Investigating the mechanism by which Poldip2 activates Ect2 and the downstream consequences of this activation in vascular smooth muscle cells Público

Abstract

During atherogenesis, cell migration, proliferation, and extracellular matrix secretion are mis-regulated in vascular smooth muscle cells (VSMCs). Polymerase δ interacting protein 2 (Poldip2) is a critical regulator of these processes and many functions of Poldilp2 are mediated by its interactions with NADPH oxidases (Noxes) and RhoA. In the vasculature, Noxes serve as important enzymatic sources of reactive oxygen species (ROS) and at physiological levels ROS derived from Noxes function as signaling molecules. While the mechanism remains unclear, one consequence of Poldip2-generated ROS is activation of RhoA. The Rho family of GTPases are critical regulators of VSMC function; however, under pathophysiological conditions, their downstream effectors promote cardiovascular pathologies. In this study, we investigated the mechanism by which Poldip2 activates RhoA in VSMCs.

Canonically, Rho GTPases are activated by Rho guanine nucleotide exchange factors (GEFs). Thus, we first performed RhoA(17A) pulldowns for active GEFs in cells overexpressing Poldip2 or vector control. Analyzing these pulldowns by mass spectrometry, and confirming this by immunoblotting, we identified Ect2 as a GEF activated by Poldip2 and began to investigate the mechanism for this activation. After finding Poldip2 does not regulate Ect2 expression or subcellular localization, we hypothesized that ROS-derived from Poldip2/Nox signaling may be involved in activating Ect2. We began by testing the effect of ROS on Ect2 and found that exogenous H2O2 activates Ect2. Next, using VSMCs with genetic deletion of Nox1 or Nox4, we found that Nox4 is not required for Poldip2 to activate Ect2, but instead Nox1 is likely required. The effect of Poldip2 on Nox1 activity remains unreported; however, our data suggest that Poldip2 acts as a positive regulator of Nox1 activity. Finally, while considering the downstream consequences of this signaling, we used siRNA to probe the role of Polidp2 and Ect2 in VSMC proliferation, and showed that both of these molecules promote VSMC proliferation. Interestingly, Poldip2 and Ect2 may promote VSMC proliferation by inhibiting senescence and we are currently investigating the effect of Poldip2 and Ect2 knockdown on VSMC senescence. Thus, we propose Poldip2 signals through Nox1 to activate Ect2 in a ROS-dependent manner and enhances VSMC proliferation by inhibiting senescence.

Table of Contents

Abbreviations.........................................................................................................................1 Background............................................................................................................................3 Materials and Methods.............................................................................................................11 Results..................................................................................................................................16 Discussion..............................................................................................................................24 Work Cited.............................................................................................................................28 Figures..................................................................................................................................33

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Physiology
Palavra-chave	RhoGEF redox signaling Poldip2 NADPH oxidase Ect2 vascular smooth muscle cells
Committee Chair / Thesis Advisor	Griendling, Kathy, Emory University
Committee Members	Lassegue, Bernard P, Emory University Cafferty, Patrick William, Emory University

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