Expression of NOX in Human and Rabbit Lens Epithelial Cells

Abstract

Purpose: : Reactive oxygen species (ROS, including H₂O₂, superoxide anion and hydroxyl radical) may function as second messengers or regulators of signal transduction when produced at low concentration. We have reported that both human and rabbit lens epithelial cells produced superoxide anion upon PDGF and arachidonic acid addition. NADPH oxidase was the source of superoxide. NOX protein is one of the two membrane-bound subunits of NADPH oxidase. There are five isoforms of NOX family (i.e. NOX1, -2, -3, -4, and -5). The purpose of this study was to determine which isoform(s) of NOX were expressed in human and rabbit lens epithelial cells.

Methods: : RNA was isolated from human lens epithelial B3 cells (HLE B3) and rabbit lens epithelial cells N/N 1003A by TriZol reagent according to manufacturer’s direction. RNA samples were treated with DNase-I. RT-PCR reactions were performed with specific primers of NOX1 through NOX5 using Qiagen OneStep RT-PCR kit. Negative control reactions were run in the absence of the reverse transcriptase. Total RNA isolated from HEK 293 cells were performed RT-PCR as positive control. The reaction products were detected on ethidium-stained 1.5% agarose gels. Reaction products were identified by sequencing of the products. Cell lysate fractions were separated into soluble and particulate fractions by centrifugation at 29,000g for 30 minutes at 4°C. The pellets were resuspended in modified RIPA buffer. Equal amounts of protein were run on 8% SDS-PAGE and were detected on the immunoblots by chemiluminescence. Blots were reprobed after the antibodies were removed by stripping buffer. Antibodies used in this study included: NOX2, NOX3, NOX4, and NOX5.

Results: : RT-PCR primers designed to detect mRNAs of the five isoforms of NOX proteins documented that mRNAs encoding NOX1, -2, -3, -4, and -5 were constitutively present in both human HLE B3 cells and rabbit N/N 1003A cells. The sequence of HLE B3 cells were 95% identical with N/N 1003A cells. NOX2, -3, -4 and -5 proteins were detected in both types of lens epithelial cells by western blot. NOX2 expression was much less in N/N 1003A cells compared with that in HLE B3 cells.

Conclusions: : Five isoforms of NOX family (i.e. NOX1, NOX2, NOX3, NOX4, and NOX5) were constitutively expressed in human lens epithelial B3 cells as well as in rabbit lens epithelial cells N/N 1003A.

Keywords: enzymes/enzyme inhibitors