The transforming growth factor (TGF)-␤/Smad3 signaling pathway is considered a central mediator of pathological organ fibrosis; however, contribution of Smad2/3-independent TGF-␤ 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-␤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

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... ing the molecular mechanisms underlying the profibrotic program in this model. We demonstrate that the TGF-␤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-␤RI, (ii) a TGF-␤RI mutant defective in Smad2/3 activation, ALK5(3A), potently stimulated collagen production, (iii) elevation of TGF-␤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-␤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␤-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-␤ signaling that may operate during chronic stages of fibrosis in SSc. Downloaded from FIGURE 4. AdTGF-␤RI overexpression stimulates CTGF promoter activity in an ERK1/2-Sp1-dependent manner. A, graphical representation of data obtained from transient transfection of the WT-CTGF-luc promoter in cells transduced with the control adenovirus (AdGo), AdTGF-␤RI and AdALK5(3A) at m.o.i. 25 (*, p Ͻ 0.05). B, UO126 (10 M) inhibited the potent stimulation of TGF-␤RI overexpression on CTGF promoter activity (*, p Ͻ 0.05). C, blockade of Smad1 via AdSmad1 siRNA abrogated the stimulatory effect of AdTGF-␤RI on CTGF promoter activity. D, transient transfection data showing that the mutation of the Sp1 site proximal to the start site abrogated the stimulatory effect of AdTGF-␤RI (RI) overexpression on CTGF promoter activity, whereas mutation of the Smad binding site had no effect (*, p Ͻ 0.05). E, transient transfection data showing that Smad1 mediates the effect of TGF-␤RI overexpression on CTGF promoter activity.