Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR

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.Remodeling of extracellular matrix (ECM) is an important feature of normal growth and developmental processes. Consequently, an imbalance of ECM synthesis and degradation is associated with many diseases. Although changes in the synthesis of ECM may play a certain role in dysregulation of matrix turnover, recent studies have underlined the paramount role of ECM-degradative systems. The main proteases regulating physiological degradation of ECM are the matrix metalloproteinases (MMPs), a family of zinc-dependent enzymes, including the interstitial collagenases, stromelysins, elastases, membrane-type MMPs, and gelatinases (8,38,61,62). Tight regulation of most of these proteases is accomplished by different mechanisms, including the regulation of MMP gene expression, the processing and conversion of the inactive zymogens by other proteases, and, finally, the inhibition of active MMPs by endogenous inhibitors of MMPs, the tissue inhibitors of metalloproteinases (for a review, see reference 38).Cultured mesangial cells (MC) respond to proinflammatory cytokines such as tumor necrosis factor alpha (TNF-␣) or interleukin-1 beta (IL-1␤) with the production of several MMPs, including MMP-9 (gelatinase-B), mainly by an increase of gene expression (20,65). In addition to MMPs, MC exposed to cytokines produce high levels of NO through the expression of the inducible NO synthase (iNOS) gene (30,42). Whereas the early and rapid actions of NO signaling affect pos...

Matrix metalloproteinase 9 expression: new regulatory elements

“…Eberhardt and coauthors were the first who revealed the role of cis-regulatory AU-rich elements (ARE) in the 3′-untranslated region of MMP-9 mRNA in the regulation of mRNA stability. Their studies showed that mRNAstabilizing factor HuR and the presence of NO regulated MMP-9 mRNA stability [26,35]. In addition to AREs, we found other types of regulatory elements in the 3′-UTR, i.e., targets for miRNAs affecting efficiency of gene expression.…”

“…The UTR446 fragment contains previously described AU-rich cis-regulatory elements (ARE, Fig. 1a) which target host mRNAs towards rapid degradation and therefore may affect the efficiency of MMP-9 expression [25,26]. We then asked whether UTR446 inhibitory properties that we observed could be explained exclusively by ARE inhibitory activity.…”

Matrix metalloproteinases and their endogenous inhibitors in neuronal physiology of the adult brain

“…There are a number of gene regulatory sequences driving the MMP-9 expression, with AP-1 and NF-jB being the most prominent [5]. Once produced, the MMP-9 mRNA is subjected to regulation at its stability level by nitric oxide (NO) [6]; as well it can apparently be translocated in neurons towards activated dendrites [7]. Notably, MMP-9 contains ARE-like sequences that have been suggested to be of pivotal importance for the translocation in other messages [8].…”

Nitric oxide inhibits exocytosis of cytolytic granules from lymphokine-activated killer cells

“…How does NO regulate Ras mRNA stability? NO can disrupt the stability of mRNA encoding other proteins, such as matrix metalloproteinase 9, by interfering with proteins that normally bind to AU-rich elements; AU elements are contained in the 3Ј untranslated region of ras (39). NO decreases steady-state levels of K-Ras and N-Ras but not H-Ras (Fig.…”