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.
Meng
Dan
D
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.
Lv
Dan-Dan
DD
Fang
Jing
J
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