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Importance of Glucose-6-phosphate Dehydrogenase Activity for Cell Growth*

  • Wang-Ni Tian
    Footnotes
    Affiliations
    Renal Division and Department of Medicine, Beth Israel Deaconess Medical Center, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02115
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  • Leigh D. Braunstein
    Affiliations
    Renal Division and Department of Medicine, Beth Israel Deaconess Medical Center, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02115
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  • Jiongdong Pang
    Affiliations
    Renal Division and Department of Medicine, Beth Israel Deaconess Medical Center, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02115
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  • Karl M. Stuhlmeier
    Affiliations
    Renal Division and Department of Medicine, Beth Israel Deaconess Medical Center, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02115
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  • Qiong-Chao Xi
    Affiliations
    Renal Division and Department of Medicine, Beth Israel Deaconess Medical Center, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02115
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  • Xiaoni Tian
    Affiliations
    Renal Division and Department of Medicine, Beth Israel Deaconess Medical Center, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02115
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  • Robert C. Stanton
    Correspondence
    To whom reprint requests should be addressed: Renal Div., Joslin Diabetes Center, One Joslin Pl., Boston, MA 02215. Tel.: 617-632-0522; Fax: 617-632-1861
    Affiliations
    Renal Division and Department of Medicine, Beth Israel Deaconess Medical Center, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02115
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  • Author Footnotes
    * This work was supported in part by American Cancer Society Grant BE-131A (to R. C. S.).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.
    ‡ Recipient of National Service Research Award DK09265-02.
Open AccessPublished:April 24, 1998DOI:https://doi.org/10.1074/jbc.273.17.10609
      The intracellular redox potential, which is determined by the level of oxidants and reductants, has been shown to play an important role in the regulation of cell growth. The principal intracellular reductant is NADPH, which is mainly produced by the pentose phosphate pathway through the actions of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and by 6-phosphogluconate dehydrogenase. Previous research has suggested that an increase in G6PD activity is important for cell growth. In this article, we suggest that G6PD activity plays a critical role in cell growth by providing NADPH for redox regulation. The results show the following: 1) inhibition of G6PD activity abrogated growth factor stimulation of [3H]thymidine incorporation in all cell lines tested; 2) overexpression of G6PD stimulated cell growth, as measured by an increase in [3H]thymidine incorporations as compared with cells transfected with vector alone; 3) inhibition of G6PD caused cells to be more susceptible to the growth inhibitory effects of H2O2; 4) inhibition of G6PD led to a 30–40% decrease in the NADPH/NADP ratio; and 5) inhibition of G6PD inhibited cell anchorage and significantly decreased the growth-related stimulation of tyrosine phosphorylation.
      Intracellular redox regulation is important for the regulation of cell growth (
      • Wiese A.G.
      • Pacifici R.E.
      • Davies K.J.
      ,
      • Sundaresan M.
      • Yu Z.-X.
      • Ferrans V.J.
      • Irani K.
      • Finkel T.
      ,
      • Burdon R.H.
      • Alliangana D.
      • Gill V.
      ). A critical modulator of the redox potential is NADPH, the principal intracellular reductant. Glucose-6-phosphate dehydrogenase (G6PD),
      The abbreviations used are: G6PD, glucose-6-phosphate dehydrogenase; DHEA, dehydroepiandrosterone; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; PGD, 6-phosphogluconate dehydrogenase; PPP, pentose phosphate pathway; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; R5-P, ribose 5-phosphate; HPLC, high performance liquid chromatography.
      1The abbreviations used are: G6PD, glucose-6-phosphate dehydrogenase; DHEA, dehydroepiandrosterone; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; PGD, 6-phosphogluconate dehydrogenase; PPP, pentose phosphate pathway; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; R5-P, ribose 5-phosphate; HPLC, high performance liquid chromatography.
      the rate-limiting enzyme of the pentose phosphate pathway (PPP, Fig. 1), determines the production of NADPH by controlling the metabolism of glucose via the PPP (
      • Kletzien R.F.
      • Harris P.K.W.
      • Foellmi L.A.
      ,
      • Pandolfi P.P.
      • Sonati F.
      • Rivi R.
      • Mason P.
      • Grosveld F.
      • Luzzatto L.
      ,
      • Wood T.
      ).
      Figure thumbnail gr1
      Figure 1Diagram of the pentose phosphate pathway. The rate-limiting enzyme is G6PD. Activation of the two dehydrogenase enzymes, G6PD and PGD, results in the production of NADPH, H+ ions, and ribose 5-phosphate.
      Previous research has shown an association between the stimulation of cell growth and increased activity of the PPP that occurs over hours to days. For example, 1) kidney hypertrophy due to a variety of growth stimuli (e.g. unilateral nephrectomy or diabetes mellitus) is associated with an increased activity of the PPP due to increased G6PD activity (
      • Farquhar J.K.
      • Scott W.N.
      • Coe F.L.
      ,
      • Sochor M.
      • Kunjara S.
      • Greenbaum A.L.
      • McLean P.
      ), 2) epidermal growth factor and insulin stimulated cell growth and increased G6PD activity in rat liver cells in culture (
      • Yoshimoto K.
      • Nakamura T.
      • Ichihara A.
      ), 3) growth hormone stimulated cell growth and increased G6PD activity in rat liver cells in culture (
      • Schaefer W.T.
      ), and 4) a wide variety of cancers and cultured tumor cells exhibit large increases in G6PD activity (
      • Sulis E.
      ,
      • Weber G.
      ). These findings suggest that G6PD activity is important for cell growth.
      Research from our laboratory and others have shown that, in addition to the long term stimulation of G6PD by growth factors, there is a stimulation of G6PD activity that occurs within seconds to minutes following exposure to growth factors (
      • Tian W.-N.
      • Pignatare J.N.
      • Stanton R.C.
      ,
      • Stanton R.C.
      • Seifter J.L.
      • Boxer D.C.
      • Zimmerman E.
      • Cantley L.C.
      ,
      • Stanton R.C.
      • Seifter J.L.
      ,
      • Reed B.Y.
      • King M.T.
      • Gitomer W.L.
      • Veech R.L.
      ,
      • Conricode K.M.
      • Ochs R.S.
      ). Specifically, our laboratory has shown that following stimulation of rat renal cells to grow using epidermal growth factor (EGF), an increase in the activity of G6PD was observed within seconds, maximal at 1 min, and back to base-line level in 60 min (
      • Stanton R.C.
      • Seifter J.L.
      • Boxer D.C.
      • Zimmerman E.
      • Cantley L.C.
      ). In a search for the mechanism of this rapid activation of G6PD, we discovered that G6PD, an enzyme thought to exist unbound in the cytoplasm, is probably bound to an intracellular structure and translocates following growth factor stimulation (
      • Tian W.-N.
      • Pignatare J.N.
      • Stanton R.C.
      ,
      • Stanton R.C.
      • Seifter J.L.
      • Boxer D.C.
      • Zimmerman E.
      • Cantley L.C.
      ). Using a permeabilized cell system, we demonstrated that EGF and platelet-derived growth factor (PDGF) stimulate the release of G6PD from permeabilized cells (
      • Tian W.-N.
      • Pignatare J.N.
      • Stanton R.C.
      ,
      • Stanton R.C.
      • Seifter J.L.
      • Boxer D.C.
      • Zimmerman E.
      • Cantley L.C.
      ), We have further shown that the PDGF stimulation of G6PD translocation is dependent on tyrosine phosphorylation of the PDGF receptor (
      • Tian W.-N.
      • Pignatare J.N.
      • Stanton R.C.
      ) and is likely mediated by the signal transduction proteins phosphatidylinositol 3-kinase and phospholipase C-γ (
      • Tian W.-N.
      • Pignatare J.N.
      • Stanton R.C.
      ).
      The previous work implies that there may be a mechanistic relationship between cell growth and G6PD activity. This report is designed to more directly assess the importance of G6PD activity on cell growth. The effects of both increases in G6PD activity via overexpression of G6PD and decreases in G6PD activity via the use of a G6PD inhibitor were used to address the importance and possible roles that G6PD may play in cell growth. The data suggest that proper G6PD activity is important for regulation of intracellular redox level during cell growth. The data further suggest that G6PD activity is important for proper cell anchorage and growth factor-stimulated tyrosine phosphorylation.

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