Posttranslational modification of the AU-rich element binding protein HuR by PKC elicits angiotensin II-induced stabilization and nuclear export of COX-II mRNA

pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany

^* To whom correspondence should be addressed. Email: w.eberhardt{at}em.uni-frankfurt.de.

	Abstract

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 (RNAi) 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 (UTR) of COX-2 we found that the increase in COX-2 mRNA stability is attributable to a distal class III-type of ARE. Likewise, RNA immunoprecipitation assay showed an AngII-induced binding of HuR to this ARE. Using RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeletal-bound polysomes indicative for an active ribonucleoprotein complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by an increased nucleo-cytoplasmic HuR shuttling and depends on PKC which physically interacts with nuclear HuR thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKC-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKC represents an important novel mode of HuR activation implied in renal COX-2 regulation.