Expression screening identifies a novel negative regulator (SM22/transgelin) of 92 kDa type IV collagenase expression

Abstract

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Over expression of the 92-kDa type IV collagenase (MMP-9) contributes to the progression of several malignancies. However, to date, there are relatively few known regulators of expression of this metalloproteinase. We therefore employed an expression library comprised of 500,000 cDNA clones to screen for novel regulators of MMP-9 expression. Towards this end, HT1080 cells were transiently co-transfected with a MMP-9 promoter-luciferase reporter and pools of the cDNA expression library. Positive pools were subdivided in secondary and tertiary screens after which regulatory cDNAs were identified by DNA sequencing. Of several identified putative regulators, one cDNA was homologous to the calponin-related protein, SM22/transgelin. We were particularly intrigued with SM22 since its expression is down-regulated in several cancers known to overexpress MMP-9. Transient co-transfection of HT1080 cells with a SM22 expression vector and an MMP-9 promoter-driven luciferase reporter showed a dose-dependent decrease in luciferase expression. To determine the effect of SM22 on expression of the endogenous MMP-9 gene, HT1080 cells, which show high MMP-9 activity and low SM22 expression, were made to stably express SM22. HT-1080 cells, stably transfected with the SM22-GFP expression construct, demonstrated substantially lower MMP-9 enzymatic activity/mRNA as compared with the parental/empty vector cells. Further, PMA-induced MMP-9 expression was effectively countered by SM22 expression. Studies with 5’-deleted MMP-9 promoter fragments suggested that SM22 repressed MMP-9 expression at least in part through the AP-1 motifs present in the promoter. Moreover, nuclear extract from SM22-expressing cells showed reduced transcription factor binding to the MMP-9 promoter AP-1 motifs when compared with nuclear extract derived from empty vector-transfected cells. In conclusion, we have defined SM22 as a novel repressor of MMP-9 expression and determined that the repression is mediated, in part, via reduced AP-1 binding to the MMP-9 promoter.