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Originally published In Press as doi:10.1074/jbc.M103034200 on August 1, 2001

J. Biol. Chem., Vol. 276, Issue 40, 37594-37601, October 5, 2001
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A Ca2+-activated NADPH Oxidase in Testis, Spleen, and Lymph Nodes*

Botond BánfiDagger §, Gergely Molnár§, Andres Maturana||, Klaus Steger**, Balázs HegedûsDagger Dagger , Nicolas Demaurex§§, and Karl-Heinz KrauseDagger ¶¶

From the Dagger  Biology of Aging Laboratory, Department of Geriatrics, Geneva University Hospitals, Ch. du Petit-Bel-Air 2, CH-1225 Geneva, Switzerland, the  Department of Physiology and the || Foundation for Medical Research, Geneva Medical School, Rue de Michel-Servet 1, CH-1211 Geneva, Switzerland, the § Department of Physiology, Semmelweis University, Puskin utca 9, H-1444 Budapest 8, Hungary, the ** Institute of Veterinary Anatomy, Frankfurter Strasse 98, D-35392 Giessen, Germany, and the Dagger Dagger  Department of Biological Physics, Eotvos University, Pazmany Peter Setany 1A, H-1117 Budapest, Hungary

Received for publication, April 5, 2001, and in revised form, July 17, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91phox subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca2+ concentration it generated large amounts of superoxide. Upon Ca2+ activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H+ channel in a Ca2+-dependent manner. NOX5 is likely to be involved in Ca2+-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Over the last 10 years, our concept of the biological role of reactive oxygen species (ROS)1 has remarkably evolved. In addition to their long known toxic effects, which are involved in the killing of microorganisms, aging, and cancerogenesis, ROS are now recognized as physiologically relevant signaling molecules. They have been shown to regulate a large number of biological processes including gene expression (1), kinase activation (2), oxygen sensing (3), regulation of vascular diameter (4), bone resorption (5), cell growth (6), and even mediation of sperm-oocyte fusion (7).

Superoxide generation by phagocytes plays an important role in the killing of microorganisms during host defense. It is catalyzed by the phagocyte NADPH oxidase, an enzyme complex that has been known and studied for a long time (8-10). Upon assembly, the NADPH oxidase generates electron currents (11) that flow from intracellular NADPH to extracellular (or phagosomal) oxygen resulting in superoxide generation. The main subunit of the phagocyte NADPH oxidase, gp91phox forms a transmembrane heterodimer with p22phox and functions as an electron transport chain containing four NADPH binding regions, an FAD binding site, and two heme groups anchored by four histidines (12, 13). However, association of cytosolic proteins, including p47phox, p67phox, and Rac2, with the transmembrane subunits is crucial for the activation of the electron flow (9).

Over the last several years it has become apparent that nonphagocytic cells (e.g. vascular smooth muscle (4), endothelial (4), mesangial (14), thyroid (15), fibroblasts (16), lymphocytes (17), (18), and spermatozoa (19)) also generate superoxide. The subsequent search for nonphagocyte NADPH oxidases led to the discovery of two families of gp91phox homologues. The NADPH oxidase (NOX) family members have approximately the same length as gp91phox (i.e. ~560-580 amino acids), while the dual oxidase (DUOX) family members are markedly longer (~1550 amino acids) because of their N-terminal extensions consisting of two EF hand (i.e. presumed Ca2+ binding) motifs, an additional transmembrane helix, and a peroxidase homology domain (20). The NOX family includes NOX1 (initially referred to as Mox1 or NOH-1, Refs. 21 and 22), which is predominantly expressed in colon, NOX3 (also referred to as gp91-3, Ref. 23) cloned from fetal kidney, and NOX4 found in kidney cortex (also termed Renox, Refs. 24 and 25) and in osteoclasts (26). The DUOX family includes DUOX1 (initially termed Thox1, Refs. 27 and 28) expressed in the thyroid gland and DUOX2 (also designated as Thox2, Ref. 29), which is found in thyroid, small intestine, and colon. NOX1 and NOX4 have been shown to produce low amounts of superoxide in a constitutively active manner (21, 24, 25). Hitherto no functional evidence for superoxide generation by NOX3, DUOX1, and DUOX2 has been obtained. The novel gp91phox homologues have been proposed to regulate cell growth (NOX1), participate in host defense (NOX1), mediate in oxygen sensing (NOX4), or play a role in thyroid hormone synthesis (DUOX1 and -2); however, so far no conclusive evidence concerning these proposals exists.

Activation of DUOX enzymes by elevations of the cytosolic free Ca2+ concentration, [Ca2+]c, has been suggested based on their EF hand motifs and the previously described Ca2+-activated superoxide generation in thyroid cells (30). But hitherto no direct experimental evidence exists. [Ca2+]c elevations do not activate NOX4 (24), and there is no evidence that they would activate NOX1 or NOX3. gp91phox can be activated by Ca2+, although most likely not because of a direct effect of Ca2+ on gp91phox but rather because of the activation of Ca2+-sensitive second messenger systems (31-33) and Ca2+-dependent exocytosis of gp91phox-containing granules.

Electron export by an NADPH oxidase has important electrophysiological consequences: loss of negative charges and accumulation of H+ ions lead to plasma membrane depolarization (34, 35) and to cytosolic acidification (36). There is now growing evidence that, in addition to their electron transport function, gp91phox and its homologues may also function as H+ channels, allowing charge and pH compensation (35, 37). Moreover, there is an alternatively spliced short form of NOX1, which is devoid of elements of the electron transport chain. For this protein, H+ conduction may even be the only physiological role (22).

Here we describe a distant relative of the members of NOX and DUOX families, NADPH oxidase 5 (NOX5). NOX5, which is found in testis and lymphoid organs, contains an N-terminal extension with three EF hands and is able to generate superoxide and to conduct H+ ions in response to cytosolic free [Ca2+] elevations.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of Human NOX5-encoding cDNA-- BLAST nucleotide searches were conducted in the High Throughput Genomic Sequences data base of GenBankTM with the C terminus of human gp91phox. The exons of the found gene, including the one containing the stop codon, were predicted either based on their homology with gp91phox or with the GENSCAN software (genes.mit.edu/oldGENSCAN.html). Primers were designed from the first predicted exon and from the exon encoding the predicted stop codon (5'-GCT CCG GTG GGT GAC TCA GCA GTT T-3' and 5'-ATG GTG TGG ACT TGG GGC AGA GCT T-3'), and PCR was performed on several human cDNAs prepared from different organs (Multiple Tissue cDNA Panel II, CLONTECH, Basel, Switzerland) with Taq polymerase using the "Q solution" (Qiagen, Basel, Switzerland) to help the melting of the GC-rich regions. The 5'-ends of the cDNAs were obtained by 5'-RACE PCR with the outer primer 5'-CTC CTG GAG GGT GAT GGT GCC ACT T-3' and then with the nested primer 5'-GGT GAT GGT GCC ACT TCT ATC GGA G-3' using the SMART RACE cDNA Amplification kit (CLONTECH), but random primers were applied instead of oligo(dT) for the reverse transcription of spleen and testis mRNAs.

Northern Blot Analysis-- Human Multiple Tissue Northern Blots (CLONTECH) were hybridized with a 32P-labeled NOX5beta cDNA fragment corresponding to amino acids 460-625. The hybridization was performed under high stringency conditions using the ExpressHyb hybridization solution (CLONTECH).

In Situ Hybridization-- For in situ hybridization experiments the in vitro transcription of digoxigenin-labeled cRNA was performed using the RNA-DIG Labeling Mix (Roche Molecular Biochemicals) and RNA polymerases T7 and SP6. Prior to cRNA synthesis, the vector containing the NOX5beta insert had been digested with ApaI or PstI (New England Biolabs, Frankfurt, Germany) for the production of sense cRNA or antisense cRNA. In situ hybridization was performed as reported previously (38). Briefly, 7-µm sections from Bouin-fixed and paraffin-embedded tissue samples of human testis, spleen, and lymph node were mounted on slides coated with 2% aminopropyltriethoxysilane (Sigma). Deparaffinized and rehydrated tissue sections were digested with proteinase K (20 µg/ml of 1× phosphate-buffered saline) for 30 min at 37 °C and prehybridized in 20% glycerol for 30 min. Sections were then incubated with the digoxigenin-labeled sense and antisense cRNA probes at a dilution of 1:100 in hybridization buffer containing 50% deionized formamide, 10% dextran sulfate, 2× SSC (0.3 M NaCl, 0.03 M trisodium citrate), 1× Denhardt's solution, 10 µg/ml salmon sperm DNA, and 10 µg/ml yeast tRNA. Hybridization was performed overnight at 37 °C in a humid chamber containing 50% formamide in 2× SSC. Posthybridization washes were performed as reported previously (39). Tissue samples were incubated overnight at 4 °C with an anti-digoxigenin Fab antibody conjugated to alkaline phosphatase (Roche Molecular Biochemicals). Staining was visualized with NBT/5-bromo-4-chloro-3-indolyl phosphate (KPL, Gaithersburg, MD) in a humid chamber protected from light. Finally, sections were mounted in glycerol gel and examined under bright-field microscopy.

Cell Culture and Transfection-- COS-7, HEK293, and HeLa cells were maintained in Dulbecco's modified Eagle's medium, and HL-60 and PLB-985 cells were maintained in RPMI 1640 medium. Both culture media were supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and 4 mmol/liter L-glutamine. HL-60 and PLB-985 cells were differentiated for 5 days with 1.2% Me2SO and 0.5% dimethylformamide, respectively. Human neutrophils were purified as described previously (40). For transfection, the coding regions of human NOX5beta cDNA and mouse NOX4 cDNA (both with an inserted Kozak sequence) were subcloned into pcDNA3.1 (Invitrogen, Groningen, Netherlands). COS-7, HEK293, and HeLa cells were transfected with those constructs using the Effectene transfection system (Qiagen). To obtain stable clones, NOX5beta -transfected HEK293 cells were selected with 400 µg/ml G418 starting on the 2nd day after the transfection, and 13 surviving colonies were isolated 10 days after the transfection.

RT-PCR Amplification of NOX5-- Total RNA of the cells were prepared with Trizol reagent (Life Technologies, Inc.). The RT-PCR for NOX5 was performed with GeneAmp (PerkinElmer Life Sciences) using the following primers: 5'-CAC TAT AGA CCT GGT GAC TA-3' and 5'-CAT GCT CAG AGG CAA AGA T-3'. Either the cDNAs for the PCRs were purchased from CLONTECH (Human Blood Fractions Panel) or total RNA was reverse transcribed with the Superscript reverse transcriptase (Life Technologies, Inc.). NOX5 was amplified with the following primers: 5'-ATG AGT GGC ACC CCT TCA CCA TCA G-3' and 5'-GTC AGC AGG CTC ACA AAC CAC TCG AA-3'.

Measurement of Generation of Reactive Oxygen Species-- ROS generation was measured by the peroxidase-dependent luminol-amplified chemiluminescence technique on a Luminometer Wallac 1420 Multilabel Counter (PerkinElmer Life Sciences). Cells were plated 18 h before the measurement in 96-well plates. Measurements were performed in Hanks' balanced salt solution (containing 1.26 mM Ca2+) supplemented with 6 units/ml horseradish peroxidase and 250 µM luminol. To elevate intracellular Ca2+ concentration 0.1-3 µM ionomycin (Sigma) was used. Chemiluminescence was measured once per minute at 37 °C. After the measurements cells were counted, and the results were normalized to 20,000 cells.

Extracellular superoxide production was measured in 96-well microplates at 550 nm as the SOD-sensitive reduction of 100 µM ferricytochrome C. The O&cjs1138;2 production was calculated using an absorption coefficient of 21.1 mM-1 cm-1 and normalized to 1 min and 106 cells (40).

To measure both intra- and extracellular superoxide generation a quantitative NBT test was used (41). The cells were plated on 48-well plates using 200,000 polymorphonuclear granulocytes or HL-60 cells/well or 450,000 HEK293 cells/well and incubated in Hanks' balanced salt solution containing 0.5 mg/ml NBT with or without stimuli (100 nM phorbol 12-myristate 13-acetate or 1 µM ionomycin) and with or without 800 units/ml SOD. After 12 min the cells were fixed and washed with methanol to remove the nonreduced NBT. The reduced formazan was then dissolved in 230 µl of 2 M potassium hydroxide and in 280 µl of dimethyl sulfoxide, and the absorption was measured at 630 nm. The absorption of dissolved formazan reduced by the stimulated cells was regarded as 100%, while the absorption of dissolved formazan reduced by the resting cells was regarded as 0%.

Patch-clamp Recordings-- Whole-cell patch-clamp recordings were performed as described previously (22) using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA) in the voltage clamp mode. Patch pipettes were pulled from borosilicate glass with a P-87 Brown-Flaming Micropipette Puller (Sutter Instrument Company, Novato, CA). Pipette resistance ranged between 3 and 7 megaohms, seal resistance ranged between 2 and 20 gigaohms, and the mean access resistance varied between 10 and 30 megaohms. Cells were voltage-clamped at a holding potential of -60 mV and depolarized to various test potentials as indicated. The currents were filtered at 1 kHz and sampled at 200 Hz using the pClamp 6.0 software. Leak currents were small compared with the whole-cell H+ currents and were subtracted only to allow calculation of the whole-cell conductance. The bath solution contained (in mM): CsCl 75, HEPES 100, CsOH 50, MgCl2 1, EGTA 0.2, HEDTA 0.5, pH = 7.5; the pipette solution contained (in mM): CsCl 35, PIPES 100, CsOH 132, MgCl2 1, EGTA 0.2, HEDTA 0.5, and 2 mM ATP, pH = 6.5, free [Ca2+] <10 nM. The high Ca2+ solutions contained 0.55 and 0.65 mM CaCl2 in the bath and pipette, respectively, to achieve a free [Ca2+] of 75 µM. For H+ selectivity experiments, the pipette pH was buffered to pH = 5.7 with 100 mM MES or pH = 7.5 with 100 mM HEPES. Recordings were performed 15 min after achieving the whole-cell configuration to allow the equilibration of the cytosol with the pipette solution.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NADPH Oxidase Homologues in Testis and Spleen-- A search of the High Throughput Genomic Sequences data base (www.ncbi.nlm.nih.gov/BLAST/) with the gp91phox sequence yielded a human gene (GenBankTM accession number AC026512) encoding possible exons of a novel NADPH oxidase. From the predicted exons, we designed PCR primers and screened cDNAs of several tissues by PCR. Prominent bands around 2 kb were obtained in spleen and testis. Sequencing of the PCR products demonstrated the expected homology with gp91phox and an in-frame stop codon. A 495-base pair fragment of the PCR product was then used as a probe for Northern blot analysis of tissue distribution of the novel NADPH oxidase. The Northern blot experiment showed an ~2.7-kb transcript in spleen and an ~2.9-kb mRNA in testis (Fig. 1A). Additional Northern blot experiments (not shown) did not detect the mRNA of the novel NADPH oxidase in the following tissues: brain, heart, skeletal muscle, kidney, liver, placenta, and lung. The Northern blots yielded the following information. (i) No hybridization was detected in peripheral blood leukocytes, colon (Fig. 1A), and kidney (data not shown), suggesting a restricted tissue distribution and no cross-hybridization of the probe with gp91phox, NOX1, or NOX4 mRNAs. (ii) The length of the transcripts was longer than the mRNAs of previously described NOX family members (~2.4-2.5 kb) but shorter than the transcripts of DUOX family members (~5.7-6.4 kb) (27). (iii) The length of the transcripts of the novel NADPH oxidase was different in testis and in spleen.


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  		Fig. 1.   Tissue distribution and 5'-RACE PCR of a novel gp91phox homologue. A, Northern blot analysis of various human tissue RNAs with a radiolabeled cDNA probe (495 nucleotides) revealed transcripts in spleen and testis with slightly different length. The probe was derived from an initial PCR fragment (2 kb) amplified with primers designed from the exons of the gp91phox homologue gene. B, PCR detection of cDNAs encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (upper panel) and NOX5 (lower panel) from different cell types and tissues. C, the different 5'-ends of spleen and testis transcripts of the novel gp91phox homologue were obtained by 5'-RACE PCR. Primers for the 5'-RACE PCR were designed from the 5'-end of the 2-kb PCR fragment. HUVEC, human umbilical vein endothelial cells; bp, base pairs.

The mRNA of the novel NADPH oxidase was present in spleen but was undetectable by Northern blot in peripheral blood leukocytes. To verify this result, we applied the more sensitive PCR technique to detect possible transcripts in different subsets of lymphocytes derived from blood and in myeloid cell lines. However, even after 35 PCR cycles no transcripts could be detected in circulating blood lymphocytes or in two myeloid cell lines, HL-60 and PLB-985 (Fig. 1B). PCR amplification, however, detected transcripts in testis, uterus, bone marrow, and vascular smooth muscle (Fig. 1B).

RACE PCR Amplification of 5'-Ends-- To obtain the start codon of the novel gp91phox homologue, primers were designed from the 5'-end of the 2-kb-long initial PCR product. The 5'-RACE PCR performed with those primers demonstrated a 220-base pair difference of the 5'-ends of the testis and spleen transcripts (Fig. 1B). Thus, the difference observed with the 5'-RACE PCR was in accordance with the length difference of the testis and spleen mRNAs detected by Northern blot.

Predicted Amino Acid Sequence of the Novel NADPH Oxidase (NOX5)-- To amplify the coding region of the detected mRNAs we designed primers from the sequence located in the 5' direction from the start codon (identified by the 5'-RACE PCR) and the 3' direction from the stop codon (identified by the initial PCR). The PCR products amplified with those primers demonstrated that, with the exception of the 5'-regions, the cDNA sequences of the spleen and the testis isoforms were identical, strongly suggesting that both transcripts were the products of the same gene. The 220-base pair difference at the 5'-end of the RNAs was mainly due to different 5'-untranslated regions. In addition, however, the sequence analysis predicts that the shorter RNA of spleen encodes a protein 18 amino acids longer than the protein encoded by the longer mRNA of testis (Fig. 2A). The comparison of the amino acid sequences with gp91phox revealed that the regions crucial for NADPH oxidation and electron transport (NADPH and FAD binding sequences, presumed heme-anchoring histidines) and the all-over structure of the protein (predicted with TMpred at www.ch.embnet.org/software/TMPRED_form.html) were conserved (Fig. 2, A and B). However, the sequence identity with gp91phox was relatively low (27%, as compared with 56% for NOX1, and 37% for NOX4), and unlike all the other NOX enzymes the novel NADPH oxidase showed an extended N terminus containing three EF hand (e.g. presumed Ca2+ binding) motifs. Two EF hand motifs were also found in DUOX1 and DUOX2 but hitherto not in any of the NOX family members. In contrast to the DUOX enzymes, however, the novel enzyme does not have a peroxidase homology domain. Thus, we will refer to the enzyme as NOX5. The spleen and testis isoforms will be referred to as NOX5alpha and NOX5beta , respectively (GenBankTM accession numbers AF353088 and AF325189). Further sequencing revealed two additional splice variants referred to as NOX5gamma and NOX5delta (GenBankTM accession numbers AF353089 and AF325190, respectively). Note also that there are presently five additional GenBankTM entries of NOX5-related sequences: (i) two human cDNA sequences that do not show the N-terminal EF hand regions (accession numbers AAG33638 as NOX5 (42) and AK026011 as an unnamed protein), (ii) two short porcine expressed sequence tag clones (accession numbers BF444388 and AW416159), and (iii) a Drosophila melanogaster protein sequence predicted from the gene (accession number AE003807) that shows a high degree of similarity with human NOX5, including the presence of the N-terminal EF hands.


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  		Fig. 2.   The gene and gene products of the gp91phox homologue NOX5. A, comparison of the deduced amino acid sequence of human NOX5alpha (spleen, GenBankTM accession number AF353088) and NOX5beta (testis, GenBankTM accession number AF325189) with human gp91phox. Both NOX5 isoforms contain N-terminal regions that are distinct from gp91phox and contain three predicted EF hand motifs (EF1-3). The amino acids of the six putative transmembrane regions (I-VI) are in bold. The asterisks label the presumed heme-anchoring histidines; the gray and open boxes indicate the FAD and NADPH binding sites, respectively. B, model of transmembrane topology and functional domains of NOX5: EF hands, transmembrane helices, conserved histidines, heme groups, FAD and NADPH binding sites are indicated. C, genomic organization of the NOX5 gene. Center scheme (NOX5 gene), exons and introns are shown as vertical and horizontal bars, respectively. 19 exons could be identified. Upper and lower schemes, exon usage by NOX5alpha (exons 3-19) and NOX5beta (exons 1, 2, and 4-19). Boxes represent exons. Boxed numbers indicate the number of nucleotides of the given exon. Gray shading indicates 5'-untranslated regions.

Genomic Organization of NOX5-- Based on the genomic sequence found in the GenBankTM data base we analyzed the genomic organization of NOX5. The NOX5 gene has a size of at least 30 kb (the genome fragment encoding the NOX5 gene contains gaps in its sequence) and contains at least 19 exons. Exons 3-19 generate the NOX5alpha mRNA, while exons 1, 2, and 4-19 encode the NOX5beta mRNA (Fig. 2C). The predicted start codon for NOX5alpha is in exon 3, while NOX5beta is predicted to use a start codon in exon 4. This explains why the shorter mRNA is predicted to generate a larger protein. The exon-intron boundaries of NOX5 are very different from members of the NOX and DUOX families. This is in striking contrast with the conserved exon-intron boundaries within the NOX and DUOX families (for example, see Ref. 22). Thus, there appears to be a considerable evolutionary distance between NOX5 and other members of the NOX and the DUOX families.

Localization of NOX5 by in Situ Hybridization-- We next localized NOX5 by in situ hybridization. In testis, the antisense NOX5 probe strongly labeled pachytene spermatocytes (Fig. 3A, arrows) and more weakly labeled round spermatids (Fig. 3A, arrowheads). The sense probe did not give any signal proving the specificity of the antisense hybridization (Fig. 3B). In spleen, the antisense NOX5 probe strongly hybridized with the mantle zone surrounding the germinal centers and also with the periarterial lymphoid sheaths of the white pulp (Fig. 3C), while the sense probe did not produce any signal (Fig. 3D). Thus, areas rich in mature B-lymphocytes (mantle zone) as well as areas rich in T-lymphocytes (periarterial lymphoid sheaths) were labeled. To investigate whether in other lymphoid organs NOX5 mRNA has similar localization, human lymph node sections were hybridized with the antisense probe. Similarly to spleen, NOX5 mRNA was found around the germinal centers in the mantle zone but also in the deep cortex and paracortex of the lymph nodes (Fig. 3, E and F). Note that occasionally some labeled lymphocytes were seen within the germinal centers both in spleen and in lymph nodes, while macrophages and dendritic cells within the germinal centers were consistently negative.


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  		Fig. 3.   Detection of NOX5 mRNA in testis, spleen, and lymph node by in situ hybridization. A and B, seminiferous tubules of testis hybridized with antisense (A) and sense (B) probes of NOX5 shown at ×20 magnification. The dashed lines highlight the lamina propria of the tubules. A, the antisense probe produces strong hybridization signals in pachytene spermatocytes (arrows) and weaker signals in round spermatids (arrowheads). B, the sense probe does not hybridize with the RNA of the cells. C and D, spleen samples probed with the antisense (C) and sense (D) NOX5 probes are shown at ×10 magnification. The signal given only by the antisense probe localizes NOX5 mRNA to the white pulp, being the most abundant in the mantle zone of folliculi (C). Note that the green appearing structures are erythrocytes. E and F, lymph node hybridized with the antisense probe shown at ×5 (E) and ×20 (F) magnifications. The cortex is stained the strongest around the germinal center in the mantle zone, although some cells inside the germinal center are also positive (F); the paracortical region exhibits some labeling as well (E).

Ca2+-dependent Superoxide Generation by NOX5-- To investigate the function of NOX5, we subcloned the NOX5beta -encoding cDNA in pcDNA3.1 vector. This construct was used to generate transiently transfected COS-7, HEK293, and HeLa cells and stable clones of HEK293 cells. The presence of NOX5 transcript in these cells was confirmed by RT-PCR (Fig. 4, A and B). Control cells were obtained with the transfection of empty pcDNA3.1 vector. The absence of the NOX5 mRNA in control cells is demonstrated by the negative RT-PCR results (Fig. 4, A and B).


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  		Fig. 4.   NOX5 mRNA expression in transiently and stably transfected cells. A, HeLa, COS-7, and HEK293 cells were transiently transfected either with a NOX5-containing expression vector or with an empty control vector. NOX5 mRNA was detected by RT-PCR. B, stable HEK293 cell clones transfected with NOX5 expression vector (a1-c2) or empty control vector (b10 and c10) were established. The expression of NOX5 mRNA was detected by RT-PCR. bp, base pairs.

In analogy with gp91phox, NOX5 might be a superoxide-producing NADPH oxidase. Its EF hand-containing N terminus hints toward Ca2+ activation of the enzyme. We therefore measured generation of reactive oxygen species in NOX5- and empty vector-transfected COS-7 cells using a peroxidase-dependent luminol-amplified chemiluminescence assay (43). In the absence of a stimulus, neither NOX5- nor control-transfected cells gave a chemiluminescence signal. However, after addition of ionomycin (a Ca2+ ionophore that raises the [Ca2+]c), an immediate respiratory burst was observed in NOX5- but not in control-transfected cells (Fig. 5A). These observations were confirmed by measurements in transiently NOX5-transfected HeLa and HEK293 cells (Fig. 5B). Similar results were obtained with the NOX5-transfected HEK293 stable clones: all of the tested clones produced ROS in response to [Ca2+]c elevations, some of them in striking amounts, while control clones were devoid of ROS generation (Fig. 5C).


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  		Fig. 5.   Ca2+-dependent superoxide generation in NOX5 transfectants. A, superoxide generation in COS-7 cells transfected with either NOX5 or empty vector was measured by the peroxidase-dependent luminol-amplified chemiluminescence technique. After 4-min baseline measurements, the Ca2+ ionophore ionomycin (1 µM) was added. Data represent the average of three samples of a typical experiment. B, statistical analysis of experiments as shown in panel A. Peak superoxide production of NOX5- and vector-transfected HeLa, COS-7, and HEK293 cells in response to 1 µM ionomycin (n = 3-9). C, peak superoxide generation of NOX5- and vector-transfected stable HEK293 clones and wild-type HEK293 cells in response to 1 µM ionomycin (n = 2-5). The signals in relative light units (RLU) are normalized for 20,000 cells.

Next we investigated the respiratory burst response of NOX5-transfected COS-7 cells to different ionomycin concentrations. Activation of NOX5 by ionomycin was dose-dependent (Fig. 6A) and reached a maximum around 1-3 µM. To investigate whether NOX5 generated superoxide or hydrogen peroxide we used the superoxide-specific SOD-sensitive ferricytochrome c assay. After ionomycin activation the NOX5-transfected cells of the stable clone b2 reduced ferricytochrome c that could be blocked by the flavoprotein inhibitor diphenylene iodonium (Fig. 6B). However, the control clone b10 failed to reduce the ferricytochrome c. These results indicate that the primary product of activated NOX5 is superoxide and that the amount of superoxide generated by the b2 clone is of similar magnitude as that seen with differentiated HL-60 cells. Both assays described so far (luminol-amplified chemiluminescence and ferricytochrome c reduction) only detect extracellular superoxide generation. To investigate whether there is also intracellular superoxide generation, we applied quantitative NBT measurements (41). NBT is reduced by the poorly membrane-permeable superoxide ion (44) but is capable itself of permeating the plasma membrane (45). Thus, as described previously, SOD-resistant NBT reduction is a measure of intracellular superoxide generation (46). The ability of NBT to detect both intra- and extracellular superoxide was first confirmed with control experiments using polymorphonuclear granulocytes and HL-60 cells. The SOD-insensitive phorbol 12-myristate 13-acetate (100 nM)-stimulated NBT reduction (i.e. intracellular superoxide generation) was over 50% in neutrophil granulocytes but very low in specific granules lacking HL-60 cells (Fig. 6C). This is in an accordance with previous reports in the literature (47). In NOX5-transfected HEK cells, the SOD-insensitive NBT reduction was 49% of the total ionomycin-induced signal, suggesting intracellular superoxide production in these cells.


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  		Fig. 6.   Characterization of superoxide generation of NOX5 transfectants. A, superoxide production of NOX5-transfected COS-7 cells in response to different ionomycin concentrations. B, superoxide production detected by the ferricytochrome c reduction assay in NOX5-transfected HEK293 cell stable clone b2 (black bars) and in control clone b10 (gray bars) using no stimulus (control), 3 µM ionomycin, and 3 µM ionomycin and 5 µM diphenylene iodonium (DPI) (n = 5). C, effect of 800 units/ml SOD (gray bars) on stimulus-dependent reduction of NBT by differentiated HL-60 cells stimulated with 100 nM phorbol 12-myristate 13-acetate, by polymorphonuclear granulocytes (PMN) stimulated with 100 nM phorbol 12-myristate 13-acetate, and by clone b2 stimulated with 1 µM ionomycin. The black bars (100%) represent the NBT reduction of the stimulated cells without SOD (n = 4-8). D, comparison of superoxide generation of NOX5-, NOX4-, and vector-transfected COS-7 cells in the presence or absence of 1 µM ionomycin (n = 3-9). The signals in relative light units (RLU) are normalized for 20,000 cells. diff., differentiated.

To investigate whether activation by a Ca2+ ionophore was a specific feature of the EF hand-containing NOX5 enzyme, we compared ROS generation by NOX5-transfected cells with that of NOX4-transfected cells. NOX4 was chosen because as opposed to gp91phox it functions without the need of additional cytosolic subunits (24, 25). NOX4 was transiently transfected into COS-7 cells, and RT-PCR was used to confirm its efficient transfection (data not shown). The chemiluminescence in unstimulated cells was significantly higher in NOX4 transfectants as compared with NOX5 or control transfectants (Fig. 6D). However, upon addition of ionomycin, only NOX5 transfectants but not NOX4- or control-transfected cells increased their ROS generation (Fig. 6D).

Ca2+-activated H+ Currents through NOX5-- As discussed in the Introduction, gp91phox and NOX1 also function as H+ channels. We therefore applied the patch-clamp technique to investigate a putative H+ channel function of NOX5. Fig. 7, A and B, show two typical traces acquired with the NOX5-transfected HEK293 cell stable clone a1 (see Fig. 4). Under resting conditions (i.e. low Ca2+ concentration in the pipette solution) only very small background currents were detected (Fig. 7, A and F). However, with high pipette Ca2+ concentrations, large H+ currents were measured (Fig. 7, B and F). No Ca2+ activation was observed in the parental, nontransfected HEK293 cells (Fig. 7, C, D, and F). To investigate whether the NOX5 currents were indeed carried by H+ ions, the selectivity of the Ca2+-activated currents was investigated by tail current analysis (48). The reversal potential of the currents (Erev) followed the calculated equilibrium potential of H+ ions (Fig. 7E), demonstrating the H+ selectivity of the NOX5 currents. Finally, the H+ current density was compared with that of a gp91phox-expressing HEK293 cell stable clone. The gp91phox-transfected cells had already relatively large currents under low Ca2+ conditions (see also Ref. 37), but the H+ currents were not significantly enhanced under high Ca2+ conditions (Fig. 7F). Thus, similar to gp91phox and NOX1, NOX5 functions as a H+ channel. However, as opposed to gp91phox, NOX5 conducts H+ ions only upon elevations of [Ca2+]c.


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  		Fig. 7.   Ca2+-activated H+ currents in NOX5-expressing cells. NOX5-transfected HEK293 cells (stable clone a1, A and B) and wild-type (WT) HEK293 cells (C and D) were perfused with either a low Ca2+ pipette solution (A and C, free [Ca2+] <10 nM and pCa2+ >8) or a high Ca2+ pipette solution (B and D, free [Ca2+] 75 µM and pCa2+~4). Currents were elicited by 3-s depolarizing voltage pulses ranging from -60 mV to +80 mV. The bath solution pH was 7.5, and the pipette solution was pH 6.5. E, H+ selectivity of NOX5 currents. Reversal potentials of tail currents, measured with the high Ca2+ pipette solution at different repolarizing voltages following a 2-s-long activating pulse to +60 mV, are plotted against the pipette pH. Values are mean ± S.E. of five to seven experiments. The dotted line is the calculated H+ equilibrium potential. F, mean current densities measured with low Ca2+ pipette solutions (white bars) or high Ca2+ pipette solutions (dark gray bars) in wild-type HEK293 cells, NOX5-expressing cells, or gp91phox-expressing cells. The leak-subtracted currents were recorded at +60 mV and normalized for the cell capacitance. Data are mean ± S.E. of 5-12 experiments. pA/pF, picoamperes/picofarad.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we describe the novel superoxide-producing NADPH oxidase NOX5. It is distantly related to the NOX and the DUOX families of NADPH oxidases and characterized by three N-terminal EF hand motifs. Both the superoxide production and the H+ conductance of NOX5 is activated in response to [Ca2+]c elevation. Because of its high level expression in testis and in lymphoid tissues, it is likely to play a role in sperm and lymphocyte biology.

Despite its evolutionary distance, NOX5 resembles gp91phox more closely than any other gp91phox homologues from a functional point of view. (i) NOX5 is a second messenger-activated enzyme, while presently available data suggest that NOX1 and NOX4 are constitutively active. (ii) Heterologously expressed NOX5 generates large amounts of superoxide, while NOX1 and NOX4 appear to generate only low levels (21, 24, 25) (see also Fig. 6). From a biological point of view, this makes sense: regulated NADPH oxidases may generate high amounts of superoxide for a limited period of time, while constitutively active enzymes should produce lower amounts to avoid toxicity.

However, while gp91phox needs to assemble several other subunits for its activation, it appears that in NOX5 the regulatory and catalytic modules are combined within one protein. Indeed, heterologously expressed NOX5 generates superoxide upon [Ca2+]c elevations in HEK293, COS-7, and HeLa cells, and at least one of these cell lines (HEK293) is completely devoid of p47phox and p67phox (data not shown). We can, however, not exclude a role for p22phox in NOX5 activity as low levels of p22phox mRNA were found in all three cell lines used for transfections (data not shown). Similarly, the existence of hitherto undefined, widely distributed NOX5-interacting proteins cannot be excluded at this point.

Similar to gp91phox and NOX1, NOX5 acts as a H+ channel. Thus, many (possibly all) gp91phox homologues fulfill a double function: transport of electrons and proton conductance. However, while the NOX5 H+ channel is quiescent in nonstimulated cells and activated only upon [Ca2+]c increase, the gp91phox and NOX1 H+ channels conduct protons without a need for any activator when heterologously expressed in nonphagocytic cells (22, 37, 50).

The NOX5 gene has two major mRNA products, one expressed in testis and one expressed in spleen. The two isoforms use different sites of the NOX5 gene for transcription initiation, suggesting different promoter usage in the different tissues. The difference of the two mRNA products is mainly restricted to the 5'-untranslated region, suggesting differences in mRNA handling in spleen and testis as it has been described for other 5' isoforms (51). A study published since submission of this manuscript (42), has described a fetal kidney-derived NOX5 cDNA that does not contain the N-terminal EF hands. In our studies, NOX5 without EF-hands does not generate superoxide.2

NOX5 mRNA is most abundantly found in pachytene spermatocytes. Thus, NOX5 might have a function in the early stages of spermatogenesis such as cell division, induction of apoptosis, or DNA compaction. However, the mRNAs for proteins of mature spermatozoa are synthesized early in spermatogenesis because later DNA compaction prevents gene transcription (52). Thus, we favor the hypothesis that the NOX5 protein plays a role in the function of mature spermatozoa. Indeed, although superoxide generation is typically associated with phagocytes, mammalian spermatozoa were one of the first cells in which a respiratory burst was detected (53). It was later shown that generation of reactive oxygen species by spermatozoa is activated by [Ca2+]c elevations (54) suggesting the presence of a Ca2+-activated NADPH oxidase. This ROS generation is thought to be an important mediator of the acrosome reaction and sperm-oocyte fusion as those two functions of the mature sperm are inhibited by ROS scavengers (7). Thus, NOX5 might couple [Ca2+]c elevations during sperm activation to spermatozoa effector functions.

The NOX5 mRNA is also highly expressed in cortical and paracortical lymphocytes of lymph nodes and in the white pulp of spleen. In the cortical area of lymph nodes, NOX5 mRNA is expressed in the mantle zone surrounding the germinal centers suggesting that the NOX5-expressing cells are mature memory B-lymphocytes. The abundant expression of NOX5 in the mostly T-cell-containing paracortex suggests that T-lymphocytes also express NOX5. Similarly in spleen, NOX5 mRNA was found both in the mature B-lymphocyte-rich area (mantle zone of folliculi) and in the T-lymphocyte-rich area (periarterial lymphoid sheaths). What might be the possible function of a Ca2+-activated superoxide-producing enzyme in follicular memory B-cells and in splenic and lymph node T-cells? Although no definitive answer can be given at this point, three pieces of information suggest a reasonable working hypothesis. (i) Both memory B-cells and T-lymphocytes express Ca2+-mobilizing receptors, the B-cell receptor and the T-cell receptor. Thus, upon exposure to their specific antigen, the lymphocytes raise [Ca2+]c, which leads to activation: memory B-cells divide rapidly and develop large clusters of plasmoblasts (55). The T-lymphocytes also proliferate and differentiate into memory and effector cells. (ii) The rise of [Ca2+]c may activate NOX5, which generates both intra- and extracellular superoxide at least in the transfected cells. Superoxide spontaneously (or catalyzed by SOD) dismutates to H2O2. Hydrogen peroxide is able to cross biological membranes and influence redox-sensitive processes. (iii) Proliferation and differentiation of both B- and T-cells involve redox-sensitive processes, in particular the NFkappa B/Ikappa B kinase pathway (49, 56, 57) and the c-Jun N-terminal kinase pathway (17, 18). Also, ROS generation appears to be involved in interleukin-6 secretion by B-lymphocytes (17). Thus, NOX5 might play a role as a bridge between the B-cell and T-cell receptor activation and the proliferation and differentiation of B- and T-lymphocytes.

    ACKNOWLEDGEMENTS

We are grateful to Prof. Dr. M. Bergmann, Institute of Veterinary Anatomy of the University, Giessen, for providing the testicular biopsies; to Dr. Woenckhaus, Institute of Human Pathology of the University, Giessen, for providing the spleen and lymph node biopsies; and to Prof. Dr. U. T. Ruegg and Dr. P. Lhote, Department of Pharmacology of the University of Lausanne, for providing human vascular smooth muscle cells. We thank A. Hild for skillful technical assistance in in situ hybridization, and we thank S. Jaconi for excellent technical assistance in RNA preparations and PCRs. We also thank for Prof. B. Borisch for help with the microscopic analysis of lymphoid tissues.

    FOOTNOTES

* This research was supported by Swiss National Foundation Grants 31-55344.98 and 31-56802.99; by a grant allocated by the Swiss Foundation of Aging Research (AETAS), and the foundation Hans Wilsdorf, Geneva; and by United States Public Health Service, National Institutes of Health Grants AI20866 and AG19519.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF353088, AF325189, AF353089, and AF325190.

§§ A fellow of the Dr. Max Cloetta Foundation.

¶¶ To whom correspondence should be addressed: Dept. of Geriatrics, Geneva University Hospitals, 2, Ch. du Petit-Bel-Air, CH-1225 Geneva, Switzerland. Tel.: 41-22-305-5450; Fax: 41-22-305-5455; E-mail: kkrause@cmu.unige.ch.

Published, JBC Papers in Press, August 1, 2001, DOI 10.1074/jbc.M103034200

2 B. Bánfi, G. Molnár, A. Maturana, K. Steger, B. Hegedûs, N. Demaurex, and K.-H. Krause, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; [Ca2+]c, cytosolic free Ca2+ concentration; DUOX, dual oxidase; gp91phox, 91-kDa glycoprotein subunit of the phagocyte NADPH oxidase; kb, kilo base; NBT, nitro blue tetrazolium; NOX, NADPH oxidase; NOX5, NADPH oxidase 5; RACE, rapid amplification of cDNA ends; SOD, superoxide dismutase; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; PIPES, 1,4-piperazinediethanesulfonic acid; HEDTA, N-hydroxyethylethylenediaminetriacetic acid; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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