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W2008360112 abstract "Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5–6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD50. In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5–6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD50. In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. aldoketoreductases aldose reductase murine aldose reductase mouse vas deferens protein polyacrylamide gel electrophoresis isocaproaldehyde reductase Chinese hamster ovary Aldoketoreductases (AKRs)1 are monomeric oxidoreductases that catalyze the NADPH-dependent conversion of aldehydes and ketones to their corresponding alcohols (1Bohren K.M. Bullock B. Wermuth B. Gabbay K.H. J. Biol. Chem. 1989; 264: 9547-9551Abstract Full Text PDF PubMed Google Scholar). Among the AKR superfamily, aldose reductase (AR) has been a focus of interest because of its potential role in the development of secondary diabetic complications (2Gabbay K.H. Annu. Rev. Med. 1975; 26: 521-536Crossref PubMed Scopus (264) Google Scholar). In agreement with its broad substrate specificity, AR is thought to accomplish varying physiological roles in osmotic homeostasis (3Bagnasco S. Balaban R. Fales H. Yang Y.M. Burg M. J. Biol. Chem. 1986; 261: 5872-5877Abstract Full Text PDF PubMed Google Scholar), steroid conversion (4Warren J.C. Murdock G.L. Ma Y. Goodman S.R. Zimmer W.E. Biochemistry. 1993; 32: 1401-1406Crossref PubMed Scopus (83) Google Scholar), and detoxification against xenobiotic and endogenous aldehydes (5Grimshaw C.E. Biochemistry. 1992; 31: 10139-10145Crossref PubMed Scopus (90) Google Scholar). To date, the AKR superfamily consists of at least 42 members that differ in their primary structure, substrate specificities, and catalytic properties. Adult mouse vas deferens contains a large amount of a major protein (mouse vas deferens protein (MVDP)) with an apparent molecular mass of 34,500 Da (6Taragnat C. Berger M. Jean C. J. Reprod. Fert. 1988; 83: 835-842Crossref PubMed Scopus (42) Google Scholar). Recently, it has been shown that MVDP expression is not restricted to the vas deferens, and high levels of MVDP mRNA were found in the adrenal glands (7Lau E. Cao D. Lin C. Chung S.K. Chung S.S. Biochem. J. 1995; 312: 609-615Crossref PubMed Scopus (52) Google Scholar). Androgens have been shown to be the primary regulating factors of MVDP expression in the vas deferens (8Martinez A. Pailhoux E. Berger M. Jean C. Mol. Cell. Endocrinol. 1990; 72: 201-211Crossref PubMed Scopus (33) Google Scholar); and recent studies suggest that cAMP is a key regulator of adrenal MVDP expression (9Aigueperse C. Martinez A. Lefrançois-Martinez A.-M. Veyssière G. Jean C. J. Endocrinol. 1998; 160: 147-154Crossref Scopus (38) Google Scholar). MVDP shares ∼70% amino acid identity with human, rabbit, rat, and mouse ARs (10Pailhoux E. Martinez A. Veyssière G. Jean C. J. Biol. Chem. 1990; 265: 19932-19936Abstract Full Text PDF PubMed Google Scholar, 11Daoudal S. Berger M. Pailhoux E. Tournaire C. Veyssière G. Jean C. Life Sci. Adv. (Steroid Biochem.). 1995; 14: 45-58Google Scholar), and MVDP gene structure is similar to that of the human AR gene (12Pailhoux E. Veyssière G. Fabre S. Tournaire C. Jean C. J. Steroid Biochem. Mol. Biol. 1992; 42: 561-568Crossref PubMed Scopus (18) Google Scholar). However, MVDP is more closely related to the mouse fibroblast growth factor-regulated protein FR-1 (13Donohue P.J. Alberts G.F. Hampton B. Winkles J.A. J. Biol. Chem. 1994; 269: 8604-8609Abstract Full Text PDF PubMed Google Scholar), Chinese hamster ovary reductase (14Hyndman D.J. Takenoshita R. Vera N.L. Pang S.C. Flynn T.G. J. Biol. Chem. 1997; 272: 13286-13291Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), the human AR-like geneALR1 (15Cao D. Fan S.T. Chung S.S.M. J. Biol. Chem. 1998; 273: 11429-11435Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), and human small intestine reductase (16Hyndman D.J. Flynn T.G. Biochim. Biophys. Acta. 1998; 1399: 198-202Crossref PubMed Scopus (87) Google Scholar). These proteins share ∼80–90% sequence identities and form a distinct subgroup within the AKR1B group according to the new nomenclature proposed by Jez et al. (17Jez J.M. Flynn T.G. Penning T.M. Biochem. Pharmacol. 1997; 54: 639-647Crossref PubMed Scopus (338) Google Scholar). The enzymatic nature of MVDP (AKR1B7 in agreement with the proposed nomenclature (17Jez J.M. Flynn T.G. Penning T.M. Biochem. Pharmacol. 1997; 54: 639-647Crossref PubMed Scopus (338) Google Scholar)) has not yet been demonstrated. In this study, we have compared the enzymatic characteristics and substrate specificity of MVDP with those of mAR, and we have focused on its physiological role in adrenal glands. The data indicate that MVDP acts as a major reductase for isocaproaldehyde formed during steroidogenesis. Tolrestat was a gift from Wyet-Ayerst, Sorbinil from Pfizer, and Imirestat from Alcon Laboratories. Isocaproaldehyde was prepared according to the method of Matsuura et al.(18Matsuura K. Deyashiki Y. Bunai Y. Ohya I. Hara A. Arch. Biochem. Biophys. 1996; 328: 265-271Crossref PubMed Scopus (48) Google Scholar). 4-Hydroxynonenal was provided by Interchim. All other chemicals were purchased from Sigma. MVDP and mAR cDNAs were obtained by polymerase chain reaction amplification with MVDP pUC13 (10Pailhoux E. Martinez A. Veyssière G. Jean C. J. Biol. Chem. 1990; 265: 19932-19936Abstract Full Text PDF PubMed Google Scholar) and mAR pGEMT (pmAR13) (11Daoudal S. Berger M. Pailhoux E. Tournaire C. Veyssière G. Jean C. Life Sci. Adv. (Steroid Biochem.). 1995; 14: 45-58Google Scholar) as templates, respectively, and with outside primers containing engineered 5′- BamHI and 3′-EcoRI sites (5′-BamHI primer, 5′-GCAGGATCCATGGCCACCTTCGTGGAACTC-3′; and 3′-EcoRI primer, 5′-ATTGAATTCTCAGTGTTACCATACTACATGC-3′). Recombinant MVDP and mAR were expressed in Escherichia coli by inserting their respective cDNAs into the BamHI and EcoRI sites of pET28a (Novagen) to produce N-terminal fusions with six histidine residues. Recombinant MVDP and mAR were produced in BL21(DE3) pLysS cells upon isopropyl-β-d-thiogalactopyranoside induction and purified by nickel affinity chromatography according to the manufacturer's instructions (Novagen). For each protein, column fractions were analyzed by SDS-PAGE, and those containing the purified protein were pooled and stored at 4 °C. The pCR3 vector (Invitrogen) harboring the neomycin resistance gene was used to express MVDP antisense RNA in murine adrenocortical Y1 cells (19Yasumura Y. Buonassisi V. Sato G. Cancer Res. 1966; 26: 529-535PubMed Google Scholar) in stable transfection experiments. The complete MVDP cDNA was obtained by XbaI/EcoRI digestion of pET-MVDP and inserted in antisense orientation in pCR3. This construct was named pCR3-AS. Y1 cells were transfected with either 5 μg of pCR3-AS or pCR3-EV (empty control plasmid) vector by lipofection usingN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts (Roche Molecular Biochemicals). After a 48-h recovery period, 400 μg/ml G418 was added to the medium. G418-resistant cells were selected, isolated by limiting dilution, and then propagated. Cellular clones harboring either the pCR3-AS or pCR3-EV vector were named AS and EV cells, respectively. The presence of the pCR-AS vector (6.3 kilobases) in the neomycin-resistant selected Y1 cells was identified by Southern blot analysis after EcoRI digestion of cellular genomic DNA. The Southern blot was hybridized with a MVDP probe (1226-base pair XbaI/EcoRI fragment covering the complete MVDP cDNA) labeled by random priming. Y1 cells were routinely cultured as described previously in Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented with 10% fetal calf serum (9Aigueperse C. Martinez A. Lefrançois-Martinez A.-M. Veyssière G. Jean C. J. Endocrinol. 1998; 160: 147-154Crossref Scopus (38) Google Scholar). AS or EV stably transfected Y1 cellular clones were routinely cultured in the same medium containing 200 μg/ml G418. 24 h prior to treatment with various hormones or drugs, G418 was removed. Human H295R adrenocortical cells were cultured as described previously (9Aigueperse C. Martinez A. Lefrançois-Martinez A.-M. Veyssière G. Jean C. J. Endocrinol. 1998; 160: 147-154Crossref Scopus (38) Google Scholar). 24 h before harvest, cells were cultured under basal conditions or in the presence of 10−5mforskolin or 75 mm NaCl. Adrenal glands were removed from adult male CD1 mice. Tissues or cells were immediately homogenized in 0.1 m sodium phosphate buffer (pH 6.6) containing 1 mm dithiothreitol. The homogenates were centrifuged at 105,000 × g for 1 h, and the supernatants were collected and immediately used for enzymatic assay or subsequent SDS-PAGE analysis. The standard reaction mixture for the reductase activities contained 0.1 m sodium phosphate buffer (pH 6.6), 0.4 m ammonium sulfate, and appropriate amounts of NAD(P)H, substrate, and enzyme or protein extracts as indicated for each experiment. The reaction was carried out at 25 °C, and the decrease in NAD(P)H was monitored by spectrophotometer at 340 nm. Reactions were routinely started by the addition of enzyme or protein extracts. Controls without substrate or without enzyme were run simultaneously. One enzyme unit is defined as the change at 340 nm corresponding to the oxidation of 1 μmol of NAD(P)H. Northern bolt analysis of total RNAs isolated from Y1 cells was performed according to the method previously described (9Aigueperse C. Martinez A. Lefrançois-Martinez A.-M. Veyssière G. Jean C. J. Endocrinol. 1998; 160: 147-154Crossref Scopus (38) Google Scholar). Frozen cell or tissue samples were homogenized in 250 mm Tris-HCl (pH 7.5) and 0.4 mm phenylmethylsulfonyl fluoride. Soluble tissue or cell extracts (20 μg of proteins) or recombinant MVDP or AR was subjected to SDS-PAGE or NEpHGE and transferred to nitrocellulose membranes. Blots were treated as previously described (9Aigueperse C. Martinez A. Lefrançois-Martinez A.-M. Veyssière G. Jean C. J. Endocrinol. 1998; 160: 147-154Crossref Scopus (38) Google Scholar) and then incubated with either primary anti-MVDP monoclonal antibody (ascites B263) at a 1:700 dilution or rabbit anti-rat AR serum at a 1:3000 dilution for 1 h at room temperature. When NEpHGE was performed, blots were treated with primary rabbit anti-MVDP serum at 1:5000 dilution. Peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies were added at a 1:15,000 dilution for 1 h at room temperature. Peroxidase activity was detected with the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech). Stably transfected Y1 cellular clones (AS19 or EV4 cells) were plated in 1 ml of fresh medium in 2-cm2 culture wells at a concentration of 5 × 104 cells/ml. After a recovery period, cells were cultured under basal conditions or with 10−5m forskolin alone or with 5 × 10−4m aminoglutethimide for 24 h. When the LD50 of isocaproaldehyde was assayed, cells were exposed to increasing concentrations of isocaproaldehyde for 4 h. After treatments, cells were resuspended, and their viability was immediately estimated by 0.2% (w/v) trypan blue exclusion. Results are given as means ± S.D. Statistical significance of the differences between treatment groups was determined by Student's t test. To compare the enzymatic activities and substrate specificity of MVDP with those of mAR, both cDNAs were expressed in bacteria to obtain recombinant proteins. The kinetic constants of both proteins, determined for various substrates, are summarized in TableI. Similar to native AR purified from tissues, recombinant mAR has the ability to reduce aldo sugars, glyceraldehyde being the best substrate among them. Except for relatively lower K m values with glucose, values for recombinant mAR were in accordance with those reported for AR isolated from other species (20Iwata N. Inazu N. Satoh T. Arch. Biochem. Biophys. 1990; 282: 70-77Crossref PubMed Scopus (32) Google Scholar, 21Nishimura C. Yamaoka T. Mizutami M. Yamashita K. Akera T. Tanimoto T. Biochim. Biophys. Acta. 1991; 1078: 171-178Crossref PubMed Scopus (107) Google Scholar, 22Bohren K.M. Page J.L. Shankar R. Henry S.P. Gabbay K.H. J. Biol. Chem. 1991; 266: 24031-24037Abstract Full Text PDF PubMed Google Scholar, 23Carper D.A. Hohman T.C. Old S.E. Biochim. Biophys. Acta. 1995; 1246: 67-73Crossref PubMed Scopus (28) Google Scholar). By comparison with other compounds, isocaproaldehyde, methylglyoxal, and 4-hydroxynonenal were the best substrates. MVDP showed kinetic properties distinct from those of mAR. As shown in Table I, common substrates for AKR, including aldo sugars, were poor substrates for recombinant MVDP. For all compounds studied, the k cat values were always lower than those measured for mAR (Table I), even tested under various pH conditions (data not shown). Aldose reductases are able to use NADPH and also NADH, but with less efficiency (24Boghosian R.A. McGuiness E.T. Biochim. Biophys. Acta. 1979; 567: 278-286Crossref PubMed Scopus (33) Google Scholar, 25Hadler A.B. Crabbe M.J.C. Biochem. J. 1984; 219: 33-39Crossref PubMed Scopus (36) Google Scholar, 26Wermuth B. Bürgisser H. Bohren K. Von Wartburg J.P. Eur. J. Biochem. 1982; 127: 279-284Crossref PubMed Scopus (114) Google Scholar). Other AKRs such as aldehyde or carbonyl reductases display a strict NADPH-dependent activity (27Turner A.J. Tipton K.F. Biochem. J. 1972; 130: 765-772Crossref PubMed Scopus (80) Google Scholar, 28Wermuth B. Münch J.D.B. Von Wartburg J.P. J. Biol. Chem. 1977; 252: 3821-3828Abstract Full Text PDF PubMed Google Scholar). To determine the MVDP requirements for its cofactor, we also determined the kinetic constants for MVDP with NADH as a cofactor. When NADH was used instead of NADPH, thek cat values were 2–5-fold higher than those measured using NADPH, indicating that MVDP has a strong preference for NADH. Even though this enzyme shows a strong affinity for 4-hydroxynonenal, on the basis of comparison of the catalytic efficiencies (k cat), isocaproaldehyde seems to be the preferred substrate catalyzed by MVDP. TableII lists the effects of various compounds known as inhibitors of AR. Recombinant MVDP was not inhibited by Sorbinil and p-chloromercuribenzoate and was moderately diminished by Imirestat and Tolrestat, differing in this respect from recombinant mAR.Table IKinetic constants for recombinant mAR and MVDPSubstratesmAR/NADPHMVDP/NADPHMVDP/NADHK mk catk cat/K mK mk catk cat/K mK mk catk cat/K mμms −1s −1 mm −1μms −1s −1 mm −1μms −1s −1 mm −1dl-Glyceraldehyde831.08132620.0410.1533,0000.170.005Glucose27000.370.14175,0000.0754.2 × 10−4ND1-aNot detected.Xylose33,3003.280.1143,0000.0422.7 × 10−4NDGlucuronate26,2000.940.035NDNDMenadioneNDNDND5α-DihydrocortisolNDNDNDMethylglyoxal481.4229.528.50.0351.227000.180.064-Hydroxynonenal620.6510.6100.0424.2620.101.7Isovalcraldehyde1462.617.8170.0251.44760.100.22Isocaproaldehyde531.324.5950.0900.943200.381.2Diacetyl770.9412.2700.0540.7719000.280.147NADH1921-bFor determination of the K m values for NADPH and NADH, isocaproaldehyde was held constant at 150 and 400 μm, respectively.1011-bFor determination of the K m values for NADPH and NADH, isocaproaldehyde was held constant at 150 and 400 μm, respectively.NADPH12.31-bFor determination of the K m values for NADPH and NADH, isocaproaldehyde was held constant at 150 and 400 μm, respectively.1.91-bFor determination of the K m values for NADPH and NADH, isocaproaldehyde was held constant at 150 and 400 μm, respectively.The activity was assayed with 0.1 mm NADPH or 0.15 mm NADH as cofactor in 100 mm phosphate buffer (pH 6.6). The k cat values were calculated using molecular masses of 39.5 and 42 kDa for recombinant mAR and MVDP, respectively. Each value is the mean of at least six distinct experiments.1-a Not detected.1-b For determination of the K m values for NADPH and NADH, isocaproaldehyde was held constant at 150 and 400 μm, respectively. Open table in a new tab Table IIEffect of inhibitors on recombinant mAR and MVDPInhibitorRemaining activitymAR/NADPHMVDP/NADPHMVDP/NADH %Sorbinil (1 μm)15100100Imirestat (10 μm)0.18284Tolrestat (10 μm)0.18140p-Chloromercuribenzoate (10 μm)0.1187100Aliquots of the inhibitor solution at the final concentrations indicated were added to the reaction mixture containing 100 mm phosphate buffer (pH 6.6), the enzymes, and isocaproaldehyde as substrate. Open table in a new tab The activity was assayed with 0.1 mm NADPH or 0.15 mm NADH as cofactor in 100 mm phosphate buffer (pH 6.6). The k cat values were calculated using molecular masses of 39.5 and 42 kDa for recombinant mAR and MVDP, respectively. Each value is the mean of at least six distinct experiments. Aliquots of the inhibitor solution at the final concentrations indicated were added to the reaction mixture containing 100 mm phosphate buffer (pH 6.6), the enzymes, and isocaproaldehyde as substrate. Isocaproaldehyde, the best substrate identified for MVDP, is a product of the side-chain cleavage of cholesterol, the first step of steroid biosynthesis. On the basis of Western blot analysis, both MVDP and AR were detected in adrenal glands. By comparison with known amounts of recombinant proteins, the concentrations of MVDP and AR were found to be very similar, ∼1% of total soluble proteins (Fig.1). It has been shown that in murine adrenocortical cells, MVDP expression is up-regulated by forskolin (9Aigueperse C. Martinez A. Lefrançois-Martinez A.-M. Veyssière G. Jean C. J. Endocrinol. 1998; 160: 147-154Crossref Scopus (38) Google Scholar). If isocaproaldehyde is reduced by MVDP in adrenocortical cells, it would be predicted that isocaproaldehyde reductase (ICR) activity should be enhanced by the addition of forskolin. However, since AR has been described as a major reductase for isocaproaldehyde in adrenal glands (18Matsuura K. Deyashiki Y. Bunai Y. Ohya I. Hara A. Arch. Biochem. Biophys. 1996; 328: 265-271Crossref PubMed Scopus (48) Google Scholar), we have developed a test to discriminate between AR and MVDP ICR activities. As shown in Fig. 2, treatment of Y1 cells for 24 h with 10−5m forskolin strongly increased MVDP expression, whereas a 24-h exposure of the cells to hypertonic medium had no effect on MVDP levels. Conversely, AR levels were not affected by forskolin and were enhanced after exposure to hypertonic medium. As shown in TableIII, no variation in NADPH-linked ICR activity was observed in cells stimulated by forskolin or exposed to hypertonic medium. When we used NADH instead of NADPH, a strong activity was measured in unstimulated cells. No stimulating effect was observed in cells exposed to hypertonic medium; however, when cells were exposed to forskolin treatment, NADH-linked ICR activity was strongly enhanced (∼5-fold), and this effect was not abolished by the presence of Sorbinil and correlated with forskolin-induced MVDP expression. Similar experiments showed that NADH-linked 4-hydroxynonenal reductase activity was higher than NADPH-linked 4-hydroxynonenal reductase activity, but was not increased after forskolin exposure (Table III). Using the same experimental procedures, NADH-linked ICR activity in adult murine adrenal cytosolic extracts was estimated to ∼150 μmol/min/mg of protein, and NADPH-linked ICR activity was determined to ∼25 μmol/min/mg of protein extract.Figure 2Effect of forskolin and hyperosmotic stress on MVDP and mAR protein levels in adrenocortical Y1 cells. Cell cultures were untreated or treated with either forskolin- or hypertonic NaCl-supplemented medium for 24 h. Cell extracts were prepared, and 20 μg of cytosolic soluble proteins were subjected to SDS-PAGE and Western blotting using either an anti-AR polyclonal antiserum (A) or an anti-MVDP monoclonal antibody (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIIICR and 4-hydroxynonenal reductase activities in the murine adrenocortical Y1 cell lineParameterControlForskolin (10−5m)NaCl (75 mm)Specific activity (μmol/min/mg protein) NADH-linked ICR1070 ± 4155830 ± 3181140 ± 495 NADPH-linked ICR41.5 ± 12.525.2 ± 2.935.2 ± 13 NADH-linked HNR3-a4-Hydroxynonenal reductase.1250 ± 206825 ± 1381250 ± 152 NADPH-linked HNR31.5 ± 937.5 ± 10.856.8 ± 11Inhibition (%) by 1 μm Sorbinil on NADH- linked ICR<0.1<0.1<0.1 1 μm Sorbinil on NADPH-linked ICR100100100 1 μm Sorbinil on NADH- linked HNR<0.1<0.1<0.1 1 μmSorbinil on NADPH-linked HNR100100100The activities were assayed on Y1 cell cytosolic fractions with 0.12 mm NADPH or 0.216 mm NADH as cofactor in 100 mm phosphate buffer (pH 6.6). Values are the mean of four independent experiments.3-a 4-Hydroxynonenal reductase. Open table in a new tab The activities were assayed on Y1 cell cytosolic fractions with 0.12 mm NADPH or 0.216 mm NADH as cofactor in 100 mm phosphate buffer (pH 6.6). Values are the mean of four independent experiments. Besides MVDP, the AKR1B subgroup contains proteins such as FR-1 (13Donohue P.J. Alberts G.F. Hampton B. Winkles J.A. J. Biol. Chem. 1994; 269: 8604-8609Abstract Full Text PDF PubMed Google Scholar) and human small intestine reductase (16Hyndman D.J. Flynn T.G. Biochim. Biophys. Acta. 1998; 1399: 198-202Crossref PubMed Scopus (87) Google Scholar), which are expressed in adrenal glands. To better understand the role of MVDP in adrenal glands, Y1 cells were stably transfected with the pCR3 vector containing MVDP antisense cDNA to prevent MVDP expression or with the empty vector as a control. Nine positive clones stably transfected with the pCR3 vector containing antisense cDNA and 12 clones transfected with the empty vector were selected after Southern blot analysis (data not shown). As expected, high levels of MVDP (Fig.3) and strong NADH-linked ICR activity (Table IV) were observed after forskolin treatment in Y1 cells transfected with the empty vector (EV2 and EV4 cells). In contrast, MVDP and its mRNA were undetectable in Y1 cells transfected with MVDP antisense cDNA (AS19 cells) cultured either under basal conditions or with forskolin (Fig. 3, Aand B). As MVDP and mAR display high homology in their nucleotide sequences, the presence of intact mAR protein levels was checked in these clones. As shown in Fig. 3 A, mAR levels were not altered by MVDP antisense mRNA. In Y1 cells, in which antisense RNA completely abolishes MVDP expression (AS19 cells), no NADH-linked ICR was detected, indicating that ICR activity in adrenal glands is due mainly to MVDP (Table IV). Because basal NADH-linked ICR activity was totally blocked in AS19 cells, the presence of MVDP in EV4 cells cultured under basal conditions was checked to account for its involvement in NADH-linked ICR activity. Immunodetection using a highly titrated rabbit anti-MVDP antiserum demonstrated the presence of MVDP in EV4 cells cultured under basal conditions, whereas no MVDP expression was detected in AS19 cells cultured in the presence of forskolin (Fig. 3 C).Table IVNADH- and NADPH-linked ICR activities in Y1 clones expressing or not MVDP antisense RNASpecific activityControlForskolin (10−5m)NaCl (75 mm) μmol/min/mg proteinNADH-linked activity in EV21010 ± 1254540 ± 300925 ± 130NADH-linked activity in EV4800 ± 753700 ± 175701 ± 57.5NADH-linked activity in AS19ND4-aActivity not detected.NDNDNADPH-linked activity in EV25.5 ± 1.56.5 ± 116.25 ± 1.6NADPH-linked activity in EV420.7 ± 3.519 ± 433.5 ± 5.5NADPH-linked activity in AS1918.5 ± 411.1 ± 551.5 ± 4.25The activities were assayed on cytosolic fractions of Y1 cells stably transfected with an empty vector (EV) or MVDP antisense RNA-expressing vector (AS). Values are the mean of at least three independent experiments.4-a Activity not detected. Open table in a new tab The activities were assayed on cytosolic fractions of Y1 cells stably transfected with an empty vector (EV) or MVDP antisense RNA-expressing vector (AS). Values are the mean of at least three independent experiments. Because forskolin is a potent activator of steroidogenesis in Y1 cells and therefore a potent activator of isocaproaldehyde production, we measured the viability of AS19 cells lacking MVDP ICR activity when treated with forskolin alone or with aminoglutethimide, an inhibitor of the first step of steroid synthesis. The viability of MVDP-nonexpressing AS19 cells was significantly reduced after a 24-h forskolin exposure compared with EV4 cells (Fig.4), and this effect was totally blocked when aminoglutethimide was simultaneously added to the medium, suggesting that forskolin is not toxic by itself, but works rather by stimulating the endogenous production of isocaproaldehyde through the enhancement of P450scc activity. LD50 was determined in EV4 and AS19 cells by adding increasing amounts of exogenous isocaproaldehyde (Fig.5). Under our experimental conditions, LD50 was significantly decreased from 4.9 to 3 mm in cells lacking MVDP, demonstrating that MVDP ICR activity is important for detoxifying isocaproaldehyde. As MVDP was also previously described to be expressed under forskolin control in the human adrenocortical H295R cell line (9Aigueperse C. Martinez A. Lefrançois-Martinez A.-M. Veyssière G. Jean C. J. Endocrinol. 1998; 160: 147-154Crossref Scopus (38) Google Scholar), ICR activity was therefore investigated under the same experimental conditions described for Y1 cells (Table V). In H295R cells, as observed in Y1 cells, NADH-linked ICR activity was higher than NADPH-linked activity and was also enhanced by forskolin treatment, suggesting that in human adrenocortical cells, MVDP ICR activity is also required for detoxifying isocaproaldehyde.Table VIsocaproaldehyde reductase activity in the human H295R cell lineSpecific activityControlForskolin (10−5m) μmol/min/mg proteinNADH-linked ICR activity1500 ± 5503687 ± 192NADPH-linked ICR activity24 ± 613.87 ± 6.3The activities were assayed on cytosolic fractions with 0.12 mm NADPH or 0.216 mm NADH as cofactor in 100 mm phosphate buffer (pH 6.6). Values are the mean of three independent experiments. Open table in a new tab The activities were assayed on cytosolic fractions with 0.12 mm NADPH or 0.216 mm NADH as cofactor in 100 mm phosphate buffer (pH 6.6). Values are the mean of three independent experiments. Here we demonstrate that recombinant MVDP was active when tested with a variety of common substrates for AR. However, MVDP displayed kinetic properties distinct from those of classical AR. 1) MVDP has a strong preference for NADH; and 2) its enzymatic activity was highly insensitive to most AR inhibitors tested. Based on amino acid sequence identities, MVDP is more homologous to FR-1 (13Donohue P.J. Alberts G.F. Hampton B. Winkles J.A. J. Biol. Chem. 1994; 269: 8604-8609Abstract Full Text PDF PubMed Google Scholar), CHO reductase (14Hyndman D.J. Takenoshita R. Vera N.L. Pang S.C. Flynn T.G. J. Biol. Chem. 1997; 272: 13286-13291Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar),ALR1 (15Cao D. Fan S.T. Chung S.S.M. J. Biol. Chem. 1998; 273: 11429-11435Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), and human small intestine reductase (16Hyndman D.J. Flynn T.G. Biochim. Biophys. Acta. 1998; 1399: 198-202Crossref PubMed Scopus (87) Google Scholar) than to AR (11Daoudal S. Berger M. Pailhoux E. Tournaire C. Veyssière G. Jean C. Life Sci. Adv. (Steroid Biochem.). 1995; 14: 45-58Google Scholar). The kinetic properties determined for some of these enzymes indicate that they have different enzymatic activities over a range of substrates. This observation contrasts with the apparent conservation of the amino acid residues known to be involved in AR enzymatic activities. The key amino acids proposed to be involved in hydrogen transfer in AR (Tyr48, Asp43, Lys77, and Hist110) (23Carper D.A. Hohman T.C. Old S.E. Biochim. Biophys. Acta. 1995; 1246: 67-73Crossref PubMed Scopus (28) Google Scholar, 29Tarle I. Borhani D.W. Wilson D.K. Quiocho F.A. Petrash J.M. J. Biol. Chem. 1993; 268: 25687-25693Abstract Full Text PDF PubMed Google Scholar, 30Bohren K.M. Grimshaw C.E. Lay C.J. Harrisson D.J. Ringe D. Petsko G.A. Gabbay K.H. Biochemistry. 1994; 33: 2021-2032Crossref PubMed Scopus (183) Google Scholar) and the 18 residues reported to interact with NADPH (Thr19, Trp20, Lys21, Asp43, Ser159, Asn160, Glu183, Tyr209, Ser210, Leu212, Ser214, Lys262, Ser263, Val264, Thr265, Arg268, Glu271, and Asn272) (31Wilson D.K. Bohren K.M. Gabbay K.H. Quiocho F.A. Science. 1992; 257: 81-84Crossref PubMed Scopus (394) Google Scholar) are conserved within the five sequences, except a Glu at position 21 in CHO reductase. It has been shown that deletion of the last 13 amino acid residues at the C-terminal end of AR decreased catalytic effectiveness, suggesting that this region is crucial to proper orientation of substrates in the active pocket site (32Petrash J.M. Harter T.M. Devine C.S. Olins P.O. Bhatnagar A. Lui S. Srivastava S.K. J. Biol. Chem. 1992; 267: 24833-24840Abstract Full Text PDF PubMed Google Scholar). The C-terminal domains were more divergent among mAR, FR-1, CHO reductase, ALR1, human small intestine reductase, and MVDP, suggesting that these enzymes exhibit different substrate specificities in relation to their tissue distribution. Whereas AR is present in many tested tissues, expression of MVDP, FR-1, CHO reductase, ALR1, and human small intestine reductase is restricted to a limited number of tissues. Some of these proteins have been shown to be induced by different factors, including hyperosmotic stress or an excess of galactose (AR) (3Bagnasco S. Balaban R. Fales H. Yang Y.M. Burg M. J. Biol. Chem. 1986; 261: 5872-5877Abstract Full Text PDF PubMed Google Scholar, 33Bekhor I. Shi S. Unakar N.J. Mol. Cell. Biochem. 1990; 95: 55-60Crossref PubMed Scopus (11) Google Scholar), growth factors (FR-1) (13Donohue P.J. Alberts G.F. Hampton B. Winkles J.A. J. Biol. Chem. 1994; 269: 8604-8609Abstract Full Text PDF PubMed Google Scholar), chemical factors (CHO reductase) (14Hyndman D.J. Takenoshita R. Vera N.L. Pang S.C. Flynn T.G. J. Biol. Chem. 1997; 272: 13286-13291Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), or hormonal factors (MVDP) (8Martinez A. Pailhoux E. Berger M. Jean C. Mol. Cell. Endocrinol. 1990; 72: 201-211Crossref PubMed Scopus (33) Google Scholar, 9Aigueperse C. Martinez A. Lefrançois-Martinez A.-M. Veyssière G. Jean C. J. Endocrinol. 1998; 160: 147-154Crossref Scopus (38) Google Scholar). At present, it is not known whether ALR1 and human small intestine reductase, the human homologs, are also inducible. Then, the differences observed in kinetic properties, pattern of expression, and mechanisms of induction indicate that all these related proteins are probably involved in different physiological functions. The first step of steroidogenesis is the removal of the cholesterol side chain, resulting in the formation of pregnenolone and isocaproaldehyde (4-methylpentanal), which is metabolized to isocaproic acid and isocapryl alcohol (34Hall P.F. Johnson A.D. Gomes W.R. Vandemark N.L. The Testis. Academic Press, New York1970: 1-72Crossref Google Scholar). 4-Hydroxynonenal is a reactive aldehyde formed via peroxidative damage to polyunsaturated fatty acids in membrane phospholipids (35Esterbauer H. Schaur R.J. Zollner H. Free Radical Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5936) Google Scholar). On the basis of the catalytic efficiencies obtained with various substrates, isocaproaldehyde and 4-hydroxynonenal seem to be the preferred substrates catalyzed by recombinant MVDP. Since MVDP and mAR were expressed in similar amounts in adrenal glands, both enzymes may be responsible for the reduction of isocaproaldehyde and 4-hydroxynonenal generated by cellular metabolism. However, several lines of evidence suggest that MVDP, rather than AR, is a major reductase for isocaproaldehyde in murine adrenocortical cells. First, NADH-linked ICR activity (ascribed to MVDP) was higher than NADPH-linked ICR activity (attributed to AR). Second, NADH-linked ICR activity was not inhibited by specific AR inhibitors. Third, NADH-linked ICR activity was strongly enhanced by forskolin, which stimulates MVDP expression, but not by hyperosmotic stress inducing AR overexpression. Fourth, in Y1 cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity induced by forskolin was completely abolished. Our results differ from those of Matsuuraet al. (18Matsuura K. Deyashiki Y. Bunai Y. Ohya I. Hara A. Arch. Biochem. Biophys. 1996; 328: 265-271Crossref PubMed Scopus (48) Google Scholar), who have shown that in human, monkey, dog, and rabbit adrenal glands, AR is a major reductase for isocaproaldehyde; but both NADH- and NADPH-linked ICR activities measured in the experiments of Matsuura et al. (18Matsuura K. Deyashiki Y. Bunai Y. Ohya I. Hara A. Arch. Biochem. Biophys. 1996; 328: 265-271Crossref PubMed Scopus (48) Google Scholar) are lower than those from our assay in cytosolic extracts. Some of the possible reasons for these dissimilar results include differences concerning species and experimental procedures. ICR activity has been previously measured in frozen adrenal extracts; in this study, we used fresh extracts of intact adrenal glands and fresh extracts of adrenocortical Y1 cells. Strikingly, in vitro k cat /K m values suggest that mAR/NADPH at equal concentration as is found in adrenal glands should be the main isocaproaldehyde reductase. Ex vivo experiments in Y1 cells contrast with this observation, suggesting that when produced in vitro from a bacterial expression system, MVDP is in a less effective state than when produced endogenously. In agreement with the observations of Grimshaw et al. (36Grimshaw C.E. Bohren K.M. Lai C.J. Gabbay K.H. Biochemistry. 1995; 34: 14366-14373Crossref PubMed Scopus (39) Google Scholar) that activated or oxidized forms of human placenta aldose reductase result in an increase in K m, a net decrease in k cat/K m fordl-glyceraldehyde, and an insensitivity to Sorbinil inhibition, one possibility could be that in vitro produced MVDP exists in an oxidized form. Similarly, in Y1 cells lacking MVDP, NADH-linked 4-hydroxynonenal reductase activity is strongly reduced (data not shown). The results suggest that one function for MVDP, rather than mAR, in adrenocortical cells may be detoxification for protection against endogenous harmful aldehydes, including isocaproaldehyde and 4-hydroxynonenal. The biosynthesis of steroids is acutely and chronically stimulated by trophic hormones through the intermediary of cAMP (37Simpson E.R. Waterman E.R. Annu. Rev. Physiol. 1988; 50: 427-440Crossref PubMed Scopus (425) Google Scholar). Interestingly, chronic effects of trophic hormones result from increased transcription of the genes that encode the steroidogenic enzymes (37Simpson E.R. Waterman E.R. Annu. Rev. Physiol. 1988; 50: 427-440Crossref PubMed Scopus (425) Google Scholar) as well as MVDP (9Aigueperse C. Martinez A. Lefrançois-Martinez A.-M. Veyssière G. Jean C. J. Endocrinol. 1998; 160: 147-154Crossref Scopus (38) Google Scholar), thereby maintaining optimal capacity for both steroid production and reduction of isocaproaldehyde. Immunodetection of MVDP in Leydig cell cultures 2A.-M. Lefrançois-Martinez, C. Tournaire, A. Martinez, M. Berger, S. Daoudal, D. Tritsch, G. Veyssière, and C. Jean, unpublished data. suggests that MVDP plays this role in other steroidogenic cells. We thank Dr. D. A. Carper for the gift of the rabbit anti-rat aldose reductase antiserum. We are especially grateful to Dr. Veschambre for preparing isocaproaldehyde. We thank Drs. Jean-François Biellmann, Alain Van Dorsselaer, Alberto Podjarny, Patrick Barth, Hélène Rogniaux, and Benoit Viollet for helpful discussions and advice. We thank Dr. Laurent Morel for critical reading of the text. We thank Alain Halère and Jean-Paul Saru for technical assistance." @default.
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