Phyllis LuValle

The Raf-1/MEK/ERK Pathway Regulates the Expression of the p21Cip1/Waf1 Gene in Chondrocytes

The gene encoding the cyclin-dependent kinase inhibitor p21Cip1/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.

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.

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

We have determined the full‐length cDNA sequence of the human αl(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 αl(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.

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.

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.

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.

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.

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. J. Cell. Biochem. 72:549–557, 1999.

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. J. Cell. Biochem. 70:507–516, 1998.

Role of Non-Fibrillar Collagens in Matrix Assemblies

Extracellular matrices are composed of macromolecular components that interact to form highly complex supramolecular structures. Among these components are collagen fibrils. The fibrils represent tissue elements of high tensile strength, and their tissue-dependent three-dimensional patterns provide tissues with characteristic mechanical properties. Understanding the molecular composition of collagen fibrils and the interactions that define their specific tissue organization is, therefore, an essential aspect of molecular biomechanics.