Matrix‐metabolizing enzymes as prime targets of redox regulation by nitric oxide

Abstract

Introduction Dysregulation of extracellular matrix (ECM) turnover is an important feature of many inflammatory processes in the kidney. Rat mesangial cells (MCs) express high levels of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 (MMP‐9) in response to interleukin‐1β (IL‐1β) (Pfeilschifter & Schwarzenbach 1990; Eberhardt et al. 2000a). The aim of our studies was to elucidate the mechanisms of NO‐dependent modulation of MMP‐9 expression.Methods The expression level of MMP‐9 was detected by zymography and by Northern blot analysis, respectively. The influence of NO on mRNA stability was measured by use of actinomycin‐D and by Luciferase reporter gene assays using heterologous MMP‐9 reporter gene constructs. The effects of NO on MMP‐9 transcripts were monitored by in vitro degradation assays and RNA‐binding activity determined by electrophoretic mobility shift assays (EMSA).Results MMP‐9 (gelatinase‐B) is a metalloproteinase that is synthesized and secreted in high levels by the glomerular MCs upon treatment with the proinflammatory cytokine IL‐1β mainly via an increase in MMP‐9 transcription (Eberhardt et al. 2000a). The increase in MMP‐9 expression by cytokines is partially due to generation of superoxide and involves the activation of different MAPK pathways (Eberhardt et al. 2000b). Furthermore, we demonstrate that NO substantially inhibits the cytokine‐induced expression of MMP‐9 without affecting MMP‐9 enzyme activity (Eberhardt et al. 2000a). By using actinomycin‐D, we found that NO reduces the half‐life of MMP‐9 mRNA (Akool et al. 2003). The reduction of mRNA stability is attributable to the 3′‐untranslated region of MMP‐9 since introduction of this region confered a negative NO responsiveness to an otherwise nonresponding heterologous MMP‐9 reporter luciferase gene (Akool et al. 2003). By EMSA, we found that cytoplasmic fractions display the consitutive RNA binding of complexes containing the ELAV‐like protein HuR (HuA). Moreover, the RNA binding to three putative AU‐rich elements (AREs) is strongly attenuated by different NO donors (Akool et al. 2003). Correspondingly, the decay of MMP‐9 transcripts was accelerated by cytoplasmic extracts from NO‐treated MCs as shown by in vitro degradation assay (Akool et al. 2003).Conclusion NO negatively regulates cytokine‐induced MMP‐9 expression by a post‐transcriptional mechanism which involves inhibition of the mRNA stabilizing ELAV‐like protein HuR.