Abnormal Collagen Fibrils in Cartilage of Matrilin-1/Matrilin-3-deficient Mice

Abstract

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1-and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1 ؊/؊ /Matn3 ؊/؊ and Matn3 ؊/؊ mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.Hyaline cartilage is a unique tissue, which contains relatively few cells (chondrocytes) embedded in an abundant extracellular matrix (ECM).3 The cartilage ECM assembles from collagens, proteoglycans, and non-collagenous glycoproteins that interact with each other and are organized into molecular networks. Heterotypic collagen fibrils, containing collagens II, IX, and XI, and the proteoglycan aggrecan, which forms large aggregates with hyaluronic acid, constitute the major structural components of the matrix providing tissue tensile strength and resistance against compressive forces, respectively. In addition to fibril forming collagens, articular cartilage contains beaded microfilament-forming collagen VI, while hypertrophic chondrocytes secrete collagen X. Other cartilage-specific ECM proteins play a role in matrix assembly by regulating collagen fibrillogenesis via binding to the surface of collagen fibers or by mediating interactions between the matrix and the chondrocytes.The matrilin family of ECM proteins is comprised of four structurally related members (1, 2). They contain one (matrilin-3) or two von Willebrand factor A (VWA) domain(s) (matrilin-1, -2, and -4), at least one epidermal growth factor (EGF)-like motif, and a C-terminal coiled-coil domain, which allows the formation of homo-and hetero-oligomers of variable stoichiometry. In mouse, matrilin-1 and matrilin-3 are expressed almost exclusively in cartilaginous tissues (3, ...

Abnormal bone quality in cartilage oligomeric matrix protein and matrilin 3 double‐deficient mice caused by increased tissue inhibitor of metalloproteinases 3 deposition and delayed aggrecan degradation

“…Even though the total amount of type II collagen seemed to be unchanged, the findings of both the antibody staining and the extractability experiments were markedly changed in the absence of COMP and matrilin 3. We speculate that the fibril structure is altered, since both COMP and matrilin 3 bind to collagens and are known to influence fibrillogenesis and collagen volume density, respectively (4,6,9,10,12). Moreover, consistent with the histologic findings, the physical bone parameters obtained from the peripheral QCT measurements showed alterations in the metaphysis, with an elevation of trabecular bone mineral content and density (Table 1).…”

Matrilin-1 Is Essential for Zebrafish Development by Facilitating Collagen II Secretion

“…Even mice double deficient for matrilin-1 and matrilin-3, which are both highly expressed in cartilage, did not show any overt phenotype. However, in depth analysis of the cartilage matrix of such mice revealed moderately increased collagen fibril diameters and an increased collagen volume density, which could, although less marked, be detected also in the corresponding single mutant mice (10). Taken together the results demonstrate that matrilin-1 and matrilin-3 modulate cartilage collagen fibrillogenesis and that functional compensation between the two proteins may occur.…”

“…Protein samples were obtained by sequential extraction as described (10). 1 volume of 6ϫ SDS sample buffer (2% (w/v) SDS, 10% (v/v) glycerol, 0.04% (w/v) bromphenol blue, 80 mM Tris-HCl, pH 6.8) was added to the protein extract and the sample subjected to 4 -12% (w/v) nonreducing SDS-PAGE.…”

“…The zebrafish extracellular matrix and the secretion and assembly of its components may be different from that in mammals. Despite the lack of severe phenotypes in either of the two matrilin-1-deficient mouse lines, both strains displayed alterations in collagen II fibril formation (7,10), pointing to some extent of conservation of matrilin-1 function between the two species.…”

Cartilage Collagen Fibril Network in Newborn Transgenic Mice Analyzed by Electron Microscopic Stereology

“…In the above and in most other studies examining the structure of extracellular matrix and collagen fibrils, the microscopic analyses have been qualitative in nature depending on qualitative visual estimation [Metsäranta et al, 1992;Garofalo et al, 1993;Thur et al, 2001;Barbieri et al, 2003;Nicolae et al, 2007]. Quantitative analysis of the collagen network can be performed either at the light microscopic or ultrastructural level.…”