α1 Adrenergic Agonist Induction of p21 waf1/cip1 mRNA Stability in Transfected HepG2 Cells Correlates with the Increased Binding of an AU-rich Element Binding Factor

Jie Liu,Xuening Shen,Van-Anh Nguyen,George Kunos,Bin Gao

doi:10.1074/jbc.275.16.11846

Jie Liu, Xuening Shen + Show 3 more

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https://doi.org/10.1074/jbc.275.16.11846

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Abstract

Stimulation of transfected HepG2 cells (TFG2) with the alpha(1)-adrenergic agonist phenylephrine (PE) significantly activated p21(waf1/cip1) gene expression without affecting p53 gene expression. Northern blotting and reporter assay demonstrated that this induction was due to PE stimulation of p21(waf1/cip1) mRNA stability. To further define the underlying mechanism, we prepared a chloramphenicol acetyltransferase (CAT)-p21(waf1/cip1) 3'-untranslated region (3'-UTR) hybrid construct by inserting the 3'-UTR of p21(waf1/cip1) mRNA just downstream from the CAT coding sequence and transfected it into TFG2 cells. PE treatment enhanced the activity of this construct by 6-fold. Deletion analyses indicated that an AU-rich element (AURE) located between 553 to 625 within the p21(waf1/cip1) 3'-UTR was required for this induction. RNA gel shift assays demonstrated that this AURE bound an RNA-binding protein. This protein has been purified 5000-fold from PE-treated TFG2 cells by heparin-Sepharose and RNA affinity chromatography. SDS-polyacrylamide gel electrophoresis, UV cross-linking, and Northwestern analyses indicated the molecular mass of this protein as 24 and 52 kDa. Finally, PE treatment markedly enhanced this RNA-protein binding by a p42/44 mitogen-activated protein kinase-dependent mechanism. These data suggest that the AURE located 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 stability.

Highlights

␣1-Adrenergic receptors (␣1AR)1 are G-protein-coupled receptors that play an important role in key components of the sympatho-adrenal response to stress, such as peripheral vasoconstriction, increased cardiac contractility, and hepatic glycogenolysis [1,2,3]
No HuR protein was detectable in Western blots using crude extracts of PE-treated transfected HepG2 cells (TFG2) cells. These findings indicate that the protein induced by PE in TFG2 cells that binds to the AU-rich element (AURE) within the p21waf1/cip1 3Ј-UTR is distinct from HuR
Posttranscriptional mechanisms have been implicated in the regulation of p21waf1/cip1 gene expression by a number of conditions listed in the Introduction (18 –24)

Summary

Introduction

␣1-Adrenergic receptors (␣1AR)1 are G-protein-coupled receptors that play an important role in key components of the sympatho-adrenal response to stress, such as peripheral vasoconstriction, increased cardiac contractility, and hepatic glycogenolysis [1,2,3]. TFG2 cells were treated with PE for 5 h (PE treatment significantly enhanced the binding; see below), after which crude cell extracts were prepared and applied to heparin-Sepharose column and eluted by a step gradient of KCl. Five-microliter aliquots of the 1-ml fractions collected were assayed by RMSA using 32P-labeled e5 RNA probe.

Results

Conclusion