TGFbeta and PTHrP control chondrocyte proliferation by activating cyclin D1 expression.

Exact coordination of growth plate chondrocyte proliferation is necessary for normal endochondral bone development and growth. Here we show that PTHrP and TGFbeta control chondrocyte cell cycle progression and proliferation by stimulating signaling pathways that activate transcription from the cyclin D1 promoter. The TGFbeta pathway activates the transcription factor ATF-2, whereas PTHrP uses the related transcription factor CREB, to stimulate cyclin D1 promoter activity via the CRE promoter element. Inhibition of cyclin D1 expression with antisense oligonucleotides causes a delay in progression of chondrocytes through the G1 phase of the cell cycle, reduced E2F activity, and decreased proliferation. Growth plates from cyclin D1-deficient mice display a smaller zone of proliferating chondrocytes, confirming the requirement for cyclin D1 in chondrocyte proliferation in vivo. These data identify the cyclin D1 gene as an essential component of chondrocyte proliferation as well as a fundamental target gene of TGFbeta and PTHrP during skeletal growth.

Cell cycle control in growth plate chondrocytes.

Growth of endochondral bones is regulated by the coordinated proliferation and differentiation of chondrocytes in the epiphyseal growth plates. Many skeletal diseases are caused by pathogenic disruptions of these two processes. While the intracellular mechanisms regulating chondrocyte proliferation and differentiation are poorly understood, recent evidence from studies using genetically altered mice and from experiments in cultured chondrocytes point to a prominent role of cell cycle proteins in this context. This article summarizes our current understanding of the expression, regulation, and function of cell cycle genes in chondrocytes.

Activating transcription factor 2 is necessary for maximal activity and serum induction of the cyclin A promoter in chondrocytes.

Endochondral bone growth is regulated through the proliferation and differentiation of growth plate chondrocytes. Mice deficient for the activating transcription factor 2 (ATF-2) gene show reduced proliferation of chondrocytes. Here we demonstrate that the cyclin A gene is a target of ATF-2 in chondrocytes. Serum stimulation of chondrogenic rat chondrosarcoma cells induces cyclin A expression. A cyclic AMP response element (CRE) is necessary for optimal activity and serum inducibility of the cyclin A promoter and confers regulation by ATF-2. Phosphorylation and activity of ATF-2 are enhanced dramatically upon serum stimulation of rat chondrosarcoma cells. Mutation of the CRE or overexpression of dominant-negative ATF-2 inhibits serum induction of the cyclin A promoter. Chondrocytes from ATF-2-deficient mice display reduced and delayed induction of cyclin A upon serum stimulation. The ATF-2-related transcription factor CRE-binding protein contributes to the activity of the cyclin A CRE in chondrocytes, whereas c-Jun and c-Fos regulate the promoter independently of the CRE. Our data suggest that the reduction in cyclin A levels in chondrocytes from ATF-2-deficient mice contributes to their phenotype of reduced chondrocyte proliferation and dwarfism.

The Raf-1/MEK/ERK pathway regulates the expression of the p21(Cip1/Waf1) gene in chondrocytes.

The gene encoding the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) is up-regulated in many differentiating cells, including maturing chondrocytes. Since strict control of chondrocyte proliferation is essential for proper bone formation and since p21 is likely involved in this control, we initiated analyses of the mechanisms regulating expression of p21 in chondrocytes. p21 expression and promoter activity was strongly increased during the differentiation of chondrogenic MCT cells. We have identified a 68-base pair fragment conferring transcriptional up-regulation of the p21 gene in chondrocytes. The activity of this fragment required active Raf-1 in MCT cells as well as in primary mouse chondrocytes. Inhibition of downstream factors of Raf-1 (MEK1/2, ERK1/2, and Ets2) also repressed the activity of the 68-base pair fragment in MCT cells. The chemical MEK1/2 inhibitor PD98059 reduced protein levels of p21 in MCTs and primary mouse chondrocytes. These data suggest that signaling through the Raf-1 pathway is necessary for the optimal expression of p21 in chondrocytes and may play an important role in the control of bone formation.

Serum induction of the collagen X promoter requires the Raf/MEK/ERK and p38 pathways.

The collagen X gene is expressed exclusively by differentiated, hypertrophic chondrocytes. The mechanisms controlling collagen X expression remain largely unknown. Here we show that collagen X promoter activity can be induced by serum stimulation of chondrogenic MCT cells. The serum response is conferred by a 462 nucleotide promoter fragment. Both the c-Raf/MEK/ERK and p38 MAP kinase pathways are required for this effect, whereas phosphatidylinositol-3-kinase and protein kinase A repress promoter activation. These data are the first to demonstrate serum inducibility of the collagen X promoter and to identify signal transduction pathways involved.

Cell cycle genes in chondrocyte proliferation and differentiation.

Coordinated proliferation and differentiation of growth plate chondrocytes controls longitudinal growth of endochondral bones. While many extracellular factors regulating these processes have been identified, much less is known about the intracellular mechanisms transducing and integrating these extracellular signals. Recent evidence suggests that cell cycle proteins play an important role in the coordination of chondrocyte proliferation and differentiation. Our current knowledge of the function and regulation of cell cycle proteins in endochondral ossification is summarized.

Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chondrocytes.

Endochondral bone growth is regulated by the rates of chondrocyte proliferation and differentiation. However, the intracellular mechanisms regulating these processes are poorly understood. Recently, interruption of the gene encoding the transcription factor activating transcription factor 2 (ATF-2) was shown to inhibit proliferation of chondrocytes in mice [Reimold, A. M., et al. (1996) Nature (London) 379, 262-265]. The target genes of ATF-2 that are responsible for this phenotype remain unknown. Here we report that the cyclin D1 gene is a direct target of ATF-2 in chondrocytes. ATF-2 is present in nuclear extracts from chondrogenic cell lines and binds, as a complex with a CRE-binding protein (CREB)/CRE modulator protein, to the cAMP response element (CRE) in the cyclin D1 promoter. Mutation of the cyclin D1 CRE caused a 78% reduction in the activity of the promoter in chondrocytes. Overexpression of ATF-2 in chondrocytes enhanced activity of the cyclin D1 promoter 3. 5-fold. In contrast, inhibition of endogenous ATF-2 or CREB by expression of dominant-negative inhibitors of CREB and ATF-2 significantly reduced the activity of the promoter in chondrocytes through the CRE. In addition, levels of cyclin D1 protein are greatly reduced in the chondrocytes of ATF-2-deficient mice. These data identify the cyclin D1 gene as a direct target of ATF-2 in chondrocytes and suggest that reduced expression of cyclin D1 contributes to the defective cartilage development of these mice.

Raf signaling stimulates and represses the human collagen X promoter through distinguishable elements.

Endochondral bone growth is regulated through the rates of proliferation and differentiation of growth plate chondrocytes. While little is known about the intracellular events controlling these processes, the protein kinase c-Raf, a central component of the cellular signal transduction machinery, has recently been shown to be expressed only by differentiated, hypertrophic chondrocytes. The involvement of c-Raf in the transcriptional regulation of the hypertrophic chondrocyte-specific collagen X gene was investigated using cotransfections of collagen X reporter plasmids and expression vectors for mutant c-Raf proteins. Both activated and dominant-negative forms of c-Raf reduced the activity of the collagen X promoter to approximately 30%. The element mediating the repressing effect of activated c-Raf was located between nucleotides -2864 and -2410 of the promoter, whereas the effect of the dominant-negative form of c-Raf was conferred by the 462 nucleotides immediately upstream of the transcription start site. Inhibition of MEK1/2 and ERK1/2, downstream components of Raf-signaling, also caused repression of basal collagen X promoter activity. These data suggest that c-Raf regulates collagen X promoter activity positively and negatively through different cis-acting elements and represent the first evidence of c-Raf activity described in hypertrophic chondrocytes.

A BMP responsive transcriptional region in the chicken type X collagen gene.

Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promote both chondrogenesis and chondrocyte hypertrophy. BMPs have recently been found to activate a membrane serine/threonine kinase signaling mechanism in a variety of cell types, but the downstream effectors of BMP signaling in chondrocyte differentiation remain unidentified. We have previously reported that BMP-2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that type X collagen mRNA levels in chondrocytes cultured under serum-free (SF) conditions are elevated 3- to 5-fold within 24 h. To better define the molecular mechanisms of induction of chondrocyte hypertrophy by BMPs, we examined the effect of BMPs on type X collagen production by 15-day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. Two populations of chondrocytes were used: one representing resting cartilage isolated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna. BMP-2, BMP-4, and BMP-7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline phosphatase, diminished levels of type II collagen, and induction of the hypertrophic chondrocyte-specific marker, type X collagen. To test whether BMP control of type X collagen expression occurs at the transcriptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5' flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. A 533 bp region located 2.4-2.9 kb upstream from the type X collagen transcriptional start site was both necessary and sufficient for strong BMP responsiveness in cells destined for hypertrophy, but not in chondrocytes derived from the lower sterna.

Proximal DNA elements mediate repressor activity conferred by the distal portion of the chicken collagen X promoter.

Collagen X is expressed specifically in hypertrophic chondrocytes within cartilage that is undergoing endochondral ossification. The chicken collagen X gene is transcriptionally regulated, and under the control of multiple cis elements within the distal promoter region (-4,442 to -558 base pairs from the transcription start) as well as the proximal region (-558 to +1). Our previous data (LuValle et al., [1993] J. Cell Biol. 121:1173-1179) demonstrated that the proximal sequence directed high reporter gene activity in the three cell types tested (hypertrophic chondrocytes, immature chondrocytes, and fibroblasts), while distal elements acted in an additive manner to repress the effects of the proximal sequence on reporter gene activity in non-collagen X expressing cells only (immature chondrocytes and fibroblasts). We show here that elements within the proximal sequence (nucleotides -557 to -513) are necessary for the cell-specific expression of type X collagen by hypertrophic chondrocytes. These elements bind to proteins of 100 kDa in all three cell types, and 47 kDa in non-collagen X expressing cells. Reporter gene activity in hypertrophic chondrocytes is reduced to the levels seen in non-collagen X-expressing cells in the absence of these elements.

Ascorbate differentially regulates elastin and collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts by pretranslational mechanisms.

Ascorbate contributes to several metabolic processes including efficient hydroxylation of hydroxyproline in elastin, collagen, and proteins with collagenous domains, yet hydroxyproline in elastin has no known function. Prolyl hydroxylation is essential for efficient collagen production; in contrast, ascorbate has been shown to decrease elastin accumulation in vitro and to alter morphology of elastic tissues in vivo. Ascorbate doses that maximally stimulated collagen production (10-200 microM) antagonized elastin biosynthesis in vascular smooth muscle cells and skin fibroblasts, depending on a combination of dose and exposure time. Diminished elastin production paralleled reduced elastin mRNA levels, while collagen I and III mRNAs levels increased. We compared the stability of mRNAs for elastin and collagen I with a constitutive gene after ascorbate supplementation or withdrawal. Ascorbate decreased elastin mRNA stability, while collagen I mRNA was stabilized to a much greater extent. Ascorbate withdrawal decreased collagen I mRNA stability markedly (4.9-fold), while elastin mRNA became more stable. Transcription of elastin was reduced 72% by ascorbate exposure. Differential effects of ascorbic acid on collagen I and elastin mRNA abundance result from the combined, marked stabilization of collagen mRNA, the lesser stability of elastin mRNA, and the significant repression of elastin gene transcription.

Variability in the upstream promoter and intron sequences of the human, mouse and chick type X collagen genes.

The type X collagen gene is specifically expressed in hypertrophic chondrocytes during endochondral ossification. Transcription of the type X collagen gene by these differentiated cells is turned on at the same time as transcription of several other cartilage specific genes is switched off and before mineralization of the matrix begins. Analysis of type X collagen promoters for regulatory regions in different cell culture systems and in transgenic mice has given contradictory results suggesting major differences among species. To approach this problem, we have determined the nucleotide sequences of the two introns and upstream promoter sequences of the human and mouse type X collagen genes and compared them with those of bovine and chick. Within the promoter regions, we found three boxes of homology which are nearly continuous in the human gene but have interruptions in the murine gene. One of these interruptions was identified as a complex 1.9 kb repetitive element with homology to LINE, B1, B2 and long terminal repeat sequences. Regulatory elements of the human type X collagen gene are located upstream of the region where the repetitive element is inserted in the mouse gene, making it likely that the repetitive element is inserted between the coding region and regulatory sequences of the murine gene without interfering with its expression pattern. We also compared the sequences of the introns of both genes and found strong conservation. Comparisons of the mammalian sequences with promoter and first intron sequences of the chicken type X collagen gene revealed that only the proximal 120 nucleotides of the promoter were conserved, whereas all other sequences displayed no obvious homology to the murine and human sequences.

Phenotypic stability and variation in cells of the porcine aorta: collagen and elastin production.

The extracellular matrix of the developing vasculature varies in composition as a function of time and position. Cellular models of vascular biology and pathology depend on the assumption that stable phenotypic characteristics of vascular cells can be propagated through several generations of in vitro cultivation. We show that the positional and developmental heterogeneity of matrix phenotypes in the porcine aorta are expressed by explanted vascular smooth muscle cell (SMC) and adventitial cell populations for a limited number of passages. Elastin was expressed most highly by thoracic SMC while interstitial collagen production was usually maximal in abdominal segments. Parallel gradients of collagen types I, III and V, detected by specific ELISA assays, were expressed in early-passage SMC. Adventitial cell populations from the abdominal aorta of the neonatal pig accumulated significant levels of collagen, while these fibroblasts produced less than 10% of the elastin made by SMC. All cell populations expressed alpha-smooth muscle actin in vitro. Gradients of collagen and elastin expression were evident for no more than three passages, and direct outgrowth of cells without limited digestion of the matrix further reduced phenotypic stability. Variation and decline of the elastin phenotype could be due to hypermethylation of regulatory sequences in the elastin gene or trans-acting factors, but elastin production was dose-dependently stimulated to a similar extent (100%; 10 microM 5-azacytidine) in all segmental SMC populations at early (p1) and late (p3) passage. These data indicated that faithful reflection of in vivo SMC behavior was limited to a few population doublings, at least under standard culture conditions. Modification of the cellular environment by reducing serum factors, changing matrix, or adding mechanical stimulation may increase phenotypic stability.

Spondylometaphyseal dysplasia in mice carrying a dominant negative mutation in a matrix protein specific for cartilage-to-bone transition.

The vertebrate skeleton is formed primarily by endochondral ossification, starting during embryogenesis when cartilage anlagens develop central regions of hypertrophic cartilage which are replaced by bony trabeculae and bone marrow. During this process chondrocytes express a unique matrix molecule, type X collagen. We report here that mice carrying a mutated collagen X transgene develop skeletal deformities including compression of hypertrophic growth plate cartilage and a decrease in newly formed bone, as well as leukocyte deficiency in bone marrow, reduction in size of thymus and spleen, and lymphopenia. The defects indicate that collagen X is required for normal skeletal morphogenesis and suggest that mutations in COL10A1 are responsible for certain human chondrodysplasias, such as spondylometaphyseal dysplasias and metaphyseal chondrodysplasias.

Type X collagen is transcriptionally activated and specifically localized during sternal cartilage maturation.

Type X collagen is an extracellular matrix protein which is synthesized by chondrocytes when they undergo hypertrophy. We present evidence here that the expression of type X collagen in the developing chick sternum is controlled primarily by transcriptional mechanisms. Using chondrocyte nuclei isolated from 15-, 16-, 17- and 18-day chick embryonic sterna, nuclear run-off assays demonstrate that type X collagen gene transcription begins at day 16 in chondrocytes isolated from the cephalic portion. This occurs two days prior to mineralization of this tissue as observed by alizarin red staining. The rate of type X transcription increases dramatically through days 17 and 18. Western blot analyses of extracts of freshly isolated sternal chondrocytes from the same stages show that intracellular levels of the type X protein follow the same time course. Immunostaining with a monoclonal antibody specific for type X collagen demonstrates that the initial appearances of hypertrophic cells and pericellular type X collagen occur at embryonic day 16 in the cephalic portion of sterna. Observation of immunostained cephalic sternal sections from day 18 embryos by confocal microscopy reveals that type X collagen is localized in a capsule-like configuration around each hypertrophic chondrocyte.

Genomic organization and full-length cDNA sequence of human collagen X.

We have determined the full-length cDNA sequence of the human alpha 1(X) collagen gene by sequence analysis of a genomic clone ERG [(1991) Dev. Biol. 148, 562-572], and of cDNA fragments generated from a reverse transcribed as alpha 1(X) mRNA by PCR. We defined the promoter region, the transcription initiation site and the full-length 5'-untranslated region. We also report the exon/intron boundaries of the transcript and the complete 3'-untranslated region as well as a 3'-flanking sequence containing two additional polyadenylation signals. The promoter region is homologous to chicken and mouse type X promoters within several highly conserved regions. The genomic organization shows high homologies to chicken and mouse.

Transcriptional regulation of type X collagen during chondrocyte maturation.

The 18-day embryonic chick sternum expresses type X collagen only in the cephalic, presumptive mineralization zone which consists primarily of hypertrophic chondrocytes. The smaller chondrocytes of the caudal, permanent cartilaginous zone do not express this protein. Run-off transcriptions of nuclei isolated directly from cephalic and caudal 18-day embryonic sterna demonstrate that type X collagen is specifically transcribed by cells of the cephalic zone. In addition, type X mRNA is localized to the cephalic zone as detected by in situ hybridization. The results suggest that the cell-specific expression of the type X collagen gene is due entirely to transcriptional regulation.

The type X collagen gene. Intron sequences split the 5'-untranslated region and separate the coding regions for the non-collagenous amino-terminal and triple-helical domains.

Type X collagen, expressed by hypertrophic chondrocytes, consists of homotrimeric molecules with subunits that are only about one-half the size of the polypeptides of fibrillar collagens. In this report we describe for the first time the complete primary structure of type X collagen, based on cloning and sequencing of cDNA and genomic DNA. A comparison between the nucleotide sequences of the cDNA and genomic DNA clones has also allowed determination of the complete exon structure of the type X collagen gene. Our results demonstrate that the primary translation product of the chicken type X collagen mRNA is 682 amino acid residues long with a calculated molecular mass of 67,317 Da for the nonhydroxylated form. This calculated molecular mass is in excellent agreement with the observed electrophoretic mobility of cell-free translation products with both poly(A)+ RNA isolated from chondrocytes as well as RNA transcribed in vitro from a full length cDNA construct. It is also in agreement with the observed size of type X collagen polypeptides isolated from the media of cultured hypertrophic chondrocytes. Thus, our data exclude the possibility of a high molecular weight precursor form of type X collagen. Our results also confirm that the chicken type X gene has a most unusual exon structure when compared to other vertebrate collagen genes. The gene has only three exons. One exon (97 base pairs (bp)), codes for most of the 5'-untranslated region of the mRNA, a second exon (159 bp) codes for the signal peptide and a short non-triple-helical domain, while the third exon (2136 bp) contains the coding region for the entire triple-helix and a large non-triple-helical carboxyl domain.