NADPH oxidase expression and production of superoxide by human corneal stromal cells

[摘要] Purpose: Superoxide (O2.-)may function as a second messenger or regulator of signal transductionwhen produced at low concentrations in the proper locations withincells. The purpose of these studies was to determine whether humancorneal stromal (HCS) fibroblasts are capable of producing O2.-via nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, afamily of protein complexes believed to be responsible for thelocalized and limited production of O2.- withregulatory activity. Methods: HCS cells, grown as primary andlow-passage cultures of fibroblasts, were used as the sources of RNAfor reverse transcriptase PCR, with primers specific for mRNAs encodingthe proteins that comprise NADPH oxidases. Small interfering (si)RNAswere used to knockdown specific NOX mRNAs. Proteins composingthe NADPH oxidase complexes were identified using western blots. Theproduction of O2.- by whole cells and cell-freepreparations was assessed by measurement of NADPH-dependent superoxidedismutase-inhibitable cytochrome c reduction. Results: Whole cells and cell-freeextracts of corneal stromal fibroblasts produced O2.-in an NADPH-dependent manner. These fibroblasts constitutively producedmRNAs encoding eight proteins known to comprise NADPH oxidasecomplexes. mRNAs encoding NOX1, NOX4, NOX5, p22phox, p47 phox, p67 phox, and p40 phox aswell as Rac were expressed. Treatment of HCS fibroblasts withsiRNA pools specific for each of these three NOXs significantly reducedthe steady state levels of the respective mRNAs. Western blotsconfirmed the existence of all the proteins required for O2.-production. Rac 1, a regulator of the activity of some forms of NADPHcomplexes was present in membranous cell fractions containing theoxidase proteins. Conclusions: HCS fibroblasts produced O2.-in a NADPH-dependent manner via at least three isoforms of NADPHoxidase. These cells expressed NOX1, NOX4, NOX5,p22 phox, p47 phox, p67 phox, and p40 phoxas well as Rac. SiRNAs directed against each of the threeputative isoforms of NOX significantly reduced the steady state levelsof the appropriate NOX mRNA pools, thus confirming theexistence of the three isoforms. The O2.-produced by the NADPH oxidases in HCS fibroblasts is a potentialcontributor to signal transduction pathways and a regulator of geneexpression as well as a potential participant in processes that occurduring inflammation.

[学科分类] 生物化学/生物物理