Role of Protein Kinase C Isoforms in Phorbol Ester-induced Vascular Endothelial Growth Factor Expression in Human Glioblastoma Cells

Shu-Ching Shih,Andrew Mullen,Kristin Abrams,Debabrata Mukhopadhyay,Kevin P Claffey

doi:10.1074/jbc.274.22.15407

Abstract

Aberrant expression of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), has been demonstrated to be associated with most human solid tumors. Both transcriptional and post-transcriptional mechanisms have been shown to modulate VEGF expression in a multitude of cell types. Here we report that when protein kinase C (PKC) pathways were activated in human glioblastoma U373 cells by phorbol 12-myristate 13-acetate (PMA), VEGF mRNA expression was up-regulated via a post-transcriptional mRNA stabilization mechanism. PMA treatment exhibited no increase in VEGF-specific transcriptional activation as determined by run-off transcription assays and VEGF promoter-luciferase reporter assays. However, PMA increased VEGF mRNA half-life from 0.8 to 3.6 h which was blocked by PKC inhibitors but not by protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. When U373 cells were transfected with antisense oligonucleotide sequences to the translation start sites of PKC-alpha, -beta, -gamma, -delta, -epsilon, or -zeta isoforms, both PKC-alpha and -zeta antisense oligonucleotides showed substantial inhibition of PMA-induced VEGF mRNA. In addition, overexpression of PKC-zeta resulted in a strong constitutive up-regulation of VEGF mRNA expression. This study demonstrates for the first time that specific PKC isoforms regulate VEGF mRNA expression through post-transcriptional mechanisms.

Highlights

Aberrant expression of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), has been demonstrated to be associated with most human solid tumors
These results suggest that phorbol 12-myristate 13-acetate (PMA) strongly up-regulates VEGF mRNA expression in U373 cells, and the activation requires prolonged PMA treatment with maximum VEGF mRNA levels reached at 8 h
We show that induction of VEGF expression by PMA treatment and protein kinase C (PKC) activation in human U373 glioblastoma cells is mediated through mRNA stabilization as opposed to transcriptional activation

Summary

Introduction

Aberrant expression of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), has been demonstrated to be associated with most human solid tumors Both transcriptional and post-transcriptional mechanisms have been shown to modulate VEGF expression in a multitude of cell types. We report that when protein kinase C (PKC) pathways were activated in human glioblastoma U373 cells by phorbol 12myristate 13-acetate (PMA), VEGF mRNA expression was up-regulated via a post-transcriptional mRNA stabilization mechanism. The protein kinase C (PKC) family consists of 12 phospholipiddependent serine/threonine kinases that mediate signals from the cell surface to the nucleus and play key roles in cellular signaling pathways [4, 5] These isoenzymes have been grouped into three functional classes based on the structures of their regulatory domains and their requirements for activation by phosphatidylserine, calcium, and diacylglycerol (DAG) [4, 5]. This study indicates that PKC isoform activation, especially PKC-␣ and -␨, are important upstream events resulting in increased VEGF mRNA stabilization

Methods

Results

Conclusion