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Hypoxia Induces Phosphorylation of the Cyclic AMP Response Element-binding Protein by a Novel Signaling Mechanism*

  • Dana Beitner-Johnson
    Correspondence
    To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, University of Cincinnati College of Medine, P.O. Box 67-0576, Cincinnati, OH 45267-0576. Tel.: 513-558-6009; Fax: 513-558-5738;
    Affiliations
    Department of Cellular and Molecular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576
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  • David E. Millhorn
    Affiliations
    Department of Cellular and Molecular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant RH37 L 33831 (to D. E. M.), an American Cancer Society grant (to D. B. J.), and a Parker B. Francis Foundation fellowship (to D. B. J.).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.
      To investigate signaling mechanisms by which hypoxia regulates gene expression, we examined the effect of hypoxia on the cyclic AMP response element-binding protein (CREB) in PC12 cells. Exposure to physiological levels of hypoxia (5% O2, ∼50 mm Hg) rapidly induced a persistent phosphorylation of CREB on Ser133, an event that is required for CREB-mediated transcriptional activation. Hypoxia-induced phosphorylation of CREB was more robust than that induced by any other stimulus tested, including forskolin, depolarization, and osmotic stress. Furthermore, this effect was not mediated by any of the previously known signaling pathways that lead to phosphorylation of CREB, including protein kinase A, calcium/calmodulin-dependent protein kinase, protein kinase C, ribosomal S6 kinase-2, and mitogen-activated protein kinase-activated protein kinase-2. Hypoxic activation of a CRE-containing reporter (derived from the 5′-flanking region of the tyrosine hydroxylase gene) was attenuated markedly by mutation of the CRE. Thus, a physiological reduction in O2 levels induces a functional phosphorylation of CREB at Ser133 via a novel signaling pathway.
      Hypoxic/ischemic trauma is a primary factor in the pathology of many disease states. Even brief periods of localized oxygen deprivation can result in severe cellular and tissue damage, such as that produced by cerebral or myocardial infarction. Severe hypoxia results in depletion of cellular ATP levels and cessation of oxidative phosphorylation, which results in profound deficiencies in cellular function (
      • Gibson G.E.
      • Pulsinelli W.
      • Blass J.P.
      • Duffy T.E.
      ). It is therefore not surprising that sophisticated mechanisms have evolved which allow the cell to adapt to moderate levels of hypoxia long before ATP depletion occurs (
      • Hochachka P.W.
      • Buck L.T.
      • Doll C.J.
      • Land S.C.
      ,
      • Guillemin K.
      • Krasnow M.A.
      ). In recent years, the mechanisms underlying adaptation of mammalian cells to hypoxia have begun to be elucidated. A major component of this adaptation is regulation of gene expression. A number of genes have been identified, including erythropoietin, vascular endothelial growth factor, hypoxia-inducible factor-1, and tyrosine hydroxylase (TH),
      The abbreviations used are: TH, tyrosine hydroxylase; CRE, cyclic AMP response element; CREB, cyclic AMP response element-binding protein; PKA, protein kinase A; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl; MEK, MAP/ERK kinase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; RSK, ribosomal S6 kinase; MAPKAP kinase-2, mitogen-activated protein kinase-activated protein kinase-2.
      1The abbreviations used are: TH, tyrosine hydroxylase; CRE, cyclic AMP response element; CREB, cyclic AMP response element-binding protein; PKA, protein kinase A; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl; MEK, MAP/ERK kinase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; RSK, ribosomal S6 kinase; MAPKAP kinase-2, mitogen-activated protein kinase-activated protein kinase-2.
      which are involved in the adaptive response to hypoxia (
      • Semenza G.L.
      • Wang G.L.
      ,
      • Goldberg M.A.
      • Schneider T.J.
      ,
      • Norris M.L.
      • Millhorn D.E.
      ,
      • Bunn H.F.
      • Poyton R.O.
      ). However, the primary mechanism(s) by which cells sense changes in oxygen levels and transduce this signal into the molecular events associated with changes in gene expression remain unknown.
      Rat pheochromocytoma (PC12) cells are a catecholaminergic cell line that has proven to be a useful system to study hypoxia-regulated gene expression. An acute reduction in oxygen tension triggers a variety of cellular responses in PC12 cells, including depolarization (
      • Zhu W.H.
      • Conforti L.
      • Czyzyk-Krzeska M.F.
      • Millhorn D.E.
      ) and inhibition of an oxygen-sensitive K+ channel conductance (
      • Conforti L.
      • Millhorn D.E.
      ). Prolonged (>3 h) exposure to hypoxia also leads to an induction of TH gene expression and mRNA stability (
      • Czyzyk-Krzeska M.F.
      • Furnari B.A.
      • Lawson E.E.
      • Millhorn D.E.
      ) and a stimulation of the immediate early genes c-fos and junB (
      • Norris M.L.
      • Millhorn D.E.
      ) in PC12 cells. The specific intracellular signaling pathways by which reduced oxygen stimulates expression of these genes are not understood.
      Multiple signaling pathways converge at the level of the cyclic AMP response element-binding protein (CREB), a transcription factor that regulates expression of CRE-containing genes (for reviews, see Refs.
      • Frank D.A.
      • Greenberg M.E.
      and ). CREB mediates cellular responses to a variety of physiological signals, including neurotransmitters, depolarization, synaptic activity, mitogenic and differentiative factors, and stressors (
      • Sheng M.
      • McFadden G.
      • Greenberg M.E.
      ,
      • Ginty D.D.
      • Bonni A.
      • Greenberg M.E.
      ,
      • Bonni A.
      • Ginty D.D.
      • Dudek H.
      • Greenberg M.E.
      ,
      • Deisseroth K.
      • Bito H.
      • Tsien R.W.
      ,
      • Pende M.
      • Fisher T.L.
      • Simpson P.B.
      • Russell J.T.
      • Blenis J.
      • Gallo V.
      ,
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ,
      • Iordanov M.
      • Bender K.
      • Ade T.
      • Schmid W.
      • Sachsenmaier C.
      • Engel K.
      • Gaestel M.
      • Rahmsdorf H.J.
      • Herrlich P.
      ). Upon phosphorylation at Ser133, CREB can facilitate transcriptional activation of genes containing the CRE motif (
      • Gonzalez G.A.
      • Montminy M.R.
      ). Several protein kinases, including protein kinase A; calcium/calmodulin-dependent protein kinase-I, -II, and -IV; protein kinase C; RSK-2 (also termed MAPKAP kinase-1β); and MAPKAP kinase-2, have been shown to mediate phosphorylation of CREB (
      • Ginty D.D.
      • Bonni A.
      • Greenberg M.E.
      ,
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ,
      • Yamamoto K.K.
      • Gonzalez G.A.
      • Biggs III, W.H.
      • Montminy M.R.
      ,
      • Sheng M.
      • Thompson M.A.
      • Greenberg M.E.
      ,
      • Xie H.
      • Rothstein T.L.
      ,
      • Xing J.
      • Ginty D.D.
      • Greenberg M.E.
      ). Moreover, phosphorylation of CREB at Ser133 regulates expression of the c-fos, somatostatin, and TH genes in PC12 cells (
      • Sheng M.
      • McFadden G.
      • Greenberg M.E.
      ,
      • Gonzalez G.A.
      • Montminy M.R.
      ,
      • Sheng M.
      • Thompson M.A.
      • Greenberg M.E.
      ,
      • Nagamoto-Combs K.
      • Piech K.M.
      • Best J.A.
      • Sun B.
      • Tank A.W.
      ). Given the role for CREB in regulating genes that mediate a multitude of cellular responses, we investigated whether physiological levels of hypoxia regulate CREB phosphorylation and function.

      EXPERIMENTAL PROCEDURES

       Cell Culture

      All tissue culture reagents were obtained from Life Technologies, Inc. PC12 cells, obtained from the American Type Culture Collection, were cultured in Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 15 mmHEPES, pH 7.4, 10% fetal bovine serum, and with penicillin (100 units/ml) and streptomycin (100 μg/ml). PKA-deficient (123.7) PC12 cells, kindly provided by Dr. J. A. Wagner (Cornell University Medical College, New York), were grown in high glucose Dulbecco's modified Eagle's medium supplemented with 15 mm HEPES, pH 7.4, 10% fetal bovine serum, 5% heat-inactivated horse serum, and 0.1 mg/ml G418. To compare wild-type PC12 cells with 123.7 cells, PC12 cells were grown in the same media in the absence of G418. When cells reached 85–90% confluence in 35-mm tissue culture dishes (Corning), they were exposed either to continued normoxia or placed in an oxygen-regulated incubator (Forma Scientific, Marietta, OH) in an environment of 5% O2, 5% CO2, balanced with N2, for various times. In previous studies, we have shown that the partial pressure of oxygen in the media of cells exposed to 5% O2is in the range of 30–50 mm Hg (
      • Czyzyk-Krzeska M.F.
      • Furnari B.A.
      • Lawson E.E.
      • Millhorn D.E.
      ). For experiments in which cells were pretreated with drugs or vehicle, cells were switched to serum-free Dulbecco's modified Eagle's medium and Ham's F-12 medium containing either drugs at the indicated concentrations, or the corresponding vehicle, for the indicated times before the start of hypoxia.

       Immunoblotting

      For immunoblotting analysis, cells were harvested by scraping in 0.25 ml of 1% SDS and were sonicated briefly with a microtip ultrasonic cell disruptor (Kontes, Vineland, NJ). In some experiments, samples were solubilized in an ice-cold nondenaturing lysis buffer containing 10 mm Tris, pH 7.4, 1% Triton X-100, 0.2 mm sodium vanadate, 10 mm sodium fluoride, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin. In these experiments, lysates were centrifuged 10 min at 14,000 ×g at 4 °C to remove any Triton-insoluble material. Both solubilization protocols gave similar experimental results. Samples containing 50–100 μg of protein were then run on 9% SDS-polyacrylamide gels (Protogel, National Diagnostics, Atlanta, GA) and transferred to nitrocellulose membranes (Schleicher & Schuell) using standard electrophoresis and electroblotting procedures. Nitrocellulose membranes were blocked with 3% nonfat dry milk in a buffer containing 10 mm sodium phosphate, pH 7.2, 140 mm NaCl, and 0.1% Tween 20. Blots were then imunolabeled overnight at 4 °C with antibodies specific for either Ser133 phospho-CREB (1:1,000, New England Biolabs, Beverly, MA) or which recognize equally phospho- and dephospho-CREB (1:1,000, New England Biolabs). Immunolabeling was detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) according to the manufacturer's recommended conditions. In some cases, blots were stripped and reprobed with another antibody. Blots were stripped by incubation for 1 h at 50 °C in a solution containing 62.5 mm Tris-HCl, pH 6.7, 2% SDS, and 0.7% β-mercaptoethanol. Blots were then washed for 1 h in several changes of phosphate-buffered saline/Tween 20 at room temperature and probed with ECL to confirm that antibodies had been removed completely. Blots were then reblocked and immunolabeled as described above. Immunoreactivity was quantified using densitometric analysis with an ImagePro digital analysis system (Media Cybernetics, Silver Spring, MD). Immunoreactivity for CREB was found to be linear over a 5-fold range of protein concentrations.

       CAT Assays

      The TH-CAT reporter plasmids (−272TH)CAT and (−272CRE)CAT (
      • Yoon S.O.
      • Chikaraishi D.M.
      ), were generously provided by Dr. Dona Chikaraishi (Duke University, Durham, NC). PC12 cells were transfected in 35-mm dishes with 2 μg of either (−272TH)CAT or (−272CRE)CAT using LipofectAMINE, using the manufacturer's recommended conditions (Life Technologies, Inc.). Beginning 24 h after transfection, cells were exposed for 24 h to either hypoxia (5% O2) or normoxia (21% O2). Cells were then lysed in 0.5 ml, and samples containing 120 μg of protein were analyzed in duplicate for CAT levels by enzyme-linked immunosorbent assay (Boehringer Mannheim, GmBH). Data are normalized per total μg of protein in each sample. In each experiment, cells were transfected in quadruplicate dishes, and duplicate samples were analyzed for CAT activity. In each experiment, the variance in the range of CAT values was less than 15% from the mean value.

       PKA Enzyme Assays

      After exposure to hypoxia or normoxia, cells were assayed for PKA enzyme activity exactly as described previously (
      • Beitner-Johnson D.
      • Leibold J.
      • Millhorn D.E.
      ). PKA specific activity was calculated as the difference in 8-bromo-cAMP-stimulated phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and phosphorylation found in the presence of PKI6–22, a specific inhibitor of PKA. 8-Bromo-cAMP-stimulated activity averaged 20-fold higher than that observed in the presence of PKI6–22. In each experiment, PKA activity levels were normalized per μg of protein. PKA enzyme activity was found to be linear over a 30-fold range of protein concentrations (between 0.5 and 15 μg of protein/assay).

       MAPKAP Kinase-2 Enzyme Assays

      PC12 cells were grown to 90% confluence in 100-mm dishes. The medium was replaced with serum-free medium, and cells were exposed for various times, between 20 min and 6 h, to hypoxia (5% O2) or 300 mmsorbitol. In some experiments, cells were pretreated for 1 h with 20 μm SB203580 (kindly provided by Dr. John C. Lee, Smith Kline Beecham, King of Prussia, PA) before exposure to sorbitol. Cells were harvested by scraping in 0.4 ml of an ice-cold nondenaturing lysis buffer containing 10 mm Tris, pH 7.4, 1% Triton X-100, 0.2 mm sodium vanadate, 10 mm sodium fluoride, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin. Lysates were incubated 20 min at 4 °C and then centrifuged 10 min at 14,000 ×g to remove Triton-insoluble material. Aliquots containing 1 mg of total protein were immunoprecipitated for 2 h at 4 °C with 5 μg of an anti-MAPKAP kinase-2 polyclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY) coupled to protein G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). MAPKAP kinase-2 enzyme activity was then assayed in an immunocomplex kinase assay, using 10 μg of a specific MAPKAP kinase-2 substrate peptide from a MAPKAP kinase-2 immunoprecipitation kinase kit, exactly as described in the manufacturer's recommended protocol (Upstate Biotechnology).

      RESULTS

       Effect of Hypoxia on CREB Phosphorylation

      PC12 cells were exposed to hypoxia (5% O2) for various times between 0 and 24 h, as shown in Fig. 1. Whole cell extracts were immunolabeled with an antibody specific for Ser133-phosphorylated CREB. Hypoxia induced a dramatic increase in phospho-CREB immunoreactivity (Fig. 1 A). The effect peaked at 6 h but persisted strongly at 24 h of exposure to hypoxia. This effect was not caused by changes in the total amount of CREB, as shown in Fig. 1 B, where cell extracts were immunolabeled with an antibody that recognizes phospho- and dephospho-CREB equally. The effect of osmotic stress on CREB phosphorylation was also examined. In Fig. 1 C, it can be seen that exposure of PC12 cells to 300 mm sorbitol also rapidly induces Ser133 phosphorylation of CREB. Unlike hypoxia, which required several hours to produce a maximal effect, sorbitol-stimulated CREB phosphorylation was maximal at the earliest time point examined (20 min). Exposure of SK-N-MC cells to arsenite, another stressor, has been reported to induce CREB phosphorylation (
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ).
      Figure thumbnail gr1
      Figure 1Effect of hypoxia on CREB phosphorylation in PC12 cells. PC12 cells were exposed to either hypoxia (5% oxygen) or normoxia (controls, 21% oxygen) for various times, between 0 and 24 h, as indicated. Whole cell lysates were immunolabeled with an antibody that specifically recognizes Ser133 phospho-CREB (panel A) or an antibody that equally recognizes phospho- and dephospho-CREB (panel B) as described under “Experimental Procedures.” Panel C, phospho-CREB immunoreactivity in PC12 cells exposed to 300 mm sorbitol for various times between 0 and 6 h. Representative immunoblots are shown, with n = 3–12 dishes of cells at each time point, performed in at least two separate experiments.
      To evaluate the relative effect of hypoxia on CREB phosphorylation, we compared the ability of hypoxia to stimulate phosphorylation of CREB with that of forskolin and KCl-induced depolarization. As shown in Fig.2, hypoxia induced a robust Ser133 phosphorylation of CREB, with an average effect greater than that produced by either forskolin- or KCl-induced depolarization, two prototypical stimuli used to activate CREB.
      Figure thumbnail gr2
      Figure 2Effect of hypoxia versusforskolin and depolarization-induced phosphorylation of CREB. PC12 cells were exposed to either normoxia (C), hypoxia (H, 5% O2 for 6 h), forskolin (FK, 10 μm for 1 h), or KCl (60 mm, for 30 min). Whole cell lysates were immunolabeled with anti-phospho-CREB, as described. Panel A, representative immunoblot comparing the effect of hypoxia, forskolin, and KCl on phospho-CREB immunoreactivity. Panel B, levels of phospho-CREB immunoreactivity as quantitated by densitometric analysis. Data are expressed as the average percent of control ± S.E. and represent n = 5 individual dishes in each group, each performed in two separate experiments.
      Multiple protein kinases are known to phosphorylate CREB at Ser133, including PKA (
      • Gonzalez G.A.
      • Montminy M.R.
      ). To determine whether the induction of CREB phosphorylation was mediated by PKA, we tested the effect of hypoxia on CREB phosphorylation in PKA-deficient PC12 cells (123.7) (
      • Ginty D.D.
      • Glowacka D.
      • DeFranco C.
      • Wagner J.A.
      ). These cells express mutant regulatory subunits of PKA which constitutively inactivate the catalytic activity of both type I and type II PKA (
      • Ginty D.D.
      • Glowacka D.
      • DeFranco C.
      • Wagner J.A.
      ). PKA enzyme assays confirmed that 123.7 cells had negligible 8-bromo-cAMP-stimulated PKA activity compared with parental PC12 cells (Table I). PKA-deficient PC12 cells were exposed to hypoxia (5% O2) for 0, 1, 6, or 24 h. We found that the effect of hypoxia on CREB phosphorylation persisted in the absence of PKA (Fig. 3); that is, hypoxia induced an increase in phospho-CREB immunoreactivity (Fig. 3 A) but did not alter total CREB immunoreactivity (Fig. 3 B). It can also be seen in Fig. 3 A that two major CREB-immunoreactive proteins were detected in 123.7 cells. The lower band corresponds to ATF-1, a structurally related CRE-binding transcription factor that cross-reacts with this antibody (
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ). Interestingly, phosphorylation of ATF-1 was also induced by hypoxia.
      Table IPKA activity in PKA-deficient (123.7) PC12 cells versus parental PC12 cells
      Cells+ 8-Bromo-cAMP (10 μm)+ PKI (10 μm)
      pmol/min/mg proteinpmol/min/mg protein
      PC12 (parental)72 ± 153.8 ± 0.8
      PC12 (123.7)3.2 ± 0.92.5 ± 0.2
      Parental and 123.7 PC12 cells were grown and assayed for PKA activity as described under “Experimental Procedures.” The data shown are from a single experiment and are expressed as average enzyme activity ± S.E. and represent four dishes in each group.
      Figure thumbnail gr3
      Figure 3CREB phosphorylation by hypoxia persists in PKA-deficient (123.7) PC12 cells. 123.7 cells were exposed either to hypoxia (5% oxygen) or normoxia (controls, 21% oxygen) for 0, 1, 6, or 24 h, as indicated. Whole cell lysates were immunolabeled either with an antibody specific for Ser133 phospho-CREB (panel A) or with an antibody that equally recognizes phospho- and dephospho-CREB (panel B), as described under “Experimental Procedures.” Representative immunoblots are shown, with n = 4 dishes of cells at each time point analyzed in each of three separate experiments.
      Ca2+-dependent protein kinases, including calcium/calmodulin-dependent protein kinase- I, -II, and -IV and PKC, are also known to stimulate phosphorylation of CREB (
      • Yamamoto K.K.
      • Gonzalez G.A.
      • Biggs III, W.H.
      • Montminy M.R.
      ,
      • Sheng M.
      • Thompson M.A.
      • Greenberg M.E.
      ,
      • Matthews R.P.
      • Guthrie C.R.
      • Wailes L.M.
      • Zhao X.
      • Means A.R.
      • McKnight G.S.
      ,
      • Sun P.
      • Enslen H.
      • Myung P.
      • Maurer R.A.
      ). To test whether a Ca2+-dependent protein kinase mediates phosphorylation of CREB by hypoxia, PC12 cells were exposed for 6 h to either normoxia (21% O2) or hypoxia (5% O2) in the presence or absence of Ca2+. In Fig.4 A, PC12 cells were preloaded with 100 μm BAPTA-AM, a membrane-permeable Ca2+ chelator, in Ca2+-free medium supplemented with 1 mm EGTA and were then exposed to hypoxia. The hypoxia-induced phosphorylation of CREB persists in the absence of both extracellular and intracellular Ca2+ (Fig. 4 A). Identical results were obtained when cells were exposed to hypoxia in the absence of only extracellular Ca2+ (i.e.Ca2+-free medium supplemented with 1 mm EGTA but without preloading cells with BAPTA-AM; data not shown). Pretreatment of cells with either chelerythrine chloride or Ro 31-8220, two inhibitors of PKC, also failed to inhibit hypoxia-induced phosphorylation of CREB (Fig. 4, B and C). Both of these drugs were found to inhibit PKC enzyme activity effectively in PC12 cell extracts (data not shown). Nerve growth factor-induced phosphorylation of CREB has been shown to be mediated by RSK-2 (
      • Ginty D.D.
      • Bonni A.
      • Greenberg M.E.
      ,
      • Xing J.
      • Ginty D.D.
      • Greenberg M.E.
      ). It has been shown recently that in addition to its inhibitory effects on PKC, Ro 31-8220 is also a potent inhibitor of RSK-2 and p70S6K (
      • Alessi D.R.
      ). Neither Ro 31-8220 nor PD-098059, a selective inhibitor of activation of MEK, which is an upstream activator of RSK-2 (
      • Alessi D.R.
      • Cuenda A.
      • Cohen P.
      • Dudley D.T.
      • Saltiel A.
      ), affected the ability of hypoxia to induce phosphorylation of CREB (Fig. 4, C and D), although these drugs did block nerve growth factor-stimulated CREB phosphorylation (data not shown). Similarly, hypoxia-induced phosphorylation persisted in the presence of rapamycin (Fig. 4 E), an inhibitor of p70S6K activation (
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ). It has also been shown that MAPKAP kinase-2, an enzyme that lies downstream of the stress-activated protein kinase p38/RK (
      • Stokoe D.
      • Campbell D.G.
      • Nakeilny S.
      • Hidaka H.
      • Leevers S.J.
      • Marshall C.
      • Cohen P.
      ,
      • Rouse J.
      • Cohen P.
      • Trigon S.
      • Morange M.
      • Alonso-Llamazares A.
      • Zamanillo D.
      • Hunt T.
      • Nebreda A.R.
      ,
      • Cuenda A.
      • Rouse J.
      • Doza Y.N.
      • Meier R.
      • Cohen P.
      • Gallagher T.F.
      • Young P.R.
      • Lee J.C.
      ), can phosphorylate CREB in vitro and that fibroblast growth factor or arsenite-induced CREB phosphorylation in SK-N-MC cells can be blocked by the selective p38 inhibitor SB203580 (
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ). However, SB203580 also did not inhibit phosphorylation of CREB by hypoxia (Fig. 4 F), although SB203580 did inhibit completely sorbitol-induced stimulation of MAPKAP kinase-2 activity in PC12 cells (see below). Hypoxia-induced phosphorylation of CREB was also not attenuated by either wortmannin or LY294002, two inhibitors of phosphatidylinositol 3-kinase (data not shown). Finally, there is some evidence that cGMP-dependent protein kinase (protein kinase G) can, under certain conditions, mediate CREB phosphorylation in vitro (
      • Colbran J.L.
      • Roach P.J.
      • Fiol C.J.
      • Dixon J.E.
      • Andrisani O.M.
      • Corbin J.D.
      ,
      • Gudi T.
      • Huvar I.
      • Meinecke M.
      • Lohmann S.M.
      • Boss G.R.
      • Pilz R.B.
      ). Hypoxia-induced phosphorylation of CREB persisted strongly in the presence of each of the three inhibitors of protein kinase G, including KT-5823, methylene blue, and 8-bromo-cGMP (data not shown).
      Figure thumbnail gr4
      Figure 4CREB phosphorylation by hypoxia does not require Ca2+, PKC, RSK-2, or p38. Cells were pretreated with various drugs or vehicle (0.1% dimethyl sulfoxide) as indicated. Cells were then exposed to either normoxia (C, 21% O2) or hypoxia (H, 5% O2) for 6 h, and whole cell lysates were immunolabeled with an antibody specific for Ser133 phospho-CREB, as described. Panel A, cells were preincubated in serum-free medium in either (−) the presence of Ca2+ [Ca2+]free = 1 mm and vehicle or (+) serum-free in the absence of Ca2+ (Ca2+-free medium supplemented with 1 mm EGTA and 100 μm BAPTA-AM) for 40 min. After 40 min, the external Ca2+-containing (−) or Ca2+-free (+) medium was replaced (not including vehicle or BAPTA-AM), and cells were exposed to either normoxia or hypoxia as described. Panel B, cells were pretreated for 40 min in serum-free medium with either (−) vehicle or (+) 20 μmchelerythrine chloride (CHL) and exposed to either normoxia or hypoxia as described. Panel C, cells were pretreated for 40 min in serum-free medium with either (−) vehicle or (+) 0.3 μm Ro 31-8220 and exposed to either normoxia or hypoxia as described. Panel D, cells were pretreated for 40 min in serum-free medium with either (−) vehicle or (+) 50 μmPD-098059 and exposed to either normoxia or hypoxia as described. Panel E, cells were pretreated for 40 min in serum-free medium with either (−) vehicle or (+) 10 nm rapamycin and exposed to either normoxia or hypoxia as described. Panel F, cells were pretreated for 1 h in serum-free medium with either (−) vehicle or (+) 20 μm SB 203580 and exposed to either normoxia or hypoxia as described. In all of the experiments shown above, hypoxia did not alter total levels of CREB, as determined by immunolabeling samples with an antibody that equally recognizes phospho- and dephospho-CREB (data not shown). Each experiment was performed at least two times, and the results shown are representative of those obtained in n = 6–12 dishes in each group. Note that the apparent differences in basal levels of phospho-CREB in the various panels result from differences in exposure time of the blots to film.

       Effect of Hypoxia and Osmotic Stress on MAPKAP Kinase-2

      Various stressors, including sodium arsenite, osmotic stress, UV light, and inflammatory cytokines, have been shown to activate MAPKAP kinase-2 via a p38-dependent pathway (
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ,
      • Iordanov M.
      • Bender K.
      • Ade T.
      • Schmid W.
      • Sachsenmaier C.
      • Engel K.
      • Gaestel M.
      • Rahmsdorf H.J.
      • Herrlich P.
      ,
      • Rouse J.
      • Cohen P.
      • Trigon S.
      • Morange M.
      • Alonso-Llamazares A.
      • Zamanillo D.
      • Hunt T.
      • Nebreda A.R.
      ,
      • Cuenda A.
      • Rouse J.
      • Doza Y.N.
      • Meier R.
      • Cohen P.
      • Gallagher T.F.
      • Young P.R.
      • Lee J.C.
      ). Furthermore, at least two isoforms of p38, stress-activated protein kinase-3 (SAPK-3) and SAPK-4, have been identified which are not sensitive to inhibition by SB203580 (
      • Cuenda A.
      • Cohen P.
      • Buee-Scherrer V.
      • Goedert M.
      ,
      • Kumar S.
      • McDonnell P.C.
      • Gum R.J.
      • Hand A.T.
      • Lee J.C.
      • Young P.R.
      ). Thus, we considered the possibility that MAPKAP kinase-2 could mediate hypoxia-induced phosphorylation of CREB via a member of the p38 family which was not sensitive to SB203580. PC12 cells were exposed for various times between 0 and 6 h to 5% O2 or for 20 min to 300 mm sorbitol. MAPKAP kinase-2 was immunoprecipitated, and enzyme activity was assayed in an immune complex kinase assay. As shown in Fig. 5, hypoxia had no effect on MAPKAP kinase-2 enzyme activity, whereas MAPKAP kinase-2 was strongly activated by sorbitol. Activation of MAPKAP kinase-2 by sorbitol was blocked completely by pretreatment of cells with SB203580. Taken together with the fact that hypoxia-induced phosphorylation of CREB was not inhibited by SB203580 (Fig.4 F), these results show that the effects of hypoxia on CREB are clearly not mediated by either MAPKAP kinase-2 or another p38-dependent protein kinase. Thus, it appears that a novel stress-activated signaling pathway (independent of MAPKAP kinase-2) exists and that this pathway mediates robust phosphorylation of CREB in response to hypoxia in PC12 cells.
      Figure thumbnail gr5
      Figure 5Lack of effect of hypoxia on MAPKAP kinase-2 activity. PC12 cells were pretreated for 1 h with either 20 μm SB203580 or vehicle (0.1% dimethyl sulfoxide) in serum-free medium. Cells were then exposed to either hypoxia (5% O2) for various times, as indicated, or 300 mmsorbitol for 20 min, in the presence or absence of 20 μmSB203580. MAPKAP kinase-2 was then immunoprecipitated from whole cell lysates, and MAPKAP kinase-2 enzyme activity was measured in an immunocomplex kinase assay as described under “Experimental Procedures.” The data shown were obtained in one experiment, with similar results obtained in a separate experiment.

       Role of the CRE in Hypoxia-stimulated Gene Expression

      Phosphorylation of CREB on Ser133 is necessary, but not sufficient, to activate CREB-mediated transcription (,
      • Gonzalez G.A.
      • Montminy M.R.
      ,
      • Sun P.
      • Enslen H.
      • Myung P.
      • Maurer R.A.
      ,
      • Sun P.
      • Lou L.
      • Maurer R.A.
      ). Therefore, we next asked whether the Ser133 phosphorylation of CREB resulted in functional activation of a hypoxia-regulated gene. Expression of the TH gene has been shown to be activated in response to physiological levels of hypoxia both in vivo (
      • Czyzyk-Krzeska M.F.
      • Bayliss D.A.
      • Lawson E.E.
      • Millhorn D.E.
      ) and in vitro (
      • Czyzyk-Krzeska M.F.
      • Furnari B.A.
      • Lawson E.E.
      • Millhorn D.E.
      ). The rat TH gene contains a consensus CRE localized at −45 to −38 nucleotides relative to the transcriptional start site (
      • Fung B.F.
      • Yoon S.O.
      • Chikaraishi D.M.
      ). To test for a possible role for CREB in activation of the TH gene by hypoxia, we compared the effect of hypoxia on a TH reporter plasmid (−272TH)CAT and a similar construct in which the CRE had been mutated, (−272CRE)CAT (
      • Yoon S.O.
      • Chikaraishi D.M.
      ,
      • Fung B.F.
      • Yoon S.O.
      • Chikaraishi D.M.
      ). Hypoxia activated wild-type TH-CAT activity by approximately 3-fold, an effect similar to that of forskolin or KCl on this construct (
      • Nagamoto-Combs K.
      • Piech K.M.
      • Best J.A.
      • Sun B.
      • Tank A.W.
      ,
      • Fung B.F.
      • Yoon S.O.
      • Chikaraishi D.M.
      ,
      • Lazaroff M.
      • Patankar S.
      • Yoon S.O.
      • Chikaraishi D.M.
      ). As others have reported previously (
      • Nagamoto-Combs K.
      • Piech K.M.
      • Best J.A.
      • Sun B.
      • Tank A.W.
      ,
      • Lazaroff M.
      • Patankar S.
      • Yoon S.O.
      • Chikaraishi D.M.
      ), we found that mutation of the CRE resulted in a significant inhibition (∼3-fold) of basal levels of TH-CAT reporter activity (Fig. 6). We also found that the stimulation of TH-CAT reporter activity by hypoxia was attenuated, although not ablated, in (−272CRE)CAT compared with wild-type (−272TH)CAT, as shown in Fig. 6. Thus, a CREB-CRE interaction is involved in stimulation of TH gene expression by hypoxia. The fact that some transcriptional activation persisted in (−272CRE)CAT is consistent with previous findings, which showed that an AP1 element located at −199/205 also plays a critical role in mediating hypoxia- and depolarization-induced activation of TH gene expression (
      • Norris M.L.
      • Millhorn D.E.
      ,
      • Nagamoto-Combs K.
      • Piech K.M.
      • Best J.A.
      • Sun B.
      • Tank A.W.
      ), as discussed further below.
      Figure thumbnail gr6
      Figure 6CRE is involved in activation of TH gene expression by hypoxia. PC12 cells were transfected with 2 μg of either (−272TH) CAT or (−272CRE)CAT, as described under “Experimental Procedures.” Beginning 24 h after transfection, cells were exposed for 24 h to either hypoxia (5% O2, shaded bars) or normoxia (21% O2, black bars). CAT levels were analyzed by enzyme-linked immunosorbent assay in duplicate samples each containing 120 μg of total protein, as described. The data shown are from a single experiment and are expressed as the average ± S.E. and represent n = 4 dishes in each group. Similar results were obtained in three separate experiments.

      DISCUSSION

      The intracellular pathways involved in cellular responses and adaptation to hypoxia are only poorly understood. PC12 cells are an oxygen-sensitive cell line that has been shown to respond to hypoxia with depolarization, Ca2+ influx, and dopamine release (
      • Zhu W.H.
      • Conforti L.
      • Czyzyk-Krzeska M.F.
      • Millhorn D.E.
      ,
      • Raymond R.
      • Millhorn D.
      ). A primary mediator of depolarization and Ca2+-regulated gene expression is the transcription factor CREB (). Interestingly, the CREB coactivator CBP/p300 has recently been shown to interact with hypoxia-inducible factor-1α and participate in transcriptional regulation of hypoxia-responsive genes (
      • Arany Z.
      • Huang L.E.
      • Eckner R.
      • Bhattacharya S.
      • Jiang C.
      • Goldberg M.A.
      • Bunn H.F.
      • Livingston D.M.
      ). These studies were undertaken to evaluate the role of CREB in cellular signaling mechanisms during hypoxia. The level of hypoxia used in these studies is moderate (5% O2, ∼50 mm Hg) and within the range of physiological blood levels of O2 in rats exposed to hypoxia in vivo (
      • Czyzyk-Krzeska M.F.
      • Furnari B.A.
      • Lawson E.E.
      • Millhorn D.E.
      ,
      • Czyzyk-Krzeska M.F.
      • Bayliss D.A.
      • Lawson E.E.
      • Millhorn D.E.
      ).
      Exposure of PC12 cells to hypoxia was found to induce phosphorylation of CREB at Ser133, an event that is required for CREB-mediated transcriptional activation (
      • Bonni A.
      • Ginty D.D.
      • Dudek H.
      • Greenberg M.E.
      ,
      • Gonzalez G.A.
      • Montminy M.R.
      ). To our knowledge, this is the first evidence for regulation of CREB by hypoxia. This response appeared to be biphasic. A modest level of CREB phosphorylation was first detected after a 20-min exposure to hypoxia, and the effect peaked at 6 h, although high levels of phospho-CREB immunoreactivity persisted up to at least a 24-h exposure to hypoxia. The relatively slow onset of this effect is distinct from that produced by sorbitol (Fig. 1) and other stimuli known to induce CREB phosphorylation (e.g. forskolin, depolarization, and growth factors), which typically elicit a rapid response that then declines (
      • Bonni A.
      • Ginty D.D.
      • Dudek H.
      • Greenberg M.E.
      ). This suggests that the phosphorylation of CREB may be one mechanism by which PC12 cells adapt to prolonged exposure to hypoxia, although hypoxia may also induce CREB-dependent immediate early effects. The biphasic nature of this response raises the issue of whether either phase of the hypoxia-induced CREB phosphorylation requires new protein synthesis. However, interpretation of this experiment is confounded because inhibition of protein synthesis itself is a stressor that induces phosphorylation of CREB via activation of the p38 and downstream MAPKAP kinase families of enzymes (
      • Clifton A.D.
      • Young P.A.
      • Cohen P.
      ). Thus, clarification of the mechanism by which hypoxia induces both rapid and prolonged phosphorylation of CREB will require identification of the specific signaling pathway by which this occurs.
      Strikingly, the effect of hypoxia on CREB phosphorylation was equally or more robust than that produced by the prototypical stimuli used to activate CREB, including forskolin, depolarization, or sorbitol. The hypoxia-induced phosphorylation of CREB was not mediated by any of the previously known pathways that activate CREB, including PKA- and Ca2+-dependent protein kinases, because phosphorylation of CREB by hypoxia persisted completely in PKA-deficient PC12 cells and in the absence of both extracellular and intracellular Ca2+. Hypoxia-induced phosphorylation of CREB was also not attenuated by preincubation of cells with either Ro 31-8220 or chelerythrine chloride, which inhibits both Ca2+- and non-Ca2+-dependent isoforms of PKC (
      • Herbert J.M.
      • Augereau J.M.
      • Gleye J.
      • Maffrand J.P.
      ). Interestingly, unlike phosphorylation of CREB, hypoxia-induced activation of TH gene expression is blocked completely by either removal of Ca2+ or by preincubation with chelerythrine chloride in PC12 cells (
      • Raymond R.
      • Millhorn D.
      ). Taken together, these results demonstrate that even though a reduction in O2levels induces depolarization in PC12 cells (
      • Zhu W.H.
      • Conforti L.
      • Czyzyk-Krzeska M.F.
      • Millhorn D.E.
      ), hypoxia can regulate gene expression via both Ca2+-dependent and Ca2+-independent signaling pathways.
      The pp90rsk family of kinases, including RSK-1, RSK-2, and RSK-3, are growth factor-activated serine/threonine protein kinases that are phosphorylated and activated by p42/p44 mitogen-activated protein kinase (
      • Blenis J.
      ,
      • Zhao Y.
      • Bjørbæk C.
      • Moller D.E.
      ). Several laboratories have reported that pp90rsk can mediate phosphorylation of CREB (
      • Pende M.
      • Fisher T.L.
      • Simpson P.B.
      • Russell J.T.
      • Blenis J.
      • Gallo V.
      ,
      • Xing J.
      • Ginty D.D.
      • Greenberg M.E.
      ,
      • Bohm M.
      • Moellmann G.
      • Cheng E.
      • Alvarez-Franco M.
      • Wagner S.
      • Sassone-Corsi P.
      • Halaban R.
      ,
      • Sato N.
      • Kaminom K.
      • Tateishi K.
      • Satoh T.
      • Nishiwaki Y.
      • Yoshiiwa A.
      • Ogihara T.
      ). The RSK-2 isoform specifically has been shown to phosphorylate CREB in response to nerve growth factor stimulation (,
      • Xing J.
      • Ginty D.D.
      • Greenberg M.E.
      ). Several lines of evidence strongly suggest that the effect of hypoxia is not mediated by RSK-2 or other members of the pp90rsk family of kinases. First, hypoxia-induced phosphorylation of CREB persisted in the presence of Ro 31-8220, which has recently been shown to be as equally potent an inhibitor of RSK-2 as PKC (
      • Alessi D.R.
      ). Second, in contrast to the study of Pende et al. (
      • Pende M.
      • Fisher T.L.
      • Simpson P.B.
      • Russell J.T.
      • Blenis J.
      • Gallo V.
      ), who found that growth factor- and Ca2+-induced phosphorylation of CREB was blocked by PD-098059, the effect of hypoxia on CREB persisted in the presence of PD-098059. PD-098059 is a selective inhibitor of MEK activation, which thereby also inhibits the activation of downstream effectors of MEK, including MAPK and the pp90rsk enzymes (
      • Alessi D.R.
      • Cuenda A.
      • Cohen P.
      • Dudley D.T.
      • Saltiel A.
      ,
      • Zhao Y.
      • Bjørbæk C.
      • Moller D.E.
      ). Finally, exposure to the same level of hypoxia used in this study stronglyinhibits p42/p44 MAPK activity in PC12 cells.
      P. Conrad, D. Millhorn, and D. Beitner-Johnson, submitted for publication.
      Thus, this effect does not appear to be mediated by RSK-2 or any other MAPK-dependent pp90rsk isoforms.
      Two recent studies have shown that activation of the stress-activated protein kinase p38 by cellular stressors (
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ,
      • Iordanov M.
      • Bender K.
      • Ade T.
      • Schmid W.
      • Sachsenmaier C.
      • Engel K.
      • Gaestel M.
      • Rahmsdorf H.J.
      • Herrlich P.
      ) and fibroblast growth factor (
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ) can mediate Ser133 phosphorylation of CREB and that this can be blocked by the highly specific p38 inhibitor SB203580 (
      • Cuenda A.
      • Rouse J.
      • Doza Y.N.
      • Meier R.
      • Cohen P.
      • Gallagher T.F.
      • Young P.R.
      • Lee J.C.
      ). CREB is not phosphorylated by p38 itself, but it can be phosphorylated in vitro by the downstream substrate of p38, MAPKAP kinase-2 (
      • Tan Y.
      • Rouse J.
      • Zhang A.
      • Cariati S.
      • Cohen P.
      • Comb M.J.
      ). In contrast, in our studies, hypoxia-induced phosphorylation of CREB was not diminished by SB203580, although the drug effectively blocked sorbitol-induced activation of MAPKAP kinase-2. Furthermore, hypoxia did not stimulate MAPKAP kinase-2 enzyme activity. We propose that a novel hypoxia-activated protein kinase mediates phosphorylation of CREB in response to a reduction in O2 levels. Recent studies have identified at least two isoforms of p38 which are insensitive to SB203580: SAPK-3 and SAPK-4 (
      • Cuenda A.
      • Cohen P.
      • Buee-Scherrer V.
      • Goedert M.
      ,
      • Kumar S.
      • McDonnell P.C.
      • Gum R.J.
      • Hand A.T.
      • Lee J.C.
      • Young P.R.
      ). Like p38, the major known downstream effector of these kinases is MAPKAP kinase-2, suggesting that SAPK-3 and SAPK-4 also do not mediate the effects of hypoxia on CREB. However, we cannot exclude the possibility that other downstream targets (e.g. MAPKAP kinase-3) could mediate hypoxia-induced phosphorylation of CREB.
      It has been established previously that TH gene expression is induced by hypoxia both in vivo (
      • Czyzyk-Krzeska M.F.
      • Bayliss D.A.
      • Lawson E.E.
      • Millhorn D.E.
      ) and in PC12 cells (
      • Czyzyk-Krzeska M.F.
      • Furnari B.A.
      • Lawson E.E.
      • Millhorn D.E.
      ). The TH gene contains a CRE that has been shown to be critical for cAMP- and Ca2+-induced activation of gene expression (
      • Nagamoto-Combs K.
      • Piech K.M.
      • Best J.A.
      • Sun B.
      • Tank A.W.
      ,
      • Lazaroff M.
      • Patankar S.
      • Yoon S.O.
      • Chikaraishi D.M.
      ,
      • Kilbourne E.J.
      • Nankova B.B.
      • Lewis E.J.
      • McMahon A.
      • Osaka H.
      • Sabban D.B.
      • Sabban E.L.
      ,
      • Kim K.-S.
      • Lee M.K.
      • Carroll J.
      • Joh T.H.
      ). We found that the hypoxia-induced activation of the TH gene was attenuated significantly in a TH-CAT reporter plasmid in which the CRE had been mutated. The TH gene contains several elements that participate in hypoxia-induced activation of gene expression, including AP-1 and hypoxia-inducible factor-1 elements located in the region between −284 to −190 nucleotides relative to the transcription initiation site (
      • Norris M.L.
      • Millhorn D.E.
      ). Thus, one or more of these upstream regulatory elements presumably mediates the residual activation of the TH gene in the absence of the CRE, and CREB per se is likely to be insufficient to mediate this entire effect. Our results suggest that hypoxia may induce activation of other CRE-containing genes, including c-fos, which is known to be stimulated by hypoxia (
      • Norris M.L.
      • Millhorn D.E.
      ,
      • Webster K.A.
      • Discher D.J.
      • Bishopric N.H.
      ).
      In summary, hypoxia induces a robust and persistent phosphorylation of CREB in PC12 cells. Hypoxia-induced phosphorylation of CREB is involved in functional activation of TH, a gene that is known to be induced in response to hypoxia (
      • Czyzyk-Krzeska M.F.
      • Furnari B.A.
      • Lawson E.E.
      • Millhorn D.E.
      ,
      • Czyzyk-Krzeska M.F.
      • Bayliss D.A.
      • Lawson E.E.
      • Millhorn D.E.
      ). Unlike other stimuli, which induce peak CREB phosphorylation relatively rapidly and then decline (
      • Bonni A.
      • Ginty D.D.
      • Dudek H.
      • Greenberg M.E.
      ), the effects of hypoxia on CREB phosphorylation are maximal with a 3–6-h exposure to hypoxia and persist for at least 24 h. This suggests that phosphorylation of CREB and activation of a subset of hypoxia-responsive genes may be an adaptive cellular response to a reduction in O2 levels. Interestingly, it has been reported that phospho-CREB immunoreactivity is increased in rat dentate granule cells and neocortex 1–2 days after hypoxic-ischemic induced brain damage (
      • Walton M.
      • Sirimanne E.
      • Williams C.
      • Gluckman P.
      • Dragunow M.
      ). In this study, phospho-CREB immunoreactivity was induced selectively in ischemia-resistant cells, but not CA1 pyramidal cells, which undergo neuronal death after hypoxia-ischemia in vivo, suggesting that phospho-CREB may be involved in the process of neuroprotection. Current studies are under way to identify the signaling pathway by which CREB phosphorylation is induced during hypoxia. Such studies will further our understanding of the neuronal response to oxygen deprivation and, in turn, shed light on molecular mechanisms underlying the pathology of hypoxic and/or ischemic trauma.

      ACKNOWLEDGEMENTS

      We thank Dr. Dona Chikaraishi for providing the (−272TH)CAT and (−272CRE)CAT plasmids, and Dr. John Lee for providing SB203580. We also thank Dr. Nelson Horseman for a critical reading of the manuscript and Glenn Doerman for assistance in preparation of the figures.

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