Insulin-like growth factor-I induces reactive oxygen species production and cell migration through Nox4 and Rac1 in vascular smooth muscle cells.
OBJECTIVE
We showed previously that insulin-like growth factor-I (IGF-I)-induced vascular smooth muscle cells (VSMCs) proliferation through the production of reactive oxygen species (ROS). However, how IGF-I-induced ROS was unknown. The aim of this study is to investigate the mechanisms by which IGF-I induces ROS production in VSMCs. Methods results Reverse transcription-PCR, real-time PCR, immunoblotting, and confocal microscopic image analysis were employed to determine protein expression, small Rho-GTPase Rac1 activation, and ROS production. Inhibition of NADPH oxidase 4 (Nox4) or Rac1 was performed by means of siRNA technology. Inhibition of Rac1 activity was accomplished using dominant-negative form of Rac1 (N17Rac1) plasmid. VSMCs from Sprague-Dawley rat thoracic aortas were used in this work.IGF-I enhanced ROS production in rat VSMCs. IGF-I increased the protein level of Nox4 but had little effect on its mRNA level. IGF-I induced the activation of Rac1. Either knockdown of Nox4 or inactivation of Rac1 impaired IGF-I-induced ROS. Over-expression of Nox4 increased NADPH oxidase activity, which was not influenced by inactivation of Rac1. Neither over-expression nor knockdown of Rac1 influenced Nox4 expression. Knockdown of Nox4 did not affect IGF-I-induced activation of Rac1. IGF-I increased matrix metalloproteinase (MMP)-2 and 9 activity and promoted VSMC migration, which was inhibited by knockdown of Nox4 and inactivation of Rac1.


CONCLUSIONS
Our results suggest that Nox4 and Rac1 mediate IGF-I-induced ROS production and cell migration in VSMCs and that Nox4 is not regulated by Rac1.
The Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Tai-Yuan Road, Shanghai 200031, People's Republic of China.
Dan
D
Meng
Dan-Dan
DD
Lv
Jing
J
Fang
D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013997:Time Factors; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D007334:Insulin-Like Growth Factor I; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat; D020778:Matrix Metalloproteinase 2; C522363:Mmp2 protein, rat; D020780:Matrix Metalloproteinase 9; C499134:Rac1 protein, rat; D020830:rac1 GTP-Binding Protein