ATF-1 Mediates Protease-activated Receptor-1 but Not Receptor Tyrosine Kinase-induced DNA Synthesis in Vascular Smooth Muscle Cells

Salil K Ghosh,Laxmisilpa Gadiparthi,Zhao-Zhu Zeng,Manjula Bhanoori,Carmen Tellez,Menashe Bar-Eli,Gadiparthi N Rao

doi:10.1074/jbc.m201608200

Abstract

Previously we have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) and induction of DNA synthesis in response to receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists require NADH/NADPH-like oxidase activity in vascular smooth muscle cells (VSMC). Here we tested the role of p38 MAPK in RTK and GPCR agonist-induced DNA synthesis in VSMC. Platelet-derived growth factor (PDGF)-BB and thrombin (RTK and GPCR agonists, respectively) activated p38 MAPK in a time-dependent manner in VSMC. Inhibition of p38 MAPK led to a 50% decrease in the DNA synthesis induced by thrombin but not PDGF-BB. ATF-1 was found to be the predominant member of the cyclic AMP response element (CRE)-DNA complex formed in VSMC in response to PDGF-BB and thrombin, and both agonists induced its phosphorylation. Regardless of this, inhibition of p38 MAPK reduced only thrombin- but not PDGF-BB-induced ATF-1 phosphorylation. Similarly, inhibition of p38 MAPK caused a 50% decrease in thrombin- but not PDGF-BB-induced CRE promoter-dependent transcription. Ectopic expression of an inhibitory anti-ATF-1 single-chain antibody fragment, ScFv, significantly interfered with DNA synthesis induced by thrombin but not PDGF-BB. Together, these results suggest the following conclusions. 1) Both RTK and GPCR agonists activate p38 MAPK and induce CRE promoter-dependent transcription; 2) both RTK and GPCR agonists induce ATF-1 phosphorylation, and ATF-1 is a predominant member in the CRE-DNA complexes formed in response to these agents; and 3) p38 MAPK-dependent ATF-1 phosphorylation and CRE promoter-mediated transcription are associated with GPCR agonist-induced VSMC growth.

Highlights

Increased vascular smooth muscle cell growth is a contributing factor in the pathogenesis of atherosclerosis and restenosis [1]
We show that 1) both receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists activate p38 mitogen-activated protein kinase (MAPK) in vascular smooth muscle cells (VSMC); 2) activation of p38 MAPK is not required for platelet-derived growth factor (PDGF)-BB-induced DNA synthesis, it is involved in thrombin-induced DNA synthesis; 3) thrombin- but not PDGF-BB-induced p38 MAPK activation is required for ATF-1 phosphorylation and cyclic AMP response element (CRE) promoter-dependent transcription; 4) thrombin-stimulated p38 MAPK-dependent ATF-1 phosphorylation and CRE promoter-mediated transcription are associated with growth in VSMC; and 5) disruption of ATF-1 activity by intracellular expression of an inhibitory anti-ATF-1 single-chain antibody fragment, ScFv, interfered with only thrombin but not PDGF-BB-induced DNA synthesis in VSMC
Lysates of VSMC that were treated with and without PDGF-BB (20 ng/ml) or thrombin (0.1 unit/ml) for 30 min were immunoprecipitated with anti-p38 MAPK antibodies and the immunocomplexes were assayed for p38 MAPK activity using recombinant ATF-2 and [␥-32P]ATP as substrates in the presence and absence of SB203580 (10 ␮M), a potent inhibitor of p38 MAPK

Summary

Introduction

Increased vascular smooth muscle cell growth is a contributing factor in the pathogenesis of atherosclerosis and restenosis [1]. Extracellular signal-regulated kinases have been reported to respond preferentially to agents that induce cell growth and differentiation [15,16,17,18,19,20], whereas Jun N-terminal kinases and p38 MAPK have been reported to be potently activated by cellular stressors and cytokines [21,22,23,24] Despite these differences in responsiveness, cross-talk between different groups of MAPK has been observed in mediating cellular responses to certain agonists [25]. AP-1, activator protein-1; ATF-1, activating transcription factor-1; CRE, cyclic AMP response element; CREB, cyclic AMP response element-binding protein; DMEM, Dulbecco’s modified Eagle’s medium; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; FGF, fibroblast growth factor; GPCR, G protein-coupled receptor; p38 MAPK, p38 mitogen-activated protein kinase; RTK, receptor tyrosine kinase; VSMC, vascular smooth muscle cell(s). These findings reveal that ATF-1 plays a role in thrombin- but not PDGF-BB-induced VSMC growth

Methods

Results

Conclusion