Cartilage proteoglycan aggregate and fibronectin can modulate the expression of type X collagen by embryonic chick chondrocytes cultured in collagen gels

Chick embryo sternal chondrocytes from the caudal and cephalic regions were cultured within type I collagen gels and type I collagen/proteoglycan aggregate composite gels in normal serum. Caudal region chondrocytes were also cultured within type I collagen gels in the presence of fibronectindepleted serum. There was a marked stimulation of type X collagen synthesis by the caudal region chondrocytes after 9 days in the presence of fibronectin-depleted serum and after 14 days in the presence of proteoglycan aggregate. These results provide evidence for the ability of chondrocytes from a zone of permanent cartilage to synthesise type X collagen and for the involvement of extracellular matrix components in the control of type X collagen gene expression.     

Deposition of type X collagen in the cartilage extracellular matrix

In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.     

A comparison of the effects of different substrata on chondrocyte morphology and the synthesis of collagen types IX and X

Summary Embryonic chick sternal chondrocytes were cultured either within three dimensional gels of type I collagen, type II collagen or agar, or as monolayers on plastic dishes coated with air-dried films of these matrix macromolecules. It was observed that cell shape and cell growth varied markedly between the different culture conditions. Flattened monolayers of cells on plastic or films of type I or type II collagen, proliferated more rapidly and reached a higher final cell density per culture than the more rounded cells found in the cultures on agar films or within three-dimensional gels. Biosynthetic studies demonstrated that in addition to the synthesis of type II collagen, all the cultures were producing collagen types IX and X. Chondrocytes cultured on plastic or films of the different matrix macromolecules all showed a similar expression of types IX and X collagen, independent of whether they displayed a flattened or round cell morphology. In contrast, marked variations in the proportions of the minor collagens, particularly type X collagen, were observed when the cells were cultured within three-dimensional gels. The data suggest that direct interaction of the cell surface with matrix constituents displaying a particular spatial array could be an important aspect in the control of type IX and X collagen expression by chondrocytes. The financial support of the Arthritis & Rheumatism Council and the Medical Research Council is gratefully acknowledged.     

Subepithelial type II collagen deposition during embryonic chick limb development

Summary Type II collagen is a major component of hyaline cartilage but recent studies have demonstrated the presence of this protein in a variety of interfaces that separate epithelia from mesenchyme, particularly in early stages of embryonic chick development. In the present study an immunohistochemical analysis of the distribution of type II collagen was performed on closely staged wing buds of early chick embryo. This report describes how using two different monoclonal antibodies against type II collagen and the peroxidase or fluorescence staining technique reveals that deposition of type II collagen at the ectoderm-mesenchyme interface occurs in the proximal part of the limb coincidentally with the appearance of this protein in the proximal core region, where chondrogenesis begins (stage 25). Then the staining in the subepithelial region spreads distallly with time, following the progression of the formation of cartilage rudiments. At about 7 days of development type II collagen is present under the apical ectoderm ridge and surrounds completely the wing bud underneath the epithelium. At the same time, another antibody directed against the cartilage-specific proteoglycan core protein only stains the chondrogenic central core of the limb and not the subepithelium. Although type II collagen and cartilage-specific proteoglycan are closely associated in the cartilage, the observations presented here suggest that the deposition of these proteins can be regulated independently during limb formation. The role of type II collagen at the epithelium-mesenchyme interface during limb formation is still to be determined.     

Developmental acquisition of type X collagen in the embryonic chick tibiotarsus

The temporal and spatial distribution of short chain skeletal (Type X) collagen was immunohistochemically examined in the chick tibiotarsus from 6 days of embryonic development to 1 day posthatching. The monoclonal antibody employed (AC9) was recently produced and characterized as being specific for an epitope located within the helical domain of the type X collagen molecule (T. M. Schmid and T. F. Linsenmayer, J. Cell Biol., in press). The earliest detectable appearance of type X collagen was at 7.5 days, at which time it was restricted to a middiaphyseal location (i.e., in the primary center of ossification). This was in marked contrast to type II collagen, which appears earlier and is distributed throughout the cartilaginous anlagen. With increasing embryonic age, the reactivity with the type X antibody progressively extended toward the epiphyses, lagging somewhat behind the progression of chondrocyte hypertrophy. The anti-type X collagen antibody also reacted with the bony matrix itself, but the immunofluorescent signal produced by this source was considerably less than that produced by cartilage. At 19 days of development, a new small site of type X deposition was initiated in an epiphyseal location, which subsequently enlarged in circumference. These results are consistent with our previous biochemical studies suggesting that, in cartilage, type X collagen is specifically a product of that population of chondrocytes which have undergone hypertrophy.     

Immunohistochemical localization of collagen type XI alpha1 and alpha2 chains in human colon tissue.

In previous studies, collagen XI mRNA has been detected in colon cancer, but its location in human colon tissue has not been determined. The heterotrimeric collagen XI consists of three alpha chains. While it is known that collagen XI plays a regulatory role in collagen fibril formation, its function in the colon is unknown. The characterization of normal human colon tissue will allow a better understanding of the variance of collagen XI in abnormal tissues. Grossly normal and malignant human colon tissue was obtained from pathology archives. Immunohistochemical staining with a 58K Golgi marker and alpha1(XI) and alpha2(XI) antisera was used to specifically locate their presence in normal colon tissue. A comparative bright field microscopic analysis showed the presence of collagen XI in human colon. The juxtanuclear, dot-like collagen XI staining in the Golgi apparatus of goblet cells in normal tissue paralleled the staining of the 58K Golgi marker. Ultra light microscopy verified these results. Staining was also confirmed in malignant colon tissue. This study is the first to show that collagen XI is present in the Golgi apparatus of normal human colon goblet cells and localizes collagen XI in both normal and malignant tissue. Although the function of collagen XI in the colon is unknown, our immunohistochemical characterization provides the foundation for future immunohistopathology studies of the colon.     

Anti-citrullinated collagen type I antibody is a target of autoimmunity in rheumatoid arthritis

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, but its autoimmune mechanisms are not clearly understood. Recently, anti-citrullinated peptide antibodies have been specifically observed in sera of RA patients. Furthermore, we identified RA-susceptible variant in a gene encoding citrullinating enzyme, peptidylarginine deiminase type 4 (PADI4). Therefore, we hypothesized that proteins which are modified in RA synovium by PADI4 act as autoantigens. Subsequently, we obtained human collagen type I (huCI) as one of the autoantigens using a RA synoviocyte cDNA library by immunoscreening. We also investigated that the levels of anti-citrullinated huCI were significantly higher in RA patient sera than in normal control sera with high specificity (99%) and positively correlated with the levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies. We concluded that huCI is a novel substrate protein of PADIs and that citrullinated huCI is a candidate autoantigen of RA.     

Liquid crystalline phases of sonicated type I collagen

The assembly properties of concentrated solutions of type I collagen molecules are compared before and after a 5-min sonication, breaking the 300-nm triple helices into short segments of about 20 nm, with a strong polydispersity. The collagen concentration of these solutions, sonicated or not, was increased up to 100 mg/ml by slow evaporation of the solvent. Whereas the non-sonicated solutions remain isotropic, the sonicated solutions transform after a few hours into a twisted liquid crystalline phase, well recognizable in polarizing microscopy. The evidence of a twisted assembly of collagen triple helices in vitro is new and relevant in a biological context since it was reported in various collagen matrices.     

Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and × collagen and matrix mineralization

In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis.     

Double thermal transitions of type I collagen in acidic solution

Contributed equally to this work. To further understand the origin of the double thermal transitions of collagen in acidic solution induced by heating, the denaturation of acidic soluble collagen was investigated by micro-differential scanning calorimeter (micro-DSC), circular dichroism (CD), dynamic laser light scattering (DLLS), transmission electron microscopy (TEM), and two-dimensional (2D) synchronous fluorescence spectrum. Micro-DSC experiments revealed that the collagen exhibited double thermal transitions, which were located within 31–37?°C (minor thermal transition, T _s?～?33?°C) and 37–55?°C (major thermal transition, T _m?～?40?°C), respectively. The CD spectra suggested that the thermal denaturation of collagen resulted in transition from polyproline II type structure to unordered structure. The DLLS results showed that there were mainly two kinds of collagen fibrillar aggregates with different sizes in acidic solution and the larger fibrillar aggregates (T _p2?=?40?°C) had better heat resistance than the smaller one (T _p1?=?33?°C). TEM revealed that the depolymerization of collagen fibrils occurred and the periodic cross-striations of collagen gradually disappeared with increasing temperature. The 2D fluorescence correlation spectra were also applied to investigate the thermal responses of tyrosine and phenylalanine residues at the molecular level. Finally, we could draw the conclusion that (1) the minor thermal transition was mainly due to the defibrillation of the smaller collagen fibrillar aggregates and the unfolding of a little part of triple helices; (2) the major thermal transition primarily arose from the defibrillation of the larger collagen fibrillar aggregates and the complete denaturation of the majority part of triple helices.     

Expression of type X collagen mRNA levels in embryonic chick sternum during development

Embryonic chick sternum cartilage exhibits profound spatial and temporal changes in Type X collagen biosynthesis during development. Production of this collagen is confined to the presumptive calcification region and its expression is not acquired until stage 43. To examine the mechanisms responsible for regulation of developmental changes in biosynthetic expression of Type X collagen, we determined the levels of translatable Type X procollagen mRNA employing a cell-free translation system. We found that mRNA capable of directing Type X collagen synthesis was present exclusively in cartilage destined to undergo calcification and that its levels were nearly equivalent at all stages of development. These findings suggest that expression of Type X collagen in embryonic chick sternum is determined at the translational level.     

Cloning and expression of type XII collagen isoforms during bovine adipogenesis

In order to isolate candidate genes involved in bovine adipocyte differentiation, we have constructed a subtraction library from a clonal bovine intra-muscular pre-adipocyte (BIP) cell line using the suppression subtractive hybridization method. We have isolated a set of subtracted cDNA fragments whose respective mRNA levels are up-regulated during the adipogenic differentiation of BIP cells, and cloned cDNAs from a differentiated BIP-lambda ZAP II cDNA library. Two cDNA clones were highly homologous to the sequence of mouse and human type XII collagen alpha-1, determined by a BLAST homology search. As type XII collagen has been reported to have four types of splicing isoform, two clones were determined to be XII-1 and XII-2 splicing isoforms, respectively, because of a difference in the C-terminal NC1 domain. From the expression analysis of type XII collagen, the XIIA-2 isoform was mainly expressed in differentiated BIP cells and adipose tissues. Although the function of type XII collagen has not been established as yet, these results suggest that type XII collagen may be associated with adipocyte differentiation and adipose formation in cattle and is a potentially useful marker for adipogenesis.     

Retinoic acid treatment induces type X collagen gene expression in cultured chick chondrocytes

The vitamin A derivative retinoic acid (RA) is widely thought to be involved in cartilage development, but its precise roles and mechanisms of action in this complex process remain unclear. We have tested the hypothesis that RA is involved in chondrocyte maturation during endochondral ossification and, in particular, is an inducer of maturation-associated traits such as type X collagen and alkaline phosphatase. Immature chondrocytes isolated from the caudal region of Day 19 chick embryo sterna were seeded in secondary monolayer cultures and treated either with a high dose (100 nM) or with physiological doses (10-35 nM) of RA for up to 3 days. We found that after an initial lag of about 24 h, physiological doses of RA indeed induced type X collagen gene expression in the immature cells. This induction was not accompanied by obvious changes in expression of the type II collagen and large aggregating proteoglycan core protein genes. As revealed by immunocytochemistry, 30-35% of the cells in cultures treated with RA for 3 days were engaged in type X collagen production. Interestingly, these cells were relatively similar in size to chondrocytes in which no type X collagen was detected, suggesting that chondrocytes can initiate type X collagen production independent of cell hypertrophy. RA treatment also led to increased alkaline phosphatase activity occurring as early as 24 h after the start of treatment. The data in this study indicate that RA may have a role in endochondral ossification as an inducer/promoter of maturation-associated traits during chondrocyte maturation.     

High density micromass cultures of embryonic limb bud mesenchymal cells: An in vitro model of endochondral skeletal development

Summary To study the mechanisms regulating endochondral skeletal development, we examined the characteristics of long-term, high density micromass cultures of embryonic chicken limb bud mesenchymal cells. By culture Day 3, these cells underwent distinct chondrogenesis, evidenced by cellular condensation to form large nodules exhibiting cartilage-like morphology and extracellular matrix. By Day 14, extensive cellular hypertrophy was seen in the core of the nodules, accompanied by increased alkaline phosphatase activity, and the limitation of cellular proliferation to the periphery of the nodules and to internodular areas. By Day 14, matrix calcification was detected by alizarin red staining, and calcium incorporation increased as a function of culture time up to 2 to 3 wk and then decreased. X-ray probe elemental analysis detected the presence of hydroxyapatite. Analogous to growth cartilage developing in vivo, these cultures also exhibited time-dependent apoptosis, on the basis of DNA fragmentation detected in situ by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), ultrastructural nuclear morphology, and the appearance of internucleosomal DNA degradation. These findings showed that cellular differentiation, maturation, hypertrophy, calcification, and apoptosis occurred sequentially in the embryonic limb mesenchyme micromass cultures and indicate their utility as a convenient in vitro model to investigate the regulatory mechanisms of endochondral ossification.     

Immunoelectron microscopy of type X collagen: supramolecular forms within embryonic chick cartilage

To determine the supramolecular forms in which avian type X collagen molecules assemble within the matrix of hypertrophic cartilage, we performed immunoelectron microscopy with colloidal gold-labeled monoclonal antibodies. In addition double-labeled analyses were performed for the molecule and type II collagen, employing two monoclonal antibodies attached to different size gold particles. Both in situ limb cartilages and the extracellular matrix of chondrocyte cultures were examined. We observed in both systems that the type X collagen is present in two forms. One is as fine filaments (less than 5 nm in diameter) within mats which are found predominantly in the pericellular matrix of the hypertrophic chondrocytes. The second form is in association with the fibrils (10-20 nm in diameter) which also react with the antibody for type II collagen. It seems that the filamentous mats represent a form in which the type X collagen is initially secreted from the cell. The type X associated with the striated fibrils most likely represents a secondary association of the molecule with preexisting type II/IX/XI fibrils. The data are consistent with our previously proposed hypothesis that type X collagen is involved in, and perhaps even "targets," certain matrix components for degradation and removal.     

Antibodies against the CB10 fragment of type II collagen in rheumatoid arthritis

Antibodies against intact type II collagen (CII) are a feature of rheumatoid arthritis (RA) but have limited diagnostic value. Here we assess whether either of the two major cyanogen bromide fragments of CII, namely CB10 or CB11, are more sensitive substrates for the detection of antibodies in RA. Cleavage of bovine CII with cyanogen bromide yielded CB10 and CB11; these were purified by column chromatography for use in an enzyme-linked immunosorbent assay. Serum antibodies were measured in patients with RA, psoriatic arthritis (PsA), osteoarthritis (OA) and blood donors. Results were compared with those using intact CII. Antibodies against CB10 were found in as many as 88% of 96 patients with long-standing RA, but only 12% of 33 patients with PsA, 6% of 34 patients with OA and 3% of 93 control sera. Lower frequencies for these diseases were obtained on testing for antibodies against CB11: 50%, 6%, 21% and 2%, respectively. The sensitivity of detection in RA of antibodies against CB10 compared with antibodies against intact CII (88% versus 24%) was not at the expense of specificity, which remained high at 94%. The much higher frequency of antibodies against CB10 in RA than in other rheumatic diseases or control sera indicates that CB10 is clearly a more sensitive substrate than the intact collagen molecule and, combined with other assays (rheumatoid factor, anti-cyclic citrullinated peptide [anti-CCP]), might comprise a panel with a highly reliable predictive value. Moreover, our findings should encourage renewed interest in the role of collagen autoimmunity in the pathogenesis of RA.     

Analysis of type II collagen RNA localization in chick wing buds by in situ hybridization

Type II collagen is a major component of cartilage extracellular matrix. Differentiation of mesenchyme into cartilage involves the cessation of type I collagen synthesis and the onset of type II collagen synthesis. Solution hybridization of mRNA isolated from chick limb buds with a cDNA probe to type II collagen mRNA showed the presence of small amounts of type II collagen message in mesenchymal chick limbs. We have examined the localization of type II collagen mRNA in mesenchymal chick wing buds by in situ hybridization using single stranded RNA probes. Our results show a small but detectable amount of type II collagen RNA distributed uniformly in early limbs until the first precartilage condensations form at stage 22. This is interesting because it is known that mesenchyme isolated from chick wing buds has the capacity to undergo chondrogenesis in culture, even if taken from nonchondrogenic areas of the limb. At stage 23, type II collagen mRNA is found at significantly increased levels in the cells of the precartilage condensation when compared to the other limb cells. As chondrogenesis proceeds, the amount of type II collagen RNA increases even more in cells of the cartilage elements. The signal in the peripheral tissue is indistinguishable from background. These results show that type II collagen message exists at low levels in cells throughout the mesenchymal chick wing bud, until the formation of the condensation results in an elevation of type II mRNA in the prechondrogenic cells found in the core of the limb.