| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Mesenchymal cells such as fibroblasts and myofibroblasts are the principle matrix-producing cells during pulmonary fibrogenesis. TGF β signaling plays an important role in stimulating the expression of type I collagen, the major component of extracellular matrix. Recently, a member of TGFβ superfamily, bone morphogenetic protein (BMP)-7, was reported to oppose TGFβ1 in fibrogenesis. Here, we have addressed the effects of gene transfer of BMP-7 on fibrogenic reaction using pulmonary myofibroblastic cell lines, in which collagen promoter activity was detected by luciferase assay. To do so, we first established cell lines from lung tissues of transgenic mice harboring the collagen 1A2 upstream sequence. These cell lines showed spindle shape, and expressed vimentin and aSMA, but not E-cadherin, which demonstrates that they are derived from lung myofibroblasts. They showed luciferase activity that was dose-dependent on TGFβ1 stimulation. This activity was also up-regulated by overexpression of Smad3 and down-regulated by that of Smad7. These cell lines were used to evaluate the effect of BMP-7 for following examination. In immunocytochemical examination, ectopic expression of BMP-7 (Ad-BMP-7) led to nuclear localization of phospho-Smadl/5/8 and suppressed that of Smad3. Ad-BMP-7 decreased collagen 1A2 promoter-dependent luciferase activity. Real-time RT PCR showed that Ad-BMP-7 suppressed the expression of collagen 1A2, tissue inhibitor of metalloproteinase and increased that of inhibitor of differentiation (Id) 2 and 3, which opposes TGFβ signaling. Furthermore, ectopic expression of Id2 and ID decreased collagen 1A2 promoter-dependent luciferase activity in pulmonary myofibroblasts. In conclusion, these data demonstrate that BMP-7 antagonizes TGFβ-dependent fibrogenesis by expression of 1d2 in mouse pulmonary myofibroblast cell lines.
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