Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I Npropeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than AD-AMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.Collagens consist of the major structural proteins of the extracellular matrix (ECM) 1 and exist in both fibril-forming (e.g. collagens I-III, V, and XI) and nonfibrillar forms (1, 2). Molecules belonging to both categories are homotrimeric (e.g. collagen II) or heterotrimeric (e.g. collagen I) assemblies of specific ␣ chains, each the product of a single gene (1, 2). The molecular types of collagen, as well as the specific supramolecular aggregates they form, are often tissue-specific and provide a specialized function. For example, collagen I, the principal collagen of skin, is arranged in randomly oriented bundles in the dermis but in parallel bundles in tendons. Collagen II, a specific component of cartilage ECM, is arranged in an open meshwork that traps proteoglycans and facilitates resistance to compression. The synthesis, secretion, and assembly of collagens into specific supramolecular aggregates is a complex, multistep process (3, 4). Fibrillar collagens I-III are synthesized as a soluble procollagen monomer comprising a long triple helical "collagenous" region with smaller polypeptide extensions (propeptides) at the amino and carboxyl ends (4). Removal of the propeptides by specific enzymes, the N-and C-propeptidases (proteinases), is a prerequisite for the correct assembly of collagens I and II into growing fibrils (3, 4). The procollagen C-propeptidase is identical to bone morphogenetic protein-1 and processes all three of these fibrillar collagens (5). Biochemically distinct N-propeptidases with specificity for procollagens I and II or procollagen III are known (6). The bovine and human procollagen I N-propeptidases have been cloned (7,8). This enzyme (designated ADAMTS-2, EC 3.4.24.14), is a zinc...

These data demonstrate that Fgf18 can act as a trophic factor for elastic chondrocytes and their progenitors in vivo and articular chondrocytes cultured in vitro. Expression of Fgf18 and the genes for two of its receptors in chondrocytes suggests that Fgf18 may play an autocrine role in the biology of normal articular cartilage.

Molecular mechanisms controlling the assembly of cartilage-specific types II, IX and XI collagens into a heteropolymeric network of uniformly thin, unbanded fibrils are not well understood, but collagen XI has been implicated. The present study on cartilage from the homozygous chondrodysplasia (cho/cho) mouse adds support to this concept. In the absence of α1(XI) collagen chains, thick, banded collagen fibrils are formed in the extracellular matrix of cho/cho cartilage. A functional knock-out of the type XI collagen molecule has been assumed. We have re-examined this at the protein level to see if rather than a complete knock-out, alternative type XI chain assemblies were formed. Mass spectrometry of purified triple helical collagen from the rib cartilage of cho/ cho mice identified α1(V) and α2(XI) chains. These chains were recovered in roughly equal amounts based on Coomassie blue staining of SDS-PAGE gels, in addition to α1(II)/α3(XI) collagen chains. Using telopeptide-specific antibodies and Western blot analysis, it was further shown that type V/ XI trimers were present in the matrix cross-linked to each other and to type II collagen molecules to form heteropolymers. Cartilage from heterozygous (cho/+) mice contained a mix of α1(V) and α1 (XI) chains and a mix of thin and thick fibrils on transmission electron microscopy. In summary, the results imply that native type XI collagen molecules containing an α1(XI) chain are required to form uniformly thin fibrils and support a role for type XI collagen as the template for the characteristic type II collagen fibril network of developing cartilage.

Until now, no biological tools have been available to determine if a cross-linked collagen fibrillar network derived entirely from type IIA procollagen isoforms, can form in the extracellular matrix (ECM) of cartilage. Recently, homozygous knock-in transgenic mice (Col2a1+ex2, ki/ki) were generated that exclusively express the IIA procollagen isoform during post-natal development while type IIB procollagen, normally present in the ECM of wild type mice, is absent. The difference between these Col2a1 isoforms is the inclusion (IIA) or exclusion (IIB) of exon 2 that is alternatively spliced in a developmentally regulated manner. Specifically, chondroprogenitor cells synthesize predominantly IIA mRNA isoforms while differentiated chondrocytes produce mainly IIB mRNA isoforms. Recent characterization of the Col2a1+ex2 mice has surprisingly shown that disruption of alternative splicing does not affect overt cartilage formation. In the present study, biochemical analyses showed that type IIA collagen extracted from ki/ki mouse rib cartilage can form homopolymers that are stabilized predominantly by hydroxylysyl pyridinoline (HP) cross-links at levels that differed from wild type rib cartilage. The findings indicate that mature type II collagen derived exclusively from type IIA procollagen molecules can form hetero-fibrils with type XI collagen and contribute to cartilage structure and function. Heteropolymers with type XI collagen also formed. Electron microscopy revealed mainly thin type IIA collagen fibrils in ki/ki mouse rib cartilage. Immunoprecipitation and mass spectrometry of purified type XI collagen revealed a heterotrimeric molecular composition of α1(XI)α2(XI)α1(IIA) chains where the α1(IIA) chain is the IIA form of the α3(XI) chain. Since the N-propeptide of type XI collagen regulates type II collagen fibril diameter in cartilage, the retention of the exon 2-encoded IIA globular domain would structurally alter the N-propeptide of type XI collagen. This structural change may subsequently affect the regulatory function of type XI collagen resulting in the collagen fibril and cross-linking differences observed in this study.