Role of TGF-beta and CTGF Signaling Transgenic Mouse Models of Scleroderma

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Scleroderma or systemic sclerosis (SSc) is a disorder of the connective tissues affecting various organ systems. The disease is complex and is characterized by excessive accumulation of collagen and other extracellular matrix components in the skin and internal organs. Because increased signaling by TGF-beta has been implicated in this disease we recently established a novel mouse model in which the TGF-beta Receptor1 is constitutively activated in fibroblasts post-natally (TBR1CA; Col1a2-CreER). These mice recapitulated the major features of human SSc, showing pronounced and generalized fibrosis of the dermis, thinner epidermis, loss of hair follicles, and fibrotic thickening of small blood vessel walls in lung and kidney. Primary skin fibroblasts of these mice showed elevated expression of downstream TGF-beta targets, reproducing the hallmark biochemical phenotype of explanted SSc dermal fibroblasts. In particular there was a marked increase in connective tissue growth factor (CTGF) expression. Since increased expression of CTGF has been implicated to play a key role in the disease process, we generated transgenic mice that over-express CTGF in fibroblasts (Col1a2-CTGF) by using a fibroblast-specific promoter/enhancer from the pro-D2(l) collagen gene. The animals exhibit a severe loss of hair. Initial histological examination of skin biopsies showed pronounced and generalized fibrosis of the dermis, thicker epidermis and inflammatory infiltrates in the area of the skin fibrosis. Preliminary analysis of mouse embryonic fibroblasts derived from these transgenic mice showed elevated expression of collagen type I and Timp-3. We propose to characterize the Col1a2-CTGF mice as well as their explanted skin fibroblasts. To understand the mechanisms by which increased expression of CTGF in fibroblasts causes a fibrotic disease, we will also perform a comparison of the molecular phenotypes of the skin fibroblasts of these mice with those of TBR1CA; Col1a2-CreER mice and with those of specific human scleroderma patients. To further examine these mechanisms we propose to attenuate the fibrotic phenotypes of the skin fibroblasts of the two transgenic mouse models of scleroderma by pharmacological inhibitors of specific signaling pathways or siRNAs. We also will attempt to inhibit the fibrotic phenotypes of these transgenic mice in vivo by crossing null mutations in either SmadS or integrin D6 in these mice. Finally, we will examine the mechanisms that cause increased sensitivity to bleomycin-induced lung fibrosis in TBR1CA; Col1a2-CreER mice and test whether a similar sensitivity occurs in Col1a2-CTGF mice. These studies should give information about the relative importance of TGF-beta and CTGF in causing fibrotic diseases.