The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA.We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.     

Localization of the expression of types I, III, and IV collagen, TGF-beta 1 and c-fos genes in developing human calvarial bones

Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.     

Temporal and spatial expression of genes for cartilage extracellular matrix proteins during avian mandibular arch development

We have examined the temporal expression of genes for extracellular matrix proteins (type I collagen, type II collagen, and the cartilage specific proteoglycan core protein) during the development of the avian mandibular arch. We detected low levels of type II collagen mRNA in the mandibular arch as early as stage 15. Type II collagen mRNA remained low but increased slightly as development progressed from stage 15 to stage 25. More dramatic increases occurred after stage 25 coincident with overt chondrogenesis. In contrast, mRNA for the core protein of cartilage specific proteoglycan was not detected prior to the onset of chondrogenesis, appeared at stage 25, and increased thereafter. Type I collagen mRNA was also present as early as stage 15 and dramatically increased after stage 28/29, coincident with initiation of osteogenesis. Using in situ hybridization, we found that type II collagen mRNA became detectable in the center of the mandible around stage 24/25 coincident with the initiation of chondrogenesis. At later stages (26-32) type II collagen mRNA was localized in the cartilaginous rudiment. The pattern of hybridization observed with the proteoglycan core protein probe at later stages of development was essentially identical to that observed with the type II collagen probe. In contrast, the probe for the alpha 1 (I) collagen mRNA was localized over the perichondrium, over differentiated bone, and in areas within the mandibular arch where bone formation had been initiated.     

Differential expression of fibrillar collagen genes during callus formation

An experimental fracture healing model in the rat tibio-fibular bone was employed to study the appearance of messenger RNAs for types I, II and III collagens during endochondral fracture repair. Total RNA was extracted from normal bone and from callus tissue at various time points. The total RNAs were analyzed in Northern hybridization for their contents of procollagen mRNAs using specific cDNA clones. The results show that during the first week of fracture repair type III collagen mRNA is increased to the greatest extent, followed by type II collagen mRNA during the second week. The 28-day callus resembles bone by containing mainly type I collagen mRNAs and very little type II or III collagen mRNA.     

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.     

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.     

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.     

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.     

Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system

Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in the gene expression of collagens, fibronectin, integrin and proteoglycans during matrix-induced bone morphogenesis

Subcutaneous implantation of demineralized bone matrix in rat results in the local cartilage and bone development. This in vivo model of bone formation was used to examine the expression patterns of cartilage and bone specific extracellular matrix genes. The steady state levels of mRNA in implants for cartilage specific type II collagen, type IX collagen, proteoglycan link protein and cartilage proteoglycan core protein (aggrecan) were increased during chondrogenesis and cartilage hypertrophy. Fibronectin mRNA levels were high during mesenchymal cell migration, attachment and chondrogenesis. Integrin (beta 1 chain) mRNA was expressed throughout the endochondral bone development. Type I collagen mRNA levels in implants increased as early as day 3, reached its peak during osteogenesis. These gene markers will be useful in the study of the mechanism of action of bone morphogenetic proteins present in the demineralized bone matrix.     

Similar, but not identical, modulation of expression of extracellular matrix components during in vitro and in vivo aging of human skin fibroblasts.

Regulation of the synthesis of procollagen and other extracellular matrix components was examined in human skin fibroblasts obtained from donors of various ages, from fetal to 80 years old (in vivo aged), and in fetal fibroblasts at varying passage levels (in vitro aged). Growth rates and saturation densities of fibroblasts decreased with increasing age of the donor and after passage 20 of fetal fibroblasts. The rates of collagen and proteoglycan synthesis also decreased during both types of aging to about 10-25% of the rate in early passage fetal fibroblasts, whereas the synthesis of total noncollagenous proteins was not greatly affected. Decreased collagen synthesis in both types of aging was correlated with lower steady-state levels of mRNAs for the two subunits of type I procollagen mRNA, although their regulation was not coordinate. Type III collagen mRNA levels also declined in both types of aging. The concentration of fibronectin mRNA also decreased during in vitro aging but more rapidly than the collagen mRNAs, whereas in fibroblasts from 51-80-year-old donors, it was similar to or higher than in early passage fetal fibroblasts. This study suggests that the decreased synthesis of procollagen and proteoglycans in in vivo aged fibroblasts represents changes that are responsible for intrinsic degenerative changes that occur in human skin during aging. Furthermore, although in vitro and in vivo aging were similar in many respects, they were not equivalent, as evidenced by the differences in regulation of fibronectin expression.     

Altered beta-actin gene expression in phorbol myristate acetate-treated chondrocytes and fibroblasts.

Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.     

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.