Protein Kinase C-ζ Regulates Transcription of the Matrix Metalloproteinase-9 Gene Induced by IL-1 and TNF-α in Glioma Cells via NF-κB

Abstract

The regulation of matrix metalloproteinase-9 (MMP-9) expression in glioma cells is one of the key processes in tumor invasion through the brain extracellular matrix. Although some studies have demonstrated the implication of classic protein kinase C (PKC) isoforms in the regulation of MMP-9 production by phorbol esters or lipopolysaccharide, the involvement of specific PKC isoforms in the signaling pathways leading to MMP-9 expression by inflammatory cytokines remains unclear. Here we report that the atypical PKC-isoform participates in the induction of MMP-9 expression by interleukin-1 (IL-1) and tumor necrosis factor-␣ (TNF-␣) in rat C6 glioma cells. Indeed, zymography and semi-quantitative reverse transcriptase-PCR analysis showed that pretreatment of C6 cells with PKC-pseudosubstrate abolished MMP-9 activity and gene expression induced by IL-1 or TNF-␣. Accordingly, IL-1 and TNF-␣ were able to induce PKC-activity, as demonstrated by in vitro kinase assay using immunoprecipitated PKC-. Furthermore, stable C6 clones overexpressing PKC-, but not PKC-⑀, displayed an up-regulation of MMP-9 constitutive expression as well as an increase of mmp-9 promoter activity. These processes were inhibited by an NF-B-blocking peptide and completely prevented by NF-B-binding site mutation in the mmp-9 promoter. Taken together, these results indicate that PKC-plays a key role in the regulation of MMP-9 expression in C6 glioma cells through NF-B.Glioma cells have the ability to invade brain tissues by secreting matrix metalloproteinases (MMPs), 1 a family of proteases able to degrade different components of the extracellular matrix including collagen, fibronectin, and proteoglycans. One of these MMPs, MMP-9, has received much attention as its expression correlates with the progression of glioma (1). Furthermore, MMP-9 seems to be essential for the invasiveness of glioma cells, as it was recently reported that the inhibition of MMP-9 expression by antisense gene transfer strongly reduced the invasion of glioblastoma cells in vitro and in vivo (2). Therefore, understanding the role of the molecules implicated in the signaling pathways leading to mmp-9 gene expression in glioma cells is important in order to identify new therapeutic targets. Several studies (3-5) have focused on the implication of protein kinase C (PKCs) in the regulation of mmp-9 gene expression, most notably by testing the effect of phorbol 12-myristate 13-acetate (PMA) on different types of cells, including human glioma cells. Members of the PKC family are divided into the following three groups of isoenzymes: the conventional PKC isoforms, which are activated by calcium and diacylglycerol (␣, ␤I, ␤II, and ␥); the novel PKCs, which are activated by diacylglycerol but are calcium-insensitive (␦, ⑀, , and ); and the atypical PKCs, which are calcium-and diacylglycerol-insensitive ( and /). Despite the fact that a large number of studies (5-7) have established a link between PKCs and MMP-9 expression using PKC inhibitors, very few studies have addressed the implicat...

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