In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5′-O-(3-thio)triphosphate–induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKCα efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKCα in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKCα compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKCα with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKCα. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E₂ synthesis. Regulation of HuR via PKCα-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.

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.

Glucocorticoids and their synthetic analogs exert potent antiinflammatory actions that, in most cases, are due to an inhibition of the expression of inflammatory genes. In this study, we elucidated the mechanisms of dexamethasone-mediated suppression of matrix metalloproteinase-9 (MMP-9) expression triggered by IL-1beta in rat mesangial cells. Treatment of mesangial cells with dexamethasone markedly reduced the gelatinolytic content of conditioned media due to a decrease in MMP-9 expression. Cloning of a 1.3-kb fragment of the rat MMP-9 gene promoter and subsequent site- directed mutagenesis revealed that a nuclear factor kappaB (NF-kappaB) site at -561 to -550 and a region from -511 to -497 bearing a distal activator protein 1 site adjacent to an Ets-binding site are essentially involved in the IL-1beta-mediated transactivation of MMP-9. Inhibition of MMP-9 expression by dexamethasone resides in a promoter region downstream of -597. The IL-1beta-caused increase in DNA binding of both NF-kappaB and Ets-1 immunopositive complexes was substantially suppressed by dexamethasone as shown by EMSA. This was paralleled with a reduced abundance of p65 and Ets-1 proteins in cell nuclei concomitantly with a reduced inhibitor of kappaB (IkappaB) degradation. In addition to NF-kappaB, we suggest a pivotal role for the Ets binding site, in concert with a distal activator protein-1 element, in the transcriptional suppression of cytokine-induced MMP-9 expression by glucocorticoids.

Stabilization of mRNA by the ubiquitous RNA binding protein human antigen R (HuR), a member of the embryonic lethal abnormal vision (ELAV) protein family, requires canonical binding to AU-rich element (ARE)-bearing target mRNA and export of nuclear HuR-mRNA complexes to the cytoplasm. In human mesangial cells (HMC) both processes are induced by angiotensin II (AngII) via protein kinase Cδ (PKCδ)-triggered serine phosphorylation of HuR. By testing different point-mutated Flag-tagged HuR proteins, we found that Ser 318 within RNA recognition motif 3 (RRM3) is essential for AngII-induced binding to ARE-bearing mRNA but irrelevant for nucleocytoplasmic HuR shuttling. Conversely, mutation at Ser 221 within the HuR hinge region prevents AngII-triggered HuR export without affecting mRNA binding of HuR. Using phosphorylation state-specific antibodies, we found a transient increase in HuR phosphorylation at both serines by AngII. Functionally, PKCδ mediates the AngII-induced stabilization of prominent HuR target mRNAs, including those of cyclin A, cyclin D₁, and cyclooxygenase-2 (COX-2), and is indispensable for AngII-triggered migration and wound healing of HMC. Our data suggest a regulatory paradigm wherein a simultaneous phosphorylation at different domains by PKCδ coordinates mRNA binding and nucleocytoplasmic shuttling of HuR, both of which events are essentially involved in the stabilization of HuR target mRNAs and relevant cell functions.