GLOMERULAR MESANGIAL CELL-SPECIFIC TRANSACTIVATION OF MATRIX METALLOPROTEINASE-2 TRANSCRIPTION IS MEDIATED BY YB-1

Mesangial cell (MC) activation plays a pivotal role in the development of the end stage sclerotic lesion characteristic of most forms of chr onic glomerular disease. We have previously demonstrated that RLC acti vation is directly linked to high level expression of the matrix metal loproteinase-2 (MMP-2) enzyme (Turck, J., Pollock, A. S., Lee, L., Mar ti, H.-P., and Lovett, D. H. (1996) J. Biol. Chem. 25, 15074-15083), t he transcription of which is regulated in a tissue-specific fashion. R ecent studies (Harendza, S., Pollock, A. Mertens, P. R., and Lovett, D . H. (1995) J. Biol. Chem. 270, 18786-18796) delineated a strong cis-a cting enhancer element, designated MMP-2 RE1, within the 5'-flanking r egion of the rat MMP-2 gene. Gel shift, DNA footprint, and transcripti onal analyses mapped the enhancer element to a unique 40-base pair (bp ) sequence located at -1322 to -1282 bp relative to the translational start site. Bromodeoxyuridine-substituted UV cross-linking of the 40-b p enhancer element with MC nuclear extracts yielded a single protein o f 52 kDa, while Southwestern blot analysis with MMP-2 RE1 demonstrated three hybridizing nuclear proteins of 52, 62, and 86 kDa size. Screen ing of a human MC cDNA expression library with MMP-2 RE1 exclusively y ielded clones with the identical sequence of the transcription factor YB-1. Western blot and supershift gel analysis of MC nuclear extracts with an anti-YB-1 antibody confirmed the presence of YB-1 within the s hifted complex. Examination of the MMP-2 RE1 sequence revealed an inco mplete Y-box sequence (CTGCTGGGCAAG), which specifically interacted wi th recombinant YB-1 on DMS protection footprinting analysis. YB-1 prot ein preferentially bound the single-stranded components of the 40-bp M MP-2 RE1 and, with increasing concentrations, formed multimeric comple xes. Co-transfection of YB-1 in MC increased the enhancer activity wit hin the context of the native MMP-2 promoter, while transfection of no n-MMP-2-synthesizing glomerular epithelial cells with YB-1 led to tran scriptional suppression. This study indicates that YB-1 is a major, ce ll type-specific transactivator of MMP-2 transcription by glomerular m esangial cells.