These reports indicated that elevated ROS production is one of the causative factors of recurrent epithelial damage in fibrotic lungs. Therefore, SPARC may be involved in epithe etc lial cell injury through enhanced H2O2 production from activated fibroblasts. This hypothesis is supported by our results indicating that knockdown of SPARC expression level by siRNA mitigated the decrease in viability of A549 epithelial cells in coculture with TGF B stimulated fibro blasts. This reduction in A549 cell viability was alleviated in the presence of NAC. In addition, interference with SPARC expression by siRNA reduced H2O2 release from fi broblasts treated with TGF B. SPARC has been shown to play an important role in ECM accumulation.

In addition to this role of SPARC in the pathogenesis of fibrosis, our findings indicated a possible contribution of SPARC to epithelial cell damage through regulation of ROS production. We demonstrated the involvement of ILK in the mech anism underlying enhanced ROS production by SPARC, which was supported by a number of observations. First, knockdown of SPARC with siRNA diminished ILK activa tion in TGF B stimulated fibroblasts. Second, siRNA against ILK significantly reduced extracellular H2O2 generation in TGF B stimulated fibroblasts. Our findings were consistent with those of previous studies indicating that SPARC activates ILK in fibroblasts and that activation of ILK by high pressure leads to ROS produc tion in vessels through Rac 1 mediated NAD H oxidase activation.

In isolated cardiomyocytes, ILK is activated by stromal cell derived factor 1 and is necessary for SDF 1 triggered activation of Rac 1, NAD H oxidase, and release of ROS. ILK interacts with the cytoplasmic domain of the integrin B1/B3 subunits, which is important for cell adhesion, differentiation, and survival. Blocking of SPARC integrin Brefeldin_A B1 interaction by function blocking anti integrin B1 antibody impairs ILK activation, suggesting that SPARC ILK signaling is mediated at least in part by integrin B1. NADPH oxidase family of proteins is comprised of five members, including NADPH oxidase 1 to 5. In the present study, knockdown of NOX4 using siRNA almost completely blocked TGF B induced H2O2 production in HFL 1 cells, suggesting NOX4 is a major NADPH oxidase involved in TGF B induced H2O2 production. It has been known that NOX4 is a constitutively active NADPH oxidase isoform and NOX4 activity is regulated, at least in part, at the transcriptional level. NOX4 expression is increased by TGF B stimulation in fibroblasts. Consistent with these reports, our study showed that NOX4 was upre gulated by TGF B in inhibitor price HFL 1 cells.