zhang, Z. - Leir, S. H. - Harris, A.
Journal: Mol Cell Biol

An airway-selective DNase hypersensitive site (DHS) at -35kb 5' to the cystic fibrosis transmembrane conductance regulator (CFTR) gene is evident in many lung cell lines and primary human tracheal epithelial cells but is absent from intestinal epithelia. DHS-35kb contains an element with enhancer activity in 16HBE14o- airway epithelial cells and is enriched for mono-methylated H3K4 histones (H3K4me1). We now define a 350 bp region within DHS-35kb, which has full enhancer activity and binds interferon regulatory factor-1 (IRF1) and nuclear factor Y (NF-Y) in vitro and in vivo. SiRNA-mediated depletion of IRF1 or overexpression of IRF2, an antagonist of IRF1, reduces CFTR expression in 16HBE14o- cells. NF-Y is critical for maintenance of H3K4me1 enrichment at DHS-35kb since depletion of NF-YA, a subunit of NF-Y, reduces H3K4me1 enrichment at this site. Moreover, depletion of SETD7, an H3K4 monomethyl transferase, reduces both H3K4me1 and NF-Y occupancy suggesting a requirement of H3K4me1 for NF-Y binding. NFY depletion also represses Sin3A and reduces its occupancy across the CFTR locus, which is accompanied by an increase in p300 enrichment at multiple sites. Our results reveal that the DHS-35kb airway selective enhancer element plays a pivotal role in regulation of CFTR expression by two independent regulatory mechanisms.

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Testosterone-Dependent Interaction between Androgen Receptor and Aryl Hydrocarbon Receptor Induces Liver Receptor Homolog-1 Expression in Rat Granulosa Cells.

Publication Date: 2013 May 20 PMID: 23689136
Authors: Wu, Y. - Baumgarten, S. C. - Zhou, P. - Stocco, C.
Journal: Mol Cell Biol

Androgens play a major role in the regulation of normal ovarian function, however, they are also involved in the development of ovarian pathologies. These contrasting effects may involve a differential response of granulosa cells to the androgens, testosterone (T) and dihydrotestosterone (DHT). To determine the molecular pathways that mediate the distinct effects of T and DHT, we studied the expression of liver receptor homolog-1 (LRH-1), a gene differentially regulated by these steroids. We found that although both T and DHT stimulate androgen receptor (AR) binding to the LRH-1 promoter, DHT prevents T mediated stimulation of LRH-1 expression. T stimulated the expression of aryl hydrocarbon receptor (AHR) and its interaction with the AR. T also promoted the recruitment of the AR/AHR complex to the LRH-1 promoter. These effects were not mimicked by DHT. We also observed that the activation of extracellular regulated kinases by T is required for AR and AHR interaction. In summary, T, but not DHT, stimulates AHR expression and the interaction between AHR and AR leading to the stimulation of LRH-1 expression. These findings could explain the distinct response of granulosa cells to T and DHT and provide a molecular mechanism by which DHT negatively affects ovarian function.

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Insights into the mechanism of ribosomal incorporation of mammalian L13a protein during ribosome biogenesis.

Publication Date: 2013 May 20 PMID: 23689135
Authors: Das, P. - Basu, A. - Biswas, A. - Poddar, D. - Andrews, J. - Barik, S. - Komar, A. A. - Mazumder, B.
Journal: Mol Cell Biol

In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the Arginine residue at position 68 of L13a as essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S pre-ribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S pre-ribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extra-ribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis.

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Interaction and Antagonistic Roles of NF-kappaB and Hes6 in the Regulation of Cortical Neurogenesis.

Publication Date: 2013 May 20 PMID: 23689134
Authors: Methot, L. - Hermann, R. - Tang, Y. - Lo, R. - Al-Jehani, H. - Jhas, S. - Svoboda, D. - Slack, R. S. - Barker, P. A. - Stifani, S.
Journal: Mol Cell Biol

The involvement of nuclear factor-kappaB (NF-kappaB) in several processes in the postnatal and adult brain, ranging from neuronal survival to synaptogenesis and plasticity, has been documented. In contrast, little is known about the functions of NF-kappaB during embryonic brain development. It is shown here that NF-kappaB is selectively activated in neocortical neural progenitor cells in the developing mouse telencephalon. Blockade of NF-kappaB activity leads to premature cortical neuronal differentiation and depletion of the progenitor cell pool. Conversely, NF-kappaB activation causes decreased cortical neurogenesis and expansion of the progenitor cell compartment. This effect is antagonized by the pro-neuronal transcription factor Hes6, which physically and functionally interacts with RelA-containing NF-kappaB complexes in cortical progenitor cells. In turn, NF-kappaB exerts an inhibitory effect on the ability of Hes6 to promote cortical neuronal differentiation. These results reveal previously uncharacterized functions, and modes of regulation, for NF-kappaB and Hes6 during cortical neurogenesis.

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Impaired epidermal permeability barrier in mice lacking the Elovl1 gene responsible for very long-chain fatty acid production.

Publication Date: 2013 May 20 PMID: 23689133
Authors: Sassa, T. - Ohno, Y. - Suzuki, S. - Nomura, T. - Nishioka, C. - Kashiwagi, T. - Hirayama, T. - Akiyama, M. - Taguchi, R. - Shimizu, H. - Itohara, S. - Kihara, A.
Journal: Mol Cell Biol

The sphingolipid backbone ceramide (Cer) is a major component of lipid lamellae in the stratum corneum of epidermis and has a pivotal role in epidermal barrier formation. Unlike Cers in other tissues, Cers in epidermis contain extremely long fatty acids (FAs). Decreases in epidermal Cer levels, as well as changes in their FA chain-lengths, cause several cutaneous disorders. However, the molecular mechanisms that produce such extremely long Cers and determine their chain-lengths are poorly understood. We generated mice deficient in the Elovl1 gene, encoding the FA elongase responsible for producing C20-28 FAs. The Elovl1 knockout mice died shortly after birth due to epidermal barrier defects. The lipid lamellae in the stratum corneum were largely diminished in these mice. In the epidermis of the Elovl1-null mice, the levels of Cers with >/=C26 FAs were decreased, while those of Cers with
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Regulation of FAK activation, breast cancer cell motility and amoeboid invasion by the RhoA GEF Net1.

Publication Date: 2013 May 20 PMID: 23689132
Authors: Carr, H. S. - Zuo, Y. - Oh, W. - Frost, J. A.
Journal: Mol Cell Biol

Net1 is a RhoA GEF that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits LPA-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with FAK, localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid-type invasion and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high beta1-integrin activity and MT1-MMP expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during ECM invasion.

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Phospholipase D1 has a pivotal role in IL-1beta-driven chronic autoimmune arthritis through regulation of NFkappaB, HIF-1alpha and FoxO3a.

Publication Date: 2013 May 20 PMID: 23689131
Authors: Kang, D. W. - Park, M. K. - Oh, H. J. - Lee, D. G. - Park, S. H. - Choi, K. Y. - Cho, M. L. - Min, D. S.
Journal: Mol Cell Biol

IL-1beta is a potent proinflammatory and immune-regulatory cytokine playing an important role in the progression of rheumatoid arthritis (RA). However, the signaling network of IL-1beta in synoviocytes from RA is still poorly understood. Here, we show for the first time that phospholipase D1 (PLD1) but not PLD2, is selectively upregulated in the IL-1beta-stimulated synoviocytes as well as synovium from RA patients. IL-1beta enhanced the binding of NFkappaB and ATF-2 to PLD1 promoter, thereby enhancing PLD1 expression. PLD1 inhibition abolished IL-1beta-induced expression of proinflammatory mediators and angiogenic factors by suppressing the binding of NFkappaB or HIF-1alpha to the promoter of its target genes as well as IL-1beta-induced proliferation or migration. However, suppression of PLD1 activity promoted cell cycle arrest via transactivation of FoxO3a. Furthermore, PLD1 inhibitor significantly suppressed joint inflammation and destruction in IL-1 receptor antagonist deficient mice (IL-1Ra-/-), a model of spontaneous arthritis. Taken together, these results suggest that the abnormal upregulation of PLD1 may contribute to the pathogenesis of IL-1beta-induced chronic arthritis, and a selective PLD1 inhibitor might provide a potential therapeutic molecule for the treatment of chronic inflammatory autoimmune disorders.

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Rpd3 and Spt16-mediated nucleosome assembly and transcriptional regulation on yeast rDNA genes.

Publication Date: 2013 May 20 PMID: 23689130
Authors: Johnson, J. M. - French, S. L. - Osheim, Y. N. - Li, M. - Hall, L. - Beyer, A. L. - Smith, J. S.
Journal: Mol Cell Biol

Ribosomal DNA (rDNA) genes in eukaryotes are organized into multi-copy tandem arrays and transcribed by RNA polymerase I. During cell proliferation, approximately 50% of these genes are active and have a relatively open chromatin structure characterized by elevated accessibility to psoralen cross-linking. In Saccharomyces cerevisiae, transcription of rDNA genes becomes repressed and chromatin structure closes when cells enter the diauxic shift and growth dramatically slows. In this study we found that nucleosomes are massively depleted from the active rDNA genes during log phase and reassembled during the diauxic shift, largely accounting for the differences in psoralen accessibility between active and inactive genes. The Rpd3L histone deacetylase complex was required for diauxic shift-induced H4 and H2B deposition onto rDNA genes, suggesting involvement in assembly or stabilization of the entire nucleosome. The Spt16 subunit of FACT, however, was specifically required for H2B deposition, suggesting specificity for the H2A/H2B dimer. Miller chromatin spreads were used for electron microscopic visualization of rDNA genes in an spt16 mutant, which was found to be deficient in the assembly of normal nucleosomes on inactive genes and the disruption of nucleosomes on active genes, consistent with an inability to fully re-activate Pol I transcription when cells exit stationary phase.

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Editorial board.

Publication Date: 2013 Jun PMID: 23657217
Authors:
Journal: Mol Cell Biol

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Articles of significant interest selected from this issue by the editors.

Publication Date: 2013 Jun PMID: 23657216
Authors:
Journal: Mol Cell Biol

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Novel insights into the role of Neurospora crassa NDUFAF2, an evolutionarily conserved mitochondrial complex I assembly factor.

Publication Date: 2013 May 6 PMID: 23648483
Authors: Pereira, B. - Videira, A. - Duarte, M.
Journal: Mol Cell Biol

Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it exerts its function remains unknown. Herein, we demonstrate that the Neurospora crassa NDUFAF2 homologue, the 13.4L protein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the connecting part but lacking the N-module of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed by the subunits 13.4 (NDUFA12), 18.4 (NDUFS6) and 21 kDa (NDUFS4). Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals.

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MEN1 is a Melanoma Tumor Suppressor that Preserves Genomic Integrity by Stimulating Transcription of Genes that Promote Homologous Recombination-Directed DNA Repair.

Publication Date: 2013 May 6 PMID: 23648481
Authors: Fang, M. - Xia, F. - Mahalingam, M. - Virbasius, C. M. - Wajapeyee, N. - Green, M. R.
Journal: Mol Cell Biol

Multiple endocrine neoplasia type 1 is a familial cancer syndrome resulting from loss-of-function mutations in the MEN1 gene. We previously identified the tumor suppressor MEN1 as a gene required for oncogene-induced senescence in melanocytes, raising the possibility that MEN1 is a melanoma tumor suppressor. Here we show that MEN1 expression is lost in a high percentage of human melanomas and melanoma cell lines. We find that melanocytes depleted of MEN1 are deficient in homologous recombination (HR)-directed DNA repair, which is accompanied by increased non-homologous end joining activity. Following DNA damage, MEN1 levels increase resulting from phosphorylation by the DNA damage kinase ATM/ATR. Most importantly, we show that MEN1 functions by directly stimulating transcription of several genes, including BRCA1, RAD51 and RAD51AP1, that encode proteins involved in HR. MEN1 and its coactivator, the histone methyltransferase MLL, are recruited to the BRCA1, RAD51 and RAD51AP1 promoters by estrogen receptor 1, resulting in increased histone H3-lysine 4 trimethylation and transcription. Collectively, our results indicate that MEN1 is a melanoma tumor suppressor that functions by stimulating transcription of genes involved in HR-directed DNA repair.

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The protein level of PGC-1alpha, a key metabolic regulator, is controlled by NADH-NQO1.

Publication Date: 2013 May 6 PMID: 23648480
Authors: Adamovich, Y. - Shlomai, A. - Tsvetkov, P. - Umansky, K. B. - Reuven, N. - Estall, J. L. - Spiegelman, B. M. - Shaul, Y.
Journal: Mol Cell Biol

PGC-1alpha is a key transcription coactivator regulating energy metabolism in a tissue-specific manner. PGC-1alpha expression is tightly regulated, it is a highly labile protein, and it interacts with various proteins- the known attributes of intrinsically disordered proteins (IDPs). In this study, we characterize PGC-1alpha as an IDP and demonstrate that it is susceptible to 20S proteasomal degradation by default. We further demonstrate that PGC-1alpha degradation is inhibited by NQO1, a 20S-gatekeeper protein. NQO1 binds and protects PGC-1alpha from degradation in an NADH-dependent manner. Using different cellular physiological settings we also demonstrate that NQO1-mediated PGC-1alpha protection plays an important role in controlling both basal and physiologically induced PGC-1alpha protein level and activity. Our findings link NQO1, a cellular redox sensor, to the metabolite-sensing network that tunes PGC-1alpha expression and activity in regulating energy metabolism.

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Efficient initiation of DNA replication in eukaryotes requires Dpb11/TopBP1--GINS interaction.

Publication Date: 2013 Apr 29 PMID: 23629628
Authors: Tanaka, S. - Komeda, Y. - Umemori, T. - Kubota, Y. - Takisawa, H. - Araki, H.
Journal: Mol Cell Biol

Dpb11/Cut5/TopBP1 is evolutionarily conserved and is essential for the initiation of DNA replication in eukaryotes. The Dpb11 of budding yeast has four BRCT domains (BRCT1-4). The N-terminal pair (BRCT1-2) and the C-terminal pair (BRCT3-4) bind to CDK-phosphorylated Sld3 and Sld2, respectively. These phosphorylation-dependent interactions trigger the initiation of DNA replication. BRCT1-2 and BRCT3-4 of Dpb11 are separated by a short stretch of approximately 100 amino acids. It is unknown whether this inter-BRCT region functions in DNA replication. Here, we showed that the inter-BRCT region is a GINS-interaction domain that is essential for cell growth, and that mutations in this domain cause replication defects in budding yeast. We found the corresponding region in the vertebrate ortholog, TopBP1, and showed that the corresponding region also interacts with GINS and is required for efficient DNA replication. We propose that the inter-BRCT region of Dpb11 is a functionally conserved GINS-interaction domain that is important for the initiation of DNA replication in eukaryotes.

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The Poly-C Binding Proteins, alphaCPs, Act as Global Regulators of Alternative Polyadenylation.

Publication Date: 2013 Apr 29 PMID: 23629627
Authors: Ji, X. - Wan, J. - Vishnu, M. - Xing, Y. - Liebhaber, S. A.
Journal: Mol Cell Biol

We have previously demonstrated that the KH-domain protein, alphaCP, binds to a 3' UTR C-rich motif of the nascent halpha-globin transcript and enhances the efficiency of 3' processing. Here we assess the genome-wide impact of alphaCP RNP complexes on 3' processing with specific focus on its role in alternative polyA site utilization (APA). The major isoforms of alphaCP were acutely depleted from a human hematopoietic cell line and the impact on mRNA representation and polyA site utilization was determined by direct RNA sequencing (DRS). Bioinformatic analysis revealed 357 significant alterations in polyA site utilization that could be specifically linked to the alphaCP depletion. These APA events correlated strongly with the presence of C-rich sequences in close proximity to the impacted polyA addition sites. The most significant linkage was the presence of a C-rich motif within a window 30-40 bases 5' to polyA signals (AAUAAA) that were repressed upon alphaCP depletion. This linkage is consistent with a general role for alphaCPs as enhancers of 3' processing. These findings predict a role for alphaCPs in post-transcriptional control pathways that can alter the coding potential and/or levels of expression of subsets of mRNAs in the mammalian transcriptome.

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Wnt7a regulates multiple steps of neurogenesis.

Publication Date: 2013 Apr 29 PMID: 23629626
Authors: Qu, Q. - Sun, G. - Murai, K. - Ye, P. - Li, W. - Asuelime, G. - Cheung, K. - Shi, Y.
Journal: Mol Cell Biol

Although Wnt7a has been implicated in axon guidance and synapse formation, investigations of its role in the early steps of neurogenesis have just begun. We show here that Wnt7a is essential for neural stem cell self-renewal and neural progenitor cell cycle progression in adult mouse brains. Loss of Wnt7a expression dramatically reduced the neural stem cell population and increased the rate of cell cycle exit in neural progenitors in the hippocampal dentate gyrus of adult mice. Furthermore, Wnt7a is important for neuronal differentiation and maturation. Loss of Wnt7a expression led to a substantial decrease in the number of newborn neurons in the hippocampal dentate gyrus. Wnt7a-/- dentate granule neurons exhibited dramatically impaired dendritic development. Moreover, Wnt7a activated beta-catenin and its downstream target genes to regulate neural stem cell proliferation and differentiation. Wnt7a stimulated neural stem cell proliferation by activating the beta-catenin-cyclin D1 pathway, and promoted neuronal differentiation and maturation by inducing the beta-catenin-neurogenin 2 pathway. Thus Wnt7a exercised critical control over multiple steps of neurogenesis by regulating genes involved in both cell cycle control and neuronal differentiation.

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SAD-A Kinase Enhances Insulin Secretion as a Downstream Target of GLP-1 Signaling in Pancreatic beta-cells.

Publication Date: 2013 Apr 29 PMID: 23629625
Authors: Nie, J. - Lilley, B. N. - Pan, Y. A. - Faruque, O. - Liu, X. - Zhang, W. - Sanes, J. R. - Han, X. - Shi, Y.
Journal: Mol Cell Biol

Type 2 diabetes is characterized by defective glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells, which can be restored by GLP-1, an incretin hormone commonly used for the treatment of type 2 diabetes. However, molecular mechanisms by which GLP-1 affects glucose responsiveness in islet beta-cells remain poorly understood. Here we investigated a role of SAD-A, an AMPK-related kinase, in regulating GSIS in mice with conditional SAD-A deletion. We show that selective deletion of SAD-A in pancreas impaired incretin's effect on GSIS, leading to glucose intolerance. Conversely, overexpression of SAD-A significantly enhanced GSIS, and further potentiated GLP-1's effect on GSIS from isolated mouse islets. In support of SAD-A as a mediator of incretin response, SAD-A is exclusively expressed in pancreas and brain, the primary targeting tissues of GLP-1 action. Additionally, SAD-A kinase is activated in response to stimulation by GLP-1 through [cAMP]/[Ca2+]-dependent signaling pathways in islet beta-cells. Furthermore, we also identified Thr443 as a key auto-inhibitory phosphorylation site which mediates SAD-A's effect on incretin response in islet beta-cells. Consequently, ablation of Thr443 significantly enhanced GLP-1's effect on GSIS from isolated mouse islets. Together, these findings identified SAD-A kinase as a pancreas-specific mediator of incretin response in islet beta-cells.

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Delineation of the key aspects in the regulation of epithelial monolayer formation.

Publication Date: 2013 Apr 22 PMID: 23608536
Authors: Aschauer, L. - Gruber, L. N. - Pfaller, W. - Limonciel, A. - Athersuch, T. J. - Cavill, R. - Khan, A. - Gstraunthaler, G. - Grillari, J. - Grillari, R. - Hewitt, P. - Leonard, M. O. - Wilmes, A. - Jennings, P.
Journal: Mol Cell Biol

The formation, maintenance and repair of epithelial barriers is of critical importance for whole body homeostasis. However, the molecular events involved in epithelial tissue maturation are not fully established. To this end we investigated the molecular processes involved in renal epithelial proximal tubule monolayer maturation utilising transcriptomic, metabolomic and functional parameters. We uncovered profound dynamic alterations in transcriptional regulation, energy metabolism and nutrient utilisation over the maturation process. Proliferating cells exhibited high glycolytic rates and high transcript levels for fatty acid synthesis genes (FASN), whereas matured cells had low glycolytic rates, increased oxidative capacity and preferentially expressed genes for beta oxidation. There were dynamic alterations in the expression and localisation of several adherens (CDH1, 4 and 16) and tight junction proteins (ZO3, CLDN2 and 10). Genes involved in differentiated proximal tubule function, cilia biogenesis (BBS1) and transport (ATP1A1, ATP1B1) exhibited increased expression during epithelial maturation. Using TransAM transcription factor activity assays, we could demonstrate that p53 and FOXO-1 were highly active in matured cells, whereas HIF1A and c-MYC were highly active in proliferating cells. The data elaborated here will be invaluable in the further delineation of the complex dynamic cellular processes involved in epithelial cell regulation.

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MAPKAPK2/3 Regulate SERCA2a Expression and Fiber Type Composition to Modulate Skeletal Muscle and Cardiomyocyte Function.

Publication Date: 2013 Apr 22 PMID: 23608535
Authors: Scharf, M. - Neef, S. - Freund, R. - Geers-Knorr, C. - Franz-Wachtel, M. - Brandis, A. - Krone, D. - Schneider, H. - Groos, S. - Menon, M. B. - Chang, K. C. - Kraft, T. - Meissner, J. D. - Boheler, K. R. - Maier, L. S. - Gaestel, M. - Scheibe, R. J.
Journal: Mol Cell Biol

The MAPK-activated protein kinases 2 and 3 (MAPKAPK2/3, MK2/3) represent protein kinases downstream of the p38 mitogen-activated protein kinase (MAPK). Using MK2/3 double knockout mice (MK2/3-/-), we analyzed the role of MK2/3 in cross-striated muscle by transcriptome and proteome analyses, and by histology. We demonstrated enhanced expression of the slow oxidative skeletal muscle myofiber gene program, including the PPARgamma coactivator 1alpha (PGC-1alpha). Using reporter gene and electrophoretic gel mobility shift assays we demonstrated that MK2 catalytic activity directly regulated the promoters of the fast fiber-specific myosin heavy chain IId/x and the slow fiber-specific sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 gene. Elevated SERCA2a gene expression caused by a decreased transcription factor Egr-1 to Sp1 ratio was associated with accelerated relaxation and enhanced contractility in MK2/3-/- cardiomyocytes, concomitant with improved force parameters in MK2/3-/- soleus muscle. These results link MK2/3 to the regulation of calcium dynamics and identify enzymatic activity of MK2/3 as a critical factor for modulating cross-striated muscle function by generating a unique muscle phenotype exhibiting both, reduced fatigability and enhanced force in MK2/3-/- mice. Hence, the p38-MK2/3 axis may represent a novel target for the design of therapeutic strategies for diseases related to fiber type changes or impaired SERCA2 function.

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Degradation of newly synthesized polypeptides by ribosome-associated RACK1/JNK/eEF1A2 complex.

Publication Date: 2013 Apr 22 PMID: 23608534
Authors: Gandin, V. - Gutierrez, G. J. - Brill, L. M. - Varsano, T. - Feng, Y. - Aza-Blanc, P. - Au, Q. - McLaughlan, S. - Ferreira, T. A. - Alain, T. - Sonenberg, N. - Topisirovic, I. - Ronai, Z. A.
Journal: Mol Cell Biol

Folding of newly synthesized polypeptides (NSPs) into functional proteins is a highly regulated process. Rigorous quality control ensures that NSPs attain their native fold, during or shortly after completion of translation. Nonetheless, signaling pathways that govern degradation of NSPs in mammals remain elusive. We demonstrate that the stress-induced c-Jun N-terminal kinase (JNK) is recruited to ribosomes by the receptor for activated protein C kinase 1 (RACK1). RACK1 is an integral component of the 40S ribosome and adaptor for protein kinases. Ribosome-associated JNK phosphorylates the eukaryotic translation elongation factor 1A isoform 2 (eEF1A2) on serines 205 and 358 to promote degradation of NSPs by the proteasome. These findings establish a role for a RACK1/JNK/eEF1A2 complex in the quality control of NSPs in response to stress.

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p90RSK2 mediates anti-anoikis signals by both transcription-dependent and -independent mechanisms.

Publication Date: 2013 Apr 22 PMID: 23608533
Authors: Jin, L. - Li, D. - Lee, J. S. - Elf, S. - Alesi, G. N. - Fan, J. - Kang, H. B. - Wang, D. - Fu, H. - Taunton, J. - Boggon, T. J. - Tucker, M. - Gu, T. L. - Chen, Z. G. - Shin, D. M. - Khuri, F. R. - Kang, S.
Journal: Mol Cell Biol

How invasive and metastatic tumor cells evade from anoikis induction remains unclear. We found that knockdown of RSK2 sensitizes diverse cancer cells to anoikis induction, which is mediated through phosphorylation targets, including apoptosis signal-regulating kinase 1 (ASK1) and cAMP response element-binding (CREB). We provide evidence to show that RSK2 inhibits ASK1 by phosphorylating S83, T1109 and T1326 through novel mechanism in which phospho-T1109/T1326 inhibits ATP binding to ASK1, while phospho-S83 attenuates ASK1 substrate MKK6 binding. Moreover, RSK2-CREB signaling pathway provides anti-anoikis protection by regulating gene expression of protein effectors that are involved in cell death regulation, including anti-apoptotic protein tyrosine kinase 6 (PTK6) and pro-apoptotic inhibitor of growth protein 3 (ING3). PTK6 overexpression or ING3 knockdown in addition to ASK1 knockdown further rescued the increased sensitivity to anoikis induction in RSK2 knockdown cells. These data together suggest that RSK2 functions as a signal integrator to provide anti-anoikis protection to cancer cells in both transcription-independent and -dependent manners, in part by signaling through ASK1 and CREB, and contributes to cancer cell invasion and tumor metastasis.

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Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS.

Publication Date: 2013 Jun PMID: 23589333
Authors: Jun, J. E. - Yang, M. - Chen, H. - Chakraborty, A. K. - Roose, J. P.
Journal: Mol Cell Biol

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cgamma (PLCgamma)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation.

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BRD4 Coordinates Recruitment of Pause Release Factor P-TEFb and the Pausing Complex NELF/DSIF To Regulate Transcription Elongation of Interferon-Stimulated Genes.

Publication Date: 2013 Jun PMID: 23589332
Authors: Patel, M. C. - Debrosse, M. - Smith, M. - Dey, A. - Huynh, W. - Sarai, N. - Heightman, T. D. - Tamura, T. - Ozato, K.
Journal: Mol Cell Biol

RNA polymerase II (Pol II) and the pausing complex, NELF and DSIF, are detected near the transcription start site (TSS) of many active and silent genes. Active transcription starts when the pause release factor P-TEFb is recruited to initiate productive elongation. However, the mechanism of P-TEFb recruitment and regulation of NELF/DSIF during transcription is not fully understood. We investigated this question in interferon (IFN)-stimulated transcription, focusing on BRD4, a BET family protein that interacts with P-TEFb. Besides P-TEFb, BRD4 binds to acetylated histones through the bromodomain. We found that BRD4 and P-TEFb, although not present prior to IFN treatment, were robustly recruited to IFN-stimulated genes (ISGs) after stimulation. Likewise, NELF and DSIF prior to stimulation were hardly detectable on ISGs, which were strongly recruited after IFN treatment. A shRNA-based knockdown assay of NELF revealed that it negatively regulates the passage of Pol II and DSIF across the ISGs during elongation, reducing total ISG transcript output. Analyses with a BRD4 small-molecule inhibitor showed that IFN-induced recruitment of P-TEFb and NELF/DSIF was under the control of BRD4. We suggest a model where BRD4 coordinates both positive and negative regulation of ISG elongation.

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Macrophage Fusion Is Controlled by the Cytoplasmic Protein Tyrosine Phosphatase PTP-PEST/PTPN12.

Publication Date: 2013 Jun PMID: 23589331
Authors: Rhee, I. - Davidson, D. - Souza, C. M. - Vacher, J. - Veillette, A.
Journal: Mol Cell Biol

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as beta-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

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CAPNS1 Regulates USP1 Stability and Maintenance of Genome Integrity.

Publication Date: 2013 Jun PMID: 23589330
Authors: Cataldo, F. - Peche, L. Y. - Klaric, E. - Brancolini, C. - Myers, M. P. - Demarchi, F. - Schneider, C.
Journal: Mol Cell Biol

Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (mu-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Here we show that calpain is required for the stability of the deubiquitinating enzyme USP1 in several cell lines. USP1 modulates DNA replication polymerase choice and repair by deubiquitinating PCNA. The ubiquitinated form of the USP1 substrate PCNA is stabilized in CAPNS1-depleted U2OS cells and mouse embryonic fibroblasts (MEFs), favoring polymerase-eta loading on chromatin and increased mutagenesis. USP1 degradation directed by the cell cycle regulator APC/C(cdh1), which marks USP1 for destruction in the G1 phase, is upregulated in CAPNS1-depleted cells. USP1 stability can be rescued upon forced expression of calpain-activated Cdk5/p25, previously reported as a cdh1 repressor. These data suggest that calpain stabilizes USP1 by activating Cdk5, which in turn inhibits cdh1 and, consequently, USP1 degradation. Altogether these findings point to a connection between the calpain system and the ubiquitin pathway in the regulation of DNA damage response and place calpain at the interface between cell cycle modulation and DNA repair.

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Arsenic Inhibits Autophagic Flux, Activating the Nrf2-Keap1 Pathway in a p62-Dependent Manner.

Publication Date: 2013 Jun PMID: 23589329
Authors: Lau, A. - Zheng, Y. - Tao, S. - Wang, H. - Whitman, S. A. - White, E. - Zhang, D. D.
Journal: Mol Cell Biol

The Nrf2-Keap1 signaling pathway is a protective mechanism promoting cell survival. Activation of the Nrf2 pathway by natural compounds has been proven to be an effective strategy for chemoprevention. Interestingly, a cancer-promoting function of Nrf2 has recently been observed in many types of tumors due to deregulation of the Nrf2-Keap1 axis, which leads to constitutive activation of Nrf2. Here, we report a novel mechanism of Nrf2 activation by arsenic that is distinct from that of chemopreventive compounds. Arsenic deregulates the autophagic pathway through blockage of autophagic flux, resulting in accumulation of autophagosomes and sequestration of p62, Keap1, and LC3. Thus, arsenic activates Nrf2 through a noncanonical mechanism (p62 dependent), leading to a chronic, sustained activation of Nrf2. In contrast, activation of Nrf2 by sulforaphane (SF) and tert-butylhydroquinone (tBHQ) depends upon Keap1-C151 and not p62 (the canonical mechanism). More importantly, SF and tBHQ do not have any effect on autophagy. In fact, SF and tBHQ alleviate arsenic-mediated deregulation of autophagy. Collectively, these findings provide evidence that arsenic causes prolonged activation of Nrf2 through autophagy dysfunction, possibly providing a scenario similar to that of constitutive activation of Nrf2 found in certain human cancers. This may represent a previously unrecognized mechanism underlying arsenic toxicity and carcinogenicity in humans.

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NuMA Is Required for the Selective Induction of p53 Target Genes.

Publication Date: 2013 Jun PMID: 23589328
Authors: Ohata, H. - Miyazaki, M. - Otomo, R. - Matsushima-Hibiya, Y. - Otsubo, C. - Nagase, T. - Arakawa, H. - Yokota, J. - Nakagama, H. - Taya, Y. - Enari, M.
Journal: Mol Cell Biol

The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription.

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Establishing Links between Endoplasmic Reticulum-Mediated Hormesis and Cancer.

Publication Date: 2013 Jun PMID: 23572563
Authors: Mollereau, B.
Journal: Mol Cell Biol

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Macrophage glucose-6-phosphate dehydrogenase stimulates proinflammatory responses with oxidative stress.

Publication Date: 2013 Jun PMID: 23572562
Authors: Ham, M. - Lee, J. W. - Choi, A. H. - Jang, H. - Choi, G. - Park, J. - Kozuka, C. - Sears, D. D. - Masuzaki, H. - Kim, J. B.
Journal: Mol Cell Biol

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that regulates cellular redox potential. In this study, we demonstrate that macrophage G6PD plays an important role in the modulation of proinflammatory responses and oxidative stress. The G6PD levels in macrophages in the adipose tissue of obese animals were elevated, and G6PD mRNA levels positively correlated with those of proinflammatory genes. Lipopolysaccharide (LPS) and free fatty acids, which initiate proinflammatory signals, stimulated macrophage G6PD. Overexpression of macrophage G6PD potentiated the expression of proinflammatory and pro-oxidative genes responsible for the aggravation of insulin sensitivity in adipocytes. In contrast, when macrophage G6PD was inhibited or suppressed via chemical inhibitors or small interfering RNA (siRNA), respectively, basal and LPS-induced proinflammatory gene expression was attenuated. Furthermore, macrophage G6PD increased activation of the p38 mitogen-activated protein kinase (MAPK) and NF-kappaB pathways, which may lead to a vicious cycle of oxidative stress and proinflammatory cascade. Together, these data suggest that an abnormal increase of G6PD in macrophages promotes oxidative stress and inflammatory responses in the adipose tissue of obese animals.

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LOXL4 Is Induced by Transforming Growth Factor beta1 through Smad and JunB/Fra2 and Contributes to Vascular Matrix Remodeling.

Publication Date: 2013 Jun PMID: 23572561
Authors: Busnadiego, O. - Gonzalez-Santamaria, J. - Lagares, D. - Guinea-Viniegra, J. - Pichol-Thievend, C. - Muller, L. - Rodriguez-Pascual, F.
Journal: Mol Cell Biol

Transforming growth factor beta1 (TGF-beta1) is a pleiotropic factor involved in the regulation of extracellular matrix (ECM) synthesis and remodeling. In search for novel genes mediating the action of TGF-beta1 on vascular ECM, we identified the member of the lysyl oxidase family of matrix-remodeling enzymes, lysyl oxidase-like 4 (LOXL4), as a direct target of TGF-beta1 in aortic endothelial cells, and we dissected the molecular mechanism of its induction. Deletion mapping and mutagenesis analysis of the LOXL4 promoter demonstrated the absolute requirement of a distal enhancer containing an activator protein 1 (AP-1) site and a Smad binding element for TGF-beta1 to induce LOXL4 expression. Functional cooperation between Smad proteins and the AP-1 complex composed of JunB/Fra2 accounted for the action of TGF-beta1, which involved the extracellular signal-regulated kinase (ERK)-dependent phosphorylation of Fra2. We furthermore provide evidence that LOXL4 was extracellularly secreted and significantly contributed to ECM deposition and assembly. These results suggest that TGF-beta1-dependent expression of LOXL4 plays a role in vascular ECM homeostasis, contributing to vascular processes associated with ECM remodeling and fibrosis.

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Regulatory nexus of synthesis and degradation deciphers cellular nrf2 expression levels.

Publication Date: 2013 Jun PMID: 23572560
Authors: Suzuki, T. - Shibata, T. - Takaya, K. - Shiraishi, K. - Kohno, T. - Kunitoh, H. - Tsuta, K. - Furuta, K. - Goto, K. - Hosoda, F. - Sakamoto, H. - Motohashi, H. - Yamamoto, M.
Journal: Mol Cell Biol

Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine.

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The unfolded protein response selectively targets active smoothened mutants.

Publication Date: 2013 Jun PMID: 23572559
Authors: Marada, S. - Stewart, D. P. - Bodeen, W. J. - Han, Y. G. - Ogden, S. K.
Journal: Mol Cell Biol

The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease.

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Constitutively Active ALK2 Receptor Mutants Require Type II Receptor Cooperation.

Publication Date: 2013 Jun PMID: 23572558
Authors: Bagarova, J. - Vonner, A. J. - Armstrong, K. A. - Borgermann, J. - Lai, C. S. - Deng, D. Y. - Beppu, H. - Alfano, I. - Filippakopoulos, P. - Morrell, N. W. - Bullock, A. N. - Knaus, P. - Mishina, Y. - Yu, P. B.
Journal: Mol Cell Biol

Constitutively activating mutations in receptor kinases recruit downstream effector pathways independently of upstream signaling, with consequences ranging from developmental syndromes to cancer. Classic fibrodysplasia ossificans progressiva (FOP) is a congenital syndrome resulting from highly conserved activating mutations of the glycine-serine-rich (GS) regulatory domain of ACVR1, encoding bone morphogenetic protein (BMP) type I receptor ALK2, which lead to inappropriate signaling and heterotopic ossification of soft tissues. It is unclear if constitutively active mutant ALK2 receptors (caALK2) can function independently of signaling complexes with type II receptors and ligands. We found that ablation of BmpRII and ActRIIa abrogated BMP ligand-mediated and caALK2-mediated signaling and transcription in cells and disrupted caALK2-induced heterotopic ossification in mice. Signaling via GS domain ALK2 mutants could be restored by the expression of either BMP type II receptor. The contribution of BMP type II receptors was independent of their ligand-binding or kinase function but was dependent upon an intact cytoplasmic domain. These data demonstrate that GS domain ALK2 mutants act independently of upstream signaling but may require a nonenzymatic scaffolding function provided by type II receptors to form functional, apparently ligand-independent signaling complexes. These findings define the minimal requirements for signaling of GS domain ALK2 mutants, with implications for the therapeutic targeting of their activity in disease.

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Ablation of dihydroceramide desaturase 1, a therapeutic target for the treatment of metabolic diseases, simultaneously stimulates anabolic and catabolic signaling.

Publication Date: 2013 Jun PMID: 23547262
Authors: Siddique, M. M. - Li, Y. - Wang, L. - Ching, J. - Mal, M. - Ilkayeva, O. - Wu, Y. J. - Bay, B. H. - Summers, S. A.
Journal: Mol Cell Biol

The lipotoxicity hypothesis posits that obesity predisposes individuals to metabolic diseases because the oversupply of lipids to tissues not suited for fat storage leads to the accumulation of fat-derived molecules that impair tissue function. Means of combating this have been to stimulate anabolic processes to promote lipid storage or to promote catabolic ones to drive fat degradation. Herein, we demonstrate that ablating dihydroceramide desaturase 1 (Des1), an enzyme that produces ceramides, leads to the simultaneous activation of both anabolic and catabolic signaling pathways. In cells lacking Des1, the most common sphingolipids were replaced with dihydro forms lacking the double bond inserted by Des1. These cells exhibited a remarkably strong activation of the antiapoptotic and anabolic signaling pathway regulated by Akt/protein kinase B (PKB), were resistant to apoptosis, and were considerably larger than their wild-type counterparts. Paradoxically, Des1(-/-) cells exhibited high levels of autophagy. Mechanistic studies revealed that this resulted from impaired ATP synthesis due in part to decreased expression and activity of several complexes of the electron transport chain, particularly complex IV, leading to activation of AMP-activated protein kinase and its induction of the autophagosome. Thus, Des1 ablation enhanced starvation responses but dissociated them from the anabolic, prosurvival, and antiautophagic Akt/PKB pathways.

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Bmal1 and beta-Cell Clock Are Required for Adaptation to Circadian Disruption, and Their Loss of Function Leads to Oxidative Stress-Induced beta-Cell Failure in Mice.

Publication Date: 2013 Jun PMID: 23547261
Authors: Lee, J. - Moulik, M. - Fang, Z. - Saha, P. - Zou, F. - Xu, Y. - Nelson, D. L. - Ma, K. - Moore, D. D. - Yechoor, V. K.
Journal: Mol Cell Biol

Circadian disruption has deleterious effects on metabolism. Global deletion of Bmal1, a core clock gene, results in beta-cell dysfunction and diabetes. However, it is unknown if this is due to loss of cell-autonomous function of Bmal1 in beta cells. To address this, we generated mice with beta-cell clock disruption by deleting Bmal1 in beta cells (beta-Bmal1(-/-)). beta-Bmal1(-/-) mice develop diabetes due to loss of glucose-stimulated insulin secretion (GSIS). This loss of GSIS is due to the accumulation of reactive oxygen species (ROS) and consequent mitochondrial uncoupling, as it is fully rescued by scavenging of the ROS or by inhibition of uncoupling protein 2. The expression of the master antioxidant regulatory factor Nrf2 (nuclear factor erythroid 2-related factor 2) and its targets, Sesn2, Prdx3, Gclc, and Gclm, was decreased in beta-Bmal1(-/-) islets, which may contribute to the observed increase in ROS accumulation. In addition, by chromatin immunoprecipitation experiments, we show that Nrf2 is a direct transcriptional target of Bmal1. Interestingly, simulation of shift work-induced circadian misalignment in mice recapitulates many of the defects seen in Bmal1-deficient islets. Thus, the cell-autonomous function of Bmal1 is required for normal beta-cell function by mitigating oxidative stress and serves to preserve beta-cell function in the face of circadian misalignment.

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A Regulatory Role for MicroRNA 33* in Controlling Lipid Metabolism Gene Expression.

Publication Date: 2013 Jun PMID: 23547260
Authors: Goedeke, L. - Vales-Lara, F. M. - Fenstermaker, M. - Cirera-Salinas, D. - Chamorro-Jorganes, A. - Ramirez, C. M. - Mattison, J. A. - de Cabo, R. - Suarez, Y. - Fernandez-Hernando, C.
Journal: Mol Cell Biol

hsa-miR-33a and hsa-miR-33b, intronic microRNAs (miRNAs) located within the sterol regulatory element-binding protein 2 and 1 genes (Srebp-2 and -1), respectively, have recently been shown to regulate lipid homeostasis in concert with their host genes. Although the functional role of miR-33a and -b has been highly investigated, the role of their passenger strands, miR-33a* and -b*, remains unclear. Here, we demonstrate that miR-33a* and -b* accumulate to steady-state levels in human, mouse, and nonhuman primate tissues and share a similar lipid metabolism target gene network as their sister strands. Analogous to miR-33, miR-33* represses key enzymes involved in cholesterol efflux (ABCA1 and NPC1), fatty acid metabolism (CROT and CPT1a), and insulin signaling (IRS2). Moreover, miR-33* also targets key transcriptional regulators of lipid metabolism, including SRC1, SRC3, NFYC, and RIP140. Importantly, inhibition of either miR-33 or miR-33* rescues target gene expression in cells overexpressing pre-miR-33. Consistent with this, overexpression of miR-33* reduces fatty acid oxidation in human hepatic cells. Altogether, these data support a regulatory role for the miRNA* species and suggest that miR-33 regulates lipid metabolism through both arms of the miR-33/miR-33* duplex.

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Inactivation of the mTORC1-Eukaryotic Translation Initiation Factor 4E Pathway Alters Stress Granule Formation.

Publication Date: 2013 Jun PMID: 23547259
Authors: Fournier, M. J. - Coudert, L. - Mellaoui, S. - Adjibade, P. - Gareau, C. - Cote, M. F. - Sonenberg, N. - Gaudreault, R. C. - Mazroui, R.
Journal: Mol Cell Biol

Stress granules (SG) are cytoplasmic multimeric RNA bodies that form under stress conditions known to inhibit cap-dependent translation. SG contain translation initiation factors, RNA binding proteins, and signaling molecules. SG are known to inhibit apoptotic pathways, thus contributing to chemo- and radioresistance in tumor cells. However, whether stress granule formation involves oncogenic signaling pathways is currently unknown. Here, we report a novel role of the mTORC1-eukaryotic translation initiation factor 4E (eIF4E) pathway, a key regulator of cap-dependent translation initiation of oncogenic factors, in SG formation. mTORC1 specifically drives the eIF4E-mediated formation of SG through the phosphorylation of 4E-BP1, a key factor known to inhibit formation of the mTORC1-dependent eIF4E-eIF4GI interactions. Disrupting formation of SG by inactivation of mTOR with its specific inhibitor pp242 or by depletion of eIF4E or eIF4GI blocks the SG-associated antiapoptotic p21 pathway. Finally, pp242 sensitizes cancer cells to death in vitro and inhibits the growth of chemoresistant tumors in vivo. This work therefore highlights a novel role of the oncogenic mTORC1-eIF4E pathway, namely, the promotion of formation of antiapoptotic SG.

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Hsp27 and F-Box Protein beta-TrCP Promote Degradation of mRNA Decay Factor AUF1.

Publication Date: 2013 Jun PMID: 23530064
Authors: Li, M. L. - Defren, J. - Brewer, G.
Journal: Mol Cell Biol

Activation of the mitogen-activated protein (MAP) pathway kinases p38 and MK2 induces phosphorylation of the chaperone Hsp27 and stabilization of mRNAs containing AU-rich elements (AREs) (ARE-mRNAs). Likewise, expression of phosphomimetic mutant forms of Hsp27 also stabilizes ARE-mRNAs. It appears to perform this function by promoting degradation of the ARE-mRNA decay factor AUF1 by proteasomes. In this study, we examined the molecular mechanism linking Hsp27 phosphorylation to AUF1 degradation by proteasomes. AUF1 is a target of beta-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase Skp1-cullin-F-box protein complex, SCF(beta-TrCP). Depletion of beta-TrCP stabilized AUF1. In contrast, overexpression of beta-TrCP enhanced ubiquitination and degradation of AUF1 and led to stabilization of reporter mRNAs containing cytokine AREs. Enhanced AUF1 degradation required expression of phosphomimetic mutant forms of both Hsp27 and AUF1. Our results suggest that a signaling axis composed of p38 MAP kinase-MK2-Hsp27-beta-TrCP may promote AUF1 degradation by proteasomes and stabilization of cytokine ARE-mRNAs.

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MCJ/DnaJC15, an Endogenous Mitochondrial Repressor of the Respiratory Chain That Controls Metabolic Alterations.

Publication Date: 2013 Jun PMID: 23530063
Authors: Hatle, K. M. - Gummadidala, P. - Navasa, N. - Bernardo, E. - Dodge, J. - Silverstrim, B. - Fortner, K. - Burg, E. - Suratt, B. T. - Hammer, J. - Radermacher, M. - Taatjes, D. J. - Thornton, T. - Anguita, J. - Rincon, M.
Journal: Mol Cell Biol

Mitochondria are the main engine that generates ATP through oxidative phosphorylation within the respiratory chain. Mitochondrial respiration is regulated according to the metabolic needs of cells and can be modulated in response to metabolic changes. Little is known about the mechanisms that regulate this process. Here, we identify MCJ/DnaJC15 as a distinct cochaperone that localizes at the mitochondrial inner membrane, where it interacts preferentially with complex I of the electron transfer chain. We show that MCJ impairs the formation of supercomplexes and functions as a negative regulator of the respiratory chain. The loss of MCJ leads to increased complex I activity, mitochondrial membrane potential, and ATP production. Although MCJ is dispensable for mitochondrial function under normal physiological conditions, MCJ deficiency affects the pathophysiology resulting from metabolic alterations. Thus, enhanced mitochondrial respiration in the absence of MCJ prevents the pathological accumulation of lipids in the liver in response to both fasting and a high-cholesterol diet. Impaired expression or loss of MCJ expression may therefore result in a "rapid" metabolism that mitigates the consequences of metabolic disorders.

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Epithelial Tyrosine Phosphatase SHP-2 Protects against Intestinal Inflammation in Mice.

Publication Date: 2013 Jun PMID: 23530062
Authors: Coulombe, G. - Leblanc, C. - Cagnol, S. - Maloum, F. - Lemieux, E. - Perreault, N. - Feng, G. S. - Boudreau, F. - Rivard, N.
Journal: Mol Cell Biol

Polymorphisms of PTPN11 encoding SHP-2 are biomarkers for ulcerative colitis (UC) susceptibility. However, their functional relevance is unknown. We thus investigated the role of epithelial SHP-2 in the control of intestinal homeostasis. Mice with an intestinal epithelial cell-specific SHP-2 deletion (SHP-2(IEC-KO) mice) were generated. Control and SHP-2(IEC-KO) mice were monitored for clinical symptoms and sacrificed for histological staining and Western blot analyses. Cytokines and chemokines, as well as intestinal permeability, were quantified. SHP-2 mRNA expression was evaluated in control and UC patients. SHP-2(IEC-KO) mice showed growth retardation compared to control littermates and rapidly developed severe colitis. Colon architecture was markedly altered with infiltration of immune cells, crypt abscesses, neutrophil accumulation, and reduced goblet cell numbers. Decreased expression of claudins was associated with enhanced intestinal permeability in mutant SHP-2(IEC-KO) mice. Inflammatory transcription factors Stat3 and NF-kappaB were hyperactivated early in the mutant colonic epithelium. Levels of several epithelial chemokines and cytokines were markedly enhanced in SHP-2(IEC-KO) mice. Of note, antibiotic treatment remarkably impaired the development of colitis in SHP-2(IEC-KO) mice. Finally, SHP-2 mRNA levels were significantly reduced in intestinal biopsy specimens from UC patients. Our results establish intestinal epithelial SHP-2 as a critical determinant for prevention of gut inflammation.

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Negative feedback regulation of the yeast cth1 and cth2 mRNA binding proteins is required for adaptation to iron deficiency and iron supplementation.

Publication Date: 2013 Jun PMID: 23530061
Authors: Martinez-Pastor, M. - Vergara, S. V. - Puig, S. - Thiele, D. J.
Journal: Mol Cell Biol

Iron (Fe) is an essential element for all eukaryotic organisms because it functions as a cofactor in a wide range of biochemical processes. Cells have developed sophisticated mechanisms to tightly control Fe utilization in response to alterations in cellular demands and bioavailability. In response to Fe deficiency, the yeast Saccharomyces cerevisiae activates transcription of the CTH1 and CTH2 genes, which encode proteins that bind to AU-rich elements (AREs) within the 3' untranslated regions (3'UTRs) of many mRNAs, leading to metabolic reprogramming of Fe-dependent pathways and decreased Fe storage. The precise mechanisms underlying Cth1 and Cth2 function and regulation are incompletely understood. We report here that the Cth1 and Cth2 proteins specifically bind in vivo to AREs located at the 3'UTRs of their own transcripts in an auto- and cross-regulated mechanism that limits their expression. By mutagenesis of the AREs within the CTH2 transcript, we demonstrate that a Cth2 negative-feedback loop is required for the efficient decline in Cth2 protein levels observed upon a rapid rise in Fe availability. Importantly, Cth2 autoregulation is critical for the appropriate recovery of Fe-dependent processes and resumption of growth in response to a change from Fe deficiency to Fe supplementation.

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Farnesoid x receptor inhibits the transcriptional activity of carbohydrate response element binding protein in human hepatocytes.

Publication Date: 2013 Jun PMID: 23530060
Authors: Caron, S. - Huaman Samanez, C. - Dehondt, H. - Ploton, M. - Briand, O. - Lien, F. - Dorchies, E. - Dumont, J. - Postic, C. - Cariou, B. - Lefebvre, P. - Staels, B.
Journal: Mol Cell Biol

The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4alpha (HNF4alpha). FXR interacts directly with ChREBP and HNF4alpha proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4alpha, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4alpha binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes.

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Fanconi Anemia Group J Helicase and MRE11 Nuclease Interact To Facilitate the DNA Damage Response.

Publication Date: 2013 Jun PMID: 23530059
Authors: Suhasini, A. N. - Sommers, J. A. - Muniandy, P. A. - Coulombe, Y. - Cantor, S. B. - Masson, J. Y. - Seidman, M. M. - Brosh, R. M. Jr
Journal: Mol Cell Biol

FANCJ mutations are linked to Fanconi anemia (FA) and increase breast cancer risk. FANCJ encodes a DNA helicase implicated in homologous recombination (HR) repair of double-strand breaks (DSBs) and interstrand cross-links (ICLs), but its mechanism of action is not well understood. Here we show with live-cell imaging that FANCJ recruitment to laser-induced DSBs but not psoralen-induced ICLs is dependent on nuclease-active MRE11. FANCJ interacts directly with MRE11 and inhibits its exonuclease activity in a specific manner, suggesting that FANCJ regulates the MRE11 nuclease to facilitate DSB processing and appropriate end resection. Cells deficient in FANCJ and MRE11 show increased ionizing radiation (IR) resistance, reduced numbers of gammaH2AX and RAD51 foci, and elevated numbers of DNA-dependent protein kinase catalytic subunit foci, suggesting that HR is compromised and the nonhomologous end-joining (NHEJ) pathway is elicited to help cells cope with IR-induced strand breaks. Interplay between FANCJ and MRE11 ensures a normal response to IR-induced DSBs, whereas FANCJ involvement in ICL repair is regulated by MLH1 and the FA pathway. Our findings are discussed in light of the current model for HR repair.

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MicroRNA 9-3p Targets beta1 Integrin To Sensitize Claudin-Low Breast Cancer Cells to MEK Inhibition.

Publication Date: 2013 Jun PMID: 23530058
Authors: Zawistowski, J. S. - Nakamura, K. - Parker, J. S. - Granger, D. A. - Golitz, B. T. - Johnson, G. L.
Journal: Mol Cell Biol

MEK1/2 inhibitors such as AZD6244 are in clinical trials for the treatment of multiple cancers, including breast cancer. Targeted kinase inhibition can induce compensatory kinome changes, rendering single therapeutic agents ineffective. To identify target proteins to be used in a combinatorial approach to inhibit tumor cell growth, we used a novel strategy that identified microRNAs (miRNAs) that synergized with AZD6244 to inhibit the viability of the claudin-low breast cancer cell line MDA-MB-231. Screening of a miRNA mimic library revealed the ability of miR-9-3p to significantly enhance AZD6244-induced extracellular signal-regulated kinase inhibition and growth arrest, while miR-9-3p had little effect on growth alone. Promoter methylation of mir-9 genes correlated with low expression of miR-9-3p in different breast cancer cell lines. Consistent with miR-9-3p having synthetic enhancer tumor suppressor characteristics, miR-9-3p expression in combination with MEK inhibitor caused a sustained loss of c-MYC expression and growth inhibition. The beta1 integrin gene (ITGB1) was identified as a new miR-9-3p target, and the growth inhibition seen with small interfering RNA knockdown or antibody blocking of ITGB1 in combination with MEK inhibitor phenocopied the growth inhibition seen with miR-9-3p plus AZD6244. The miRNA screen led to identification of a druggable protein, ITGB1, whose functional inhibition synergizes with MEK inhibitor.

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Structural Basis for Activation of ZAP-70 by Phosphorylation of the SH2-Kinase Linker.

Publication Date: 2013 Jun PMID: 23530057
Authors: Yan, Q. - Barros, T. - Visperas, P. R. - Deindl, S. - Kadlecek, T. A. - Weiss, A. - Kuriyan, J.
Journal: Mol Cell Biol

Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only approximately 5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 approximately 100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck.

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Arkadia, a Novel SUMO-Targeted Ubiquitin Ligase Involved in PML Degradation.

Publication Date: 2013 Jun PMID: 23530056
Authors: Erker, Y. - Neyret-Kahn, H. - Seeler, J. S. - Dejean, A. - Atfi, A. - Levy, L.
Journal: Mol Cell Biol

Arkadia is a RING domain E3 ubiquitin ligase that activates the transforming growth factor beta (TGF-beta) pathway by inducing degradation of the inhibitor SnoN/Ski. Here we show that Arkadia contains three successive SUMO-interacting motifs (SIMs) that mediate noncovalent interaction with poly-SUMO2. We identify the third SIM (VVDL) of Arkadia to be the most relevant one in this interaction. Furthermore, we provide evidence that Arkadia can function as a SUMO-targeted ubiquitin ligase (STUBL) by ubiquitinating SUMO chains. While the SIMs of Arkadia are not essential for SnoN/Ski degradation in response to TGF-beta, we show that they are necessary for the interaction of Arkadia with polysumoylated PML in response to arsenic and its concomitant accumulation into PML nuclear bodies. Moreover, Arkadia depletion leads to accumulation of polysumoylated PML in response to arsenic, highlighting a requirement of Arkadia for arsenic-induced degradation of polysumoylated PML. Interestingly, Arkadia homodimerizes but does not heterodimerize with RNF4, the other STUBL involved in PML degradation, suggesting that these two E3 ligases do not act synergistically but most probably act independently during this process. Altogether, these results identify Arkadia to be a novel STUBL that can trigger degradation of signal-induced polysumoylated proteins.

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Integration of Apoptosis Signal-Regulating Kinase 1-Mediated Stress Signaling with the Akt/Protein Kinase B-IkappaB Kinase Cascade.

Publication Date: 2013 Jun PMID: 23530055
Authors: Puckett, M. C. - Goldman, E. H. - Cockrell, L. M. - Huang, B. - Kasinski, A. L. - Du, Y. - Wang, C. Y. - Lin, A. - Ichijo, H. - Khuri, F. - Fu, H.
Journal: Mol Cell Biol

Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of kappaB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-kappaB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development.

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Deciphering Dynamic Dose Responses of Natural Promoters and Single cis Elements upon Osmotic and Oxidative Stress in Yeast.

Publication Date: 2013 Jun PMID: 23530054
Authors: Dolz-Edo, L. - Rienzo, A. - Poveda-Huertes, D. - Pascual-Ahuir, A. - Proft, M.
Journal: Mol Cell Biol

Fine-tuned activation of gene expression in response to stress is the result of dynamic interactions of transcription factors with specific promoter binding sites. In the study described here we used a time-resolved luciferase reporter assay in living Saccharomyces cerevisiae yeast cells to gain insights into how osmotic and oxidative stress signals modulate gene expression in a dose-sensitive manner. Specifically, the dose-response behavior of four different natural promoters (GRE2, CTT1, SOD2, and CCP1) reveals differences in their sensitivity and dynamics in response to different salt and oxidative stimuli. Characteristic dose-response profiles were also obtained for artificial promoters driven by only one type of stress-regulated consensus element, such as the cyclic AMP-responsive element, stress response element, or AP-1 site. Oxidative and osmotic stress signals activate these elements separately and with different sensitivities through different signaling molecules. Combination of stress-activated cis elements does not, in general, enhance the absolute expression levels; however, specific combinations can increase the inducibility of the promoter in response to different stress doses. Finally, we show that the stress tolerance of the cell critically modulates the dynamics of its transcriptional response in the case of oxidative stress.

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Erythropoiesis in the absence of adult hemoglobin.

Publication Date: 2013 Jun PMID: 23530053
Authors: Liu, S. - McConnell, S. C. - Ryan, T. M.
Journal: Mol Cell Biol

During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucleated erythrocytes. Although Hb is one of the most extensively studied proteins, the role of Hb in erythroid lineage commitment, differentiation, and maturation remains unclear. In this study, we generate mouse embryos and embryonic stem (ES) cells with all of the adult alpha and beta globin genes deleted (Hb Null). While Hb Null embryos die in midgestation, adult globin genes are not required for primitive or definitive erythroid lineage commitment. In vitro differentiation of Hb Null ES cells generates viable definitive proerythroblasts that undergo apoptosis upon terminal differentiation. Surprisingly, all stages of Hb Null-derived definitive erythroblasts develop normally in vivo in chimeric mice, and Hb Null erythroid cells undergo enucleation to form reticulocytes. Free heme toxicity is not observed in Hb Null-derived erythroblasts. Transplantation of Hb Null-derived bone marrow cells provides short-term radioprotection of lethally irradiated recipients, whose progressive anemia results in an erythroid hyperplasia composed entirely of Hb Null-derived erythroblasts. This novel experimental model system enables the role played by Hb in erythroid cell enucleation, cytoskeleton maturation, and heme and iron regulation to be studied.

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Brain-Derived Neurotrophic Factor Induces Matrix Metalloproteinase 9 Expression in Neurons via the Serum Response Factor/c-Fos Pathway.

Publication Date: 2013 Jun PMID: 23508111
Authors: Kuzniewska, B. - Rejmak, E. - Malik, A. R. - Jaworski, J. - Kaczmarek, L. - Kalita, K.
Journal: Mol Cell Biol

Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the regulation of the transcription of genes that encode proplasticity proteins. In the present study, we provide evidence that stimulation of rat primary cortical neurons with BDNF upregulates matrix metalloproteinase 9 (MMP-9) mRNA and protein levels and increases enzymatic activity. The BDNF-induced MMP-9 transcription was dependent on extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and c-Fos expression. Overexpression of AP-1 dimers in neurons led to MMP-9 promoter activation, with the most potent being those that contained c-Fos, whereas knockdown of endogenous c-Fos by small hairpin RNA (shRNA) reduced BDNF-mediated MMP-9 transcription. Additionally, mutation of the proximal AP-1 binding site in the MMP-9 promoter inhibited the activation of MMP-9 transcription. BDNF stimulation of neurons induced binding of endogenous c-Fos to the proximal MMP-9 promoter region. Furthermore, as the c-Fos gene is a known target of serum response factor (SRF), we investigated whether SRF contributes to MMP-9 transcription. Inhibition of SRF and its cofactors by either overexpression of dominant negative mutants or shRNA decreased MMP-9 promoter activation. In contrast, MMP-9 transcription was not dependent on CREB activity. Finally, we showed that neuronal activity stimulates MMP-9 transcription in a tyrosine kinase receptor B (TrkB)-dependent manner.

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Inhibition of nonsense-mediated RNA decay activates autophagy.

Publication Date: 2013 Jun PMID: 23508110
Authors: Wengrod, J. - Martin, L. - Wang, D. - Frischmeyer-Guerrerio, P. - Dietz, H. C. - Gardner, L. B.
Journal: Mol Cell Biol

Nonsense-mediated RNA decay (NMD) is an mRNA surveillance mechanism which rapidly degrades select cytoplasmic mRNAs. We and others have shown that NMD is a dynamically regulated process inhibited by amino acid deprivation, hypoxia, and other cellular stresses commonly generated by the tumor microenvironment. This inhibition of NMD can result in the accumulation of misfolded, mutated, and aggregated proteins, but how cells adapt to these aberrant proteins is unknown. Here we demonstrate that the inhibition of NMD activates autophagy, an established protein surveillance mechanism, both in vitro and in vivo. Conversely, the hyperactivation of NMD blunts the induction of autophagy in response to a variety of cellular stresses. The regulation of autophagy by NMD is due, in part, to stabilization of the documented NMD target ATF4. NMD inhibition increases intracellular amino acids, a hallmark of autophagy, and the concomitant inhibition of autophagy and NMD, either molecularly or pharmacologically, leads to synergistic cell death. Together these studies indicate that autophagy is an adaptive response to NMD inhibition and uncover a novel relationship between an mRNA surveillance system and a protein surveillance system, with important implications for the treatment of cancer.

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The Glucocorticoid Receptor and KLF15 Regulate Gene Expression Dynamics and Integrate Signals through Feed-Forward Circuitry.

Publication Date: 2013 Jun PMID: 23508109
Authors: Sasse, S. K. - Mailloux, C. M. - Barczak, A. J. - Wang, Q. - Altonsy, M. O. - Jain, M. K. - Haldar, S. M. - Gerber, A. N.
Journal: Mol Cell Biol

The glucocorticoid receptor (GR) regulates adaptive transcriptional programs that alter metabolism in response to stress. Network properties that allow GR to tune gene expression to match specific physiologic demands are poorly understood. We analyzed the transcriptional consequences of GR activation in murine lungs deficient for KLF15, a transcriptional regulator of amino acid metabolism that is induced by glucocorticoids and fasting. Approximately 7% of glucocorticoid-regulated genes had altered expression in Klf15-knockdown (Klf15(-/-)) mice. KLF15 formed coherent and incoherent feed-forward circuits with GR that correlated with the expression dynamics of the glucocorticoid response. Coherent feed-forward gene regulation by GR and KLF15 was characterized by combinatorial activation of linked GR-KLF15 regulatory elements by both factors and increased GR occupancy, while expression of KLF15 reduced GR occupancy at the incoherent target, MT2A. Serum deprivation, which increased KLF15 expression in a GR-independent manner in vitro, enhanced glucocorticoid-mediated induction of feed-forward targets of GR and KLF15, such as the loci for the amino acid-metabolizing enzymes proline dehydrogenase and alpha-aminoadipic semialdehyde synthase. Our results establish feed-forward architecture as an organizational principle for the GR network and provide a novel mechanism through which GR integrates signals and regulates expression dynamics.

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RSUME Enhances Glucocorticoid Receptor SUMOylation and Transcriptional Activity.

Publication Date: 2013 Jun PMID: 23508108
Authors: Druker, J. - Liberman, A. C. - Antunica-Noguerol, M. - Gerez, J. - Paez-Pereda, M. - Rein, T. - Iniguez-Lluhi, J. A. - Holsboer, F. - Arzt, E.
Journal: Mol Cell Biol

Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation. We now demonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulates GR transcriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating that GR SUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-induced GR SUMOylation. These results show that inhibitory and stimulatory SUMO sites are present in the GR and at higher SUMOylation levels the stimulatory one becomes dominant.

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The ubiquitin-proteasome system regulates mitochondrial intermembrane space proteins.

Publication Date: 2013 Jun PMID: 23508107
Authors: Bragoszewski, P. - Gornicka, A. - Sztolsztener, M. E. - Chacinska, A.
Journal: Mol Cell Biol

Mitochondrial precursor proteins are synthesized in the cytosol and subsequently imported into mitochondria. The import of mitochondrial intermembrane space proteins is coupled with their oxidative folding and governed by the mitochondrial intermembrane space import and assembly (MIA) pathway. The cytosolic steps that precede mitochondrial import are not well understood. We identified a role for the ubiquitin-proteasome system in the biogenesis of intermembrane space proteins. Interestingly, the function of the ubiquitin-proteasome system is not restricted to conditions of mitochondrial protein import failure. The ubiquitin-proteasome system persistently removes a fraction of intermembrane space proteins under physiological conditions, acting as a negative regulator in the biogenesis of this class of proteins. Thus, the ubiquitin-proteasome system plays an important role in determining the levels of proteins targeted to the intermembrane space of mitochondria.

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