title: c-Src and hydrogen peroxide mediate transforming growth factor-beta1-induced smooth muscle cell-gene expression in 10T1/2 cells.
abstract: OBJECTIVE
  Transforming growth factor-beta1 (TGF-beta1) controls the expression of numerous genes, including smooth muscle cell (SMC)-specific genes and extracellular matrix protein genes. Here we investigated whether c-Src plays a role in TGF-beta1 signaling in mouse embryonic fibroblast C3H10T1/2 cells.
  
  
  RESULTS
  TGF-beta1 induction of the SMC contractile protein SM22alpha gene expression was inhibited by PP1 (an inhibitor of Src family kinases) or by C-terminal Src kinase (a negative regulator of c-Src). Induction of SM22alpha by TGF-beta1 was markedly attenuated in SYF cells (c-Src(-), Yes(-), and Fyn(-)) compared with Src(++) cells (c-Src(++), Yes(-), and Fyn(-)). PP1 also inhibited the TGF-beta1-induced expression of serum response factor (SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF-beta1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD(P)H oxidase inhibitors such as apocynin inhibited the TGF-beta1-induced expression of SM22alpha. Furthermore, we demonstrate that TGF-beta1 induction of the plasminogen activator inhibitor-1 (PAI-1) gene, which is known to be dependent on Smad but not on SRF, is inhibited by PP1 and apocynin.
  
  
  CONCLUSIONS
  Our results suggest that TGF-beta1 activates c-Src and generates hydrogen peroxide through NAD(P)H oxidase, and these signaling pathways lead to the activation of specific sets of genes, including SM22alpha and PAI-1. TGF-beta1 controls the expression of numerous genes, including SM22alpha and PAI-1. We investigated whether c-Src plays a role in TGF-beta1 signaling. TGF-beta1 induction of such genes was significantly reduced in Src family tyrosine kinase-deficient cells, and Csk and pharmacological inhibitors for Src family kinases or antioxidants inhibit the effects of TGF-beta1. These results indicate that c-Src and hydrogen peroxide are required for TGF-beta1 signaling.
authors.affiliation: Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Japan.
authors.forename: Mahito
authors.initials: M
authors.lastname: Sato
authors.forename: Keiko
authors.initials: K
authors.lastname: Kawai-Kowase
authors.forename: Hiroko
authors.initials: H
authors.lastname: Sato
authors.forename: Yuko
authors.initials: Y
authors.lastname: Oyama
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Kanai
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Ohyama
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Suga
authors.forename: Toshitaka
authors.initials: T
authors.lastname: Maeno
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Aoki
authors.forename: Junichi
authors.initials: J
authors.lastname: Tamura
authors.forename: Hironosuke
authors.initials: H
authors.lastname: Sakamoto
authors.forename: Ryozo
authors.initials: R
authors.lastname: Nagai
authors.forename: Masahiko
authors.initials: M
authors.lastname: Kurabayashi
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D002374:Catalase / Q000494:pharmacology; D002460:Cell Line / Q000187:drug effects / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D004050:Ditiocarb / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000502:physiology; D005419:Flavonoids / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D018390:Gene Targeting; D006861:Hydrogen Peroxide / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008840:Microfilament Proteins / Q000096:biosynthesis* / Q000235:genetics; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000502:physiology; D009281:Naphthalenes / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D026362:Serum Response Factor / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051900:Smad3 Protein; D051901:Smad4 Protein; D051905:Smad6 Protein; D013450:Sulfones / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology* / Q000502:physiology; D053773:Transforming Growth Factor beta1; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000502:physiology
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D000098:Acetophenones; D004268:DNA-Binding Proteins; D005419:Flavonoids; D005452:Fluoresceins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009281:Naphthalenes; D017395:Plasminogen Activator Inhibitor 1; D011720:Pyrazoles; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051901:Smad4 Protein; D051905:Smad6 Protein; C497803:Smad6 protein, mouse; D013450:Sulfones; C508961:Tgfb1 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C051615:transgelin; C029569:diacetyldichlorofluorescein; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D004050:Ditiocarb; C056165:acetovanillone; D006861:Hydrogen Peroxide; D002374:Catalase; D019255:NADPH Oxidases; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; C058819:calphostin C; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000111:Acetylcysteine