NAD Kinase Levels Control the NADPH Concentration in Human Cells

Abstract

NAD kinases (NADKs) are vital, as they generate the cellular NADP pool. As opposed to three compartment-specific isoforms in plants and yeast, only a single NADK has been identified in mammals whose cytoplasmic localization we established by immunocytochemistry. To understand the physiological roles of the human enzyme, we generated and analyzed cell lines stably deficient in or overexpressing NADK. Short hairpin RNA-mediated down-regulation led to similar (about 70%) decrease of both NADK expression, activity, and the NADPH concentration and was accompanied by increased sensitivity toward H2O2. Overexpression of NADK resulted in a 4–5-fold increase in the NADPH, but not NADP+, concentration, although the recombinant enzyme phosphorylated preferentially NAD+. Surprisingly, NADK overexpression and the ensuing increase of the NADPH level only moderately enhanced protection against oxidant treatment. Apparently, to maintain the NADPH level for the regeneration of oxidative defense systems human cells depend primarily on NADP-dependent dehydrogenases (which re-reduce NADP+), rather than on a net increase of NADP. The stable shifts of the NADPH level in the generated cell lines were also accompanied by alterations in the expression of peroxiredoxin 5 and Nrf2. Because the basal oxygen radical level in the cell lines was only slightly changed, the redox state of NADP may be a major transmitter of oxidative stress. NAD kinases (NADKs) are vital, as they generate the cellular NADP pool. As opposed to three compartment-specific isoforms in plants and yeast, only a single NADK has been identified in mammals whose cytoplasmic localization we established by immunocytochemistry. To understand the physiological roles of the human enzyme, we generated and analyzed cell lines stably deficient in or overexpressing NADK. Short hairpin RNA-mediated down-regulation led to similar (about 70%) decrease of both NADK expression, activity, and the NADPH concentration and was accompanied by increased sensitivity toward H2O2. Overexpression of NADK resulted in a 4–5-fold increase in the NADPH, but not NADP+, concentration, although the recombinant enzyme phosphorylated preferentially NAD+. Surprisingly, NADK overexpression and the ensuing increase of the NADPH level only moderately enhanced protection against oxidant treatment. Apparently, to maintain the NADPH level for the regeneration of oxidative defense systems human cells depend primarily on NADP-dependent dehydrogenases (which re-reduce NADP+), rather than on a net increase of NADP. The stable shifts of the NADPH level in the generated cell lines were also accompanied by alterations in the expression of peroxiredoxin 5 and Nrf2. Because the basal oxygen radical level in the cell lines was only slightly changed, the redox state of NADP may be a major transmitter of oxidative stress. Recent investigations have established the pyridine nucleotides not only as key molecules for metabolic conversions, but also as critical regulators of major cellular events. In particular, NAD+ appears to act as a versatile molecule with both messenger and bioenergetic functions (1Berger F. Ramirez-Hernandez M.H. Ziegler M. Trends Biochem. Sci. 2004; 29: 111-118Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Whereas NAD is largely present in its oxidized state (NAD+), its phosphorylated counterpart, NADP, is predominantly found in its reduced form, as NADPH (2Burch H.B. Bradley M.E. Lowry O.H. J. Biol. Chem. 1967; 242: 4546-4554Abstract Full Text PDF PubMed Google Scholar, 3Veech R.L. Eggleston L.V. Krebs H.A. Biochem. J. 1969; 115: 609-619Crossref PubMed Scopus (420) Google Scholar). Indeed, the most prominent function of NADP appears to be the maintenance of a pool of reducing equivalents for metabolic systems that in one way or another protect the cell from damage. Most prominently, NADPH is essential to the regeneration of all known oxidative defense systems, such as glutathione, thioredoxin, and peroxiredoxins. Moreover, detoxifying pathways (for example, cytochromes P450 and catalase) as well as the NADPH oxidase, which catalyzes the “oxidative burst” as part of the immune response, depend on NADPH. Interestingly, the redox properties of the NAD+/NADH and NADP+/NADPH couples are similar, but their functions are largely divergent. Apparently, a major reason for this separation is the possibility to maintain one pool, namely NADP, in its reduced form to assure an immediate regeneration of the defense systems following oxidative assaults. In this role, NADPH is of vital importance, because survival following oxidative stress, which accompanies a multitude of pathological states such as inflammatory processes or ischemia/reperfusion injury, depends primarily on the capacity of the defense systems. Nevertheless, surprisingly little is known regarding the generation and maintenance of the cellular NADP pool, especially in mammalian cells. The only reaction known to yield NADP is the phosphorylation of NAD by NAD kinase (NADK). 2The abbreviations used are: NADK, NAD kinase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; PBS, phosphate-buffered saline; eGFP, enhanced green fluorescent protein. 2The abbreviations used are: NADK, NAD kinase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; PBS, phosphate-buffered saline; eGFP, enhanced green fluorescent protein. Accordingly, NAD kinase has been found to be an essential activity for cell survival (4Bieganowski P. Seidle H.F. Wojcik M. Brenner C. J. Biol. Chem. 2006; 281: 22439-22445Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 5Gerdes S.Y. Scholle M.D. D'Souza M. Bernal A. Baev M.V. Farrell M. Kurnasov O.V. Daugherty M.D. Mseeh F. Polanuyer B.M. Campbell J.W. Anantha S. Shatalin K.Y. Chowdhury S.A. Fonstein M.Y. Osterman A.L. J. Bacteriol. 2002; 184: 4555-4572Crossref PubMed Scopus (238) Google Scholar, 6Grose J.H. Joss L. Velick S.F. Roth J.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7601-7606Crossref PubMed Scopus (95) Google Scholar, 7Kobayashi K. Ehrlich S.D. Albertini A. Amati G. Andersen K.K. Arnaud M. Asai K. Ashikaga S. Aymerich S. Bessieres P. Boland F. Brignell S.C. Bron S. Bunai K. Chapuis J. Christiansen L.C. Danchin A. Debarbouille M. Dervyn E. Deuerling E. Devine K. Devine S.K. Dreesen O. Errington J. Fillinger S. Foster S.J. Fujita Y. Galizzi A. Gardan R. 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Marchuk D.A. Strand M.K. Mitochondrion. 2006; 6: 94-101Crossref PubMed Scopus (24) Google Scholar). In yeast and plants, three NADK isoforms have been identified and characterized (10Pollak N. Dölle C. Ziegler M. Biochem. J. 2007; 402: 205-218Crossref PubMed Scopus (502) Google Scholar). Both organisms possess at least one cytosolic isoform (4Bieganowski P. Seidle H.F. Wojcik M. Brenner C. J. Biol. Chem. 2006; 281: 22439-22445Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 11Chai M.F. Wei P.C. Chen Q.J. An R. Chen J. Yang S. Wang X.C. Plant J. 2006; 47: 665-674Crossref PubMed Scopus (76) Google Scholar, 12Turner W.L. Waller J.C. Snedden W.A. Biochem. J. 2005; 385: 217-223Crossref PubMed Scopus (57) Google Scholar, 13Shi F. Kawai S. Mori S. Kono E. Murata K. FEBS J. 2005; 272: 3337-3349Crossref PubMed Scopus (67) Google Scholar). In yeast, a mitochondrial NADK appears to be vital, and its deletion caused a drastically higher sensitivity toward oxidative stress (14Outten C.E. Culotta V.C. EMBO J. 2003; 22: 2015-2024Crossref PubMed Scopus (139) Google Scholar, 15Strand M.K. Stuart G.R. Longley M.J. Graziewicz M.A. Dominick O.C. 2003; PubMed Scopus Google deletion of a isoform in and M.F. Chen Q.J. An R. Chen Chen J. Wang X.C. Plant Mol. Biol. 2005; PubMed Scopus Google Scholar, H. A. Tanaka A. M. K. H. Plant 2006; 47: PubMed Scopus Google Scholar). that the of NADK the and is in yeast and plants, only a single NADK has been found in mammals F. M. A. Ziegler M. Biochem. PubMed Scopus Google Scholar). As the only NADK the of the human enzyme was and its activity characterized F. M. A. Ziegler M. Biochem. PubMed Scopus Google Scholar). in has only been to as which that its deletion is K.V. Marchuk D.A. Strand M.K. Mitochondrion. 2006; 6: 94-101Crossref PubMed Scopus (24) Google Scholar). The of NADK in has not been although to be cytosolic J.R. J. Biochem. PubMed Scopus Google Scholar). In the of only a single NADK isoform in the as to at least the and the be both a of the cellular NADP, but is for a physiological of NADP the mitochondrial in plants PubMed Scopus Google Scholar). NAD kinases from organisms toward NAD+ or as The of the reduced form to NADPH be as of by dehydrogenases be the the of a be an to maintain redox for NAD+/NADH and is the human enzyme is for the reduced or oxidized form of was that this enzyme is for as and also not of the pyridine F. M. A. Ziegler M. Biochem. PubMed Scopus Google Scholar). on the human form has the of this single NADK activity be to the cellular level of NADP and NADP-dependent pathways defense systems. To understand the of the known human NADK we have generated cell lines stably overexpressing human NADK or hairpin the NADK Overexpression of NADK resulted in an increase of both the the and the resulted in an which was accompanied by a similar decrease of the cellular NADK NADK was found to in the and preferentially NAD+ as the of NADK expression was primarily in the of the NADPH, and not NADP+, are with an essential of the cytoplasmic in the cellular level of NADPH. of expression of human NADK the was by the and and and An a was and expression of human NADK the was the and following and The generation of was to R. R. 2002; PubMed Scopus Google In against human NADK against and in or against in Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google were and the expression were the its and of cells in of essential with and were with the by the were in by another of lines were in with of with were on cells. The was and cells were in phosphate-buffered with and or for at was and the cells were for the of of cells were at on cell were with or in with and for the was by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium and for The was and the generated in The was a at is as of the cells. of of was is to cells is to and oxidized by to the fluorescent The cells were at on cell and were with in and for at the cells with PBS, they were with or in with and for The was by PBS, and the in a at an of and an of was to the and are to cells. of and were in 5 on and a for 5 at and the was to separation in the were against human NADK the or and the The against was a from N. V. The were enhanced and cells on were with PBS, with in PBS, with in PBS, and The were or and were or were an of NAD kinase activity in cell was by a (14Outten C.E. Culotta V.C. EMBO J. 2003; 22: 2015-2024Crossref PubMed Scopus (139) Google with of cell was to a reaction 5 in a of following for 5 at The of was a by the of 5 5 of yeast MTT, and of was at for 5 a and the of NADP by to known of NADK activity was by the of NADP and one was as the of enzyme of NADP in at of NAD+ or phosphorylation by the recombinant NADK, the enzyme was as F. M. A. Ziegler M. Biochem. PubMed Scopus Google and analyzed by was from the cell lines the to the and used for by to the and for the were the of were in a in for 5 and of for for and for were to as expression was PubMed Scopus Google Scholar). of and were by to of which were and a for on the were by the were a The nucleotides in the were by and by on of from cell were by their The of a an and a were in a at a of The was with of and the of the the was with for nucleotides were in a to and by a to The was by the concentration of to for 5 by with for 5 the NADK a the of human NADK, we analyzed the for the of that a The K. P. Trends Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google NADK to be The in are with this of a was to the or the was the but not as by the of and cells with the of NADK from mammalian was that the enzyme activity is rather because of expression rather than a activity F. M. A. Ziegler M. Biochem. PubMed Scopus Google Scholar). Indeed, of cells a NADK not The and of NAD kinases from has established that of both NAD+ and as to be for NAD+ in N. Dölle C. Ziegler M. Biochem. J. 2007; 402: 205-218Crossref PubMed Scopus (502) Google Scholar, G. G. N. Chem. 2006; 6: PubMed Scopus Google Scholar). As in and the human enzyme NAD+ as a of to NADPH was rather at a concentration of 5 that the of NADK with NAD+ was only with was used as at and NAD+ was present as phosphorylated Nevertheless, NAD+ was the in the of the at NAD+ was phosphorylated than that may at and that its phosphorylation is in the of NAD+. to a S. Kawai S. F. Murata K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google the of NAD kinases is with the of a or a in the to in the human enzyme was for example, for the NADK from that a of the by resulted in the of the enzyme to S. Kawai S. F. Murata K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Indeed, is a in the human enzyme with its to as of in cells stably overexpressing human NADK, which we cells. Moreover, to decrease the NADK activity, cells stably the NADK were also of the NADK were and cells were with a the expression of an which was against the of enhanced green fluorescent The of the was analyzed by of a and the by an As in of the reduced expression their for down-regulation of NADK. The a higher with the for the recombinant NADK and were generated as and and characterized for their NADK with to activity, and As in both cell lines NADK activity with cells overexpressing NADK a increase in the activity, and this was similar for all analyzed kinase activity and in cells with NADK expression NAD+ kinase activity in cell of cell lines was as and are as protein. is as NADP The are the of three S.D. NADK were by and to The of cell lines in be used the in a that the decrease of or increase in NADK activity was by the NADK and a similar alterations on the level with a decrease of NADK in the cells the down-regulation with were the cells which were cells. of the or caused of the cells was the NAD the of the of the NADK expression level on the cellular pyridine The nucleotides were by treatment. the reduced nucleotides are the oxidized to The was such that of the oxidized and of the reduced nucleotides were The cellular were analyzed by The pyridine and and was surprisingly similar in all cell lines overexpression of NADK caused a 4–5-fold increase in the NADPH the concentration of this was in the cells and were because human NADK rather NAD+ Because the concentration not the NADPH generated in the cells have from the by NADP-dependent least the the major are and of the the As be from was a decrease of expression of all three in the in the cells the expression was slightly of the activity that the of NADK activity in the established cell lines on the activity of this and in cells in NADK expression The of the cell lines were by as and S.D. of three to in a expression of reducing of the of the in and cells were by as and Interestingly, was in the cells regarding pyridine redox or the little metabolic of the reduced NADPH overexpression of NADK and the increase of the NADPH concentration were accompanied by a higher state of NADP and an increased NAD+/NADH The in the cells was also slightly The of of the critical of NADPH in oxidative defense was of to the of NADK expression and the ensuing in the NADPH concentration on the of The level of in the cell lines was the fluorescent The level was by in of NADK was accompanied by an increased with the or the of in the cells that found in the overexpression of NAD kinase reduced the from the cellular NADK or NADPH level the survival of cells following to oxidative stress. The cell lines were to or for was the treatment. As in NADK overexpression the cells and increased the survival of the although to than the in the NADPH have the cells were rather to the treatment. both and the survival of cells was similar to that of cells. The down-regulation of NADK by expression resulted in toward as by a in survival The and in the cellular oxidant state the expression level of a of of the expression expression, for example, of with functions Biol. 2004; PubMed Scopus Google Scholar). be that most cells are to oxidative stress and are and for example, by phosphorylation or of from the to the The in expression not and from stable shifts in the NADP redox analyzed the expression of because is a rather which has been to the and A. C. K. R. C. E. P. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. S. R. S. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google but also found in the H. S. S. R. S. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google to in the A. V. V. N. A. R. J. G. N. J. Biochem. PubMed Scopus Google Scholar). its expression be by redox the cells. As in and the expression level on the expression of NADK. NADK, and NADPH, was the was to of NADK with a of the cellular NADPH concentration led to an increase of the by The of expression were in a also the NADK level the expression of a that the expression of by with present in their T. Yang Biol. 2004; PubMed Scopus (420) Google Scholar). Whereas overexpression of NADK on the in the cells expression was enhanced a key of the known NADK in cells for the maintenance of the cellular NADPH pool. that this enzyme is essential for NADP the key functions of this and the major of this In to the of NADK the human found a a to the human NADK which of and human was generated from a we were to this Moreover, at least for the cell lines used not the of an NADK to the also that for NADK in human cells only a to the NADP pool. Both and down-regulation of NADK expression was by the NADK activity as well as the NADP In particular, the cells an of all the NADK the the NADK activity and the NADP a NADK from human was characterized Biochem. PubMed Scopus Google Scholar). The and properties of this enzyme were slightly from the recombinant NADK F. M. A. Ziegler M. Biochem. PubMed Scopus Google Scholar). The NADK activity, which was by and a also known for NADK or of plants and in N. Dölle C. Ziegler M. Biochem. J. 2007; 402: 205-218Crossref PubMed Scopus (502) Google Scholar). Because of the three NADK isoforms of A. is by the is to be by present in the but not the recombinant be that the enzyme for human to the NADK in this The of the of human NAD kinase the that the enzyme in the Overexpression of the NADK by led to the of the in this The possibility that the human NADK which be by the but be to be for example, to for the generation of the mitochondrial pool of NADP. In of NAD kinases the activity was to the cytosolic J.R. J. Biochem. PubMed Scopus Google Scholar). is for an of NADP the and the in mammalian although has been for cells PubMed Scopus Google Scholar). the cytosolic NADK expression and the cellular NADPH have an on expression, a which is primarily but also in A. C. K. R. C. E. P. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. S. R. S. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. V. V. N. A. R. J. G. N. J. Biochem. PubMed Scopus Google Scholar). The increase in the cellular NADPH concentration by NADK overexpression caused a stable down-regulation of expression, the decrease of NADPH resulted in an increased expression level of that as a redox which is to the of the regeneration of the oxidative defense systems, NADPH. be to the NADK, that NADPH to is for a of example, the the expression of the on has or not Wang S.Y. C. D. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). such has been for NADPH. the of expression also to a the cellular NADPH and of has been established as a of oxidative stress, is to the to expression of T. Yang Biol. 2004; PubMed Scopus (420) Google Scholar). of is only a oxidative stress. NADPH by NADK with only a increase of expression was found to be be to the NADPH and expression and also a in the NADP redox is to In may expression A. V. J. A. V. N. FEBS J. 2006; PubMed Scopus Google which is in with at least in the cells. In this the of the and cells a to expression which depends on the of the NADP redox in the of or to oxidative stress. An to the of NADK on the NADPH concentration and the maintenance of defense Overexpression or down-regulation of NADK the of NADPH in the the rather concentration or analyzed this have been for NAD kinases that as we found that the human enzyme is rather to physiological The enzyme to NADPH, but than phosphorylated NAD+. Moreover, the cellular of NAD+ by that of appears that generated by NADK is reduced to NADPH by the expression of the three of the both at an or expression level of NADK. that the capacity of the dehydrogenases is to the generated the the increase of NADPH was only 4–5-fold that this an appears similar to the enzyme from J.H. Joss L. Velick S.F. Roth J.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7601-7606Crossref PubMed Scopus (95) Google human NADK be by NADPH phosphorylation of NAD+ or may also NADK activity the NADPH concentration physiological overexpression of dehydrogenases resulted in an to and increase in activity for mitochondrial M.K. J.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and F. A. A. S. S. M.V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google with the increase of NADK activity in the generated overexpressing cell lines is and not to have that NADK activity be enhanced of oxidative stress to the cellular defense In of this or with of cells of the and O. C. D. M. Mol. 2005; PubMed Scopus Google Scholar). Moreover, yeast cells for were toward oxidative stress (14Outten C.E. Culotta V.C. EMBO J. 2003; 22: 2015-2024Crossref PubMed Scopus (139) Google Scholar, 15Strand M.K. Stuart G.R. Longley M.J. Graziewicz M.A. Dominick O.C. 2003; PubMed Scopus Google Scholar). increase of human NADK activity or expression be or cells were to the rather overexpression of NADK in cells only the NADPH concentration been increased be to oxidative the cells rather on the NADP pool and the capacity of in a reduced state by the activity of NADP-dependent Indeed, NADP-dependent dehydrogenases a prominent in the to oxidative stress. NADPH from NADP+, the of the which the reduced pool of in for have that dehydrogenases are of oxidative stress in human and cells M.K. J.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. J.W. J. Biol. 2004; PubMed Scopus (45) Google Scholar, O. H. Biochem. 2004; PubMed Scopus Google Scholar, M.V. A. G. S. G. Biochem. J. PubMed Scopus Google Scholar, V. 2005; PubMed Scopus Google Scholar). the reason to not increase the NADP concentration is not that an increase of the capacity to a increase in NADPH a similar of toward oxidative stress. may well be that the rather of protection is caused by the of the NADP-dependent dehydrogenases in the cells. the of NADPH regeneration is critical than the concentration of the NADPH. the of the NADP pool the as by the higher sensitivity of the cells toward for the of to the of this and for The by as well as the of by are are also to N. V. for and