P53, YB-1 and the AP-1 family of transcription factors mediate MMP-2 transcription induced by skeletal muscle ischemia

Abstract

Introduction: The expression of matrix metalloproteinases (MMP) is essential to the angiogenic response following tissue ischemia. The transcriptional regulators of MMP gene expression in ischemia are unknown. We hypothesized that the AP-1 family of transcription factors as well as factors activated by genotoxic stress (p53, YB-1) are involved in transcriptional activation of MMP-2 in skeletal muscle ischemia. Methods: Hindlimb ischemia was induced in CD-1 mice and chromatin immunoprecipitation of the AP-1 and RE-1 sites (−1140 to −1050 bp) within the mouse MMP-2 gene was performed with antibodies to known transcription factors on gastrocnemius chromatin extracts. The significance of the AP-1/RE-1 sites was defined with transgenic mice containing constructs of rat MMP-2 promoter fragments (−1687 to + 3400 bp) coupled to the beta-galactosidase gene with deletion of the −1140/−1050 segment in selected mice. Limb oxygen tension was determined by laser Doppler and beta-galactosidase activity determined by chemiluminescent assay. Results: Ischemia induced binding of the transcription factors FosB, JunB, FosC, JunC, and Fra2 to the AP-1 site and decreased binding of the negative regulator JunD. Ischemia induced p53, YB-1 binding to the RE-1 site and decreased AP-2 alpha binding. Deletion of these proximal sites decreased MMP-2 transcription (23.3 ± 5.6 RLU/mcg protein vs. 71 ± 11.5, n = 5, p < .05) with similar tissue ischemia (16.2 ± 2.3 mm O2 vs. 17.1 ± 1.3, p = N.S.). Conclusions: The AP-1 and RE-1 sites are critical for MMP-2 transcription in ischemia. Identification of the specific transcription factors responsible for gene expression will aid in the design of therapy to treat critical limb ischemia.