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Abstract
Bcl-2 is an anti-apoptotic protein that has recently been shown to regulate other cellular functions. We previously reported that Bcl-2 regulates chondrocyte matrix gene expression, independent of its anti-apoptotic function. Here, we further investigate this novel function of Bcl-2 and examine three intracellular signaling pathways likely to be associated with this function. The present study demonstrates that the activity of Sox9, a master transcription factor that regulates the gene expression of chondrocyte matrix proteins, is suppressed by Bcl-2 small interference RNA in the presence of caspase inhibitors. This effect was attenuated by prior exposure of chondrocytes to an adenoviral vector expressing sense Bcl-2. In addition, the down-regulation of Bcl-2, Sox9, and chondrocyte-specific gene expression by serum withdrawal in primary chondrocytes was reversed by expressing Bcl-2. Inhibition of the protein kinase C alpha and NFkappaB pathways had no effect on the maintenance of Sox9-dependent gene expression by Bcl-2. In contrast, whereas the MEK-ERK1/2 pathway negatively regulated the differentiated phenotype in wild type chondrocytes, inhibition of this pathway reversed the loss of differentiation markers and fibroblastic phenotype in Bcl-2-deficient chondrocytes. In conclusion, the present study identifies a specific signaling pathway, namely, MEK-ERK1/2, that is downstream of Bcl-2 in the regulation of Sox9-dependent chondrocyte gene expression and phenotype.
Highlights
Signaling pathway that activates Sp1 DNA binding in cancer cells [11]
The Level of Bcl-2 Protein Modulates Sox9-dependent Chondrocyte Matrix Gene Expression—We previously demonstrated that suppression of Bcl-2 protein level with integrated antisense constructs resulted in down-regulation of expression of mRNA coding for cartilage matrix proteins even in the presence of caspase inhibitors that blocked full apoptosis (29 –31)
Immunocytochemistry was used to demonstrate no change in the Bcl-2 level in Immortalized rat chondrocytes (IRC) chondrocytes transfected with control siRNA containing random DNA sequences (Fig. 1A), whereas there was an obvious decrease in the percentage of chondrocytes expressing Bcl-2 following transfection with the Bcl-2 siRNA (Fig. 1B)
Summary
Signaling pathway that activates Sp1 DNA binding in cancer cells [11]. Bcl-2 overexpression in melanoma cells under hypoxia conditions modulates vascular endothelial growth factor expression [2, 7]. In the hypertrophic zone where chondrocytes undergo terminal differentiation and start to express collagen type X and bone related matrix proteins, there is decreased expression of Bcl-2 and cartilage matrix proteins as well as loss of Sox expression [20, 25,26,27]. Chondrocytes constitutively expressing Bcl-2 are protected from down-regulation of aggrecan, collagen type II, and Sox following serum withdrawal [30, 31]. From these studies we hypothesize that Bcl-2 may function to regulate cartilage matrix expression in addition to or independent of its role in regulating apoptosis.
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