Intronic regulation mediates ischemia-induced MMP-2 transcription

Abstract

Introduction: MMP-2 transcription is essential to angiogenesis and arteriogenesis, and is increased in tissue ischemia. The mechanisms responsible for MMP-2 transcription remain unknown. The purpose of this study was to determine the role of intronic regulation in mediating ischemia-induced Matrix Metalloproteinase-2 (MMP-2) transcription in vivo. Methods: MMP-2 transcription was studied by beta-galactosidase assay in gastrocnemius muscle of transgenic mouse strains following femoral artery excision to induce unilateral hindlimb ischemia. Ischemia was confirmed with laser Doppler analysis of limbs. The F8 strain carried 5 Kb of the rat MMP-2 promoter coupled to beta-galactosidase, and the the F8-HGH strain is identical except for substitution of the first MMP-2 intron with a generic intron (human growth hormone). Potential transcription factor binding sites were identified with transcription factor database (TESS) analysis. Transcription factor occupancy of binding sites was confirmed by chromatin immunoprecipitation on nuclei from ischemic and non-ischemic muscle and transcription factor expression was determined by immunohistochemistry. All data are mean±SEM of 6-8 animals/group studied on day 3 of ischemia, * p<.05 by t-test or ANOVA). Results: Femoral artery excision resulted in marked hindlimb ischemia in both F8 (15.69±1 vs. 41.86±2.7 mmO2*) and F8-HGH strains (11.9±1.6 vs. 27.45±2.5 mmO2*). Beta-galactosidase assay (RLU as % of contralat. Limb) demonstrated marked MMP-2 transcription from the native promoter (F8, 226±18% of control, *), which was abolished by substitution of the first intron (F8-HGH, 113±19.7% of control, p=N.S.). TESS database analysis of the first MMP-2 intron demonstrated multiple potential binding sites for NFATc2 and AP-2 (Log Likelihood scores >16) and a single FosB site (Score=12). Chromatin immunoprecipitation demonstrated ischemia-induced NFATc2 occupancy only of a cluster of 3’ binding sites in the first MMP-2 intron, no change in FosB binding with ischemia and no AP-2 binding to the first intron. Immunohistochemistry demonstrated increased NFATc2 expression in ischemic myocytes. Conclusions: This study demonstrates a critical role for intronic regulation (by Intron I) in ischemia-induced MMP-2 transcription in vivo, and identifies a potential mechanism (NFATc2 transactivation) to mediate this requirement for the first intron. These studies define novel molecular mechanisms activated in vivo that are essential for expression of genes (i.e. MMP-2) important in the physiological responses to tissue ischemia.