The effect of Diaphanous-related formin-1 (mDia1) on α-tubulin expression and vascular smooth muscle cell migration 公开
Jo, Sally (2013)
Abstract
RATIONALE : The migration of vascular smooth muscle cells (VSMCs) is essential for numerous biological processes, such as vascular development and wound healing. However, VSMC migration can also contribute to the progression of cardiovascular disease. Diaphanous-related formin-1 (mDia1), which regulates cytoskeletal assembly in response to various extracellular signals, such as reactive oxygen species, mechanical forces, and growth factors, may affect the migration of VSMCs.
OBJECTIVE: Here, we assess whether mDia1's impact on VSMC migration is realized through its regulation of α-tubulin, a building block of microtubules.
HYPOTHESIS: Previous experiments indicate that the knockout of mDia1 increases α-tubulin expression and the migration of VSMCs. Based on these results, we hypothesized that the short interfering RNA (siRNA)-induced knockdown of mDia1 would also increase VSMC migration and α-tubulin levels by promoting the expression of the α-tubulin mRNA and/or repressing the proteasomal degradation of α-tubulin.
RESULTS: Although the knockdown of mDia1 does not have a significant effect on VSMC migration, it slightly increases the cytosolic levels of α-tubulin. Our results show that the knockdown of mDia1 is unlikely to affect the mRNA levels of α-tubulin or the proteasomal degradation of α-tubulin. Instead, the knockdown of mDia1 may prevent the translocation of α-tubulin to the membrane, thereby increasing the cytosolic levels of α-tubulin.
CONCLUSION: mDia1 may promote the translocation of α-tubulin to cellular membranes. Although the exact physiological significance of this process is unknown, these membrane tubulins may regulate the growth and contraction of VSMCs by causing the activation of ion channels.
Table of Contents
TABLE OF CONTENTS
List of Figures...i
Introduction...1
Materials and Methods...9
Cell Culture...9
siRNA Transfection...9
Migration Assay...9
Cell Lysate Preparation and Western Blot...10
RNA Extraction and qRT-PCR...11
MG132 Proteasome Inhibitor Treatment and Cell
Fractionation...11
Statistical Analysis...13
Results...14
The knockdown of mDia1 does not cause a
significant change in VSMC migration...14
The knockdown of mDia1 slightly increases α-tubulin levels in
VSMCs...14
The effect of mDia1 knockdown on α-tubulin mRNA expression
(Preliminary)...15
The effect of mDia1 knockdown on the proteasomal degradation of
α-tubulin...15
The knockdown of mDia1 may increase cytosolic levels of
α-tubulin in VSMCs by preventing the translocation of
α-tubulin to the membrane...16
Discussion...18
References...23
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