Transforming growth factor-beta receptor type I-dependent fibrogenic gene program is mediated via activation of Smad1 and ERK1/2 pathways.
The transforming growth factor (TGF)-beta/Smad3 signaling pathway is considered a central mediator of pathological organ fibrosis; however, contribution of Smad2/3-independent TGF-beta signaling has not been fully explored. The present study utilized previously a described model of scleroderma (SSc) fibrosis based on forced expression of the TGF-betaRI (ALK5) (Pannu, J., Gardner, H., Shearstone, J. R., Smith, E., and Trojanowska, M. (2006) Arthritis Rheum. 54, 3011-3021). This study was aimed at determining the molecular mechanisms underlying the profibrotic program in this model. We demonstrate that the TGF-betaRI-dependent up-regulation of collagen and CCN2 (CTGF) does not involve Smad2/3 activation but is mediated by ALK1/Smad1 and ERK1/2 pathways. The following findings support this conclusion: (i) Smad2 and -3 were not phosphorylated in response to TGF-betaRI, (ii) a TGF-betaRI mutant defective in Smad2/3 activation, ALK5(3A), potently stimulated collagen production, (iii) elevation of TGF-betaRI triggered sustained association of ALK5 with ALK1 and high levels of Smad1 phosphorylation, (iv) blockade of Smad1 via small interfering RNA abrogated collagen and CCN2 up-regulation in this model, (v) elevated TGF-betaRI led to a prolonged activation of ERK1/2, (vi) the pharmacologic inhibitor of ERK1/2 inhibited Smad1 phosphorylation and abrogated profibrotic effects of elevated TGFbeta-RI. Additional experiments demonstrated that a GC-rich response element located -6 to -16 (upstream of the transcription start site) in the CCN2 promoter mediated Smad1-dependent increased promoter activity in this model. This element was shown previously to mediate up-regulation of the CCN2 promoter in SSc fibroblasts. In conclusion, this study defines a novel ALK1/Smad1- and ERK1/2-dependent, Smad3-independent mode of TGF-beta signaling that may operate during chronic stages of fibrosis in SSc.
Pannu
Jaspreet
J
Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Nakerakanti
Sashidhar
S
Smith
Edwin
E
ten Dijke
Peter
P
Trojanowska
Maria
M
D030201:Activin Receptors, Type I / Q000235:genetics / Q000378:metabolism*; D030301:Activin Receptors, Type II / Q000378:metabolism; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D005355:Fibrosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020862:GC Rich Sequence; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis* / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D012594:Scleroderma, Localized / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051898:Smad1 Protein / Q000378:metabolism; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D015854:Up-Regulation
C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D018125:Receptors, Transforming Growth Factor beta; C497399:SMAD1 protein, human; C497790:SMAD3 protein, human; D051898:Smad1 Protein; D051899:Smad2 Protein; D051900:Smad3 Protein; D055513:Connective Tissue Growth Factor; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C425364:ACVRL1 protein, human; D030201:Activin Receptors, Type I; D030301:Activin Receptors, Type II; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human