Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation, Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

Venkatesh Kundumani-Sridharan,Nikhlesh K Singh,Sanjay Kumar,Ravisekhar Gadepalli,Gadiparthi N Rao

doi:10.1074/jbc.m113.454082

Abstract

Recent literature suggests that cyclin-dependent kinases (CDKs) mediate cell migration. However, the mechanisms were not known. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a delayed time-dependent manner. MCP1 also stimulated F-actin stress fiber formation in a delayed manner in HASMCs, as well as the migration and proliferation of these cells. Inhibition of Pak1 suppressed MCP1-induced HASMC F-actin stress fiber formation, migration, and proliferation. MCP1 induced cyclin D1 expression as well as CDK6 and CDK4 activities, and these effects were dependent on activation of NFATc1. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1-induced Pak1 phosphorylation/activation and resulted in decreased HASMC F-actin stress fiber formation, migration, and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. MCP1 also activated Rac1 in a time-dependent manner, and depletion/inhibition of its levels/activation abrogated MCP1-induced NFATc1-cyclin D1-CDK6-CDK4-Pak1 signaling and, thereby, decreased HASMC F-actin stress fiber formation, migration, and proliferation. In addition, smooth muscle-specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities, resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1-dependent, NFATc1-mediated cyclin D1 expression and CDK6 activity mediate this effect. In addition, smooth muscle-specific deletion of NFATc1 prevented the capacity of vascular smooth muscle cells for MCP-1-induced activation of the cyclin D1-CDK6-CDK4-Pak1 signaling axis, affecting their migration and proliferation in vitro and injury-induced neointima formation in vivo.

Highlights

We explore the mechanisms by which nuclear factor of activated T cells c1 (NFATc1) mediates vascular wall remodeling
We have reported that cyclin D1/CDK6 mediates the activation of the Rho GTPase effector PKN1 in mediating Monocyte chemotactic protein 1 (MCP1)-induced human aortic smooth muscle cells (HASMCs) migration and proliferation and injury-induced neointima formation in an NFATc1-dependent manner [22]
1) MCP1 activated Pak1 in a sustained manner; 2) Pak1 activation is required for MCP1-induced HASMC migration and proliferation; 3) MCP1 induced CDK6 and CDK4 activities in a time-dependent manner; 4) both CDK6 and CDK4 activities are required for MCP1-induced migration and proliferation; 5) MCP1 induced cyclin D1 expression in a time- and NFATc1dependent manner; 6) MCP1-induced CDK6 and CDK4 activities required NFATc1-dependent cyclin D1 expression; 7) MCP1-induced Pak1 activation required NFATc1 activation, cyclin D1 expression, and CDK6 and CDK4 activities; and 8) CDK4 phosphorylated recombinant Pak1 (rPak1) in vitro, and this phosphorylation is sufficient for rPak1 activity

Summary

Introduction

Results: MCP1 activates Pak in a Rac1-NFATc1-cyclin D1-CDK6-CDK4-dependent manner in the mediation of HASMC migration and proliferation. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1-induced Pak phosphorylation/activation and resulted in decreased HASMC F-actin stress fiber formation, migration, and proliferation. MCP1 activated Rac in a time-dependent manner, and depletion/inhibition of its levels/activation abrogated MCP1induced NFATc1-cyclin D1-CDK6-CDK4-Pak signaling and, thereby, decreased HASMC F-actin stress fiber formation, migration, and proliferation. Smooth muscle-specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak activities, resulting in reduced neointima formation in response to injury.

Objectives

Methods

Results

Conclusion