The Nox Family of NAD(P)H Oxidases: Host Defense and Beyond
2004; Elsevier BV; Volume: 279; Issue: 50 Linguagem: Inglês
10.1074/jbc.r400024200
ISSN1083-351X
AutoresMiklós Geiszt, Thomas L. Leto,
Tópico(s)Neonatal Health and Biochemistry
ResumoReactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; phox, phagocyte oxidase; Nox, NADPH oxidase; LPS, lipopolysaccharide; TLR, toll-like receptor; Noxo1, NADPH oxidase organizer 1; Noxa1, NADPH oxidase activator 1; EPO, erythropoietin. play diverse roles in biology, including host defense, hormone biosynthesis, fertilization, and redox signaling involved in mitogenesis, apoptosis, or oxygen sensing. Until recently, much of our understanding of the mechanisms of ROS generation and their functions in man was based on studies of phagocytic cells, where abundant microbicidal oxidants released during engulfment of microbes serve as a first line of host defense. In phagocytic cells the ROS precursor superoxide is produced by a NADPH oxidase complex (1Leto T.L. Gallin J.I. Snyderman R. Inflammation Basic Principles and Clinical Correlates. Lippincott Williams & Wilkins, Philadelphia1999: 769-787Google Scholar, 2Quinn M.T. Gauss K.A. J. Leukocyte Biol. 2004; 76: 760-781Crossref PubMed Scopus (392) Google Scholar, 3Cross A.R. Segal A.W. Biochim. Biophys. Acta. 2004; 1657: 1-22Crossref PubMed Scopus (370) Google Scholar). The phagocytic oxidase (phox) consists of a membrane-bound flavocytochrome b558, several modular cytosolic regulators (p47phox, p67phox, p40phox), and a small GTPase, Rac1 or Rac2. The cytochrome is a complex of two proteins: a flavin- and heme-binding glycoprotein (gp91phox) and a smaller subunit (p22phox). With the recent expansion of information available in human genome databases, several novel gp91phox homologues have been recognized (Fig. 1), suggesting that enzymes similar to the phagocytic oxidase function in a variety of tissues, notably the colon, kidney, thyroid gland, testis, salivary glands, airways, and lymphoid organs (4Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1284) Google Scholar, 5Geiszt M. Kopp J.B. Varnai P. Leto T.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8010-8014Crossref PubMed Scopus (715) Google Scholar, 6Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 7De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 8Shiose A. Kuroda J. Tsuruya K. Hirai M. Hirakata H. Naito S. Hattori M. Sakaki Y. Sumimoto H. J. Biol. Chem. 2001; 276: 1417-1423Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 9Banfi B. Molnar G. Maturana A. Steger K. Hegedus B. Demaurex N. Krause K.H. J. Biol. Chem. 2001; 276: 37594-37601Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). The physiological functions of these novel NADPH oxidases, now designated the Nox family, are currently under intensive investigation. Recent reviews have described in detail the molecular features of the phox system (2Quinn M.T. Gauss K.A. J. Leukocyte Biol. 2004; 76: 760-781Crossref PubMed Scopus (392) Google Scholar, 3Cross A.R. Segal A.W. Biochim. Biophys. Acta. 2004; 1657: 1-22Crossref PubMed Scopus (370) Google Scholar). This review updates our current knowledge about novel members of the Nox family. Nox1, the first recognized homologue of gp91phox, now termed Nox2, is detected in abundance in the colon and at lower levels in uterus, prostate, and vascular smooth muscle cells (4Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1284) Google Scholar). The 564-amino acid protein is 56% identical to gp91phox and contains the essential features of its phagocytic counterpart, including six transmembrane segments and several conserved motifs considered important for binding of NADPH, FAD, and two heme molecules. Two variants of the full-length isoform have been described. Bánfi et al. (10Bánfi B. Maturana A. Jaconi S. Arnaudeau S. Laforge T. Sinha B. Ligeti E. Demaurex N. Krause K.H. Science. 2000; 287: 138-142Crossref PubMed Scopus (251) Google Scholar) isolated a short mRNA product of the Nox1 gene encoding a proton-conductive channel, called NOH-1S (10Bánfi B. Maturana A. Jaconi S. Arnaudeau S. Laforge T. Sinha B. Ligeti E. Demaurex N. Krause K.H. Science. 2000; 287: 138-142Crossref PubMed Scopus (251) Google Scholar). They suggested that a unique intraexonic splicing process gives rise to the NOH-1S-coding mRNA. Recent studies showed, however, that the NOH-1S product is not transcribed from the Nox1 gene but is artifactually derived by intramolecular template switching during reverse transcription. 2Geiszt, M., Lekstrom, K., and Leto, T. (2004) 279, 51661–51668. Although RNA splicing does not account for NOH-1S synthesis, RNA splicing does generate another Nox1 transcript in normal colon and in colon carcinoma cells, where it is nearly as abundant as the full-length transcript. This transcript lacks the entire exon 11 (residues 433–482) and does not encode a functional oxidase 2Geiszt, M., Lekstrom, K., and Leto, T. (2004) 279, 51661–51668. (10Bánfi B. Maturana A. Jaconi S. Arnaudeau S. Laforge T. Sinha B. Ligeti E. Demaurex N. Krause K.H. Science. 2000; 287: 138-142Crossref PubMed Scopus (251) Google Scholar). In early work exploring the function of Nox1, heterologous overexpression of Nox1 in NIH-3T3 cells was associated with increased cell proliferation and resulted in tumor formation when these cells were injected into nude mice (4Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1284) Google Scholar). Subsequent reports proposed that hydrogen peroxide (H2O2) formation in these Nox1-expressing cells is responsible for the increased mitogenesis (11Arnold R.S. Shi J. Murad E. Whalen A.M. Sun C.Q. Polavarapu R. Parthasarathy S. Petros J.A. Lambeth J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5550-5555Crossref PubMed Scopus (423) Google Scholar). These findings were enthusiastically received because enhanced cellular ROS generation has been linked to the enhanced growth triggered by growth factor stimulation or tumorigenesis (12Sundaresan M. Yu Z.-X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2322) Google Scholar, 13Irani K. Xia Y. Zweier J. Sollott S. Der C. Rearon E. Sundaresan M. Finkel T. Goldschmidt-Clermont P. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1441) Google Scholar, 14Szatrowski T.P. Nathan C.F. Cancer Res. 1991; 51: 794-798PubMed Google Scholar). Cells overexpressing Nox1 also showed increased vascular endothelial growth factor expression, suggesting that ROS produced by Nox1 also stimulates angiogenesis in these tumors (15Arbiser J.L. Petros J. Klafter R. Govindajaran B. McLaughlin E.R. Brown L.F. Cohen C. Moses M. Kilroy S. Arnold R.S. Lambeth J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 715-720Crossref PubMed Scopus (404) Google Scholar). Further studies revealed, however, that these Nox1-transfected NIH-3T3 cell lines also carry a mutant form of Ras that can account for the enhanced proliferation and transformation (16Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2492) Google Scholar). Observations in colon-derived epithelial cells, which naturally express Nox1, do not support the proposed role of Nox1 as a mitogenic oxidase. In HT29 colon cancer cells, the suppression of Nox1 levels does not affect cell proliferation (17Geiszt M. Lekstrom K. Brenner S. Hewitt S.M. Dana R. Malech H.L. Leto T.L. J. Immunol. 2003; 171: 299-306Crossref PubMed Scopus (167) Google Scholar). Furthermore, significantly higher Nox1 expression occurs when HT29 or CaCo2 cells are induced to differentiate by interferon-γ or by calcitriol (17Geiszt M. Lekstrom K. Brenner S. Hewitt S.M. Dana R. Malech H.L. Leto T.L. J. Immunol. 2003; 171: 299-306Crossref PubMed Scopus (167) Google Scholar). Finally, a survey of various human cancerous tissues showed Nox1 expression is limited to colon tumors and the highest Nox1 expression occurs in more differentiated tumors (17Geiszt M. Lekstrom K. Brenner S. Hewitt S.M. Dana R. Malech H.L. Leto T.L. J. Immunol. 2003; 171: 299-306Crossref PubMed Scopus (167) Google Scholar). Together, these findings suggest that Nox1 serves some other specialized function in differentiated colon epithelium unrelated to mitogenesis. Several observations suggest that Nox1 could serve as a host defense oxidase. Nox1 induction, along with increased ROS release, was detected in guinea pig gastric pit cells primed with Helico-bacter pylori lipopolysaccharides (LPS) (18Kawahara T. Teshima S. Oka A. Sugiyama T. Kishi K. Rokutan K. Infect. Immun. 2001; 69: 4382-4389Crossref PubMed Scopus (161) Google Scholar). Kawahara et al. (19Kawahara T. Kuwano Y. Teshima-Kondo S. Takeya R. Sumimoto H. Kishi K. Tsunawaki S. Hirayama T. Rokutan K. J. Immunol. 2004; 172: 3051-3058Crossref PubMed Scopus (123) Google Scholar) proposed that LPS from pathogenic H. pylori strains potently stimulates ROS production through a toll-like receptor 4 (TLR4) pathway. In contrast, colon epithelial cells, which lack TLR4 receptors and do not respond to LPS, exhibit high Nox1-mediated ROS production in response to flagellin from Salmonella enteritides, acting through TLR5-dependent pathways (19Kawahara T. Kuwano Y. Teshima-Kondo S. Takeya R. Sumimoto H. Kishi K. Tsunawaki S. Hirayama T. Rokutan K. J. Immunol. 2004; 172: 3051-3058Crossref PubMed Scopus (123) Google Scholar). A role for Nox1 in innate immunity was also suggested by experiments showing that Nox1 could replace Nox2 (gp91phox) in the regulated production of superoxide, thereby partially rescuing the deficiency in superoxide production observed in chronic granulomatous disease neutrophils (17Geiszt M. Lekstrom K. Brenner S. Hewitt S.M. Dana R. Malech H.L. Leto T.L. J. Immunol. 2003; 171: 299-306Crossref PubMed Scopus (167) Google Scholar). Together, these findings demonstrate that Nox1 is functionally similar to gp91phox/Nox2. Whether Nox1 has a primary role in maintaining colon epithelial integrity or in host defense will require further investigation. Although few reports have explored the role of Nox1 in the colon, where it is most abundant, several groups have examined the possible involvement of Nox1 in ROS production in vascular tissue. Nox1 expression in cultured smooth muscle cells was described by Suh et al. (4Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1284) Google Scholar), and several other groups confirmed these findings (reviewed in Ref. 20Lassegue B. Clempus R.E. Am. J. Physiol. 2003; 285: R277-R297Crossref PubMed Scopus (21) Google Scholar). Several agonists, including platelet-derived growth factor, angiotensin II, and prostaglandin F2α, appear to up-regulate Nox1 mRNA levels in cultured smooth muscle cells (4Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1284) Google Scholar, 21Lassegue B. Sorescu D. Szocs K. Yin Q. Akers M. Zhang Y. Grant S.L. Lambeth J.D. Griendling K.K. Circ. Res. 2001; 88: 888-894Crossref PubMed Scopus (760) Google Scholar, 22Katsuyama M. Fan C. Yabe-Nishimura C. J. Biol. Chem. 2002; 277: 13438-13442Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), whereas suppression of Nox1 expression by antisense techniques inhibited superoxide production (4Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1284) Google Scholar). Thus, production of ROS by Nox1 in these cells could have roles in angiotensin II- and growth factor-induced cell hypertrophy or proliferation. Future studies on knock-out and other transgenic animals should provide better insight into the role of Nox1 in vascular tissue. Nox3, a 568-amino acid protein, also has close similarities to gp91phox/Nox2 (58% sequence identity) (23Kikuchi H. Hikage M. Miyashita H. Fukumoto M. Gene (Amst.). 2000; 254: 237-243Crossref PubMed Scopus (125) Google Scholar). Nox3 mRNA was not detected in adult or in fetal tissues by Northern blotting; however, reverse transcription-PCR experiments detected Nox3 mRNA in several fetal tissues, including kidney, liver, lung, and spleen (23Kikuchi H. Hikage M. Miyashita H. Fukumoto M. Gene (Amst.). 2000; 254: 237-243Crossref PubMed Scopus (125) Google Scholar, 24Cheng G. Cao Z. Xu X. van Meir E.G. Lambeth J.D. Gene (Amst.). 2001; 269: 131-140Crossref PubMed Scopus (707) Google Scholar). Recently, a unique role for this oxidase within the inner ear was revealed by positional cloning studies that mapped genetic lesions causing the head tilt (het) phenotype in mice (25Paffenholz R. Bergstrom R.A. Pasutto F. Wabnitz P. Munroe R.J. Jagla W. Heinzmann U. Marquardt A. Bareiss A. Laufs J. Russ A. Stumm G. Schimenti J.C. Bergstrom D.E. Genes Dev. 2004; 18: 486-491Crossref PubMed Scopus (214) Google Scholar). Mice with Nox3 mutations exhibit impaired otoconial morphogenesis and defects in perception of gravity and balance. It was proposed that this oxidase mediates ROS-dependent conformation changes in otoconin 90 involved in the nucleation of calcite crystal formation during the development of otoconia. Nox4, a 578-amino acid protein with 39% sequence identity to gp91phox/Nox2 was originally described as a renal oxidase (Renox) because its expression at high levels is limited to the kidney (5Geiszt M. Kopp J.B. Varnai P. Leto T.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8010-8014Crossref PubMed Scopus (715) Google Scholar, 8Shiose A. Kuroda J. Tsuruya K. Hirai M. Hirakata H. Naito S. Hattori M. Sakaki Y. Sumimoto H. J. Biol. Chem. 2001; 276: 1417-1423Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). However, the specific intrarenal distribution of Nox4 mRNA differs significantly in mice and humans. In situ hybridization in mouse kidney sections demonstrated the highest levels in proximal tubules (5Geiszt M. Kopp J.B. Varnai P. Leto T.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8010-8014Crossref PubMed Scopus (715) Google Scholar), whereas immunohistochemical and in situ hybridization studies on human kidneys detected Nox4 expression in distal portions of the nephron 3M. Geiszt and T. Leto, unpublished observations. (8Shiose A. Kuroda J. Tsuruya K. Hirai M. Hirakata H. Naito S. Hattori M. Sakaki Y. Sumimoto H. J. Biol. Chem. 2001; 276: 1417-1423Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). When overexpressed in NIH-3T3 fibroblasts, Nox4 increased superoxide production and induced a cellular senescence phenotype (5Geiszt M. Kopp J.B. Varnai P. Leto T.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8010-8014Crossref PubMed Scopus (715) Google Scholar, 8Shiose A. Kuroda J. Tsuruya K. Hirai M. Hirakata H. Naito S. Hattori M. Sakaki Y. Sumimoto H. J. Biol. Chem. 2001; 276: 1417-1423Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Expression of antisense Nox4 mRNA in HEK293 cells, which contain endogenous Nox4, resulted in a decreased NADH- and NADPH-dependent superoxide production in vitro (8Shiose A. Kuroda J. Tsuruya K. Hirai M. Hirakata H. Naito S. Hattori M. Sakaki Y. Sumimoto H. J. Biol. Chem. 2001; 276: 1417-1423Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). The expression pattern of Nox4 is consistent with several renal-specific functions. One proposed role in oxygen sensing and regulation of erythropoietin (EPO) synthesis (5Geiszt M. Kopp J.B. Varnai P. Leto T.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8010-8014Crossref PubMed Scopus (715) Google Scholar, 8Shiose A. Kuroda J. Tsuruya K. Hirai M. Hirakata H. Naito S. Hattori M. Sakaki Y. Sumimoto H. J. Biol. Chem. 2001; 276: 1417-1423Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar) is based on several findings. The phagocyte cell homologue of Nox4, gp91phox/Nox2, was described as an oxygen sensor in the lung (26Wang D. Youngson C. Wong V. Yeger H. Dinauer M.C. Vega-Saenz Miera E. Rudy B. Cutz E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13182-13187Crossref PubMed Scopus (158) Google Scholar). In the kidney, EPO synthesis also occurs in proximal tubules (27Loya F. Yang Y. Lin H. Goldwasser E. Albitar M. Blood. 1994; 84: 1831-1836Crossref PubMed Google Scholar), and ROS are implicated as negative feedback signals regulating EPO synthesis (28Elbert B.L. Bunn H.F. Blood. 1999; 94: 1864-1877Crossref PubMed Google Scholar). Although the hypoxia-inducible factor-1α, which regulates EPO synthesis, is regulated by proline hydroxylases (29Bruick R.K. Genes Dev. 2003; 17: 2614-2623Crossref PubMed Scopus (375) Google Scholar), other hypoxia-inducible factor-1α-independent transcription factors, such as GATA-2, are direct H2O2-sensitive targets that suppress EPO expression (30Imagawa S. Yamamoto M. Miura T. Blood. 1997; 89: 1430-1439Crossref PubMed Google Scholar). Finally, recent knock-out studies indicate that superoxide dismutase 3, which is also expressed in renal proximal tubules, has a role in erythroid responses to hypoxia (31Suliman H.B. Ali M. Piantadosi C.A. Blood. 2004; 104: 43-50Crossref PubMed Scopus (63) Google Scholar). These observations along with the high constitutive activity of Nox4 (5Geiszt M. Kopp J.B. Varnai P. Leto T.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8010-8014Crossref PubMed Scopus (715) Google Scholar, 8Shiose A. Kuroda J. Tsuruya K. Hirai M. Hirakata H. Naito S. Hattori M. Sakaki Y. Sumimoto H. J. Biol. Chem. 2001; 276: 1417-1423Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar) are consistent with a role for Nox4 in oxygen sensing. A distinct possibility related to high renal Nox4 expression is that it serves as a phox-like antimicrobial system by releasing ROS into the glomerular filtrate; this might explain significant (i.e. 100 μm) urinary hydrogen peroxide levels (32Varma S.D. Devamanoharan P.S. Free Radic. Res. 1990; 8: 73-78Crossref Scopus (56) Google Scholar). Other possible functions of the renal oxidase include roles in oxidation or detoxification of urine wastes or in renal pH or electrolyte homeostasis, based on the proton-generating and electrogenic activities of all NADPH oxidases. Finally, Gorin et al. (33Gorin Y. Ricono J.M. Wagner B. Kim N.H. Bhandari B. Ghosh Choudhury G. Abboud H.E. Biochem. J. 2004; 381: 231-239Crossref PubMed Scopus (109) Google Scholar) suggested that a Nox4-based oxidase is stimulated in response to angiotensin II in mesangial cells. Several groups have also explored possible extra-renal functions of Nox4. According to Yang et al. (34Yang S. Madyastha P. Bingel S. Ries W. Key L. J. Biol. Chem. 2001; 276: 5452-5458Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), Nox4 and gp91phox (Nox2) are present in murine osteoclasts, where they may provide an oxidative basis for bone resorption. Low Nox4 expression is detected in vascular smooth muscle cells, where it appears to be down-regulated by agonists that increase Nox1 expression (21Lassegue B. Sorescu D. Szocs K. Yin Q. Akers M. Zhang Y. Grant S.L. Lambeth J.D. Griendling K.K. Circ. Res. 2001; 88: 888-894Crossref PubMed Scopus (760) Google Scholar). Although the relative contribution of Nox4 to vascular ROS production remains unclear, Nox4 was also suggested as the major component of an endothelial NAD(P)H oxidase (35Ago T. Kitazono T. Ooboshi H. Iyama T. Han Y.H. Takada J. Wakisaka M. Ibayashi S. Utsumi H. Iida M. Circulation. 2004; 109: 227-233Crossref PubMed Scopus (436) Google Scholar). In several cell lines, growth factor or insulin receptor stimulation increases ROS production, which has an enhancing effect on tyrosine phosphorylation in part through inhibition of protein-tyrosine phosphatases (36Rhee S.G. Chang T.S. Bae Y.S. Lee S.R. Kang S.W. J. Am. Soc. Nephrol. 2003; 14: S211-S215Crossref PubMed Google Scholar). A recent study indicates that Nox4 mediates insulin-stimulated ROS production in 3T3-L1 adipocytes (37Mahadev K. Motoshima H. Wu X. Ruddy J.M. Arnold R.S. Cheng G. Lambeth J.D. Goldstein B.J. Mol. Cell. Biol. 2004; 24: 1844-1854Crossref PubMed Scopus (444) Google Scholar). Further studies in gene-targeted animals may confirm roles for Nox4 in insulin signaling. NADPH oxidase 5 (Nox5) is more distantly related to the other Nox proteins, with an overall homology to gp91phox of 27% (9Banfi B. Molnar G. Maturana A. Steger K. Hegedus B. Demaurex N. Krause K.H. J. Biol. Chem. 2001; 276: 37594-37601Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). The protein consists of 737 amino acids and contains an additional N-terminal extension comprising four calcium-binding, EF-hand motifs that appear to render the enzyme directly responsive to calcium. Superoxide production by Nox5-expressing cells is induced by the calcium ionophore ionomycin (9Banfi B. Molnar G. Maturana A. Steger K. Hegedus B. Demaurex N. Krause K.H. J. Biol. Chem. 2001; 276: 37594-37601Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Recent work demonstrated that the EF-hands engage in direct, calcium-dependent interactions with the C-terminal domain of Nox5 (38Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). In testis, Nox5 mRNA was detected in pachytene spermatocytes (9Banfi B. Molnar G. Maturana A. Steger K. Hegedus B. Demaurex N. Krause K.H. J. Biol. Chem. 2001; 276: 37594-37601Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar), raising the possibility that it may function in mature spermatozoa, such as in oxidative changes associated with human sperm capacitation or the acrosome reaction (39Baker M.A. Aitken R.J. Mol. Cell. Endocrinol. 2004; 216: 47-54Crossref PubMed Scopus (142) Google Scholar). In the spleen, Nox5 message localizes to the mantle zone surrounding germinal centers (areas rich in B cells) and to periarterial lymphoid sheets (where mostly T cells are present) (9Banfi B. Molnar G. Maturana A. Steger K. Hegedus B. Demaurex N. Krause K.H. J. Biol. Chem. 2001; 276: 37594-37601Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). T and B lymphocytes produce ROS when stimulated by some receptor agonists (40Devadas S. Zaritskaya L. Rhee S.G. Oberley L. Williams M.S. J. Exp. Med. 2002; 195: 59-70Crossref PubMed Scopus (372) Google Scholar, 41Lee J.R. Koretzky G.A. Eur. J. Immunol. 1998; 28: 4188-4197Crossref PubMed Scopus (74) Google Scholar). A role for Nox5 in cell proliferation was suggested by Brar et al. (42Brar S.S. Corbin Z. Kennedy T.P. Hemendinger R. Thornton L. Bommarius B. Arnold R.S. Whorton A.R. Sturrock A.B. Huecksteadt T.P. Quinn M.T. Krenitsky K. Ardie K.G. Lambeth J.D. Hoidal J.R. Am. J. Physiol. 2003; 285: C353-C369Crossref PubMed Scopus (226) Google Scholar), who demonstrated that antisense oligonucleotide-mediated down-regulation of Nox5 expression in DU 145 prostate cancer cells inhibits cell proliferation. Dual oxidases, originally designated thyroid oxidases (thOX or tox), were cloned from human and porcine thyroid glands and proposed to serve in iodide organification during thyroxine synthesis (6Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 7De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). The Duox (dual oxidase) nomenclature was suggested (43Edens W.A. Sharling L. Cheng G. Shapira R. Kinkade J.M. Lee T. Edens H.A. Tang X. Sullards C. Flaherty D.B. Benian G.M. Lambeth J.D. J. Cell Biol. 2001; 154: 879-891Crossref PubMed Scopus (319) Google Scholar) as these proteins contain an N-terminal extracellular peroxidase-like domain and a gp91phox-like oxidase portion (6Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 7De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 43Edens W.A. Sharling L. Cheng G. Shapira R. Kinkade J.M. Lee T. Edens H.A. Tang X. Sullards C. Flaherty D.B. Benian G.M. Lambeth J.D. J. Cell Biol. 2001; 154: 879-891Crossref PubMed Scopus (319) Google Scholar). Human Duox1 and Duox2 proteins contain 1551 and 1548 amino acids, respectively, and show 83% sequence similarity. Separating the peroxidase-like domain and the NADPH oxidase portion are an additional transmembrane segment and two EF-hand motifs (Fig. 1). The peroxidase-like domains of Duox proteins are unusual in that they lack conserved histidine residues found in all other peroxidases, considered essential for heme binding (44Daiyasu H. Toh H. J. Mol. Evol. 2000; 51: 433-445Crossref PubMed Scopus (77) Google Scholar). The presence of EF-hands suggests that calcium directly regulates these enzymes, consistent with early observations showing that calcium ionophores stimulate H2O2 production in thyroid cells (45Dupuy C. Deme D. Kaniewski J. Pommier J. Virion A. FEBS Lett. 233. 1988; : 74-78Crossref PubMed Scopus (31) Google Scholar). Heterologous Duox expression in several mammalian cell lines fails to reconstitute ROS release, suggesting other tissue-specific oxidase components are needed for Duox activity (46De Deken X. Wangm D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar). In thyroid follicles, the Duox proteins are detected on the thyrocyte apical surface, where they could supply H2O2 for thyroperoxidase-mediated iodination and cross-linking of thyroglobulin tyrosine residues. Duox2 is clearly essential for thyroxine biosynthesis because Duox2 mutations result in congenital hypothyroidism, even in heterozygotes (47Moreno J.C. Bikker H. Kempers M.J. van Trotsenburg A.S. Baas F. de Vijlder J.J. Vulsma T. Ris-Stalpers C. N. Engl. J. Med. 2002; 347: 95-102Crossref PubMed Scopus (398) Google Scholar). The role of Duox1 in thyroid tissue has not been clarified. Duox proteins are also encoded by the genomes of lower species, such as Caenorhabditis elegans (43Edens W.A. Sharling L. Cheng G. Shapira R. Kinkade J.M. Lee T. Edens H.A. Tang X. Sullards C. Flaherty D.B. Benian G.M. Lambeth J.D. J. Cell Biol. 2001; 154: 879-891Crossref PubMed Scopus (319) Google Scholar). Defective cuticle formation was observed during C. elegans development following inhibition of Duox expression by RNA interference (43Edens W.A. Sharling L. Cheng G. Shapira R. Kinkade J.M. Lee T. Edens H.A. Tang X. Sullards C. Flaherty D.B. Benian G.M. Lambeth J.D. J. Cell Biol. 2001; 154: 879-891Crossref PubMed Scopus (319) Google Scholar). It was proposed that Duox provides the ROS for oxidative cross-linking of extracellular matrix tyrosine residues in a reaction thought to involve the extracellular peroxidase-like domain. The isolated C. elegans peroxidase-like domain produced in Escherichia coli appears to catalyze cross-linking of tyrosine residues in vitro (43Edens W.A. Sharling L. Cheng G. Shapira R. Kinkade J.M. Lee T. Edens H.A. Tang X. Sullards C. Flaherty D.B. Benian G.M. Lambeth J.D. J. Cell Biol. 2001; 154: 879-891Crossref PubMed Scopus (319) Google Scholar). Novel roles for Duox enzymes in host defense were recently proposed in several non-thyroid tissues (48Geiszt M. Witta J. Baffi J. Lekstrom K. Leto T.L. FASEB J. 2003; 17: 1502-1504Crossref PubMed Scopus (419) Google Scholar). Based on the high levels of Duox in epithelial cells of salivary gland ducts and along mucosal surfaces of colon, rectum, and major airways, these enzymes were proposed to serve as sources of H2O2 supporting the anti-microbial activity of lactoperoxidase. Lactoperoxidase, abundant in milk, saliva, tears, and mucosal secretions, uses H2O2 to oxidize thiocyanate to hypothiocyanite, an oxidant effective against both Gram-negative and Gram-positive bacterial species (49Reiter B.A. Oram J.D. Nature. 1967; 216: 328-330Crossref PubMed Scopus (119) Google Scholar). Thus, the dual oxidases may represent "missing links" that complete an oxidant-dependant microbicidal system that has been long recognized in many body fluids. In phagocytic cells, gp91phox is the core catalytic component of a multi-component enzyme complex (1Leto T.L. Gallin J.I. Snyderman R. Inflammation Basic Principles and Clinical Correlates. Lippincott Williams & Wilkins, Philadelphia1999: 769-787Google Scholar, 2Quinn M.T. Gauss K.A. J. Leukocyte Biol. 2004; 76: 760-781Crossref PubMed Scopus (392) Google Scholar, 3Cross A.R. Segal A.W. Biochim. Biophys. Acta. 2004; 1657: 1-22Crossref PubMed Scopus (370) Google Scholar). However, p22phox, p47phox, and p67phox are also essential for superoxide production, as is evident in chronic granulomatous disease, where defects or the absence of any one of these components result in oxidase deficiencies. p47phox, a classic adaptor protein, recruits p67phox into the complex, whereas p67phox and Rac appear to regulate catalysis directly (50Han C.H. Freeman J.L. Lee T. Motalebi S.A. Lambeth J.D. J. Biol. Chem. 1998; 273: 16663-16668Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 51Bokoch G.M. Knaus U.G. Trends Biochem. Sci. 2003; 28: 502-508Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). An essential role for Rac in oxidase activation was
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