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Posttranslational Modification of the AU-Rich Element Binding Protein HuR by Protein Kinase C Elicits Angiotensin II-Induced Stabilization and Nuclear Export of Cyclooxygenase 2 mRNA

Anke Doller, El-Sayed Akool, Andrea Huwiler, Roswitha Müller, Heinfried H. Radeke, Josef Pfeilschifter, and Wolfgang Eberhardt^*

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

Received 22 August 2007/ Returned for modification 20 September 2007/ Accepted 6 February 2008

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 C (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.

Published ahead of print on 19 February 2008.

Present address: Institut für Pharmakologie, Universität Bern, CH-3010 Bern, Switzerland.