Matrix Biology

Treatment of mitochondrial dysfunction in patients with collagen VI mutations.

Publication Date: 2008 May PMID: 18456160
Authors:
Journal: Matrix Biol

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The extracellular matrix protein WARP is a novel component of a distinct subset of basement membranes.

Publication Date: 2008 May PMID: 18314316
Authors: Allen, J. M. - Brachvogel, B. - Farlie, P. G. - Fitzgerald, J. - Bateman, J. F.
Journal: Matrix Biol

WARP is a recently described member of the von Willebrand factor A domain superfamily of extracellular matrix proteins, and is encoded by the Vwa1 gene. We have previously shown that WARP is a multimeric component of the chondrocyte pericellular matrix in articular cartilage and intervertebral disc, where it interacts with the basement membrane heparan sulfate proteoglycan perlecan. However, the tissue-specific expression of WARP in non-cartilaginous tissues and its localization in the extracellular matrix of other perlecan-containing tissues have not been analyzed in detail. To visualize WARP-expressing cells, we generated a reporter gene knock-in mouse by targeted replacement of the Vwa1 gene with beta-galactosidase. Analysis of reporter gene expression and WARP protein localization by immunostaining demonstrates that WARP is a component of a limited number of distinct basement membranes. WARP is expressed in the vasculature of neural tissues and in basement membrane structures of the peripheral nervous system. Furthermore, WARP is also expressed in the apical ectodermal ridge of developing limb buds, and in skeletal and cardiac muscle. These findings are the first evidence for WARP expression in non-cartilaginous tissues, and the identification of WARP as a component of a limited range of specialized basement membranes provides further evidence for the heterogeneous composition of basement membranes between different tissues.

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Collagen IX-deficiency seriously compromises growth cartilage development in mice.

Publication Date: 2008 May PMID: 18313277
Authors: Dreier, R. - Opolka, A. - Grifka, J. - Bruckner, P. - Grassel, S.
Journal: Matrix Biol

For a large part, skeletal development, growth, and repair occur by endochondral ossification which comprises an orderly sequence of consecutive steps of proliferation and late differentiation of chondrocytes. After vascular invasion into hypertrophic cartilage, the tissue is remodelled into bone. At all stages, the process is under tight environmental control exerted by a combination of regulators, including nutritional supply and signalling through growth factors, hormones, and cell-matrix-interactions. Therefore, genetic elimination of collagen IX, a stabilizing component of the periphery of thin cartilage fibrils, is expected to compromise extracellular matrix properties and, hence, the chondrocyte environment required for normal cartilage development and homeostasis. Here, we have shown that growth plate cartilage morphology is markedly disturbed in mice lacking collagen IX. Abnormalities were most prominent in late proliferative, pre-hypertrophic, and hypertrophic zones whereas resting and early proliferative zones were less affected. In central epiphyseal regions of long bones, newborn animals show grossly abnormal areas with strongly reduced cell numbers, irregular distribution of glycosaminoglycans in the extracellular matrix, and a profoundly disturbed columnar arrangement of chondrocytes with an irregular beta(1) integrin immunostaining. As a result, all long bones are shorter and broader in newborn Col9a1-/- mice. Remarkably, these abnormalities are attenuated in adult mice, but the number of cells per area still is too low due to reduced cell proliferation.

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Fibroblast adhesion results in the induction of a matrix remodeling gene expression program.

Publication Date: 2008 May PMID: 18291634
Authors: Kennedy, L. - Shi-Wen, X. - Carter, D. E. - Abraham, D. J. - Leask, A.
Journal: Matrix Biol

Fibrosis is believed to occur through the failure to terminate the normal tissue remodeling program. Tissue repair intimately involves the ability of fibroblasts to attach to extracellular matrix (ECM), resulting in cell migration and ECM contraction. Elevated, activated adhesive signaling is a key phenotypic hallmark of fibrotic cells. The precise contribution of adhesion to tissue remodeling and repair and fibrotic responses in fibroblasts is unclear, but involves focal adhesion kinase (FAK). FAK signals downstream of integrin-mediates attachment of fibroblasts to extracellular matrix. In this report, we show that FAK is required for the expression of a cohort of mRNAs encoding ECM and matrix remodeling genes including CCN2, alpha-smooth muscle actin (SMA) and type I collagen. Adhesion of fibroblasts to fibronectin, a component of the provisional matrix deposited in the initial phases of tissue repair, also resulted in the induction of CCN2, alpha-SMA and type I collagen mRNAs. Endothelin-1 (ET-1), a key inducer of pro-fibrotic gene expression, was also induced upon fibroblast attachment to ECM, and antagonism of the ET-1 receptors significantly reduced the ability of adhesion to induce expression of CCN2, alpha-SMA and type I collagen mRNAs. These results suggest that adhesion of fibroblasts to matrix during the initial phases of tissue remodeling and repair may actively contribute to the tissue repair program through the induction of pro-fibrotic gene expression.

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Ameloblastin and amelogenin share a common secretory pathway and are co-secreted during enamel formation.

Publication Date: 2008 May PMID: 18281204
Authors: Zalzal, S. F. - Smith, C. E. - Nanci, A.
Journal: Matrix Biol

The epithelially-derived ameloblasts secrete two main categories of extracellular matrix proteins, amelogenins (AMEL) and nonamelogenins. These proteins assume differential distributions in the forming enamel layer and thereby regulate deposition and structuring of the mineral phase. The objective of this study was to elucidate whether their distribution results from distinctive physicochemical behaviors or differences in intracellular routing. Dual-immunogold labeling was used to visualize the presence of AMEL and ameloblastin (AMBN), the major nonamelogenin, and quantify the proportion of secretory granules containing one or both of these proteins in ameloblasts during the phase of appositional growth of the enamel layer in continuously-erupting rat incisors. Some rats were treated with brefeldin A (BFA) to generate a synchronized cohort of newly-formed secretory granules. The results show that nearly 70% of granules contain both AMEL and AMBN, 13% label only for AMBN and 1% only for AMEL. These proportions reach 98% (AMEL+AMBN) and 2% (AMBN only) following BFA treatment. The observation that AMEL is almost always packaged with AMBN suggests a functional association between these two proteins. The subpopulation of granules containing only AMBN could be responsible for augmenting its local concentration along secretory surfaces against which hydroxyapatite crystals actively elongate.

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Collagen 11a1 is indirectly activated by lymphocyte enhancer-binding factor 1 (Lef1) and negatively regulates osteoblast maturation.

Publication Date: 2008 May PMID: 18280717
Authors: Kahler, R. A. - Yingst, S. M. - Hoeppner, L. H. - Jensen, E. D. - Krawczak, D. - Oxford, J. T. - Westendorf, J. J.
Journal: Matrix Biol

Alpha 1 (XI) collagen (Col11a1) is essential for normal skeletal development. Mutations in Col11a1 cause Marshall and Stickler syndromes, both of which are characterized by craniofacial abnormalities, nearsightedness and hearing deficiencies. Despite its link to human diseases, few studies have described factors that control Col11a1 transcription. We previously identified Col11a1 as a differentially expressed gene in Lef1-suppressed MC3T3 preosteoblasts. Here we report that Lef1 activates the Col11a1 promoter. This activation is dependent upon the DNA binding domain of Lef1, but does not require the beta-catenin interaction domain, suggesting that it is not responsive to Wnt signals. Targeted suppression of Col11a1 with an antisense morpholino accelerated osteoblastic differentiation and mineralization in C2C12 cells, similar to what was observed in Lef1-suppressed MC3T3 cells. Moreover incubation with a purified Col11a1 N-terminal fragment, V1B, prevented alkaline phosphatase expression in MC3T3 and C2C12 cells. These results suggest that Lef1 is an activator of the Col11a1 promoter and that Col11a1 suppresses terminal osteoblast differentiation.

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Tension is required for fibripositor formation.

Publication Date: 2008 May PMID: 18262777
Authors: Kapacee, Z. - Richardson, S. H. - Lu, Y. - Starborg, T. - Holmes, D. F. - Baar, K. - Kadler, K. E.
Journal: Matrix Biol

Embryonic tendon cells (ETCs) have actin-rich fibripositors that accompany parallel bundles of collagen fibrils in the extracellular matrix. To study fibripositor function, we have developed a three-dimensional cell culture system that promotes and maintains fibripositors. We show that ETCs cultured in fixed-length fibrin gels replace the fibrin during ~6 days in culture with parallel bundles of narrow-diameter collagen fibrils that are uniaxially aligned with fibripositors, thereby generating a tendon-like construct. Fibripositors occurred simultaneously with onset of parallel collagen fibrils. Interestingly, the constructs have a tendon-like crimp. In initial experiments to study the effects of tension, we showed that cutting the constructs resulted in loss of tension, loss of fibripositors and the appearance of immature fibrils with no preferred orientation.

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Formation of smooth muscle alpha actin filaments in CD34+ bone marrow cells on arterial elastic laminae: Potential role of SH2 domain-containing protein tyrosine phosphatase-1.

Publication Date: 2008 May PMID: 18258420
Authors: Liu, S. Q. - Tefft, B. J. - Zhang, A. - Zhang, L. Q. - Wu, Y. H.
Journal: Matrix Biol

Arterial smooth muscle cells (SMCs) are present in the elastic lamina-containing media, suggesting that the elastic laminae may regulate the development of SMCs. Here, we investigated the role of elastic laminae in regulating the formation of SM alpha actin filaments in mouse CD34+ bone marrow cells and the role of a protein tyrosine phosphatase, SH2 domain-containing protein tyrosine phosphatase (SHP)-1, in the mediation of this process. Mouse CD34+ bone marrow cells were isolated by magnetic separation and used for assessing the influence of elastic laminae and collagen matrix on the formation of SM alpha actin filaments. CD34+ cells with transgenic SHP-1 knockout or siRNA-mediated SHP-1 knockdown were used to assess the role of SHP-1 in mediating the formation of SM alpha actin filaments. In cell culture tests, elastic laminae, but not collagen matrix, stimulated the formation of SM alpha actin filaments in CD34+ cells. The phosphatase SHP-1 mediated the stimulatory effect of elastic laminae. The interaction of CD34+ cells with elastic laminae, but not with collagen matrix, induced activation of SHP-1. The suppression of SHP-1 by transgenic SHP-1 knockout or siRNA-mediated SHP-1 knockdown significantly reduced the formation of SM alpha actin filaments in CD34+ cells cultured on elastic laminae. The in vitro observations were confirmed by using an in vivo model of implantation of elastic lamina and collagen matrix scaffolds into the aorta. These observations suggest that elastic laminae stimulate the formation of SM alpha actin filaments in CD34+ bone marrow cells and SHP-1 mediates the stimulatory effect of elastic laminae.

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First molecular characterization and immunolocalization of keratoepithelin in adult human skeletal muscle.

Publication Date: 2008 May PMID: 18249103
Authors: Sciandra, F. - Morlacchi, S. - Allamand, V. - De Benedetti, G. - Macchia, G. - Petrucci, T. C. - Bozzi, M. - Brancaccio, A.
Journal: Matrix Biol

Keratoepithelin (KE) is an extracellular matrix protein that binds collagens, fibronectin, decorin, biglycan and integrins, interconnecting extracellular matrix components with resident cells in several tissues. KE has a molecular mass of 68 kDa and harbours four FAS1 domains named after those identified in the insect cell adhesion molecule fasciclin I. In humans, KE is preferentially expressed by the corneal epithelial layer and liberated towards the corneal stroma but it was also detected in the lung and in the bladder smooth muscle. No detailed information is available on the distribution of this protein in other human tissues. In this work, we have raised a polyclonal antibody against the recombinantly expressed human fourth FAS1 domain which is able to specifically detect KE in human skeletal muscle tissue extracts. Immunofluorescence experiments indicate that KE is localized around the perimysium and endomysium of each skeletal muscle fiber. The same kind of analysis shows that in muscle sections from patients affected by different forms of muscular dystrophy KE is upregulated and widely distributed in fibrotic tissues. The muscle specific expression of KE was also demonstrated by RT-PCR. In human skeletal muscle, KE may help to build up a bridge between collagen VI and yet unidentified muscle receptor(s), adding to the complexity of the adhesive molecular network established between muscle fibers and the surrounding basement membrane.

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Calcium indirectly regulates immunochemical reactivity and functional activities of the N-domain of thrombospondin-1.

Publication Date: 2008 May PMID: 18226512
Authors: Calzada, M. J. - Kuznetsova, S. A. - Sipes, J. M. - Rodrigues, R. G. - Cashel, J. A. - Annis, D. S. - Mosher, D. F. - Roberts, D. D.
Journal: Matrix Biol

Conformational changes induced in thrombospondin-1 by removal of calcium regulate interactions with some ligands of its N-modules. Because calcium binds primarily to elements of the C-terminal signature domain of thrombospondin-1, which are distant from the N-modules, such regulation was unexpected. To clarify the mechanism for this regulation, we compared ligand binding to the N-modules of thrombospondin-1 in the full-length protein and recombinant trimeric thrombospondin-1 truncated prior to the signature domain. Three monoclonal antibodies were identified that recognize the N-modules, two of which exhibit calcium-dependent binding to native thrombospondin-1 but not to the truncated trimeric protein. These antibodies or calcium selectively modulate interactions of fibronectin, heparin, sulfatide, alpha3beta1 integrin, tumor necrosis factor-alpha-stimulated gene-6 protein, and, to a lesser extent, alpha4beta1 integrin with native thrombospondin-1 but not with the truncated protein. These results indicate connectivity between calcium binding sites in the C-terminal signature domain and the N-modules of thrombospondin-1 that regulates ligand binding and functional activities of the N-modules.

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Ablation of collagen IX and COMP disrupts epiphyseal cartilage architecture.

Publication Date: 2008 May PMID: 18191556
Authors: Blumbach, K. - Niehoff, A. - Paulsson, M. - Zaucke, F.
Journal: Matrix Biol

Chondrodysplasias are a genetically heterogeneous group of skeletal disorders. Mutations in genes coding for cartilage oligomeric matrix protein (COMP), collagen IX and matrilin-3 have been described to cause the autosomal dominantly inherited form of multiple epiphyseal dysplasia (MED). Even though there is clear evidence that these cartilage matrix proteins interact with each other, their exact functions in matrix organisation and bone development still need to be elucidated. We generated a mouse model lacking both collagen IX and COMP to study the potential complementary role of these proteins in skeletal development. Mice deficient in both proteins exhibit shortened and widened long bones as well as an altered bone structure. They display severe growth plate abnormalities with large hypocellular areas in the central parts of the tibia. In addition, chondrocytes in the proliferative and hypertrophic zones do not show their typical columnar arrangement. These phenotypical traits were not observed in mice deficient only in COMP, while mice lacking only collagen IX showed similar growth plate disturbances and shorter and wider tibiae. The contribution of COMP to the phenotype of mice deficient in both collagen IX and COMP appears minor, even though clear differences in the deposition of matrilin-3 were detected.

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