Type X collagen gene expression is transiently up-regulated by retinoic acid treatment in chick chondrocyte cultures

During endochondral ossification, resting and proliferating chondrocytes mature into hypertrophic chondrocytes that initiate synthesis of type X collagen. The mechanisms regulating the differential expression of type X collagen gene were examined in confluent Day 12 secondary cultures of chick vertebral chondrocytes in monolayer treated with the vitamin A analog retinoic acid (RA). Preliminary results showed that major effects of RA on chondrocyte gene expression occurred between 24 and 48 h of treatment. Thus in subsequent experiments cultures were treated for 24, 30, 36, 42, 48, 72, 96, and 120 h. Total RNAs were isolated and analyzed by hybridization with ³²P-labeled plasmid probes coding for five matrix macromolecules including type X collagen. We found that the steady-state levels of mRNAs for the large keratan sulfate/chondroitin sulfate proteoglycan (KS:CS-PG) core protein and type II collagen decreased several fold between 24 and 48 h of treatment compared to untreated cells, and remained low with further treatment. In sharp contrast, the level of type X collagen mRNA increased threefold by 42 h of treatment; thereafter it began to decrease and reached minimal levels by 72–120 h of treatment. The changes in steady-state mRNA levels during RA regimen paralleled similar changes in relative rates of protein synthesis. The transient up-regulation of type X collagen gene expression at 42 h of treatment was preceded by a five-fold increase in fibronectin gene expression, was followed by a several fold increase in type I collagen gene expression, and was accompanied by cell flattening and loss of the pericellular proteoglycan matrix. Thus, RA treatment leads to a unique biphasic modulation of type X collagen gene expression in maturing chondrocyte cultures. The underlying, RA-sensitive mechanisms effecting this modulation may reflect those normally regulating the differential expression of this collagen gene during endochondral ossification.     

Construction and characterization of type II collagen complementary deoxyribonucleic acid clones

The mRNA for type II collagen was purified from embryonic chick sternum or from purified sternal chondrocytes with guanidine thiocyanate as the extractant. Double-stranded cDNAs to procollagen mRNAs from sternum were synthesized and dC-tailed. After annealing with PstI-cleaved, dG-tailed pBR322, this DNA was used to transform Escherichia coli X1776. Transformed colonies were screened by colony hybridization to type I and II collagen cDNAs. Clones that preferentially hybridized to type II cDNA were characterized further. Four such cDNA clones, pCgII-2, 3, 10 and 12, with inserts of 400, 320, 260 and 750 bp, have been identified as type II collagen cDNA clones by several criteria, including their preference for hybridizing with type II rather than type I collagen mRNAs in hybrid-selected translation experiments.     

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.     

Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.     

Collagen types IX and X in the developing chick tibiotarsus: analyses of mRNAs and proteins

To examine the regulation of collagen types IX and X during the hypertrophic phase of endochondral cartilage development, we have employed in situ hybridization and immunofluorescence histochemistry on selected stages of embryonic chick tibiotarsi. The data show that mRNA for type X collagen appears at or about the time that we detect the first appearance of the protein. This result is incompatible with translational regulation, which would require accumulation of the mRNA to occur at an appreciably earlier time. Data on later-stage embryos demonstrate that once hypertrophic chondrocytes initiate synthesis of type X collagen, they sustain high levels of its mRNA during the remainder of the hypertrophic program. This suggests that these cells maintain their integrity until close to the time that they are removed at the advancing marrow cavity. Type X collagen protein in the hypertrophic matrix also extends to the marrow cavity. Type IX collagen is found throughout the hypertrophic matrix, as well as throughout the younger cartilaginous matrices. But the mRNA for this molecule is largely or completely absent from the oldest hypertrophic cells. These data are consistent with a model that we have previously proposed in which newly synthesized type X collagen within the hypertrophic zone can become associated with type II/IX collagen fibrils synthesized and deposited earlier in development (Schmid and Linsenmayer, 1990; Chen et al. 1990).     

Effect of heparin on synthesis of short chain collagen by chondrocytes and smooth muscle cells

The treatment of embryonic chick chondrocyte cultures with heparin results in a decrease in collagen synthesis. One of the collagens synthesized by hypertrophic chondrocytes, specifically type X collagen, may play an important role in cartilage mineralization and endochondral ossification. Recently a new short chain collagenous component was found in cultures of rat vascular smooth muscle cells (Majack, R. A., and P. Bornstein, 1985, J. Cell Biol., 100: 613-619). The present study was initiated to investigate heparin's effect on type X collagen in embryonic chick chondrocytes and to further evaluate the nature of the short chain component synthesized by rat vascular smooth muscle cells. Different tissues may respond differently to the administration of heparin. In chondrocyte cultures heparin decreased both total collagen synthesis as well as the synthesis of type X collagen. There was an accumulation of collagen precursors, found principally in the cell layer compartment, which appeared to be the result of heparin's inhibition of the NH2-terminal protease. In cultures of rat vascular smooth muscle cells heparin was found to increase the synthesis of a short chain collagenous component as previously reported. However, comparison with a type X collagen standard showed this to be different from type X. In all cases, the effect of heparin on collagen chain precursors, chondrocyte type X synthesis, and synthesis of a vascular smooth muscle short chain collagen was shown to be reversible. Similar effects were obtained by adding chondroitin sulfate to chondrocytes, suggesting a role for extracellular matrix components in the modulation of collagen synthesis. These findings are consistent with the concept of a group of short chain collagens with type X collagen being unique to hypertrophic chondrocytes.     

Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.     

Mechanoregulation of chondrocyte proliferation, maturation, and hypertrophy: ion-channel dependent transduction of matrix deformation signals

Mechanical stress-induced matrix deformation plays a fundamental role in regulating cellular activities; however, little is known about its underlying mechanisms. To understand the effects of matrix deformation on chondrocytes, we characterized primary chondrocytes cultured on three-dimensional collagen scaffoldings, which can be loaded mechanically with a computer-controlled "Bio-Stretch" device. Cyclic matrix deformation greatly stimulated proliferation of immature chondrocytes, but not that of hypertrophic chondrocytes. This indicates that mechanical stimulation of chondrocyte proliferation is developmental stage specific. Synthesis of cartilage matrix protein (CMP/matrilin-1), a mature chondrocyte marker, and type X collagen, a hypertrophic chondrocyte marker, was up-regulated by stretch-induced matrix deformation. Therefore, genes of CMP and type X collagen are responsive to mechanical stress. Mechanical stimulation of the mRNA levels of CMP and type X collagen occurred exactly at the same time points when these markers were synthesized by nonloading cells. This indicates that cyclic matrix deformation does not alter the speed of differentiation, but affects the extent of differentiation. The addition of the stretch-activated channel blocker gadolinium during loading abolished mechanical stimulation of chondrocyte proliferation, but did not affect the up-regulation of CMP mRNA by mechanical stretch. In contrast, the calcium channel blocker nifedipine inhibited both the stretch-induced proliferation and the increase of CMP mRNA. This suggests that stretch-induced matrix deformation regulates chondrocyte proliferation and differentiation via two signal transduction pathways, with stretch-activated channels involved in transducing the proliferative signals and calcium channels involved in transducing the signals for both proliferation and differentiation.     

Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen

Mouse endochondral chondrocytes were immortalized with a temperature- sensitive simian virus 40 large tumor antigen. Several clonal isolates as well as pools of immortalized cells were characterized. In monolayer cultures at the temperature permissive for the activity of the large tumor antigen (32 degrees C), the cells grew continuously with a doubling time of approximately 2 d, whereas they stopped growing at nonpermissive temperatures (37 degrees C-39 degrees C). The cells from all pools and from most clones expressed the genes for several markers of hypertrophic chondrocytes, such as type X collagen, matrix Gla protein, and osteopontin, but had lost expression of type II collagen mRNA and failed to be stained by alcian blue which detects cartilage- specific proteoglycans. The cells also contained mRNAs for type I collagen and bone Gla protein, consistent with acquisition of osteoblastic-like properties. Higher levels of mRNAs for type X collagen, bone Gla protein, and osteopontin were found at nonpermissive temperatures, suggesting that the expression of these genes was upregulated upon growth arrest, as is the case in vivo during chondrocyte hypertrophy. Cells also retained their ability to respond to retinoic acid, as indicated by retinoic acid dose-dependent and time- dependent increases in type X collagen mRNA levels. These cell lines, the first to express characteristic features of hypertrophic chondrocytes, should be very useful to study the regulation of the type X collagen gene and other genes activated during the last stages of chondrocyte differentiation.     

Type X collagen synthesis during in vitro development of chick embryo tibial chondrocytes

In the developing chick embryo tibia type X collagen is synthesized by chondrocytes from regions of hypertrophy and not by chondrocytes from other regions (Capasso, O., G. Tajana, and R. Cancedda, 1984, Mol. Cell. Biol. 4:1163-1168; Schmid, T. M., and T. F. Linsenmayer, 1985, Dev. Biol. 107:375-381). To investigate further the relationship between differentiation of endochondral chondrocytes and type X collagen synthesis we have developed a novel culture system for chondrocytes from 29-31-stage chick embryo tibiae. At the beginning of the culture these chondrocytes are small and synthesize type II and not type X collagen, but when grown on agarose-coated dishes they further differentiate into hypertrophic chondrocytes that synthesize type X collagen. The synthesis of type X collagen has been monitored in cultured cells by analysis of labeled collagens and in vitro translation of mRNAs. When the freshly dissociated chondrocytes are plated in anchorage-permissive dishes, most of the cells attach and dedifferentiate, as revealed by their fibroblastic morphology. Dedifferentiated chondrocytes, after several passages, can still reexpress the differentiated phenotype and continue their development to hypertrophic, type X collagen-synthesizing chondrocytes. Hypertrophic chondrocytes, when plated in anchorage permissive dishes, attach, maintaining the differentiated phenotype, and continue the synthesis of type X collagen.     

In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells

Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in fresh or conditioned culture media. A cloning efficiency of approximately 13% was obtained using medium conditioned by dedifferentiated chondrocytes. A cloning efficiency of only 1.4% was obtained when conditioned medium from hypertrophic chondrocytes was used, and efficiencies of essentially 0 were found with fresh medium or medium conditioned by J2-3T3 mouse fibroblasts. Cell clones were selected by morphological criteria and clones showing a dedifferentiated phenotype (fibroblast-like) were further characterized. Out of 38 clones analyzed, 17 were able to differentiate to the hypertrophic chondrocyte stage and reconstitute hypertrophic cartilage when placed in the appropriate culture conditions. Cells from these clones expressed the typical markers of chondrocyte differentiation, i.e., type II and type X collagens. Clones not undergoing differentiation continued to express only type I collagen. Hypertrophic chondrocytes from differentiating clones were analyzed at the single cell level by immunofluorescence; all the cells were positive for type X collagen, while approximately 50% of them showed positivity for type II collagen.     

Expression of collagen type transcripts in chick embryonic bone detected by in situ cDNA-mRNA hybridization

The development of the chick embryonic calvarium, an intramembranous bone, is characterized by direct differentiation of cranial ectomesenchymal cells into osteoblasts without the formation of a cartilage anlage. Collagen biosynthesis remains predominantly as type I in the calvaria. However, in severely calcium-deficient chick embryos maintained in shell-less (SL) culture, cartilage-specific type II collagen is synthesized by the calvaria. Immunohistochemistry localized the cells expressing type II collagen to undermineralized regions of the SL bone. In this study, collagen gene expression in bones of normal (N) and calcium-deficient SL chick embryos was examined at Incubation Day 14 by in situ cDNA-mRNA hybridization. A critical step in the procedure, which used biotinylated cDNA probes, was the selection of fixation conditions which maximized RNA retention and maintenance of tissue morphology. Tissues fixed in modified Carnoy's fixative (58% ethanol, 30% choloroform, 10% acetic acid, 2% formaldehyde) for 2-4 hr at -20 degrees C sectioned well and retained their cell morphology and cytoplasmic RNA. Other treatments important for the procedure included demineralization in 0.25 M HCl and removal of matrix by hyaluronidase digestion. In situ hybridization with type-specific collagen cDNA probes revealed that type II collagen mRNA was present in cells throughout the SL calvaria. More importantly, cells with type II collagen mRNA were also present in N calvaria which do not synthesize the protein. The overall abundance of type II-positive cells in N calvaria was not significantly different from that in SL calvaria, but their distribution throughout the bones differed. In general, the regional distribution of type II cells was inversely correlated with the extent of matrix mineralization. In the N calvaria, cells containing collagen type II mRNA were absent in the extensively mineralized superior zone, but were found in the temporal zone which showed limited mineralization. On the other hand, in the SL calvaria, which were substantially undermineralized overall, cells with type II mRNA were found throughout the tissue. Interestingly, the overall ratio of type I cells to type II cells was approximately 50% higher in N calvaria. These findings suggest that collagen type mRNA expression in the chick embryonic calvarium is correlated with, and perhaps dependent on, the extent of tissue matrix mineralization.     

Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during “in vitro” chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.Therefore anchorin CII mRNA reaches its maximum level in hypertrophic stage II chondrocytes.