Frank Beier

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.

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.

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.

Localization of silencer and enhancer elements in the human type X collagen gene

Collagen type X is a short, network‐forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5′‐flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non‐tissue‐specific positive regulatory element was present in the first intron. Third, a cell‐type‐specific enhancer element active only in hypertrophic chondrocytes was located between −2.4 and −0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291‐296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements. J. Cell. Biochem. 66:210‐218, 1997.

Type X collagen expression and hypertrophic differentiation in chondrogenic neoplasias

Abstract Little is known about matrix biochemistry and cell differentiation patterns in chondrogenic neoplasms. This is the first description of the focal expression of collagen type X by neoplastic chondrocytes in situ and its incorporation into the extracellular matrix of cartilaginous tumors. This shows that neoplastic chondrocytes have the potential to undergo the full program of cell differentiation, including hypertrophy, comparable to their physiological counterparts in the growth plate. However, only in benign osteochondromas was a zonal expression of type X collagen found similar to that observed in the growth plate, where the cells immediately above the ossification frontier are selectively positive for type X collagen. In enchondromas and chondrosarcomas, the expression was randomly distributed within the tumors. Surprisingly, in less differentiated chondrosarcomas with spindle-shaped cells and non-cartilaginous extracellular matrix, exceptional expression of collagen type X was observed, which indicates potential uncoupling of collagen type X expression from the differentiated chondrocytic phenotype in neoplastic chondrocytes in vivo.

Upregulation of type X collagen expression in osteoarthritic cartilage.

Joint destruction of articular cartilage in osteoarthritis and rheumatoid arthritis is heralded by surface fibrillation, loss of proteoglycans from the articular surface and increasing hydration and softening of the cartilage matrix (Sokoloff 1982). These initial changes are followed by chondrocyte proliferation and formation of cell clusters and extensive remodeling of the cartilage matrix, i.e. massive degradation and synthesis of new, but partially altered cartilage components.

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.