title: Testosterone induces vascular smooth muscle cell migration by NADPH oxidase and c-Src-dependent pathways.
abstract: Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs.
authors.affiliation: Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil. andreiazc@usp.br
authors.forename: Andreia Z
authors.initials: AZ
authors.lastname: Chignalia
authors.forename: Elke Z
authors.initials: EZ
authors.lastname: Schuldt
authors.forename: Lívia L
authors.initials: LL
authors.lastname: Camargo
authors.forename: Augusto C
authors.initials: AC
authors.lastname: Montezano
authors.forename: Gláucia E
authors.initials: GE
authors.lastname: Callera
authors.forename: Francisco R
authors.initials: FR
authors.lastname: Laurindo
authors.forename: Lucia R
authors.initials: LR
authors.lastname: Lopes
authors.forename: Maria Christina W
authors.initials: MC
authors.lastname: Avellar
authors.forename: Maria Helena C
authors.initials: MH
authors.lastname: Carvalho
authors.forename: Zuleica B
authors.initials: ZB
authors.lastname: Fortes
authors.forename: Rhian M
authors.initials: RM
authors.lastname: Touyz
authors.forename: Rita C
authors.initials: RC
authors.lastname: Tostes
mesh_terms: D000726:Androgen Antagonists / Q000494:pharmacology; D000728:Androgens / Q000494:pharmacology; D000818:Animals; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D005485:Flutamide / Q000494:pharmacology; D015151:Immunoblotting; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D013739:Testosterone / Q000494:pharmacology*
chemical_list: C539297:3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo(3,4-d)pyrimidin-4-amine; D000726:Androgen Antagonists; D000728:Androgens; D011500:Protein Synthesis Inhibitors; D011743:Pyrimidines; D017382:Reactive Oxygen Species; D003609:Dactinomycin; D013739:Testosterone; D005485:Flutamide; D003513:Cycloheximide; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C000614712:NOX1 protein, rat; C498911:Nox4 protein, rat; C060505:neutrophil cytosolic factor 1; D016392:Proto-Oncogene Proteins pp60(c-src)