Cellular heterogeneity in cultured human chondrocytes identified by antibodies specific for alpha 2(XI) collagen chains

Collagen type XI is a component of hyaline cartilage consisting of alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains; with 5-10% of the total collagen content, it is a minor but significant component next to type II collagen, but its function and precise localization in cartilaginous tissues is still unclear. Owing to the homology of the alpha 3(XI) and alpha 1(II) collagen chains, attempts to prepare specific antibodies to native type XI collagen have been unsuccessful in the past. In this study, we report on the preparation and use for immunohistochemistry of a polyclonal antibody specific for alpha 2(XI) denatured collagen chains. The antibody was prepared by immunization with the isolated alpha 2(XI) chain and reacts neither with native type XI collagen nor type I, II, V, or IX by ELISA or immunoblotting, nor with alpha 1(XI) or alpha 3(XI), but with alpha 2(XI) chains. Using this antibody, it was possible to specifically localize alpha 2(XI) in cartilage by pretreating tissue sections with 6 M urea. In double immunofluorescence staining experiments, the distribution of alpha 2(XI) as indicative for type XI collagen in fetal bovine and human cartilage was compared with that of type II collagen, using a monoclonal antibody to alpha 1(II). Type XI collagen was found throughout the matrix of hyaline cartilage. However, owing to cross-reactivity of the monoclonal anti-alpha 1(II) with alpha 3(XI), both antibodies produced the same staining pattern. Cellular heterogeneity was, however, detected in monolayer cultures of human chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in Chain Usage and Cross-linking Specificities of Cartilage Type V/XI Collagen Isoforms with Age and Tissue

Collagen type V/XI is a minor but essential component of collagen fibrils in vertebrates. We here report on age- and tissue-related variations in isoform usage in cartilages. With maturation of articular cartilage, the α1(V) chain progressively replaced the α2(XI) chain. A mix of the molecular isoforms, α1(XI)α1(V)α3(XI) and α1(XI)α2(XI)α3(XI), best explained this finding. A prominence of α1(V) chains is therefore characteristic and a potential biomarker of mature mammalian articular cartilage. Analysis of cross-linked peptides showed that the α1(V) chains were primarily cross-linked to α1(XI) chains in the tissue and hence an integral component of the V/XI polymer. From nucleus pulposus of the intervertebral disc (in which the bulk collagen monomer is type II as in articular cartilage), type V/XI collagen consisted of a mix of five genetically distinct chains, α1(XI), α2(XI), α3(XI), α1(V), and α2(V). These presumably were derived from several different molecular isoforms, including α1(XI)α2(XI)α3(XI), (α1(XI))₂α2(V), and others. Meniscal fibrocartilage shows yet another V/XI phenotype. The findings support and extend the concept that the clade B subfamily of COL5 and COL11 gene products should be considered members of the same collagen subfamily, from which, in combination with clade A gene products (COL2A1 or COL5A2), a range of molecular isoforms has evolved into tissue-dependent usage. We propose an evolving role for collagen V/XI isoforms as an adaptable polymeric template of fibril macro-architecture.The collagen framework of hyaline cartilages is based on a covalently cross-linked heteropolymeric network of types II, IX, and XI collagens. During development, collagen type IX molecules are covalently linked to the surface of thin, new fibrils of type II collagen polymerized on a template of type XI collagen (1–5). In fetal cartilage, type XI collagen is a heterotrimer of three genetically distinct chains, α1(XI), α2(XI), and α3(XI) in a 1:1:1 ratio (6–9). The α3(XI) chain has the same primary sequence as α1(II), but the chains differ in their post-translational processing and cross-linking properties (7–9). All three collagen subunits, II, IX, and XI, are heavily cross-linked in the same fibril through a lysyl oxidase-mediated mechanism (2, 5, 9). The location of the cross-links determined by sequence analysis of peptides prepared from proteolytically degraded fibrils reveals a high degree of chain specificity (9). Collagen XI molecules are linked to each other in a head-to-tail fashion by N-telopeptide² to helix cross-links and laterally to type II collagen molecules through α1(II) C-telopeptides (9). Isolated from mature articular cartilage, type XI collagen includes a significant pool of α1(V) chains (6), implying the presence of V/XI hybrid molecules. The ratio of type XI collagen to type II collagen is about 1 to 10 in fetal bovine and human epiphyseal cartilage when compared with 1 to 30 in adult articular cartilage. Similarly, the ratio of collagen IX to collagen II falls from about 1 to 10 to 1 to 100 between fetal and adult. In adult articular cartilage, most of the collagen IX is located in the immediate pericellular matrix (10–12).The intervertebral disc has a unique collagen architecture that combines features of ligament and cartilage in its morphology, function, and matrix biochemistry. The lamellar fabric of the outer annulus fibrosus combines collagens I and II fibrils in a complex weave with a radial gradient from mostly type I in the outermost layers and mostly type II in the interior. Nucleus pulposus, the gel-like center of the young intervertebral disc, has a similar collagen molecular phenotype to hyaline cartilage in which types II, IX, and XI collagens are the principal cross-linked fibrillar components (13–16). Collagen IX in the disc has a different protein isoform to that of hyaline cartilages. The α1(IX) chain is expressed as a short form that lacks the amino-terminal NC4 domain (16). One of the aims of the present study was to determine whether a unique pattern of type V/XI hybrid molecules is present in disc tissue when compared with articular cartilage and a more typical fibrocartilage, the knee meniscus.The results show an accumulation of collagen α1(V) chains as articular cartilage matures. A related but distinct complexity in chain usage in the type V/XI collagen of nucleus pulposus is also revealed. Such tissue diversity suggests that the different molecular isoforms produce functional differences in the type V/XI polymeric template on which the bulk fibril architecture of a tissue is built.     

Identification of the cartilage alpha 1(XI) chain in type V collagen from bovine bone

Type V collagen prepared from bovine bone was resolved into three distinct alpha-chains by high performance liquid chromatography and gel electrophoresis. Peptide mapping established two chains as alpha 1(V) and alpha 2(V) as expected and the third as the cartilage alpha 1(XI) chain (previously thought to be unique to cartilage). In adult bone, the type V collagen fraction was richer in alpha 1(XI) chains than in fetal bone (about 1/3 of the chains in the adult). How these polypeptides are organized into native molecules is not yet clear, though the stoichiometry suggests cross-type heterotrimers between the type V and XI chains.     

Collagen XXIV,a vertebrate fibrillar collagen with structural features of invertebrate collagens: selective expression in developing cornea and bone

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar alpha1(V), alpha1(XI), and alpha2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the alpha1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains alpha1(V) and alpha1(XI). However, a short imperfection in the triple helix makes alpha1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.     

Fibril-forming collagens in lamprey

Five types of collagen with triple-helical regions approximately 300 nm in length were found in lamprey tissues which show characteristic D-periodic collagen fibrils. These collagens are members of the fibril forming family of this primitive vertebrate. Lamprey collagens were characterized with respect to solubility, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carboxylmethyl-cellulose chromatography, peptide digestion patterns, composition, susceptibility to vertebrate collagenase, thermal stability, and segment long spacing-banding pattern. Comparison with fibril-forming collagens in higher vertebrates (types I, II, III, V, and XI) identified three lamprey collagens as types II, V, and XI. Both lamprey dermis and major body wall collagens had properties similar to type I but not the typical heterotrimer composition. Dermis molecules had only alpha 1(I)-like chains, while body wall molecules had alpha 2(I)-like chains combined with chains resembling lamprey type II. Neither collagen exhibited the interchain disulfide linkages or solubility properties of type III. The conservation of fibril organization in type II/type XI tissues in contrast to the major developments in type I and type III tissues after the divergence of lamprey and higher vertebrates is consistent with these results. The presence of type II and type I-like molecules as major collagens and types V and XI as minor collagens in the lamprey, and the differential susceptibility of these molecules to vertebrate collagenase is analogous to the findings in higher vertebrates.     

Biosynthesis and proteolytic processing of type XI collagen in embryonic chick sterna

The biosynthesis and proteolytic processing of type XI procollagen was examined using pulse-chase labelling of 17-day embryonic chick sterna in organ culture with [3H]proline. Products of biosynthesis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with and without prior reduction of disulfide bonds. Pro-alpha chains, intermediates, and matrix forms were identified by cyanogen bromide or Staphylococcus aureus V8 protease digestion. The results show that type XI pro-alpha chains assemble into trimeric molecules with interchain disulfide bonds. Proteolytic processing begins at least 40 min after the start of labeling which is later than that of type II procollagen (25 min). This first processing step involves the loss of the domain containing the interchain disulfide bonds which most likely is the carboxyl propeptide. In the case of the pro-alpha 3 chain, this generates the matrix form, m alpha 3, which retains its amino propeptide. For the pro-alpha 1 and pro-alpha 2 chains, this step generates intermediate forms, p alpha 1 and p alpha 2, which undergo a second proteolytic conversion to m alpha 1 and m alpha 2, and yet retain a pepsin-labile domain. The conversion of p alpha 2 to m alpha 2 is largely complete 2 h after labeling. p alpha 1 is converted to m alpha 1 very slowly and is 50% complete after 18 h of chase in organ culture. The apparent proteolytic processing within the amino propeptide, and the differential rate of processing between two chains in the same molecule are unusual and distinguish type XI from collagen types I, II, and III. It is possible that the extremely slow processing of p alpha 1 affects the formation of the heterotypic cartilage collagen fibrils and may be related to the function of type XI collagen.     

Structural model of the amino propeptide of collagen XI alpha1 chain with similarity to the LNS domains

Fibrillar collagens are the principal structural molecules of connective tissues. The assembly of collagen fibrils is regulated by quantitatively minor fibrillar collagens, types V and XI. A unique amino-terminal propeptide domain of these collagens has been attributed this regulatory role. The structure of the amino terminal propeptide has yet to be determined. Low sequence similarity necessitated a secondary structure-based method to carry out homology modeling based upon the determined structure of LNS family members, named for a common structure in the laminin LG5 domain, the neurexin 1B domain and the sex hormone binding globulin. Distribution of amino acids within the model suggested glycosaminoglycan interaction and calcium binding. These activities were tested experimentally. Sequence analyses of existing genes for collagens indicate that 16 known collagen alpha chains may contain an LNS domain. A similar approach may prove useful for structure/function studies of similar domains in other collagens with similar domains. This will provide mechanistic details of the organization and assembly of the extracellular matrix and the underlying basis of structural integrity in connective tissues. The absolute requirement for collagen XI in skeletal growth is indicated by collagen XI deficiencies such as chondrodystrophies found in the cho/cho mouse and in humans with Stickler syndrome.     

Axial structure of the heterotypic collagen fibrils of vitreous humour and cartilage

We have compared the axial structures of negatively stained heterotypic, type II collagen-containing fibrils with computer-generated staining patterns. Theoretical negative-staining patterns were created based upon the "bulkiness" of the individual amino acid side-chains in the primary sequence and the D-staggered arrangement of the triple-helices. The theoretical staining pattern of type II collagen was compared and cross-correlated with the experimental staining pattern of both reconstituted type II collagen fibrils, and fibrils isolated from adult and foetal cartilage and vitreous humour. The isolated fibrils differ markedly in both diameter and composition. Correlations were significantly improved when a degree of theoretical hydroxylysine glycosylation was applied, showing for the first time that this type of glycosylation influences the negative-staining pattern of collagen fibrils. Increased correlations were obtained when contributions from types V/XI and IX collagen were included in the simulation model. The N-propeptide of collagen type V/XI and the NC2 domain of type IX collagen both contribute to prominent stain-excluding peaks in the gap region. With decreasing fibril diameter, an increase of these two peaks was observed. Simulations of the fibril-derived staining patterns with theoretical patterns composed of proportions of types II, V/XI and IX collagen confirmed that the thinnest fibrils (i.e. vitreous humour collagen fibrils) have the highest minor collagen content. Comparison of the staining patterns showed that the organisation of collagen molecules within vitreous humour and cartilage fibrils is identical. The simulation model for vitreous humour, however, did not account for all stain-excluding mass observed in the staining pattern; this additional mass may be accounted for by collagen-associated macromolecules.     

The human rhabdomyosarcoma cell line A204 lays down a highly insoluble matrix composed mainly of alpha 1 type-XI and alpha 2 type-V collagen chains.

The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of alpha 1(XI) and alpha 2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [alpha 1(XI)]2 alpha 2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing alpha 2(V) but not alpha 1(XI) collagen chains. Electron microscopy analyses of 24-h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold-labelled anti-(type-V collagen) IgG, but not with anti-(type-XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of beta-aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti-(type-V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti-(type-XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa. These data indicate that type-V and type-XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.     

Concerted modulation of alpha 1(XI) and alpha 2(V) collagen mRNAs in bovine vascular smooth muscle cells.

We have isolated a partial cDNA for alpha 1(XI) collagen from a bovine smooth muscle cell (SMC) library. Previously, this collagen was not known to be expressed in SMCs. Comparison of the nucleotide and deduced amino acid sequence of the 2.7-kilobase bovine clone and the human alpha 1(XI) sequence indicates 92 and 98% homology, respectively. Bovine SMCs in culture were found to produce alpha 1(XI) mRNA. However, alpha 2(XI) and alpha 1(II) collagen RNA were not detectable; therefore, SMCs cannot synthesize the same type XI collagen as found in cartilage. Since type XI collagen is structurally related to type V collagen, the expression of alpha 1(XI) and alpha 2(V) collagen mRNA in SMCs was characterized. Levels of alpha 1(XI) and alpha 2(V) collagen mRNAs were low in exponentially growing SMCs and increased 3-4-fold as cells became confluent. Increased mRNA levels were also observed when exponentially growing subconfluent SMCs were incubated in medium containing 0.5% fetal bovine serum for 24 h, similar to the effects of serum deprivation on the expression of types I and III collagen genes (Kindy, M. S., Chang, C.-J., and Sonenshein, G. E. (1988) J. Biol. Chem. 263, 11426-11430). However, as cell density increased, serum deprivation resulted in very different responses for these collagen genes. Serum deprivation caused a decrease in expression of alpha 1(XI) and alpha 2(V) collagen mRNAs in cultures as they approached confluence. In contrast, at confluence alpha 1(I) and alpha 2(I) mRNA levels no longer responded to serum concentration whereas expression of alpha 1(III) mRNA remained inducible by serum deprivation. These results suggest concerted regulation of alpha 1(XI) and alpha 2(V) collagen gene expression, which is distinct from that for the chains of type I and type III collagen with respect to cell density and serum.     

Interaction between amino propeptides of type XI procollagen alpha1 chains

Type XI collagen is a quantitatively minor yet essential constituent of the cartilage extracellular matrix. The amino propeptide of the alpha1 chain remains attached to the rest of the molecule for a longer period of time after synthesis than the other amino propeptides of type XI collagen and has been localized to the surface of thin collagen fibrils. Yeast two-hybrid system was used to demonstrate that a homodimer of alpha1(XI) amino propeptide (alpha1(XI)Npp) could form in vivo. Interaction was also confirmed using multi-angle laser light scattering, detecting an absolute weight average molar mass ranging from the size of a monomer to the size of a dimer (25,000-50,000 g/mol), respectively. Binding was shown to be saturable by ELISA. An interaction between recombinant alpha1(XI)Npp and the endogenous alpha1(XI)Npp was observed, and specificity for alpha1(XI)Npp but not alpha2(XI)Npp was demonstrated by co-precipitation. The interaction between the recombinant form of alpha1(XI)Npp and the endogenous alpha1(XI)Npp resulted in a stable association during the regeneration of cartilage extracellular matrix by fetal bovine chondrocytes maintained in pellet culture, generating a protein that migrated with an apparent molecular mass of 50-60 kDa on an SDS-polyacrylamide gel.     

Cartilage contains mixed fibrils of collagen types II, IX, and XI

The distribution of collagen XI in fibril fragments from 17-d chick embryo sternal cartilage was determined by immunoelectron microscopy using specific polyclonal antibodies. The protein was distributed throughout the fibril fragments but was antigenically masked due to the tight packing of collagen molecules and could be identified only at sites where the fibril structure was partially disrupted. Collagens II and IX were also distributed uniformly along fibrils but, in contrast to collagen XI, were accessible to the antibodies in intact fibrils. Therefore, cartilage fibrils are heterotypically assembled from collagens II, IX, and XI. This implies that collagen XI is an integral component of the cartilage fibrillar network and homogeneously distributed throughout the tissue. This was confirmed by immunofluorescence.     

Characterization of type XI collagen-glycosaminoglycan interactions

Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen. Pretreatment of chondrocytes with either heparin or heparinase significantly reduced attachment to type XI collagen. Incubation of denatured and cyanogen bromide-cleaved type XI collagen with radiolabeled heparin identified sites of interaction on the alpha1(XI) and alpha2(XI) chains. NH(2)-terminal sequence data confirmed that the predominant heparin-binding peptide contained the sequence GKPGPRGQRGPTGPRGSRGAR from the alpha1(XI) chain. Using rotary shadowing electron microscopy of native type XI collagen molecules and heparin-bovine serum albumin conjugate, an additional binding site was identified at one end of the triple helical region of the collagen molecule. This coincides with consensus heparin binding motifs present at the amino-terminal ends of both the alpha1(XI) and the alpha2(XI) chains. The contribution of glycosaminoglycan-type XI collagen interactions to cartilage matrix stabilization is discussed.     

On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bone formation contained type IX collagen, whereas periosteal and membranous bones were negative. The thin collagenous fibrils in cartilage consisted of type II collagen as determined by immunoelectron microscopy. Type IX collagen was associated with the fibrils but essentially was restricted to intersections of the fibrils. These observations suggested that type IX collagen contributes to the stabilization of the network of thin fibers of the extracellular matrix of cartilage by interactions of its triple helical domains with several fibrils at or close to their intersections.     

Immunohistochemical study of collagens of the extracellular matrix in cartilage of Sepia officinalis

We used various anti-collagen antibodies to perform indirect immunofluorescent staining of cartilage sections from cuttlefish (S. officinalis). On ultrathin sections and collagen fibril preparations from the same tissue, we performed immunostaining with colloidal gold. The extracellular matrix (ECM) of S. officinalis cartilage reacted intensely and homogeneously with an antibody directed against type I-like collagen isolated from the cartilage of cuttlefish and with anti-rat type V collagen antibody. A weak reaction was observed with anti-fish and anti-chicken type I collagen antibodies, while no reaction was observed with anti-rat type I and anti calf type II collagen antibodies. Anti-chicken type II, anti calf type IX and type XI collagen antibodies reacted weakly with ECM, while stained cell bodies and cell processes reacted more intensely. A similar pattern of reaction was observed on cartilage section and isolated collagen fibrils prepared for electron microscopy. These findings suggest that ECM of cuttlefish cartilage may be composed of molecules similar to the type I, type V, type IX and type XI collagen molecules of vertebrates. Cephalopods have evolved a cartilage of structure and macromolecular organisation similar to that of vertebrate cartilage. However, the main molecular components of S. officinalis cartilage--type I-like and type V collagens--differ from those of vertebrate cartilage. We suggest that this type I-like collagen can be considered an initial step toward the evolution of type II collagen typical of vertebrates.     

Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators

Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-ECM interface. There are more than 30 collagens and collagen-related proteins but the most abundant are collagens I and II that exist as D-periodic (where D=67nm) fibrils. The fibrils are of broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair, fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin, fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular mechanisms are in place to control what is otherwise a protein self-assembly process. This review puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.     

Binding of soluble type I collagen to fibroblasts: specificities for native collagen types, triple helical structure, telopeptides, propeptides, and cyanogen bromide-derived peptides

Unlabeled collagenous proteins were quantified as inhibitors of binding of native, soluble, radioiodinated type I collagen to the fibroblast surface. Collagen types IV, V a minor cartilage isotype (1 alpha 2 alpha 3 alpha), and the collagenlike tail of acetylcholinesterase did not inhibit binding. Collagen types II and III behaved as competitive inhibitors of type I binding. Denaturation of native collagenous molecules exposed cryptic inhibitory determinants in the separated constituent alpha chains. Inhibition of binding by unlabeled type I collagen was not changed by enzymatic removal of the telopeptides. Inhibitory determinants were detected in cyanogen bromide-derived peptides from various regions of helical alpha 1 (I) and alpha 1(III) chains. The aminoterminal propeptide of chick pro alpha 1(I) was inhibitory for binding, whereas the carboxyterminal three-chain propeptide fragment of human type I procollagen was not. The data are discussed in terms of the proposal that binding to surface receptors initiates the assembly of periodic collagen fibrils in vivo.     

Developmentally regulated alternative splicing of the alpha1(XI) collagen chain: spatial and temporal segregation of isoforms in the cartilage of fetal rat long bones.

Type XI collagen is a component of the heterotypic collagen fibrils of fetal cartilage and is required to maintain the unusually thin diameter of these fibrils. The mature matrix form of the molecule retains an N-terminal variable region whose structure is modulated by alternative exon splicing that is tissue-specific and developmentally regulated. In the alpha1(XI) chain, antibodies to two of the peptides, p6b and p8, encoded by the alternatively spliced exons localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of rat long bones (humerus). Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo at embryonic Day (E) 14. P8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected throughout prechondrogenic mesenchyme and immature cartilage. After E16, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiation-specific distribution of alpha1(XI) isoforms is evidence that Type XI collagen participates in skeletal development via a mechanism that may be distinct from regulation of fibrillogenesis.     

Collagen II Is Essential for the Removal of the Notochord and the Formation of Intervertebral Discs

Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II–deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice.Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that α1(XI) and α2 (XI) chains form unstable collagen XI molecules, demonstrating that the α3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.