RESUMO
Cartilage oligomeric matrix protein (COMP) is a component of cartilage, synovium, ligament, and tendon, yet its normal function is largely unknown. To identify its function we have expressed it in 293 and HeLa cell lines and in primary human chondrocytes. We find that COMP protects these cells against death, either in the presence or absence of tumor necrosis factor alpha and is able to block activation of caspase 3, a critical effector caspase. This effect appears to be mediated by the IAP (inhibitor of apoptosis protein) family of anti-apoptotic proteins because the levels of XIAP, survivin, cIAP1 and cIAP2 are significantly elevated in the COMP-expressing cells and down-regulation of survivin and XIAP protein levels by small interfering RNAs blocks the ability of COMP to enhance survival. The mRNAs for most of the IAP family members were not increased by COMP, indicating that a translational/post-translational mechanism was involved in their induction. However, in both HeLa cells and chondrocytes, COMP induced survivin mRNA by 5-fold. Thus survivin is the first gene identified to be up-regulated transcriptionally by COMP. The carboxyl-terminal half of the protein comprising the type 3 repeats and the RGD sequence (CaCTD domain) was sufficient to promote survival and to elevate the IAPs. Further, an RGD peptide was able to block the prosurvival effect of COMP and the induction of XIAP and survivin, indicating that survival is likely mediated through integrin signaling. These data point to a new role for COMP in protecting cells against death.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/fisiologia , Proteína de Matriz Oligomérica de Cartilagem , Caspase 3/genética , Caspase 3/metabolismo , Caspase 6/genética , Caspase 6/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular , Células Cultivadas , Condrócitos/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/genética , Glicoproteínas/fisiologia , Células HeLa , Humanos , Immunoblotting , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/fisiologia , Proteínas Matrilinas , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologiaRESUMO
The Wnt family of secreted glycoproteins plays an integral role in embryonic development and differentiation. To explore the role of Wnt's in one aspect of differentiation, namely osteogenesis, we employed a retroviral gene transfer approach to express Wnt-3a in the multipotent murine embryonic mesenchymal cell line C3H10T1/2. We found that expression of Wnt-3a in these cells had a significant, positive effect on cell growth in serum-containing medium, in that the cells grew to very high densities compared to the control cells. Additionally, apoptosis was markedly inhibited by Wnt-3a. However, when the cells were grown in serum-deficient medium, the Wnt-3a-expressing cells arrested efficiently in G1 phase, indicating that serum growth factors were needed in addition to Wnt-3a for enhanced proliferation. Wnt-3a-expressing cells exhibited high levels of alkaline phosphatase gene expression and enzymatic activity, but did not show any matrix mineralization. Unexpectedly, basal expression of bone sialoprotein, osteocalcin, and osteopontin were markedly inhibited by Wnt-3a, as were other known target genes of Wnt-3a, such as Brachyury, FGF-10, and Cdx1. When Wnt-3a-expressing cells were treated with osteogenic supplements in the presence of BMP-2, alkaline phosphatase gene expression and activity were further elevated. Additionally, BMP-2 was able to reverse the inhibitory effect of Wnt-3a on osteocalcin and osteopontin gene expression. These results indicate that while Wnt-3a represses basal expression of some osteogenic genes, this repression can be partially reversed by BMP-2. Finally, the enhanced gene expression of alkaline phosphatase induced by Wnt-3a could be effectively suppressed by the combined action of dexamethasone and 1,25-dihydroxyvitamin D(3). These data show for the first time that Wnt-3a has an unusual effect on multipotential embryonic cells, in that it enhances cellular proliferation and expression of alkaline phosphatase, while it represses most other marker genes of osteogenic differentiation.
