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Presentation Number: 1975
Purpose:
By stimulating collagen synthesis and other profibrotic responses, TGF-ß plays a fundamental role in pathogenesis of scleroderma. Previously, we functionally implicated Smad2/3-mediated signal transduction in TGF-ß fibrogenic activity. Recent observations have begun to reveal novel Smad-independent pathways for TGF-ß signaling. Here, we investigated the expression, mechanism and role of the non-receptor tyrosine kinase c-Abl and its inhibition in TGF-ß-induced profibrotic responses.
Methods:
Cytotoxicity of the tyrosine kinase inhibitor imatinib methylate (Gleevec) was examined by MTT assay. Intracellular protein levels and phosphorylation were examined with Western blots. Regulation of matrix gene expression was investigated by Northern and Western analysis and transient transfection assays. In vivo, c-Abl regulation was investigated in a mouse model of scleroderma.
Results:
Detectable levels of c-Abl were found in normal and lesional scleroderma fibroblasts in vitro. Stimulation of normal fibroblasts with TGF-ß induced phosphorylation of ectopically expressed c-Abl. The response was blocked by preincubation with non-cytotoxic (10 µM) concentrations of imatinib, which had modest anti-proliferative effect. Imatinib was selective for c-Abl, as TGF-ß-dependent Smad activation was unaffected. Imatinib efficiently prevented TGF-ß-induced stimulation of collagen and ECM protein synthesis and mRNA expression. Further, it blocked stimulation of COL1A2 promoter activity in transiently transfected fibroblasts. Surprisingly, imatinib caused p38 MAP kinase activation. Ectopic expression of c-Abl resulted in up-regulation of collagen gene expression independent of ligand, and imatinib blocked this response. TGF-ß elicited rapid activation of the Smad pathway in fibroblasts from c-Abl-null mouse embryos. Smooth muscle actin stress fiber formation induced by TGF-ß was comparable in wildtype and c-Abl-null fibroblasts. In vivo, development of tissue fibrosis induced by bleomycin was associated with marked up-regulation of c-Abl protein expression and phosphorylation.
Conclusion:
The non-receptor tyrosine kinase c-Abl appears to have a novel and important function in Smad3-independent profibrotic TGF-ß responses. Mouse scleroderma is associated with activation of c-Abl in lesional fibroblasts, implicating c-Abl in TGF-ß-mediated tissue fibrosis. Imatinib, an effective anti-leukemic agent, inhibits c-Abl, blocks profibrotic TGF-ß responses, and may have utility in the treatment of scleroderma.
By stimulating collagen synthesis and other profibrotic responses, TGF-ß plays a fundamental role in pathogenesis of scleroderma. Previously, we functionally implicated Smad2/3-mediated signal transduction in TGF-ß fibrogenic activity. Recent observations have begun to reveal novel Smad-independent pathways for TGF-ß signaling. Here, we investigated the expression, mechanism and role of the non-receptor tyrosine kinase c-Abl and its inhibition in TGF-ß-induced profibrotic responses.
Methods:
Cytotoxicity of the tyrosine kinase inhibitor imatinib methylate (Gleevec) was examined by MTT assay. Intracellular protein levels and phosphorylation were examined with Western blots. Regulation of matrix gene expression was investigated by Northern and Western analysis and transient transfection assays. In vivo, c-Abl regulation was investigated in a mouse model of scleroderma.
Results:
Detectable levels of c-Abl were found in normal and lesional scleroderma fibroblasts in vitro. Stimulation of normal fibroblasts with TGF-ß induced phosphorylation of ectopically expressed c-Abl. The response was blocked by preincubation with non-cytotoxic (10 µM) concentrations of imatinib, which had modest anti-proliferative effect. Imatinib was selective for c-Abl, as TGF-ß-dependent Smad activation was unaffected. Imatinib efficiently prevented TGF-ß-induced stimulation of collagen and ECM protein synthesis and mRNA expression. Further, it blocked stimulation of COL1A2 promoter activity in transiently transfected fibroblasts. Surprisingly, imatinib caused p38 MAP kinase activation. Ectopic expression of c-Abl resulted in up-regulation of collagen gene expression independent of ligand, and imatinib blocked this response. TGF-ß elicited rapid activation of the Smad pathway in fibroblasts from c-Abl-null mouse embryos. Smooth muscle actin stress fiber formation induced by TGF-ß was comparable in wildtype and c-Abl-null fibroblasts. In vivo, development of tissue fibrosis induced by bleomycin was associated with marked up-regulation of c-Abl protein expression and phosphorylation.
Conclusion:
The non-receptor tyrosine kinase c-Abl appears to have a novel and important function in Smad3-independent profibrotic TGF-ß responses. Mouse scleroderma is associated with activation of c-Abl in lesional fibroblasts, implicating c-Abl in TGF-ß-mediated tissue fibrosis. Imatinib, an effective anti-leukemic agent, inhibits c-Abl, blocks profibrotic TGF-ß responses, and may have utility in the treatment of scleroderma.
W. Ishida, None.