Quantitative analysis of collagen expression in embryonic chick chondrocytes having different developmental fates

A quantitative determination of collagen expression was carried out in cultured chondrocytes obtained from a tissue that undergoes endochondral bone replacement (ventral vertebra) and one that does not (caudal sterna). The "short chain" collagen, type X is only expressed in the former while the other "short chain" collagen type IX, was primarily expressed in the latter. These two tissues also differ in that vertebral chondrocytes express moderate levels of both type I procollagen mRNAs which were translated into full length procollagen chains both in vivo and in vitro, while caudal sternal chondrocytes did not. The percent of collagen synthesis was about 50% in both cell types, but sternal cells expressed twice as much collagen as vertebral cells even though type II procollagen was more efficiently processed to alpha-chains in vertebral chondrocytes than in sternal chondrocytes. The number of type II procollagen mRNA molecules/cell was found to be about 2300 in vertebral chondrocytes and about 8000 in sternal cells, in good agreement with the results reported by Kravis and Upholt (Kravis, D., and Upholt, W. B. (1985) Dev. Biol. 108, 164-172). There were about 630 copies of type I procollagen mRNAs with an alpha 1/alpha 2 ratio of 1.6 in vertebral chondrocytes compared with 5100 copies and an alpha 1/alpha 2 ratio of 2.2 in osteoblasts, and less than 40 copies in sternal cells. Since the rate of type I collagen chain synthesis was 50 times greater in osteoblasts than in vertebral cells, type I procollagen mRNAs were about six times less efficiently translated in vertebral cells than in osteoblasts. The type I mRNAs in vertebral chondrocytes were polyadenylated and had 5' ends that were identical in osteoblasts, fibroblasts, and myoblasts. Moreover, type I mRNAs isolated from vertebral chondrocytes were translated into full length preprocollagen chains in vitro in rabbit reticulocyte lysates. Thus, chondrocytes isolated from cartilage tissues with different developmental fates differed quantitatively and qualitatively in total collagen synthesis, procollagen processing, and distribution of collagen types.     

Partial characterization of type X collagen from bovine growth-plate cartilage. Evidence that type X collagen is processed in vivo

Sequential extraction of bovine growth-plate cartilage with 4 M guanidinium chloride and pepsin was used to identify the intact and pepsinized forms respectively of type X collagen. This collagen occurs predominantly as the processed [alpha 1(X)]3 form in vivo, although the procollagen [pro alpha 1(X)]3 form can also be detected. The bovine pro alpha 1(X) and alpha 1(X) chains have Mr values identical to the corresponding chick species (Mr 59,000 and 49,000). However, the pepsinized alpha 1(X)p chains (Mr 47,000) are larger than those of the chick (Mr 45,000), and the bovine collagen type X is further distinguished by being disulphide-bonded within the triple-helical domain.     

Sites of stromelysin cleavage in collagen types II, IX, X, and XI of cartilage

Human recombinant stromelysin-1 was shown to cleave four types of collagen (types II, IX, X, and XI) prepared from bovine and rat cartilages at specific sites. Stromelysin-1 cleaved salt-soluble native molecules of type IX collagen into two main triple-helical fragments, COL1 and COL2,3. Protein microsequencing identified the exact cleavage sites in the NC2 domain of all three chains, alpha 1(IX), alpha 2(IX), and alpha 3(IX). Stromelysin-1 also acted as a "telopeptidase," in that it efficiently clipped intact molecules of types II and XI collagens at sites just inside their terminal cross-linking hydroxylysine residues. Native molecules of type X collagen were cleaved by stromelysin-1 within their triple helical domains at a COOH-terminal site that reduced the alpha 1(X) chain size by 10 kDa. These findings suggest an important role for stromelysin in the turnover and remodeling of the collagenous matrix of cartilage both normally and in degenerative joint disease.     

Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development

The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis- regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal death. Expression of T antigen was tissue specific and in the main characteristic of the mouse alpha 1(II) collagen gene. Chondrocyte densities and levels of alpha 1(II) collagen mRNAs were reduced in the transgenic mice. Islands of cells which express cartilage characteristic genes such as type IIB procollagen, long form alpha 1(IX) collagen, alpha 2(XI) collagen, and aggrecan were found in the articular and growth cartilages of pAL21 chimeric fetuses and neonates. But these cells, which were expressing T antigen, were not properly organized into columns of proliferating chondrocytes. Levels of alpha 1(II) collagen mRNA were reduced in these chondrocytes. In addition, these cells did not express type X collagen, a marker for hypertrophic chondrocytes. The skeletal abnormality in pAL21 mice may therefore be due to a retardation of chondrocyte maturation or an impaired ability of chondrocytes to complete terminal differentiation and an associated paucity of some cartilage matrix components.     

Areas of asbestoid (amianthoid) fibers in human thyroid cartilage characterized by immunolocalization of collagen types I,II, IX,XI and X

The distribution of type I, II, IX, XI and X collagens in and close to areas of asbestoid (amianthoid) fibers in thyroid cartilages of various ages was investigated in this study. Asbestoid fibers were first detected in thyroid cartilage from a 3-year-old male child. Areas of asbestoid fibers functionally appear to serve as guide rails for vascularization of thyroid cartilage. Alcian blue staining in the presence of 0.3 M MgCl₂ revealed a loss of glycosaminoglycans in areas of asbestoid fibers. In addition, the fibers reacted positively with antibodies against collagen types II, IX and XI, but showed no staining with antibodies to collagen types I and X. Territorial matrix of adjacent chondrocytes showed the same staining pattern. In addition to staining for type II, IX and XI collagens, asbestoid fibers showed strong immunostaining for type I collagen after puberty but not for type X collagen. However, groups of chondrocytes within areas of asbestoid fibers reacted strongly with antibodies to type X collagen, suggesting that this collagen plays an important role in matrix of highly differentiated chondrocytes. The finding that these type X collagen-positive chondrocytes also revealed immunostaining for type I collagen confirms previous studies showing that hypertrophic chondrocytes can further differentiate into cells that are characterized by the synthesis of type X and I collagens.     

Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues

Monoclonal antibodies were produced against the recently described short chain cartilage collagen (type X collagen), and one (AC9) was extensively characterized and used for immunohistochemical localization studies on chick tissues. By competition enzyme-linked immunosorbent assay, antibody AC9 was observed to bind to an epitope within the helical domain of type X collagen and did not react with the other collagen types tested, including the minor cartilage collagens 1 alpha, 2 alpha, 3 alpha, and HMW-LMW. Indirect immunofluorescence analyses with this antibody were performed on unfixed cryostat sections from various skeletal and nonskeletal tissues. Only those of skeletal origin showed detectable reactivity. Within the cartilage portion of the 13-d-old embryonic tibiotarsus (a developing long bone) fluorescence was observed only in that region of the diaphysis containing hypertrophic chondrocytes. None was detectable in adjacent regions or in the epiphysis. Slight fluorescence was also present within the surrounding sleeve of periosteal bone. Consistent with these results, the antibody did not react with the cartilages of the trachea and sclera, which do not undergo hypertrophy during the stages examined. It did, however, lightly react with the parietal bones of the head, which form by intramembranous ossification. These results are consistent with our earlier biochemical analyses, which showed type X collagen to be a product of that subpopulation of chondrocytes that have undergone hypertrophy. In addition, either it or an immunologically cross-reactive molecule is also present in bone, and exhibits a diminished fluorescent intensity as compared with hypertrophic cartilage.     

Immune interferon suppresses levels of procollagen mRNA and type II collagen synthesis in cultured human articular and costal chondrocytes

Cultured human articular and costal chondrocytes were used as a model system to examine the effects of recombinant gamma-interferon (IFN-gamma) on synthesis of procollagens, the steady state levels of types I and II procollagen mRNAs, and the expression of major histocompatibility complex class II (Ia-like) antigens on the cell surface. Adult articular chondrocytes synthesized mainly type II collagen during weeks 1-3 of primary culture, whereas types I and III collagens were also produced after longer incubation and predominated after the first subculture. Juvenile costal chondrocytes synthesized no detectable alpha 2(I) collagen chains until after week 1 of primary culture; type II collagen was the predominant species even after weeks of culture. The relative amounts of types I and II collagens synthesized were reflected in the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. In articular chondrocytes, the levels of alpha 1(I) procollagen mRNA were disproportionately low (alpha 1(I)/alpha 2(I) less than 1.0) compared with costal chondrocytes (alpha 1 (I)/alpha 2(I) approximately 2). Recombinant IFN-gamma (0.1-100 units/ml) inhibited synthesis of type II as well as types I and III collagens associated with suppression of the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. IFN-gamma suppressed the levels of alpha 1(I) and alpha 1(II) procollagen mRNAs to a greater extent than alpha 2(I) procollagen mRNA in articular but not in costal chondrocytes. Human leukocyte interferon (IFN-alpha) at 1000 units/ml suppressed collagen synthesis and procollagen mRNA levels to a similar extent as IFN-gamma at 1.0 unit/ml. In addition, IFN-gamma but not IFN-alpha induced the expression of HLA-DR antigens on intact cells. The lymphokine IFN-gamma could, therefore, have a role in suppressing cartilage matrix synthesis in vivo under conditions in which the chondrocytes are in proximity to T lymphocytes and their products.     

Biosynthesis and in vitro translation of type IV procollagens

The present paper describes how epithelial cells, cultured from bovine anterior lens capsule explants, synthesize and secrete procollagen type IV polypeptide chains alpha 1(IV) and alpha 2(IV). Metabolic labeling of these cells with [14C]proline for different time intervals and subsequent analysis by SDS/polyacrylamide gel electrophoresis revealed the presence of two polypeptide chains with apparent molecular masses of 180 kDa and 170 kDa. The procollagens were bacterial-collagenase-sensitive and were specifically immunoprecipitated by antibodies raised against the 7S domain of type IV collagen. Type IV procollagen poly(A)-rich RNA was isolated from cultured lens capsule cells and translated in a reticulocyte lysate cell-free system. Two polypeptides with apparent molecular masses of 152 kDa and 145 kDa were identified as procollagen type IV unmodified chains by gel electrophoresis, collagenase digestion and specific immunoprecipitation. During experiments in which cells were labeled in the presence of alpha, alpha'-bipyridyl, type IV procollagen appeared as one major band comigrating with a 145 kDa polypeptide on SDS-gel electrophoresis.     

Purification and characterization of the low-molecular-mass (type X) collagen from chick-embryo tibial cartilage

Type X collagen, synthesized in large amount by cultured tibial chondrocytes, is deposited in vivo in the epiphyseal cartilages of 17-day-old chick embryo tibiae. Here we report the extraction of this collagen from these cartilages by limited pepsin digestion and its purification to electrophoretic homogeneity by salt precipitation followed by agarose gel filtration. Identity of the collagen purified from cartilage with the type X collagen synthesized by cultured chondrocytes is confirmed by comparison of the amino acid compositions. The high glycosylation extent of type X collagen is reminiscent of the glycosylation extent of pericellular collagens. The possible role of type X collagen is discussed.     

Immunolocalization of type X collagen before and after mineralization of human thyroid cartilage

In this study the distribution of type X collagen in thyroid cartilages of various ages is described. Fetal and juvenile thyroid cartilage was negative for type X collagen, but showed a strong staining reaction for type II collagen. Type X collagen and calcium deposition were first detected in thyroid cartilage of 18-to 21-year-old adults. Type X collagen was restricted to large chondrocytes near or in mineralized cartilage, confirming the notion that type X collagen precedes mineralization. From these observations it was concluded that chondrocytes in thyroid cartilage undergo differentiation steps that are similar, but much slower, compared to cells in growth plate and sternal cartilage. Some type X collagen-positive areas also showed staining for type I collagen, suggesting that there is a further differentiation of chondrocytes to cells which are characterized by the simultaneous synthesis of type X and I collagen. However, a dedifferentiation process during aging of thyroid cartilage where cells switch from synthesis of type II to type I collagen cannot be excluded.     

Biosynthesis of two subunits of type IV procollagen and of other basement membrane proteins by a human tumor cell line

The major collagenous component secreted into the medium of cultured HT-1080 tumor cells was identified as type IV procollagen by specific antibodies and characteristic ratios of incorporated labeled 3-hydroxyproline and 4-hydroxyproline. The disulfide-bonded molecules consisted of two subunits, pro-alpha 1(IV) and pro-alpha 2(IV) chains with apparent molecular weights of 180 000 and 165 000. No conversion of the procollagen to collagen or to procollagen intermediates was detected in the cell cultures. The two subunits apparently represent different gene products, since enzymatic digestion of the separated chains produced quite different peptide maps. Pepsin degraded native type IV procollagen successively into several fragments, some still disulfide-linked, giving rise to a complex set of polypeptide chains (Mr = 30 000-140 000). This agrees with similar diverse patterns produced by pepsin from authentic type IV collagens. The ratio between the pro-alpha 1(IV) and pro-alpha 2(IV) chains varied in several experiments between 1.3 and 1.8, suggesting that the two chains belong to different triple-helical molecules. The cells also produced distinct amounts of fibronectin (subunit Mr = 230 000) and of the basement membrane glycoprotein laminin. The latter showed three subunits with Mr = 220 000, 210 000, and 400 000. A further disulfide-bonded, non-collagenous polypeptide (Mr = 160 000) was detected but not yet identified. Immunofluorescence demonstrated these proteins within the cells but not in a pericellular matrix. The production of basement membrane components by HT-1080 cells and lack of interstitial collagens disagree with the original classification of the cell line as a fibrosarcoma.     

Bovine cartilage types VI and IX collagens. Characterization of their forms in vivo.

1. Collagens were extracted from bovine cartilage by 4 M-guanidinium chloride in the presence of proteinase inhibitors and identified by immunoblotting with specific anti-collagen sera. 2. The collagens retained their native conformations (shown by the resistance of their triple-helical domains to pepsin digestion), and the molecular masses of their component alpha-chains indicated that the chains were intact. 3. Type VI collagen was extracted as a large-molecular-mass disulphide-bonded aggregate composed of components of molecular mass 140 kDa and 200-240 kDa, and was therefore similar to type VI collagen identified in noncartilaginous tissues. Immunoblotting established the 200-240 kDa components as intact forms of the alpha 3(VI) chain. 4. Type IX collagen consisted of three clearly separable components of molecular mass 84 kDa, 72 kDa and 66 kDa, which were assigned to the alpha 1(IX)-, alpha 3(IX)- and alpha 2(IX)-chains respectively, and a large proportion of this collagen had no covalently bound glycosaminoglycan attached to the alpha 2(IX)-chain. 5. Differences between the type IX collagen extracted from bovine cartilage and that identified in biosynthetic studies on chick cartilage are discussed.     

Characterization of recombinant human type IX collagen. Association of alpha chains into homotrimeric and heterotrimeric molecules.

As type IX collagen is a minor cartilage component, it is difficult to purify sufficient amounts of it from tissues or cultured cells to study its structure and function. Also, the conventional pepsin digestion used for fibrillar collagens cannot be utilized for purifying type IX collagen, because it contains several interruptions in its collagenous triple helix. A baculovirus expression system was used here to produce recombinant human type IX collagen by coinfecting insect cells with three viruses containing full-length cDNAs for the alpha1(IX), alpha2(IX), and alpha3(IX) collagen chains together with a double promoter virus for the alpha and beta subunits of human prolyl 4-hydroxylase. Correctly folded recombinant type IX collagen was secreted, consisting of the three alpha chains in a 1:1:1 ratio and showing the expected biphasic thermal melting profile. When the individual alpha chains were expressed, disulfide-bonded homotrimers and homodimers of the alpha chains were observed. When the cells were coinfected with the viruses for all three alpha chains, heterotrimers of alpha1(IX), alpha2(IX), and alpha3(IX) were detected in cell culture medium, and the other possible combinations were less prominent. When any two of the alpha chains were co-expressed, in addition to the homodimers and homotrimers, only alpha1(IX) and alpha3(IX) chains were disulfide-bonded. The results thus suggest that the most favored molecular species is an alpha1(IX)alpha2(IX)alpha3(IX) heterotrimer, but the chains are also able to form disulfide-bonded heterotrimers of alpha1(IX) and alpha3(IX) chains and (alpha1(IX))(3), (alpha2(IX))(3), and (alpha3(IX))(3) homotrimers.     

Identification of the type IX collagen polypeptide chains. The alpha 2(IX) polypeptide carries the chondroitin sulfate chain(s)

Type IX collagen has recently been shown to contain glycosaminoglycan chain(s) and furthermore to be immunologically identical with proteoglycan Lt (Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985) J. Biol. Chem. 260, 4758-4763). Here we demonstrate that the chondroitin sulfate carrying 115-kDa polypeptide of type IX collagen corresponds to the alpha 2(IX) chain. In addition the 84- and 68-kDa polypeptides were identified as the alpha 1(IX) and the alpha 3(IX) chains, respectively. This conclusion is based on a comparison of the tryptic fingerprints of the 84-, 115-, and 68-kDa chains of type IX collagen on high performance liquid chromatography with the similarly treated C2, C3, and C5 chains of the peptic fragment HMW. In addition, we provide evidence that both the C3 and C4 components of HMW are derived from the alpha 2(IX) chain.     

Assembly of chick and bovine lens-capsule collagen.

Chick-embryo and adult bovine lens-capsular epithelia in organ culture synthesized 4-hydroxy[3H]proline-containing polypeptides when incubated in the presence of [3H]proline. These collagenous polypeptides of apparent Mr 180 000, 175 000 and 160 000 became incorporated with time into aggregates of higher molecular size. The formation of such aggregates was inhibited when the tissues were labelled in the presence of beta-aminopropionitrile, thereby implicating lysine-derived cross-links in aggregate formation. When the tissues were incubated in the presence of tunicamycin, the collagenous polypeptides synthesized exhibited increased electrophoretic mobilities on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The addition to lens-capsule incubation medium of alpha alpha'-bipyridine led to the synthesis of underhydroxylated type IV collagen, also of increased electrophoretic mobility. Extended pulse-chase experiments indicated that such underhydroxylated collagen did not participate in aggregate formation, but was at least as stable as fully hydroxylated non-cross-linked collagen synthesized in the presence of beta-aminopropionitrile. Native type IV collagen, recovered from the culture medium when capsules were incubated with [3H]proline for 24h, was purified by ion-exchange chromatography. Separations conducted on CM-cellulose under denaturing and nondenaturing conditions suggested that the alpha 1(IV) and alpha 2(IV) chains occur in the same heterologous triple helix. Densitometric analyses of appropriate fluorograms indicated that these two polypeptides occur in a 2:1 ratio, suggesting that lens-capsule collagen is synthesized as a triple-helical molecule of composition [alpha 1(IV)]2 alpha 2(IV).     

Changes in the types of collagen synthesized during chondrogenesis of the mouse otic capsule

We have investigated the temporal relationship between the morphological differentiation of the mouse otic capsule and the pattern of collagen synthesis by mouse otocyst-mesenchyme complexes labeled in vitro. In 10.5- to 12-day embryos the mesenchyme surrounding the otocyst was loosely organized except for a few lateroventral condensations; explants from these embryos synthesized only small amounts of collagen. Collagen synthesis by whole explants increased by more than 50% between 12 and 13 days concomitant with metachromatic staining of the lateral periotic mesenchyme. Cartilage specific type II collagen was the predominant collagen synthesized by these explants as confirmed by SDS-PAGE, densitometry, CNBr cleavage, and V8 protease digestion. This biochemical expression of the cartilage phenotype preceded morphologic recognition of otic capsular cartilage by almost 2 days. Type II collagen synthesis continued to increase and predominate through Day 16 of gestation by which time the otic labyrinth was surrounded by mature cartilage. The minor cartilage collagen chains, 1 alpha, 2 alpha, and 3 alpha, first appeared on different days of gestation. The 1 alpha, and 3 alpha chains were synthesized by explants from 11-day embryos while the 2 alpha chain appeared during Day 13, just before overt differentiation of mature cartilage. These results suggested that the 1 alpha, 2 alpha, and 3 alpha chains may not form heterotrimers containing all three chains and that synthesis of the 2 alpha chain may be associated with stabilization of the cartilaginous matrix. Comparison of these data with the patterns of collagen production by mutant, diseased, or experimentally manipulated inner ear tissues may provide insights into the molecular basis of chondrogenic tissue interactions.     

Changes in the synthesis of minor cartilage collagens after growth of chick chondrocytes in 5-bromo-2'-deoxyuridine or to senescence

Analyses were made of the minor collagens synthesized by cultures of chondrocytes derived from 14-day chick embryo sterna. Comparisons were made between control cultures, cultures grown for 9 days in 5-bromo-2'-deoxyuridine (BrdU) and clones of chondrocytes grown to senescence. Separation of minor collagens from interstitial collagens was achieved by differential salt precipitation in the presence of carrier collagens in acid conditions. The precipitate at 0.9 M NaCl 0.5 M acetic acid from control cultures was shown by CNBr peptide analysis to contain only the alpha 1(II) chain of type II collagen, whereas after BrdU treatment or growth to senescence synthesis of only alpha 1(I) and alpha 2(I) chains occurred. The synthesis of type III collagen was not detected. Analysis of the precipitate at 2.0 M NaCl, 0.5 M HAc from control cultures demonstrated the synthesis of 1 alpha, 2 alpha and 3 alpha chains together with the synthesis of short chain (SC) collagen of Mr 43000 after pepsin digestion. After BrdU treatment or growth to senescence alpha chains were isolated which possessed the migration positions on polyacrylamide gel electrophoresis (PAGE), or the elution positions on CM-cellulose chromatography, of the alpha 1(V) and alpha 2(V) chains of type V collagen. In addition, for BrdU-treated but not for control cultures, intracellular immunofluorescent staining was observed with a monoclonal antibody which specifically recognizes an epitope present in the triple helix of type V collagen. Synthesis of short chain (SC) collagen was not detected after BrdU treatment or growth to senescence. These results suggest that chick chondrocytes grown in conditions known to cause switching of collagen synthesis from type II to type I collagen also undergo a switch from the synthesis of 1 alpha, 2 alpha and 3 alpha chains to the synthesis of the alpha 1(V) and alpha 2(V) chains of type V collagen. It appears that there are several cartilage-specific collagens which together undergo a regulatory control to the synthesis of collagens typical of other connective tissues.     

Occurrence in chick embryo vitreous humor of a type IX collagen proteoglycan with an extraordinarily large chondroitin sulfate chain and short alpha 1 polypeptide

We have prepared a high buoyant density proteoglycan fraction from the vitreous humor of 13-day-old chick embryos. Using immunoblot analysis coupled with chondroitinase digestion, we demonstrate that the purified preparation is composed predominantly of type IX collagen-like chondroitin sulfate proteoglycan with an alpha 1(IX) chain Mr approximately 23,000 shorter than the known alpha 1 in cartilage type IX. Also different from cartilage type IX is the size of the chondroitin sulfate chain attached to the alpha 2(IX) polypeptide; its Mr is approximately 350,000 indicating that it is approximately 10 times larger in vitreous humor than in cartilage. Examination of vitreous bodies at different developmental stages indicates that a transition occurs in the size of alpha 1(IX) in a well defined temporal pattern; at about stage 31, a cartilage-type alpha 1(IX) of Mr 84,000 is the predominant species, whereas at stage 36 and thereafter, a Mr 61,000 species appears with a concomitant disappearance of the Mr 84,000 species. Immunostaining for type IX collagen followed by electron microscopic observation of 13-day-old chick embryo vitreous humor reveals a regular D-periodic arrangement of vitreous type IX collagen proteoglycan along thin fibrils. It seems possible that the chondroitin sulfate chains of extraordinarily high viscosity and high molecular weight may extend away from the fibrils, thus contributing to structural as well as functional properties of this unique matrix.