Abstract
Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3′ untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3′ UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 553 and PF 361/1-1), the Stiftung Verum für Gesundheit und Umwelt, and a grant from the Stiftung “Nachlässe der Held und Hecker Stiftung 2002.” E.-S. Akool was supported by a grant of the Ministry of Education of the Arab Republic of Egypt.
We thank Andrew P. Levy (Technion-Israel Institute of Technology, Haifa, Israel) for kindly providing the expression plasmid pZeoSV2-HuR containing the entire coding sequence of HuR and Henry Furneaux (Memorial Sloan Kettering Cancer Center, New York, N.Y.) for kindly providing the expression plasmid pGEX-HuR. Furthermore, we thank Roswitha Müller for excellent technical assistance.