Effect of cartilage oligomeric matrix protein on mesenchymal chondrogenesis in vitro.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) mutations have been identified as responsible for two arthritic disorders, multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). However, the function of COMP in chondrogenic differentiation is largely unknown. Our investigation focuses on analyzing the function of normal COMP protein in cartilage biology. METHODS AND RESULTS: To explore the function of COMP we make use of an in vitro model system for chondrogenesis, consisting of murine C3H10T1/2 mesenchymal cells maintained as a high-density micromass culture and stimulated with bone morphogenetic protein 2 (BMP-2). Under these culture conditions, C3H10T1/2 cells undergo active chondrogenesis in a manner analogous to that of embryonic limb mesenchymal cells, and have been shown to serve as a valid model system to investigate the mechanisms regulating mesenchymal chondrogenesis. Our results indicate that ectopic COMP expression enhances several early aspects of chondrogenesis induced by BMP-2 in this system, indicating that COMP functions in part to positively regulate chondrogenesis. Additionally, COMP has inhibitory effects on proliferation of cells in monolayer. However, at later times in micromass culture, ectopic COMP expression in the presence of BMP-2 causes an increase in apoptosis, with an accompanying reduction in cell numbers in the micromass culture. However, the remaining cells retain their chondrogenic phenotype. CONCLUSIONS: These data suggest that COMP and BMP-2 signaling converge to regulate the fate of these cells in vitro by affecting both early and late stages of chondrogenesis.

Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2.

The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environment, following treatment with soluble human bone morphogenetic protein-2 (BMP-2). To enhance this process, the human BMP-2 cDNA was cloned into a retroviral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3HIOT1/2 cells with this retroviral construct resulted in an infection efficiency of 90-95% and was highly effective in converting cells in micromass culture to a chondrocyte phenotype, as assessed by positive Alcian blue staining for extracellular matrix proteoglycans, increased sulfate incorporation, increased expression of the cartilage marker genes collagen type II and aggrecan, and decreased expression of collagen type I. Interestingly, BMP-2 expression in the micromass cultures also induced the expression of the cell cycle inhibitory protein/differentiation factor p21/WAF1, suggesting its functional involvement in chondrogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected cells did not chondrify when co-cultured as a nonoverlapping micromass adjacent to BMP-2 expressing cells. These data indicate that retrovirally expressed BMP-2 is highly effective at inducing a chondrocyte phenotype in a multipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.

Regulation of scleraxis function by interaction with the bHLH protein E47.

Scleraxis is a basic helix-loop-helix (bHLH) protein whose function has been postulated to be preconfigurative of sclerotomal mesenchymal patterning during early embryonic development by regulating expression of differentiation-specific genes, particularly those involved in chondrogenesis. To gain understanding of the molecular action of scleraxis we test the hypothesis that it heterodimerizes with another bHLH protein to activate gene expression. Transient coexpression of scleraxis and E47, a candidate bHLH protein, showed that scleraxis dimerizes with E47 in vivo and that this complex binds to a classic E-box DNA sequence better than either factor alone. Further, when expressed together, scleraxis and E47 synergistically enhanced transcription from a promoter containing multiple E-box binding sites.

Tumor necrosis factor and gamma-interferon repress transcription from the c-myc P2 promoter by reducing E2F binding activity.

Transcription of the c-myc gene in HeLa cells has been shown to be repressed by the combined action of tumor necrosis factor (TNF) and gamma-interferon (gamma-INF). Shown here, these two cytokines inhibit proliferation of Hela cells with a coordinate inhibition of c-myc gene expression. It was found that these two cytokines exert their effects on the more proximal region of the c-myc P2 promoter. Using c-myc promoter:CAT constructs, it was found that the combined action of these cytokines significantly repress transcription from P2. This repression occurred through the E2F site within the promoter and not the ME1a2 or ME1a1 sites. However, these cytokines had no effect on transcription from the rous sarcoma virus promoter or the SV40 virus early promoter. Protein binding assays indicate that TNF and gamma-INF did not effect the ability of the ME1a2 factor to bind to its site but did significantly repress E2F factor binding to its DNA sequence.

Intragenic suppressors of mutant DNA topoisomerase I-induced lethality diminish enzyme binding of DNA.

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of the antitumor drug camptothecin (Cpt). Mutation of several conserved residues in yeast top1 mutants is sufficient to induce cell lethality in the absence of camptothecin. Despite tremendous differences in catalytic activity, the mutant proteins Top1T722Ap and Top1R517Gp cause cell death via a mechanism similar to that of Cpt, i.e. stabilization of the covalent enzyme-DNA intermediate. To establish the interdomainal interactions required for the catalytic activity of Top1p and how alterations in enzyme structure contribute to the cytotoxic activity of Cpt or specific DNA topoisomerase I mutants, we initiated a genetic screen for intragenic suppressors of the top1T722A-lethal phenotype. Nine single amino acid substitutions were defined that map to the conserved central and C-terminal domains of Top1p as well as the nonconserved linker domain of the protein. All reduced the catalytic activity of the enzyme over 100-fold. However, detailed biochemical analyses of three suppressors, top1C273Y,T722A, top1G295V,T722A, and top1G369D,T722A, revealed this was accomplished via a mechanism of reduced affinity for the DNA substrate. The mechanistic implications of these results are discussed in the context of the known structures of yeast and human DNA topoisomerase I.