Abstract
We analyzed whether the cyclic AMP induced down-regulation of the nitric oxide (NO) receptor soluble guanylyl cyclase (sGC) is mediated by the mRNA-protecting protein HuR. Exposure (up to 24 h) of isolated rat aortic segments to the activator of adenylyl cyclase, forskolin (10 μM), and to both activators of cAMP-stimulated protein kinase (PKA), 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole-3′,5′-cyclic monophosphorothioate, Spisomer (Sp-5,6-DCl-cBIMPS; 400 nM), and N6-phenyl-cAMP (10 μM), strongly reduced sGCα1β1 and HuR protein and mRNA expression in a time-dependent and actinomycin D (10 μM)-sensitive fashion. In vitro degradation of sGCα1 and β1 poly(A)+ mRNA by native rat aortic protein was markedly increased by pretreatment of intact aortas with forskolin. Native protein extract from rat aorta shifted the electrophoretic mobility of biotin-labeled riboprobes from the 3′-untranslated region of sGCα1 and β1 mRNA, and these bands was supershifted by a monoclonal antibody directed against the mRNA-stabilizing protein HuR. Forskolin decreased the HuR-sGCα1 and β1 mRNA interaction and HuR protein expression in rat aorta, and this was prevented by the PKA inhibitory cAMP analog 3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In cultured smooth muscle cells from rat aorta, forskolin induced a rapid increase in Fos/p-Fos protein levels and activator protein 1 (AP-1) binding activity. Inhibition of this transcription factor by an AP-1 decoy prevented the forskolin-induced down-regulation of HuR. We conclude that forskolin/cAMP decrease the expression of heterodimeric sGC in rat aortic smooth muscle cells via activation of Fos/AP-1, which decreases the expression of HuR and thus destabilizes the sGCα1 and β1 mRNA.
- Received August 13, 2003.
- Accepted February 27, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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