It has also been reported that inhibition of p38 MAPK attenuates the progression of fibrosis in the bleomycin model. SPARC may serve as one of the downstream factors of PI3K and p38 MAPK signaling in the patho genesis of fibrosis. Although PDGF is also known to be able to activate both PI3K and p38 MAPK signalling inhibitor Erlotinib pathways, SPARC upregulation was not induced by PDGF stimulation in our study. Therefore, activation of PI3K and p38 MAPK is required but is not enough for SPARC induc tion. Other signaling pathways could also be involved in upregulation of SPARC by TGF B. Conclusions Our results indicated that SPARC contributes to the extracellular H2O2 generation induced by TGF B via ILK activation in fibroblasts and can regulate the viability of adjacent epithelial cells through H2O2 generation.

In addition, SPARC expression is upregulated by TGF B, which is thought to be a key regulator for the establish ment and progression of IPF, not only in culture but also in the animal model of pulmonary fibrosis. One of the most widely accepted views regarding the pathogenesis of IPF is the recurrent damage of alveolar epithelial cells and ECM deposition from aberrant activated fibroblasts. We demonstrated that SPARC likely contributes to epithelial damage through regulation of ROS production. As SPARC is capable of exerting pleiotropic functions on the pathogenesis of IPF, SPARC inhibition may represent a potential therapeutic approach for IPF. Methods Materials TGF B, PDGF, IL 13 and IGF were purchased from R D systems. CTGF and TNF were purchased from Pepro Tech.

Endothelin 1 and angiotensin II were purchased from Sigma Aldrich. PGF2 was purchased from Enzo life science. Anti body against SPARC was purchased from Santa Cruz Biotechnology. Antibodies against SMAD3, Tubulin, p p44/42, p44/42, p AKT, AKT, p c Jun, c Jun, p p38 MAPK, p38 MAPK and ILK were purchased from Cell Signaling Technology. Antibody against ILK was purchased from Abnova. Phospho MBP was purchased from Milipore. U0126, LY294002, PI103, SB202190, SB239063 and SP600125 were purchased from Calbiochem. Diphenyliodonium and N acetylcysteine were purchased from Sigma Aldrich. Cell culture The human fetal lung fibroblast HFL 1 and the human lung adenocarcinoma epithelial cell line A549 were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% FBS and 100 U/ml penicillin/streptomycin at 37 C under 5% CO2.

Studies were performed on passage 5 to 10 of HFL 1 cells. Coculture system of epithelial cells and fibroblasts HFL 1 cells were plated on the lower wells of 24 well transwell co culture system at a density of 1 105 cells/well, and cultured at 37 C under 5% CO2 for overnight. Then cells were grown for 24 Cilengitide h in DMEM with 0. 5% FBS before http://www.selleckchem.com/products/nutlin-3a.html treatment with/without TGF B.