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Cardiovascular Research Advance Access first published online on June 20, 2008
This version [Corrected Proof] published online on July 7, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn173
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Insulin-like growth factor-I induces reactive oxygen species production and cell migration through Nox4 and Rac1 in vascular smooth muscle cells

Dan Meng, Dan-Dan Lv and Jing Fang*

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

* Corresponding author. Tel: +86 21 54920241; fax: +86 21 54920291. E-mail address: jfang{at}sibs.ac.cn

Received 4 January 2008; revised 1 June 2008; accepted 17 June 2008

Time for primary review: 23 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: 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.

Conclusion: 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.

KEYWORDS Vascular smooth muscle cells; Insulin-like growth factor-I; Reactive oxygen species; Nox4; Rac1


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) is a major contributor to the development of atherosclerotic lesions.1 Insulin-like growth factor-I (IGF-I) is a growth factor and plays an important role in regulating VSMC proliferation and migration.2 A recent in vivo study has shown that smooth muscle-targeted over-expression of IGF-I stimulates an increase in neointimal formation after carotid artery injury that is attributable to an increase in the proliferation and migration of VSMCs.3 The biological actions of IGF-I are mediated by the IGF-I receptor (IGF-IR), a transmembrane tyrosine kinase that is abundantly expressed in VSMCs.4 The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in mediating the chemotactic signals of IGF-I in VSMCs.5,6

Reactive oxygen species (ROS), including superoxide and hydrogen peroxide, are important signalling molecules that regulate proliferation and migration of VSMCs.7 We showed previously that IGF-I-induced ROS generation in rat VSMCs.8 However, the mechanisms of IGF-I-induced ROS production are not clear. Recent studies demonstrated that the major source of ROS in VSMCs is NADPH oxidase.9 In rat aortic VSMCs, the NADPH oxidase complex mainly consists of the membrane-bound subunits of Nox and p22phox, the cytosolic components p47phox, and the small Rho-GTPase Rac1. NADPH oxidase 4 (Nox4) and Nox1 but not Nox2 are mainly expressed in rat aortic VSMCs.10,11 Nox activity is modulated by a variety of mediators detected in vascular diseases such as angiotensin II, thrombin, platelet-derived growth factor, and tumour necrosis factor alpha (TNF-{alpha}).12,13 Restenosis after carotid injury is accompanied by an up-regulation of Nox proteins and an increase in ROS production.14 Nox4, unlike other members of Nox family, produces large amounts of ROS constitutively.15,16 Nox4 can form a complex with p22phox on internal membranes to produce ROS.17 Until now, the mechanisms how Nox4 is regulated remain unknown. Rac1 has been shown to be important in the activation of Nox1 and Nox2 in non-phagocytic cells.18,19 However, the role of Rac1 in Nox4 activation is not clear in VSMCs.

In this study, we have demonstrated that (i) Nox4 and Rac1 mediate IGF-I-induced ROS production in rat VSMCs, (ii) Nox4 is not regulated by Rac1, and (iii) Nox4 and Rac1 are involved in IGF-I-induced up-regulation of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) activity and VSMC migration.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

2.1 Antibodies and chemicals
IGF-I, diphenylene iodonium (DPI), lucigenin, NADPH, cycloheximide (CHX), actinomycin D, and antibodies against β-actin and myc were from Sigma (St Louis, MO). 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) were from Molecular Probes (Eugene, OR). Antibodies against Nox1 and Nox4 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against Rac1 was a product of BD Biosciences (San Jose, CA). The plasmids encoding N17Rac1 and Nox4 were kindly provided by Dr Jiang (West Virginia University, Morgantown, USA).

2.2 Cell culture
Eight-week-old male Sprague-Dawley rats were from Shanghai Experimental Animal Center of Chinese Academy of Sciences. Primary VSMCs were isolated from rat thoracic aortas and cultured as described previously.20 The cells were cultured in DMEM with 10% FBS, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. VSMCs were identified by immuno-staining with antibodies against {alpha}-smooth muscle actin and smoothelin. The cells used in our experiments were not more than six passages.

2.3 Measurement of reactive oxygen species
DCFH-DA and DHE were used to measure ROS as described.21,22 Growth-arrested VSMCs were stimulated with IGF-I. After stimulation, the medium was aspirated and VSMCs were incubated in PBS containing DCFH-DA (5 µM) for 15 min or DHE (5 µM) for 20 min in a light-protected humidified chamber at 37°C. Cells were then rinsed in PBS and fixed with 10% buffered formalin. Images were obtained with a LSM 510 laser scanning confocal microscope (Carl Zeiss Inc., Thornwood, NY). For DCFH-DA assay, excitation was at 488 nm with an emission at 540 nm. For DHE assay, excitation was at 535 nm with an emission at 610 nm. The fluorescence image was collected by a single rapid scan with identical parameters for all samples. Fluorescent levels were expressed as percent increase over the control.

2.4 NADPH oxidase assay
NADPH oxidase activity was measured by the lucigenin chemiluminescence method.23 The cells were washed five times with ice-cold PBS and scraped from the plate followed by centrifugation at 1000g at 4°C for 10 min. The cell pellets were re-suspended in lysis buffer containing 20 mM KH2PO4, pH 7.0, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, and 0.5 µg/mL leupeptin. Cell suspensions were homogenized with 100 strokes in a Dounce homogenizer on ice. Hundred microlitre of homogenate was added into 900 µL of phosphate buffer (50 mM, pH 7.0), containing 1 mM EGTA, 150 mM sucrose, 5 µM lucigenin, and 100 µM NADPH. Photon emission was measured every 15 s for 5 min in a luminometer. A buffer blank was subtracted from each reading before calculation of the data. NADPH oxidase activity was defined as relative chemiluminescence (light) units per second per milligram of protein.

2.5 Inhibition of Nox4 or Rac1 by siRNA
The sequence 5'-AACGAAGGGGTTAAACACCTC-3' was used for rat Nox4 siRNA.24 The non-specific siRNA (scramble) consisted of a non-targeting 21 nucleotides, which bears no homology with relevant rat genes. The siRNA was cloned into a pSilencer 2.1 (Ambion, Austin, TX). siRNA against rat Rac1 was synthesized by GenePharma (Shanghai, China). The sequences used for rat Rac1 siRNA are 5'-CAAACAGACGUGUUCUUAATT-3' (sense) and 5'-UUAAGAACACGUCUGUUUGCG-3' (antisense). The scrambled siRNA consisted of the same length of 21 nucleotides without homology to any rat gene sequence. Nucleofection (Amaxa, Cologne, Germany) was used for transfection of plasmid or siRNA into VSMCs. Briefly, 1 x 106 cells were suspended in 100 µL of reagent VPI-1004 (Amaxa) with plasmid or siRNA and subjected to nucleofection program A-033.

2.6 Reverse transcription-PCR and real-time PCR
Total cellular RNA was isolated from VSMCs using Trizol reagent (Invitrogen, Carlsbad, CA). Two microgram of RNA was processed directly to cDNA with Moloney Murine Leukaemia Virus (M-MLV) reverse transcriptase (Promega, Madison, WI). The primers used for amplification of the following genes were designed by Primer Express 3.0 software (Applied Biosystems, Foster City, CA). The primers for rat Nox4 were: 5'-CTGTACAACCAAGGGCCAGAA-3' (forward), and 5'-TGCAGTTGAGGTTCAGGACAGA-3 (reverse). The primers used for rat Nox1 were: 5'-AAGTGGCTGTACTGGTTGGG-3' (forward), and 5'- CCACATAAGAAAACCCCCACCG-3' (reverse). Primers for rat GAPDH are: 5'-CCATCTTCCAGGAGCGAGATC-3' (forward), and 5'-GCCTTCTCCATGGTGGTGAA-3' (reverse). Amplification reactions were performed in a 20 µL volume containing 10 pmol of primers, 2.5 mM MgCl2, 200 µM dNTP mixtures, 0.5 units of Taq DNA polymerase, cDNA template and 1.33 µM EvaGreen (Biotium, Hayward, CA). All of the reactions were performed in triplicate in an iCycler iQ System (Bio-Rad), and the thermal cycling conditions were as follows: 95°C for 3 min; 40 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 20 s; 72°C for 10 min. To confirm specificity of amplification, the PCR products from each primer pair were subjected to a melting curve analysis and electrophoresis in 2% agarose gel. The relative mRNA levels of target genes to that of GAPDH were calculated according to the methods described previously.25

2.7 Immunoblotting
Immunoblotting was performed as described previously.26

2.8 Rac1 activity assay
Rac1 activity was determined by affinity precipitation according to the modified method of Benard et al.27 VSMCs at 60–70% confluence were serum-starved for 24 h. The starved cells were stimulated with IGF-I (200 ng/mL) and lysed. Cell lysates were centrifuged for 30 min (10 000g, 4°C) and the supernatant was incubated with 50 µg of the p21-binding domain of p21-activated kinase (PAK-1) linked to glutathione S-transferase (GST-PAK-1-PBD) on glutathione–agarose beads (Amersham Pharmacia) for 3 h at 4°C. The beads were washed and the bound proteins were separated on SDS–PAGE.

2.9 Transient transfection and matrix metalloproteinase gene promoter activity assay
A 1.7 kb MMP-2 promoter in pGL2-basic luciferase reporter and a 2.2 kb MMP-9 promoter in pGL3-basic luciferase reporter were kindly provided by Dr Ke (SIBS, China). VSMCs were seeded in 12-well plates and cultured to 60–70% confluence. The cells were transiently transfected with MMP-2 or MMP-9 reporter and pCMV-β-galactosidase plasmid using Lipofectamine from Invitrogen. The transfected cells were cultured for 18–20 h, and then incubated in serum-free medium for 12 h followed by the incubation with IGF-I (200 ng/mL) for 20 h. Cells were lysed with Reporter Lysis Buffer. Luciferase (Luc) activities of the cell extracts were determined using the Luciferase Assay System from Promega. β-Galactosidase (β-gal) activity was measured, and relative Luc activity was calculated as the ratio of Luc/β-gal activity.

2.10 Gelatin zymography
The conditioned medium was analysed for gelatinase activity by zymography.28 An equal amount of medium samples from cultured VSMCs was separated by electrophoresis, which was carried out on an 8% SDS–PAGE co-polymerized with gelatin (1 mg/mL). After electrophoresis at 125 V, the gels were incubated in renaturing solution (2.5% Triton-X-100) for 30 min at room temperature and then for 24 h at 37°C in a developing buffer containing 50 mM Tris, pH 7.5, 200 mM NaCl, 4 mM CaCl2, and 0.02% Brij-35. The gels were then stained with Coomassie blue, and regions without staining were indicative of gelatin lysis. To ascertain equal loading in gels, the top 2 cm of each gel was cut off and stained with Coomassie blue for 5 min. The intensity of the bands was determined by densitometric analysis with BioRad Molecular Imager FX and Quantity One software.

2.11 Cell migration assay
Cell migration assay was performed using 24-well cell culture inserts with 8.0 µm polyethylene terephthalate Cyclopore membranes (Falcon). The inserts were coated with 0.1% gelatin. Rat VSMCs were incubated in serum-free medium for 12 h. The cells were trypsinized, washed, and re-suspended in serum-free DMEM. DMEM containing IGF-I was loaded into the lower wells of the inserts and the cells were subsequently loaded onto the upper wells. The cells were incubated for 10 h. The cells on the upper side of the membranes were removed using cotton swabs. The migratory cells on the underside were fixed and stained with haematoxylin–eosin. The inserts were examined under the microscope, and migrated cells were counted in eight randomly chosen fields of duplicate chambers for each sample.

2.12 Statistical analysis
The data represent the mean ± SEM from three independent experiments. Statistical comparisons were made using the unpaired two-tailed Student’s t-test when two groups were compared. A one-way analysis of variance was used when more than two groups were compared. The difference of data is considered significant when a P-value is <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Insulin-like growth factor-I increases reactive oxygen species production through a flavin-containing oxidase
Production of ROS was determined by DCFH-DA, a fluorescent dye that is ROS-sensitive. As shown in Figure 1A, IGF-I induced an acute production of ROS within 10 min. In 30 min, generation of ROS became moderate and sustained for >8 h. Similar results were obtained when another ROS-sensitive dye DHE was employed as substrate (data not shown). To know whether IGF-I-induced ROS is flavoprotein-dependent, DPI, an inhibitor of flavoproteins, was used. DPI attenuated IGF-I-induced ROS production (Figure 1B). These results suggest that IGF-I induces ROS production through flavin-containing proteins in VSMCs. Extracellular ROS formation by IGF-I was determined by means of cytochrome C reduction. No extracelluar ROS production could be detected (data not shown).


Figure 1
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  		Figure 1 Insulin-like growth factor-I induces reactive oxygen species production via a flavin-containing oxidase in vascular smooth muscle cells. (A) Growth-arrested vascular smooth muscle cells were stimulated with insulin-like growth factor-I (200 ng/mL) for different time as indicated. Measurement of reactive oxygen species formation by DCFH-DA method was as described in Methods. (B) Growth-arrested vascular smooth muscle cells were pretreated with DPI (5 µM) for 30 min, followed by stimulation with insulin-like growth factor-I (200 ng/mL) for 10 min. After stimulation, reactive oxygen species was determined using DHE as substrate.

 
3.2 Insulin-like growth factor-I induces the expression of Nox4
NADPH oxidase, a flavoprotein, is the main source of intracellular ROS in VSMCs. Nox1 and Nox4 were expressed in rat VSMCs.11 We therefore asked whether IGF-I influenced the expression of Nox1 and Nox4 in VSMCs. We found that IGF-I increased the protein levels of Nox4 but had little effect on that of Nox1 (Figure 2A). IGF-I had little effect on Nox4 mRNA level (Figure 2B), from 25 to 200 ng/mL (data not shown). Exposure of the cells to CHX, an inhibitor of protein synthesis, partially blocked IGF-I-induced expression of Nox4 (Figure 2C). However, actinomycin D, an inhibitor of transcription, did not influence IGF-I-induced Nox4 (Figure 2C). These results suggest that IGF-I regulate the expression of Nox4 at a post-transcriptional level.


Figure 2
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  		Figure 2 Nox4 mediates insulin-like growth factor-I-induced reactive oxygen species generation. (A) Growth-arrested vascular smooth muscle cells were incubated with insulin-like growth factor-I (200 ng/mL) for different time as indicated. Protein level was quantified by scanning photodensitometry using Bio-Rad Molecular Imager FX and Quantity One software. (B) Growth-arrested vascular smooth muscle cells were incubated with insulin-like growth factor-I (200 ng/mL). Nox4 mRNA was determined by real-time PCR. (C) Growth-arrested vascular smooth muscle cells were pre-incubated with either cycloheximide (0.5 µg/mL) or actinomycin D (0.5 µg/mL) for 30 min, and then treated with insulin-like growth factor-I (200 ng/mL) for 8 h. (D) vascular smooth muscle cells were transfected with scrambled or Nox4 siRNA. In 24 h, the cells were harvested and the mRNA levels of Nox4 and Nox1 were determined by RT–PCR. In 48 h, the cells were harvested for detecting the Nox4 and Nox1 protein. (E) and (F) vascular smooth muscle cells were transfected with Nox4 siRNA. In 48 h the cells were growth-arrested before treatment with insulin-like growth factor-I (200 ng/mL) for 8 h. reactive oxygen species production was determined using DCFH-DA (E) and DHE (F) as substrate.

 
3.3 Nox4 mediates insulin-like growth factor-I-induced reactive oxygen species generation
Next we determined whether Nox4 mediated IGF-I-induced ROS production in VSMCs. Nox4 expression was inhibited by siRNA technology. Nox4 siRNA decreased Nox4 expression by ~60%, but had no effect on Nox1 expression (Figure 2D). Because IGF-I induced maximum expression of Nox4 in 8 h, we determined the effect of Nox4 knockdown on IGF-I-induced ROS production in 8 h. As shown in Figure 2E, inhibition of Nox4 attenuated ROS generation induced by IGF-I. Similar results were obtained when DHE was used instead of DCFH-DA (Figure 2F).

3.4 Rac1 mediates insulin-like growth factor-I-induced reactive oxygen species generation
We next asked whether Rac1 was also involved in IGF-I-induced ROS production in VSMCs. As shown in Figure 3A, IGF-I induced a rapid activation of Rac1. But no activation of Rac1 could be observed in 2, 4, or 8 h (Figure 3B). IGF-I-induced activation of Rac1 was inhibited by dominant-negative form of Rac1 (N17Rac1) (Figure 3C). To know whether Rac1 contributes to ROS generation by IGF-I, VSMCs were transfected with N17Rac1. The cells were treated with IGF-I and ROS production was determined in 10 min. Inactivation of Rac1 by N17Rac1 significantly inhibited IGF-I-induced ROS production (Figure 3D). Similar results were also obtained when DCFH-DA was replaced with DHE (Figure 3E). These results suggest that Rac1 is involved in the early production of ROS induced by IGF-I.


Figure 3
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  		Figure 3 Rac1 mediates insulin-like growth factor-I-induced reactive oxygen species generation. (A) Growth-arrested vascular smooth muscle cells were incubated with insulin-like growth factor-I (200 ng/mL) for different time as indicated. Rac1 activity was determined as described in Methods. (B) vascular smooth muscle cells were treated with insulin-like growth factor-I (200 ng/mL) for 2, 4, and 8 h. (C) vascular smooth muscle cells were transfected with N17Rac1. In 24 h, the cells were growth-arrested before treatment with insulin-like growth factor-I (200 ng/mL). Rac1 activity was determined in 10 min. Anti-Myc antibody was used to confirm the expression of N17Rac1. (D) and (E) vascular smooth muscle cells were transfected with control vector or N17Rac1. In 48 h, the cells were growth-arrested before insulin-like growth factor-I (200 ng/mL) treatment. In 10 min, reactive oxygen species production was measured using DCFH-DA (D) and DHE (E). *P < 0.05 vs. control; **P < 0.01 vs. control.

 
3.5 Nox4 is not regulated by Rac1
We then asked whether Nox4 activity is Rac1-dependent. To know this, we determined the effect of dominant-negative Rac1 on Nox4 activity. Over-expression of Nox4 increased NADPH oxidase activity (Figure 4A). Inactivation of Rac1 by N17Rac1 had little effect on the increase of NADPH oxidase activity by Nox4 (Figure 4A). The effect of Nox4 on Rac1 activation by IGF-I was also determined. As shown in Figure 4B, knockdown of Nox4 did not influence IGF-I-induced Rac1 activation. Moreover, neither over-expression of Rac1 nor knockdown of Rac1 had effect on Nox4 expression (Figure 4C). These results suggest that Nox4 is not regulated by Rac1 in VSMCs.


Figure 4
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  		Figure 4 Nox4 is not regulated by Rac1. (A) vascular smooth muscle cells were transfected with control vector, Nox4, or Nox4 plus N17Rac1. In 48 h, NADPH oxidase activity was determined. Anti-Myc antibody was used to confirm the expression of N17Rac1. (B) vascular smooth muscle cells were transfected with scrambled or Nox4 siRNA. In 24 h, the cells were growth-arrested before insulin-like growth factor-I (200 ng/mL) treatment. Rac1 activity was determined in 10 min. NS, no significant difference. (C) vascular smooth muscle cells were transfected with control vector (5 µg), plasmid coding Rac1 (5 µg), scrambled siRNA (20 nM), or Rac1 siRNA (20 nM). In 48 h, Nox4 and Rac1 proteins were detected by western blotting. *indicates significant difference when compared with the control at P < 0.05.

 
3.6 Nox4 and Rac1 mediate insulin-like growth factor-I-induced increase of MMP-2/-9 activity
MMPs are required for VSMC migration as they degrade the extracellular matrix. Considering the role of ROS in VSMCs migration, we next determined whether Nox4 and Rac1 were involved in the regulation of MMP-2 and MMP-9. IGF-I increased the activities of MMP-2 and -9, which was inhibited by Nox4 siRNA (Figure 5A). IGF-I enhanced promoter reporter activities of MMP-2 and -9, which was suppressed by Nox4 siRNA (Figure 5B). Over-expression of Nox4 enhanced MMP-2 and -9 promoter reporter activities (Figure 5C). Similar results were obtained with Rac1. Increased activation of MMP-2 and -9 in response to IGF-I was attenuated by N17Rac1 (Figure 5D). Likewise, increased transcriptional activation of MMP-2 and -9 in response to IGF-I was blocked by N17Rac1 (Figure 5E).


Figure 5
Figure 5
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  		Figure 5 Nox4 and Rac1 mediate insulin-like growth factor-I-induced increase of MMP-2 and -9 activities and their transcriptional activation. (A) vascular smooth muscle cells were transfected with scrambled or Nox4 siRNA. In 24 h, the cells were growth arrested and then treated with insulin-like growth factor-I (200 ng/mL) for 24 h. The culture media were harvested for MMP-2 and -9 activity assay. (B) MMP-2 and -9 reporter and β-gal plasmids were cotransfected with scrambled or Nox4 siRNA. In 24 h, the cells were growth-arrested and then treated with insulin-like growth factor-I (200 ng/mL) for 20 h. (C) MMP-2 and -9 reporter and β-gal plasmids were cotransfected with Nox4 plasmid. In 36 h, cell lysates were harvested for luciferase assay. (D) vascular smooth muscle cells were transfected with control vector or N17Rac1 plasmid. In 24 h, the cells were growth-arrested and then treated with insulin-like growth factor-I (200 ng/mL) for 24 h. MMP-2 and -9 activity in medium was determined. (E) MMP-2 and -9 reporter and β-gal plasmids were cotransfected with control vector or N17Rac1. In 24 h, the cells were growth-arrested and then treated in the absence or presence of insulin-like growth factor-I (200 ng/Ml) for 20 h. Relative luciferase activity was determined by the ratio of luciferase/β-galactosidase activity, and normalized to that of the control. * and ** indicate significant difference when compared with the control at P < 0.05 and P < 0.01, respectively; # indicates significant difference when compared with the insulin-like growth factor-I-treated group at P < 0.05.

 
3.7 Nox4 and Rac1 mediate insulin-like growth factor-I-induced vascular smooth muscle cell migration
We showed previously that IGF-I-induced ROS played an important role in proliferation of VSMCs.8 In the present work, we determined the role of IGF-I-induced ROS in migration of VSMCs. As shown in Figure 6A, IGF-I promoted migration of VSMCs, which was inhibited by catalase or DPI. We then examined the role of Nox4 and Rac1 in this process. Both Nox4 siRNA and N17Rac1 impaired IGF-I-induced migration of VSMCs (Figure 6B).


Figure 6
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  		Figure 6 Nox4 and Rac1 mediate insulin-like growth factor-I-induced vascular smooth muscle cell migration. (A) Growth-arrested vascular smooth muscle cells were pretreated with catalase (1500 units/mL) or DPI (5 µM) for 30 min, followed by treatment with insulin-like growth factor-I (200 ng/mL) for 10 h. (B) vascular smooth muscle cells were transfected with the plasmids as indicated. In 24 h, the cells were growth-arrested followed by stimulation with insulin-like growth factor-I (200 ng/mL) for 10 h. **indicates significant difference compared with the control at P < 0.01; ##indicates significant difference when compared with the insulin-like growth factor-I-treated group at P < 0.01.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
The major findings of the present study are that (i) Nox4 and Rac1 play an important role in IGF-I-induced ROS production in VSMCs, (ii) Nox4 is not regulated by Rac1, and (iii) Nox4 and Rac1 mediate IGF-I-induced activation of MMP-2 and -9 and VSMC migration.

We reported recently that IGF-I-induced ROS production in VSMCs.8 In the present study, we demonstrated that IGF-I induced a rapid ROS production peaking at 10 min. In 30 min, generation of ROS became moderate and sustained for >8 h (Figure 1A). Until now, little is known about the mechanisms that IGF-I induces ROS. NADPH oxidase is the main source of superoxide in vasculature and the expression of NADPH oxidase is increased in vascular lesions.9,14 We found that IGF-I enhanced the protein level of Nox4 but not that of Nox1 (Figure 2A). IGF-I had little effect on mRNA level of Nox4 (Figure 2B). CHX but not actinomycin D inhibited IGF-I-induced Nox4 (Figure 2C). These results suggest that IGF-I regulates the expression of Nox4 at a post-transcriptional level. This is similar to the regulation of Nox2 and p22phox by angiotensin II. Touyz et al.23 reported that angiotensin II-regulated expression of Nox2 and p22phox at the post-transcriptional level in VSMCs from human resistance arteries. A few factors have been found to affect Nox4 expression. For example, TGF-β1 and TNF-{alpha} could induce the expression of Nox4.29,30 However, the mechanisms that these factors regulate the expression of Nox4 are poorly understood. Pedruzzi et al.31 found that 7-ketocholesterol increased Nox4 expression via the IRE-1/JNK/AP-1 signalling pathway in VSMCs.

IGF-I-induced expression of Nox4 (Figure 2A) and knockdown of Nox4 markedly inhibited IGF-I-induced ROS production (Figure 2E and F), suggesting that IGF-I-induced Nox4 attributes to ROS generation by IGF-I. We also found that IGF-I induced a rapid activation of Rac1 (Figure 3A). The induction had disappeared after 2 h (Figure 3B). Inhibition of Rac1 activity impaired the production of ROS by IGF-I (Figure 3D and E), suggesting that Rac1 activation is involved in the early production of ROS induced by IGF-I.

In VSMCs, the relationship between Nox4 and Rac1 remains unclear. Studies concerning whether Nox4 activity is Rac1-dependent are contradictory. We found that inactivation of Rac1 had little effect on the activity of Nox4 (Figure 4A). Rac1 itself did not influence Nox4 expression (Figure 4C). Moreover, Nox4 did not influence Rac1 activation by IGF-I, either (Figure 4B). These results suggest that Rac1 does not influence Nox4 expression or activity in rat VSMCs. In fact, several lines of evidence support the point that if p22phox is available, Nox4 is sufficient for superoxide production without need of membrane or cytosolic regulatory elements.17,29,32 Recent study demonstrated that there was lack of the Rac regulatory site on the Nox4.33 Nox1 was expressed in our cultured VSMCs (Figure 2A and D). It was reported that Rac1 could bind to Nox1 and regulate Nox1-dependent superoxide generation.34 Here we speculate that the activated Rac1 by IGF-I might work in conjunction with Nox1 and stimulate Nox1-dependent ROS production. In the present study, the role of NADPH oxidase subunits p22phox and p47phox in IGF-I-induced ROS production was not determined. Further investigation of their involvement in this process is needed.

In our work, we found that inhibition of Nox4 expression or inactivation of Rac1 impaired IGF-I-enhanced activity and transcriptional activation of MMP-2 and -9 (Figure 5). It is known that ROS can activate MMPs and induce transcription of MMPs.35 So, our results can be explained that inhibition of Nox4 or Rac1 attenuated IGF-I-induced ROS production, resulting in the inhibition of MMP-2 and -9 activity as well as their transcriptional activation.

ROS play an important role in VSMC migration.7 We found that IGF-I enhanced VSMC migration (Figure 6). As expected, both inhibition of Nox4 and inactivation of Rac1 impaired IGF-I-enhanced cell migration (Figure 6B). These results suggest that Nox4 and Rac1 mediate IGF-I-induced VSMC migration. We noticed that the inhibition of cell migration by N17Rac1 was more than that by Nox4 siRNA (Figure 6B). This may be due to the multiple effects of Rac1 on cell migration. Because Rac1 is not only an activator of NADPH oxidase but is also required for membrane ruffling and lamellipodia formation.36

In summary, we demonstrated that Nox4 and Rac1 mediated IGF-I-induced ROS production and cell migration, and Nox4 is not regulated by Rac1 in rat VSMCs. Given the involvement of VSMCs migration in the pathogenesis of atherosclerosis and post-angioplasty restenosis, these findings may provide an insight into the role of NADPH oxidase-derived ROS in IGF-I-induced cardiovascular diseases.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
National Natural Science Foundation of China (No.30600245) to D.M.; Knowledge Innovation Program of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (No. 2007KIP209) to D.M.; Knowledge Innovation Program of the Chinese Academy of Sciences (No. KSCX2-YW-R-114) to J.F.; Chinese Ministry of Science and Technology (No. 2007CB947100) to J.F.


   Acknowledgements
 
The authors thank Beibei Fu and Heng Sun for their assistance in performing some experiments.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

  1. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med (1999) 340:115–126.[Free Full Text]
  2. Bayes-Genis A, Conover CA, Schwartz RS. The insulin-like growth factor axis: a review of atherosclerosis and restenosis. Circ Res (2000) 86:125–130.[Abstract/Free Full Text]
  3. Zhu B, Zhao G, Witte DP, Hui DY, Fagin JA. Targeted overexpression of IGF-I in smooth muscle cells of transgenic mice enhances neointimal formation through increased proliferation and cell migration after intraarterial injury. Endocrinology (2001) 142:3598–3606.[Abstract/Free Full Text]
  4. Delafontaine P. Insulin-like growth factor I and its binding proteins in the cardiovascular system. Cardiovasc Res (1995) 30:825–834.[Free Full Text]
  5. Duan C, Bauchat JR, Hsieh T. Phosphatidylinositol 3-kinase is required for insulin-like growth factor-I-induced vascular smooth muscle cell proliferation and migration. Circ Res (2000) 86:15–23.[Abstract/Free Full Text]
  6. Imai Y, Clemmons DR. Roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in stimulation of vascular smooth muscle cell migration and deoxyriboncleic acid synthesis by insulin-like growth factor-I. Endocrinology (1999) 140:4228–4235.[Abstract/Free Full Text]
  7. Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens (2000) 18:655–673.[CrossRef][Web of Science][Medline]
  8. Meng D, Shi X, Jiang BH, Fang J. Insulin-like growth factor-I (IGF-I) induces epidermal growth factor receptor transactivation and cell proliferation through reactive oxygen species. Free Radic Biol Med (2007) 42:1651–1660.[CrossRef][Web of Science][Medline]
  9. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res (2000) 86:494–501.[Abstract/Free Full Text]
  10. Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, et al. Cell transformation by the superoxide-generating oxidase Mox1. Nature (1999) 401:79–82.[CrossRef][Medline]
  11. Sorescu D, Weiss D, Lassegue B, Clempus RE, Szocs K, Sorescu GP, et al. Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation (2002) 105:1429–1435.[Abstract/Free Full Text]
  12. Wingler K, Wünsch S, Kreutz R, Rothermund L, Paul M, Schmidt HH. Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo. Free Radic Biol Med (2001) 31:1456–1464.[CrossRef][Web of Science][Medline]
  13. Sturrock A, Cahill B, Norman K, Huecksteadt TP, Hill K, Sanders K, et al. Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol (2006) 290:L661–L673.[Abstract/Free Full Text]
  14. Szöcs K, Lassègue B, Sorescu D, Hilenski LL, Valppu L, Couse TL, et al. Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury. Arterioscler Thromb Vasc Biol (2002) 22:21–27.[Abstract/Free Full Text]
  15. Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem (2004) 279:45935–45941.[Abstract/Free Full Text]
  16. Ago T, Kitazono T, Ooboshi H, Iyama T, Han YH, Takada J, et al. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase. Circulation (2004) 109:227–233.[Abstract/Free Full Text]
  17. Martyn KD, Frederick LM, von LK, Dinauer MC, Knaus UG. Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases. Cell Signal (2006) 18:69–82.[CrossRef][Web of Science][Medline]
  18. Kawahara T, Kohjima M, Kuwano Y, Mino H, Teshima-Kondo S, Takeya R, et al. Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells. Am J Physiol Cell Physiol (2005) 288:C450–C457.[Abstract/Free Full Text]
  19. Hordijk PL. Regulation of NADPH oxidases: the role of Rac proteins. Circ Res (2006) 98:453–462.[Abstract/Free Full Text]
  20. Aoyagi M, Fukai N, Sakamoto H, Shinkai T, Matsushima Y, Yamamoto M, et al. Altered cellular responses to serum mitogens, including platelet-derived growth factor, in cultured smooth muscle cells derived from arteries of patients with moyamoya disease. J Cell Physiol (1991) 147:191–198.[CrossRef][Web of Science][Medline]
  21. Liu LZ, Hu XW, Xia C, He J, Zhou Q, Shi X, et al. Reactive oxygen species regulate epidermal growth factor-induced vascular endothelial growth factor and hypoxia-inducible factor-1alpha expression through activation of AKT and P70S6K1 in human ovarian cancer cells. Free Radic Biol Med (2006) 41:1521–1533.[CrossRef][Web of Science][Medline]
  22. Menshikov M, Plekhanova O, Cai H, Chalupsky K, Parfyonova Y, Bashtrikov P, et al. Urokinase plasminogen activator stimulates vascular smooth muscle cell proliferation via redox-dependent pathways. Arterioscler Thromb Vasc Biol (2006) 26:801–807.[Abstract/Free Full Text]
  23. Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, et al. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II. Circ Res (2002) 90:1205–1213.[Abstract/Free Full Text]
  24. Mahadev K, Motoshima H, Wu X, Ruddy JM, Arnold RS, Cheng G, et al. The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. Mol Cell Biol (2004) 24:1844–1854.[Abstract/Free Full Text]
  25. Mao F, Leung WY, Xin X. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol (2007) 7:76.[CrossRef][Medline]
  26. Zhou Q, Meng D, Yan B, Jiang BH, Fang J. Transactivation of epidermal growth factor receptor by insulin-like growth factor 1 requires basal hydrogen peroxide. FEBS Lett (2006) 580:5161–5166.[CrossRef][Web of Science][Medline]
  27. Benard V, Bohl BP, Bokoch GM. Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem (1999) 274:13198–13204.[Abstract/Free Full Text]
  28. Ke Z, Lin H, Fan Z, Cai TQ, Kaplan RA, Ma C, et al. MMP-2 mediates ethanol-induced invasion of mammary epithelial cells over-expressing ErbB2. Int J Cancer (2006) 119:8–16.[CrossRef][Web of Science][Medline]
  29. Sturrock A, Cahill B, Norman K, Huecksteadt TP, Hill K, Sanders K, et al. Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol (2006) 290:L661–L673.[Abstract/Free Full Text]
  30. Moe KT, Aulia S, Jiang F, Chua Y L, Koh TH, Wong MC, et al. Differential upregulation of Nox homologues of NADPH oxidase by tumor necrosis factor-alpha in human aortic smooth muscle and embryonic kidney cells. J Cell Mol Med (2006) 10:231–239.[Web of Science][Medline]
  31. Pedruzzi E, Guichard C, Ollivier V, Driss F, Fay M, Prunet C, et al. NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol (2004) 24:10703–10717.[Abstract/Free Full Text]
  32. Serrander L, Cartier L, Bedard K, Banfi B, Lardy B, Plastre O, et al. NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation. Biochem J (2007) 406:105–114.[CrossRef][Web of Science][Medline]
  33. Kao YY, Gianni D, Bohl B, Taylor RM, Bokoch GM. Identification of a conserved Rac binding site on NADPH oxidases supports a direct GTPase regulatory mechanism. J Biol Chem (2008) Published online ahead of print March 17, 2008.
  34. Cheng G, Diebold BA, Hughes Y, Lambeth JD. Nox1-dependent reactive oxygen generation is regulated by Rac1. J Biol Chem (2006) 281:17718–17726.[Abstract/Free Full Text]
  35. Siwik DA, Colucci WS. Regulation of matrix metalloproteinases by cytokines and reactive oxygen/nitrogen species in the myocardium. Heart Fail Rev (2004) 9:43–51.[CrossRef][Web of Science][Medline]
  36. Gerthoffer WT. Mechanisms of vascular smooth muscle cell migration. Circ Res (2007) 100:607–621.[Abstract/Free Full Text]

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NADPH Oxidase 1 Controls the Persistence of Directed Cell Migration by a Rho-Dependent Switch of {alpha}2/{alpha}3 Integrins
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