alpha(1) adrenergic agonist induction of p21(waf1/cip1) mRNA stability in transfected HepG2 cells correlates with the increased binding of an AU-richelement binding factor

Stimulation of transfected HepG2 cells (TFG2) with the alpha(1)-adrenergic agonist phenylephrine (PE) significantly activated p21(waf1/cip1) gene expr ession without affecting p53 gene expression. Northern blotting and reporte r assay demonstrated that this induction was due to PE stimulation of p21(w af1/cip1) mRNA stability. To further define the underlying mechanism, we pr epared a chloramphenicol acetyltransferase (CAT)-p21(waf1/cip1) 3'-untransl ated region (3'-UTR) hybrid construct by inserting the 3'-UTR of p21(waf1/c ip1) mRNA just downstream from the CAT coding sequence and transfected it i nto TFG2 cells. PE treatment enhanced the activity of this construct by 6-f old. Deletion analyses indicated that an AU-rich element (AURE) located bet ween 553 to 625 within the p21(waf1/cip1) 3'-UTR was required for this indu ction. RNA gel shift assays demonstrated that this AURE bound an RNA-bindin g protein. This protein has been purified 5000-fold from PE-treated TFG2 ce lls by heparin-Sepharose and RNA affinity chromatography. SDS-polyacrylamid e gel electrophoresis, UV cross-linking, and Northwestern analyses indicate d the molecular mass of this protein as 24 and 52 kDa. Finally, PE treatmen t markedly enhanced this RNA-protein binding by a p42/44 mitogen-activated protein kinase-dependent mechanism. These data suggest that the AURE locate d between 553 and 625 within the p21(waf1/cip1) mRNA 3'-UTR, which binds an RNA-binding protein, is responsible for PE-induced p21(waf1/cip1) mRNA sta bility.