Elsevier

Gene

Volume 254, Issues 1–2, 22 August 2000, Pages 237-243
Gene

NADPH oxidase subunit, gp91phox homologue, preferentially expressed in human colon epithelial cells

https://doi.org/10.1016/S0378-1119(00)00258-4Get rights and content

Abstract

The NADPH oxidases are a group of plasma membrane-associated enzymes found in a variety of cells. They catalyze the production of superoxide (O2) by a one-electron reduction of oxygen, using NADPH as the electron donor. To characterize the expression of this enzyme, two homologues of the NADPH oxidase catalytic subunit, gp91phox, were cloned from the cDNAs of a human colon cancer cell line, Caco2, and human fetal kidney, using information relating to an expressed sequence tag (EST) from a DNA database. Amino acid identity was 58% (gp91-2) and 56% (gp91-3), respectively, against the catalytic subunit (gp91-1/gp91phox) of the NADPH oxidase found in peripheral blood leukocytes. Using the reverse transcription–polymerase chain reaction (RT–PCR) method, the messenger RNA of gp91-2 was detected mainly in the colon (and also in kidney and prostate) among human adult tissues, in the thymus among human fetal tissues, and in the cancer cell lines (HepG2 and Caco2). An expression of gp91-3 was detected in the fetal kidney, and in the cancer cell line (HepG2), but not at all in adult tissues (by the RT–PCR method). In situ hybridization revealed that gp91-2 is located in the absorptive epithelial cells of the adult colon. Neither gp91-2 nor gp91-3 was expressed in peripheral blood leukocytes.

Introduction

Erythropoietin (Epo) production is triggered in kidney and liver by hypoxia, with the cortical peritubular cells in the kidney being the main source (Koury et al., 1988). The oxygen-sensing process involved in this hypoxia-mediated hormone production has been investigated intensively at the molecular level in the hepatoma cell lines Hep3B and HepG2 (Goldberg et al., 1988). Both hypoxia and cobalt chloride markedly increase the expression of Epo mRNA as well as that of biologically active Epo in these cells. Since the increased Epo production seen in hypoxia can be inhibited by CO, the involvement of a haem protein in the oxygen-sensing mechanism has been suggested (Goldberg et al., 1988). A b-type cytochrome has been proposed as a possible candidate for the oxygen sensor, since phenobarbital, an inducer of the cytochrome p-450 2B group, has been found to enhance Epo production (Goldberg et al., 1988).

NADPH oxidase is a multicomponent enzyme complex that includes an integral membrane protein (flavocytochrome b558) and four cytosolic protein components [p47phox, p67phox, p40phox, and a small GTP-binding protein (Rac)] (Babior, 1999). Flavocytochrome b558, the redox center of the electron transport complex, is a heterodimer composed of a glycosylated 91 kDa subunit, gp91phox, and a non-glycosylated 22 kDa subunit, p22phox (Babior, 1999). A spectrophotometric study of HepG2 cells revealed that HepG2 exhibits haem signals at 559 and 427 nm, suggesting the presence of a b-type cytochrome (Gorlach et al., 1993). The same authors found that this haem possessed a 22 kDa subunit and that, like NADPH oxidase, it might be part of the electron transfer chain. The generation of superoxide by NADPH oxidase is primarily associated with the microbicidal activity of phagocytic leukocytes, such as neutrophils and macrophages. However, several studies have demonstrated that a number of non-phagocytic cell types, including HepG2 cells (Gorlach et al., 1993) and human fibroblast cells (Meier et al., 1989), generate a low level of superoxide. In HepG2 cells, p47phox and p22phox have been detected by the Western blot method (Gorlach et al., 1993). In human fibroblast cells, p47phox, p67phox, and p22phox have been detected by the reverse transcription–polymerase chain reaction (RT–PCR) method. However, gp91phox has not been detected in HepG2 cells or human fibroblast cells. To test the hypothesis that an NADPH oxidase-like system is present in non-phagocytic cells, we searched for genes with a similarity to gp91phox. In this study, we identified two homologues of gp91phox (designated gp91-2 and gp91-3) in the DNA database, cloned a cDNA of gp91-2 from Caco2 cells, and showed a specific expression of gp91-2 in epithelial cells of the colon by the in situ hybridization method.

Section snippets

Reverse transcription–polymerase chain reaction

Poly(A)+ RNAs from HepG2 cells and Caco2 cells were reverse transcribed into cDNA, using the random hexanucleotide primer method described previously (Kikuchi et al., 1996). For the detection of p22phox, p47phox, and p67phox, PCR amplification was performed by the RT–PCR method, using the primer sets of NADPH oxidase subunit genes described by Jones et al. (1994).

An evaluation of the tissue-specific expression of the members of the gp91 family was carried out by the RT–PCR method using the

Identification and cloning of gp91phox homologues

In HepG2 cells, Gorlich et al. reported that haem signals could be photometrically measured with absorbances at 559 and 427 nm, suggesting the presence of a b-type cytochrome, and that p22phox and p47phox could be detected by Western blot analysis (Gorlach et al., 1993). We confirmed the expression of p22phox and p47phox in HepG2 cells by the RT–PCR method (data not shown). However, we could not detect gp91phox mRNA by the RT–PCR method when we used the set of primers for gp91phox described by

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research-C (No. 07680570), Scientific Research-B (No. 11558068) and the Sumitomo Foundation for Environmental Research (No. 983036).

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