Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA.
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.
Doller
Anke
A
Pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt am Main, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. w.eberhardt@em.uni-frankfurt.de
Akool
El-Sayed
el-S
Huwiler
Andrea
A
Müller
Roswitha
R
Radeke
Heinfried H
HH
Pfeilschifter
Josef
J
Eberhardt
Wolfgang
W
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000804:Angiotensin II / Q000494:pharmacology*; D000954:Antigens, Surface / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism; D016207:Cytokines / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins / Q000378:metabolism*; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic / Q000235:genetics
D000954:Antigens, Surface; D016207:Cytokines; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D044140:Receptor, Angiotensin, Type 1; D000804:Angiotensin II; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D051745:Protein Kinase C-delta