Stabilization of cGMP-dependent protein kinase G (PKG) expression in vascular smooth muscle cells: Contribution of 3′UTR of its mRNA

The type-I cGMP-dependent protein kinase (PKG-I) expression regulation is not yet completely understood. In this study, we examined the role of 3′-untranslated region (3′UTR)-PKG-I messenger RNA (mRNA) in the control of PKG-I expression in vascular smooth muscle cells (VSMCs). Using a 3′-rapid amplification of cDNA ends (RACE) for the amplification of complementary DNA (cDNA) ends, we generated and cloned a 1.2-kb-3′UTR mRNA PKG-I in pGL3 control vector downstream of the luciferase reporter gene. Serial deletions and functional studies revealed that among the deleted constructs, only the 1.2-kb-3′UTR PKG-I mRNA possesses the highest activity in transfected VSMC. Kinetic luciferase assays in the presence of actinomycin D showed that this construct stabilizes luciferase activity compared to the control vector. Sequence analysis of 3′UTR-PKG-I mRNA revealed the existence of four AU-rich regions (AU1 through AU4) in addition to a potential poly(A) site. Different riboprobes were generated either by 5′-end- labelling of designed ribonucleotides, containing individual AU-rich regions or by in vitro transcription assay using cloned 1.2-kb cDNA as a template. RNA-elecrophoretic mobility shift assay (EMSA) and ultra-violet cross-linking (UV-CL) assays showed that AU1, AU3, AU4 and 1.2-kb probes were able to retard cytosolic and nuclear proteins. Taken together, these data suggest that PKG-I expression is subjected to post-transcriptional regulation in VSMC through the 3′UTR of its mRNA.