Expression of nonphagocytic NADPH oxidase system in the ocularlens

[摘要] Purpose: The primary goal of this study was to characterize the RacGTPase associated, NADPH oxidase-mediated Reactive Oxygen Species(ROS)-generating system in the lens tissue.Methods: NADPH oxidase activity in lens tissue was determined byquantifying superoxide-induced lucigenin photoemission. Immunologicaland PCR/RT-PCR techniques were utilized to determine expression ofdifferent components of the NADPH oxidase system in lens tissue. Growthfactor stimulated ROS production was determined quantitatively in humanlens epithelial cells using dichlorofluorescein diacetate.Results: Lens homogenates from different species showed generationof superoxide in a lucigenin-enhanced chemiluminescence assay in thepresence of NADPH. This activity was found to be lens proteinconcentration dependent, heat sensitive, and inhibitable by superoxidedismutase and the flavoprotein inhibitor, diphenyleneiodonium (DPI). Thedistribution of superoxide generating activity in lens was confinedpredominantly to the lens epithelium, with very low levels in cortex andnone in the nucleus. Immunological assays have demonstrated the presenceof p67phox and p47phox in lens tissue, while PCR and RT-PCR reactionsamplified DNA products corresponding to the p67phox, p40phox, p22phox,gp91phox, and Rac1 components of the NADPH oxidase complex from humanand mouse lens cDNA libraries. Serum starved human lens epithelial cellsstimulated with different growth factors including EGF, b-FGF, PDGF,TGF-β, and LPA demonstrated increased production of ROS, a responsewhich was blocked by inhibitors of NADPH oxidase, such as DPI and theantioxidant-N-acetyl cysteine (NAC). RT-PCR analysis of human lens RNAconfirmed readily detectable levels of expression of low molecularweight protein tyrosine phosphatase (LMW-PTP), which is awell-characterized target of redox signaling pathway(s).Conclusions: These data demonstrate the presence of a functionalnonphagocytic NADPH oxidase system in lens that is predominantlylocalized to the lens epithelium. Several growth factors appear tostimulate the activity of lens NADPH oxidase, resulting in increasedproduction of ROS in lens epithelial cells, indicating that redoxsignaling may have an important role in growth factor effects on lensgrowth and development.

[学科分类] 生物化学/生物物理