Transforming growth factor-β 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-beta RI ( 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-beta RI-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-beta RI, ( ii) a TGF- beta RI mutant defective in Smad2/3 activation, ALK5( 3A), potently stimulated collagen production, (iii) elevation of TGF-beta RI 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-beta RI led to a prolonged activation of ERK1/2, ( vi) the pharmacologic inhibitor of ERK1/2 inhibited Smad1 phosphorylation and abrogated profibrotic effects of elevated TGF beta-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.