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(Circulation Research. 1996;78:82-90.)
© 1996 American Heart Association, Inc.

Articles

Hypoxia and Hypoxia/Reoxygenation Activate Raf-1, Mitogen-Activated Protein Kinase Kinase, Mitogen-Activated Protein Kinases, and S6 Kinase in Cultured Rat Cardiac Myocytes

Yoshinori Seko, Kazuyuki Tobe, Kohjiro Ueki, Takashi Kadowaki, Yoshio Yazaki

From the Third Department of Internal Medicine (Y.S., K.T., K.U., T.K., Y.Y.), Faculty of Medicine, University of Tokyo (Japan); the Department of Immunology (Y.S.), School of Medicine, Juntendo University, Tokyo; and the Institute for Adult Diseases (Y.S.), Asahi Life Foundation, Tokyo.

Correspondence to Yoshinori Seko, MD, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.

	Abstract

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Abstract In response to hypoxia and reoxygenation, mammalian cells are known to express a variety of genes to adapt to these external stresses or lead to further cell damage. We investigated the intracellular signaling cascades in cultured rat cardiac myocytes subjected to hypoxia or hypoxia followed by reoxygenation (hypoxia/reoxygenation). Here, we show that both hypoxia and hypoxia/reoxygenation caused rapid activation of the mitogen-activated protein kinase kinase kinase (MAPKKK) activity of Raf-1. This was followed by the sequential activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein (MAP) kinases, and S6 kinase (p90^rsk). Furthermore, hypoxia caused hyperphosphorylation of Raf-1. The maximal hyperphosphorylation of Raf-1 appeared to be accompanied by a significant decrease in MAPKKK activity. These results strongly suggest the following: (1) Intracellular signals initiated by both hypoxia and hypoxia/reoxygenation converge on Raf-1 and activate its MAPKKK activity. Then, Raf-1 activates downstream serine/threonine kinases including MAPKK, MAP kinases, and p90^rsk. (2) Raf-1 is not only located upstream from MAPKK and MAP kinases but also may be phosphorylated by MAP kinases directly or indirectly, and at least Raf-1 kinase activity may be downregulated by this feedback mechanism.

Key Words: redox • serine/threonine kinase • second messenger • phosphorylation • ischemia/reperfusion

	Introduction

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To perform continuously repetitive contraction, cardiac myocytes quickly respond and adapt to environmental stresses such as ischemia, mechanical load, and metabolic changes by expressing a number of various genes. Although there have been many studies concerning the hemodynamic and physiological mechanisms involved, little is known about the molecular mechanisms regulating cardiac myocyte response to hypoxia and reoxygenation, ie, about how hypoxia and reoxygenation stimuli are converted into intracellular signals to regulate gene expression. It is thought that nuclear proto-oncogenes act as third messengers, converting cytoplasmic signal transduction into long-term changes of gene expression. It has been reported that some of the nuclear proto-oncogenes, such as c-fos, jun B, and Egr-1, which encode DNA binding or interacting proteins known as regulators of transcription, were highly induced in porcine myocardium subjected to ischemia and reperfusion in vivo.¹ Recently, Webster and colleagues^{2 3} showed that hypoxia as well as metabolic stress highly induced c-fos, c-jun, jun B, and jun D mRNA in cultured cardiac myocytes in vitro. Sadoshima and Izumo⁴ have demonstrated that mechanical load caused rapid activation of multiple second messengers, including tyrosine kinases, p21^ras, MAP kinase, S6 kinase (pp90^RSK), PKC, and phospholipase C, which may in turn initiate a cascade of hypertrophic response of cardiac myocytes. Although there have been many studies involving inositol phosphate and cAMP pathways in response to hypoxia in various cell types including cardiac myocytes,^{5 6} there have been no reports studying the intracellular MAP kinase cascades in cardiac myocytes subjected to hypoxia and reoxygenation.

Studies of mammalian cells subjected to a hypoxic state by exposure to antioxidants revealed that transcriptional activation of the GST Ya subunit gene and the NAD(P)H dehydrogenase (quinone) reductase gene by several antioxidants can be mediated by cis-acting antioxidant responsive elements.^{7 8 9 10} Reperfusion of hypoxic tissue leads to severe oxidative injury, which is mainly mediated by reactive oxygen species such as hydrogen peroxide (H₂O₂), superoxide (O₂^- · ), and hydroxyl radicals (OH · ) produced by electron transfer reactions. Indeed, the condition of cells subjected to hypoxia followed by reoxygenation (hypoxia/reoxygenation) can be induced by exposure to oxidant H₂O₂,^{11 12} UV light,^{13 14} ionizing radiation,¹⁵ and cytokines.¹⁶ Oxidative stresses cause cell reactions involving the induction of antioxidative enzymes such as glutathione reductase, catalase, and superoxide dismutase. In mammalian cells, H₂O₂ and UV light were shown to activate immediate-early genes c-fos and c-jun, which encode proteins that participate in formation of the DNA-binding transcription factor AP-1 complex.^{11 14} The induction of the c-fos gene is mediated through activation of the serum response element in their enhancer. H₂O₂ and UV light were also shown to induce the expression and replication of human immunodeficiency virus type-1 through activation of transcription factor NF-B.^{12 13}

Although there is a lot of information concerning the transcriptional regulation by a reduction-oxidation (redox) mechanism, little is known about how such stimuli activate intracellular second messenger pathways and are transduced into the nucleus. To elucidate the mechanisms in more detail, we have investigated the intracellular signaling cascades in cultured rat cardiac myocytes subjected to hypoxia or hypoxia/reoxygenation in vitro. In the present study, we show that both hypoxia and hypoxia/reoxygenation cause rapid activation of the MAPKKK activity of Raf-1, followed by the activation of MAPKK, MAP kinases, and p90^rsk. Then, the MAPKKK activity of Raf-1 is downregulated through its hyperphosphorylation.

	Materials and Methods

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Cell Culture
Primary cultures of ventricular cardiac myocytes were prepared from neonatal rats, as previously described.¹⁷ Briefly, heart ventricles were aseptically removed from neonatal Wistar rats, minced in calcium-free PBS, and digested with 0.025% trypsin-EDTA in PBS. The isolated cardiac myocytes were washed in DMEM containing 10% FCS, dispersed into plastic dishes for 1 hour to separate the fibroblasts, and removed to new gelatin-coated culture dishes. They were cultured for 36 hours until they were confluent. After culturing in a serum-free medium (DMEM) for 24 hours, the cells were subjected to hypoxia or hypoxia/reoxygenation.

Hypoxia and Reoxygenation
Hypoxia was achieved by using an anaerobic jar (AnaeroPack Series, Mitsubishi Gas Chemical Co, Inc) equipped with an AnaeroPack, disposable O₂-absorbing and CO₂-generating agent, and an indicator to monitor oxygen depletion. The AnaeroPack jar is capable of depleting the concentration of O₂ down to <0.1% in 2 hours and of providing a 21% CO₂ atmosphere. By placing flasks, which contain serum-free medium, in a AnaeroPack jar overnight, the medium was balanced with the hypoxic atmosphere. Cultured cardiac myocytes were subjected to hypoxic conditions by immediate replacement of the medium with the hypoxic medium in the AnaeroPack jar. To keep hypoxic conditions, all the procedures were performed in an airtight glove bag filled with 95% N₂/5% CO₂. After incubating in hypoxic conditions for the time periods indicated, the cells were reoxygenated by immediate replacement of the hypoxic medium with a normoxic serum-free medium.

MBP Kinase Assays
Cardiac myocytes were subjected to hypoxia for 0, 5, 10, 15, 30, or 60 minutes or to 60 minutes of hypoxia followed by reoxygenation for 5, 10, 15, or 30 minutes (Hypox/Reoxy at 0, 5, 10, 15, 30, 60, 60/5, 60/10, 60/15, 60/30, respectively, in figures). Then the culture media were aspirated immediately, and cardiac myocytes were frozen in liquid nitrogen. The cells were lysed on ice with buffer A containing 25 mmol/L Tris-HCl, 25 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 10 mmol/L sodium pyrophosphate, 10 nmol/L okadaic acid, 0.5 mmol/L EGTA, and 1 mmol/L phenylmethylsulfonyl fluoride. After centrifugation, aliquots of the supernatants of the myocyte extracts were incubated in 40 µL of kinase buffer (25 mmol/L Tris-HCl [pH 7.4], 10 mmol/L MgCl₂, 1 mmol/L DTT, 40 µmol/L ATP, 2 µCi of [-³²P]ATP [6000 Ci/mmol, Du Pont–New England Nuclear], 2 µmol/L protein kinase inhibitor peptide, and 0.5 mmol/L EGTA) and substrates (25 µg MBP). After 10 minutes at 25°C, aliquots of the supernatant (15 µL) were spotted on 15x15-mm squares of P81 paper (Whatman), washed five times for at least 10 minutes each in 0.5% phosphoric acid, dried, and counted by the Cerenkov technique.^{18 19}

Kinase Assays in MBP-Containing SDS-PAGE
Cardiac myocytes were subjected to hypoxia or hypoxia/reoxygenation. Then the culture media were aspirated immediately, and cardiac myocytes were frozen in liquid nitrogen. The cells were lysed on ice with buffer A. The cell lysates were centrifuged, and aliquots of the supernatants were electrophoresed on an SDS–polyacrylamide gel containing 0.5 g/L MBP.^{20 21 22 23} SDS was removed from the gel by washing the gel with two changes of 100 mL each of 20% 2-propanol in 50 mmol/L Tris-HCl (pH 8.0) for 1 hour at room temperature. The enzyme was denatured by treating the gel first with two changes of 100 mL of 6 mol/L guanidine-HCl at room temperature for 1 hour and then renatured with five changes of 250 mL each of 50 mmol/L Tris-HCl (pH 8.0) containing 0.04% Tween 40 and 5 mmol/L 2-mercaptoethanol at 4°C for 3 hours. After renaturation, the gel was preincubated at 25°C for 1 hour with 5 mL of 40 mmol/L HEPES (pH 8.0) containing 2 mmol/L DTT and 10 mmol/L MgCl₂. Phosphorylation of MBP was carried out by incubating the gel at 25°C for 1 hour with 5 mL of 40 mmol/L HEPES (pH 8.0), 0.5 mmol/L EGTA, 10 mmol/L MgCl₂, 2 µmol/L protein kinase inhibitor, 40 µmol/L ATP, and 25 µCi of [-³²P]ATP. After incubation, the gel was washed with a 7% trichloroacetic acid solution until the radioactivity of the solution became negligible. The washed gel was dried and then subjected to autoradiography.

Downregulation of PKC Activity
It has been previously shown that treatment with 100 nmol/L PMA for 24 hours completely inhibited PKC activity in cardiac myocytes.²⁴ Calphostin C is a highly potent and specific inhibitor of PKC. Treatment with 1 µmol/L calphostin C has been shown to completely inhibit PKC activity.²⁵ To investigate the involvement of PKC in the increased MBP kinase activity, we preincubated cardiac myocytes with 100 nmol/L PMA for 24 hours or 1 µmol/L calphostin C for 20 minutes; then we examined the effects of hypoxia or hypoxia/reoxygenation on the MBP kinase activity.

S6 Peptide Kinase Assays
An anti-mouse S6 (rsk) kinase antibody was purchased from Upstate Biotechnology, Inc. Cardiac myocytes were subjected to hypoxia or hypoxia/reoxygenation and immediately frozen in liquid nitrogen, and then the cells were lysed on ice with buffer A. From the lysate of cardiac myocytes, we obtained the anti–S6 kinase or control rabbit IgG immunoprecipitates, which were incubated with 50 µg of S6 peptide (RRRLSSLRA) in the presence of 25 mmol/L Tris-HCl (pH 7.4), 10 mmol/L MgCl₂, 1 mmol/L DTT, 40 µmol/L ATP, 2 µCi [-³²P]ATP, 2 µmol/L protein kinase inhibitor peptide, and 0.1 mmol/L EGTA. After 10 minutes of incubation at 25°C, we added 10 mL of stopping solution containing 0.6% HCl, 0.5 mmol/L ATP, and 1% bovine serum albumin. After centrifugation, aliquots of the supernatant (15 µL) were spotted on 15x15-mm squares of P81 paper (Whatman), washed five times for at least 10 minutes each in 0.5% phosphoric acid, dried, and counted by the Cerenkov technique. To confirm that equal amounts of S6 kinase protein were immunoprecipitated in each reaction, aliquots of the samples were also immunoprecipitated and subjected to Western analysis using the anti-S6 kinase antibody.

Analyses of Raf-1 Hyperphosphorylation
After cardiac myocytes were subjected to hypoxia or hypoxia/reoxygenation, the cells were frozen in liquid nitrogen and lysed on ice with buffer A. The cell lysates were centrifuged, and aliquots of the supernatants were electrophoresed on a 10% SDS–polyacrylamide gel, blotted onto a nylon membrane, and subjected to Western analysis using a rabbit polyclonal anti–Raf-1 antibody against the C-terminal 12–amino acid peptide (CTLTTSPRLPVF) of Raf-1. The antibody-antigen complexes were visualized by alkaline phosphatase reaction.

Assays of MAPKKK Activity of Raf-1
MAPKKK activity was assayed by using a recombinant mouse MAPKK fused to GST as a substrate. The procedures for producing a recombinant mouse MAPKK were as follows: A mouse MAPKK (MEK1) cDNA was obtained by reverse-transcription PCR from mouse brain total RNA based on the sequence reported by Crews et al.²⁶ The PCR product (1.2 kb), which includes the whole coding region, was subcloned into BamHI and Xba I sites of M13 vector (mp18), and the kinase-inactive MAPKK mutant (MAPKK97KR) was generated by site-directed mutagenesis of lysine 97 to arginine by the Kunkel method. The M13 vector was digested with Xba I and generated a blunt end with Klenow treatment. The MAPKK97KR cDNA was obtained by digesting the M13 vector with BamHI and subcloned into BamHI and Sma I sites of GST bacterial expression vector (pGEX-2T). The mutation and the entire MAPKK PCR product were confirmed by DNA sequence. After cardiac myocytes were subjected to hypoxia or hypoxia/reoxygenation, the cell lysates were prepared with buffer A as described above and centrifuged. Supernatants of the cell lysates were immunoprecipitated with 5 µg of the anti–Raf-1 antibody and protein A–Sepharose (Pharmacia LKB). The immunoprecipitates were incubated with buffer B (25 mmol/L Tris-HCl [pH 7.4], 10 mmol/L MgCl₂, 1 mmol/L DTT, 2 mmol/L MnCl₂, and 0.5 mmol/L EGTA), 5 µCi [-³²P]ATP, and substrate (10 µg of GST-recombinant MAPKK) at room temperature for 30 minutes. GST-recombinant MAPKK was collected by using glutathione Sepharose 4B (Pharmacia LKB), washed, electrophoresed on 10% SDS–polyacrylamide gel, and autoradiographed. To confirm that equal amounts of Raf-1 protein were immunoprecipitated in each reaction, aliquots of the samples were also immunoprecipitated and subjected to Western analysis using the anti–Raf-1 antibody.

Assays of MAPKK Activity
MAPKK activity was assayed by using a recombinant rat MAP kinase fused to maltose binding protein as a substrate.²⁷ The procedures for producing a recombinant rat MAP kinase were described in detail previously.²⁷ After cardiac myocytes were subjected to hypoxia or hypoxia/reoxygenation, the cell lysates were prepared with buffer A as described above. To chromatographically separate MAPKK from endogenous MAP kinases, the cell lysates were applied to Q-Sepharose columns (Pharmacia LKB), and flow through fractions was immunoprecipitated with 5 µg of the rabbit polyclonal antibody against the N-terminus 16–amino acid peptide (PKKKPTPIQLNPNPEG) of Xenopus MAPKK (nMAPKK)²⁸ and protein A–Sepharose (Pharmacia LKB). The immunoprecipitates were incubated with buffer B (25 mmol/L Tris-HCl [pH 7.4], 10 mmol/L MgCl₂, 1 mmol/L DTT, 2 mmol/L MnCl₂, and 0.5 mmol/L EGTA), 5 µCi [-³²P]ATP, and substrate (100 µg of recombinant MAP kinase) at 30°C for 30 minutes. Recombinant MAP kinase was collected by using amylose resin and was electrophoresed on 10% SDS–polyacrylamide gel and autoradiographed. To confirm that equal amounts of MAPKK protein were immunoprecipitated in each reaction, aliquots of the samples were also immunoprecipitated and subjected to Western analysis using the anti-MAPKK antibody.

The kinase activity at the 73-kD or 84-kD bands was measured by densitometric scanning of the phosphoimage (Bio-image analyzer, BAS 2000, FUJI Photo Film Co, Ltd) of MAPKKK or MAPKK assay, respectively.

Statistics
Statistical comparisons of the control group with treated groups were carried out by using the unpaired t-test with P values corrected by the Bonferroni method. Values of P<.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hypoxia and Hypoxia/Reoxygenation Stimulate Kinase Activity Toward MBP
MAP kinases (or extracellular signal–regulated kinases) are a family of serine/threonine kinases, which are activated by tyrosine and threonine phosphorylation in response to a variety of growth factors^{18 20 21 29} and are also regulated in a cell cycle–dependent fashion.^{22 30} To investigate whether hypoxia or hypoxia/reoxygenation activates MAP kinase activity, supernatants of the myocyte extracts were subjected to an assay for serine/threonine kinase activity by using MBP as a substrate. This technique is the definitive method for quantifying MAP kinase activity.^{18 19} As shown in Fig 1A, hypoxia increased MBP kinase activity within 5 minutes. The activity reached a maximum level of 1.8-fold at 5 to 15 minutes and then almost returned to the control level at 30 minutes. Reoxygenation after 60 minutes of hypoxia also increased MBP kinase activity within 10 minutes. The activity reached a maximum level of 1.3-fold at 15 minutes and then almost returned to the control level at 30 minutes. Thus, both hypoxia and hypoxia/reoxygenation rapidly stimulated the MBP kinase activity. The peak level of the MBP kinase activity was higher by hypoxia than by hypoxia/reoxygenation. To examine whether or not medium change itself can activate MBP kinase activity, we also analyzed the kinase activity by simply changing the normoxic medium to hypoxic medium and the hypoxic medium to normoxic medium. We confirmed that medium change itself had no significant effects on the MBP kinase activity (data not shown). To estimate the contamination of fibroblasts, we did immunoperoxidase staining for cardiac myosin heavy chain, followed by counterstaining with hematoxylin, and we found that the contamination of fibroblasts was 10% to 20% (data not shown). We also performed MBP kinase assays using cardiac fibroblast cultures, and we had results similar to those found with cardiac myocytes (maximum level, 2.0-fold by hypoxia and 1.3-fold by hypoxia/reoxygenation; data not shown). Therefore, we determined that 10% to 20% of the effects of hypoxia as well as hypoxia/reoxygenation were due to contamination of fibroblasts and that 80% to 90% of those were due to cardiac myocytes. Taken together, we concluded that cardiac myocytes themselves as well as fibroblasts similarly respond to hypoxia and hypoxia/reoxygenation.

View larger version (45K): [in this window] [in a new window]   Figure 1. Hypoxia (Hypox) and hypoxia/reoxygenation (Reoxy) stimulate MBP kinase activity migrating at 42 and 44 kD. Serum-starved cardiac myocytes were subjected to Hypox and Reoxy for the indicated time periods and lysed in buffer A. A, Cell extracts were incubated with a substrate (MBP) in kinase buffer, and then aliquots of the supernatants were spotted onto P81 paper (Whatman), washed, dried, and counted by the Cerenkov technique. The results shown represent the mean±SEM from four experiments. The control activity (Hypox 0 or Hypox 60) is designated as 1.0. *P<.005 and **P<.001 vs control (Hypox 0); P<.01 and P<.01 vs control (Hypox 60). B and C, Cell extracts were electrophoresed on SDS–polyacrylamide gels containing MBP. SDS was removed from the gel, and after denaturation with 6 mol/L guanidine HCl and renaturation in a buffer containing 0.04% Tween 40, the gel was incubated with [-32P]ATP and Mg2+. After it was washed, the gel was dried and subjected to autoradiography. Panel B shows the time courses of MAP kinase activity stimulated by Hypox and Reoxy. The kinase activity at the 44-kD and 42-kD bands was measured by densitometric scanning of the autoradiogram. The results shown represent the mean±SEM from three experiments. The control activity (Hypox 0 or Hypox 60) is designated as 1.0. *P<.005, **P<.01, P<.001, and P<.05 vs control (Hypox 0); P<.05, P<.005, §P<.05, and §§P<.01 vs control (Hypox 60). Panel C shows a representative in-gel kinase assay of cell extracts using MBP as a substrate. The intensity of both 44-kD and 42-kD bands corresponds to MAP kinase activity.

Hypoxia and Hypoxia/Reoxygenation Stimulate MBP Kinase Activity Migrating at 42 and 44 kD
To determine the proteins responsible for the increased MBP kinase activity, we performed kinase assays in MBP-containing SDS-PAGE, followed by denaturation with 6 mol/L guanidine HCl and renaturation in a buffer containing 0.04% Tween 40 (in-gel kinase assay). We observed that both hypoxia and hypoxia/reoxygenation stimulated MBP kinase activity migrating at 42 and 44 kD. Panels B and C of Fig 1 show the time course of MBP kinase activation by hypoxia and hypoxia/reoxygenation. Hypoxia and hypoxia/reoxygenation increased MBP kinase activity migrating at 42 kD by 1.9- and 1.4-fold, respectively. Hypoxia and hypoxia/reoxygenation increased MBP kinase activity migrating at 44 kD by 1.9- and 1.6-fold, respectively. MBP kinase activity migrating at both 42 and 44 kD reached a maximum level at 5 minutes by hypoxia and at 15 minutes by reoxygenation after 60 minutes of hypoxia. Because MAP kinases have molecular sizes of 42 and 44 kD, these data confirmed that both hypoxia and hypoxia/reoxygenation rapidly stimulated the MAP kinase activity.

Effect of PKC Downregulation on Hypoxia or Hypoxia/Reoxygenation-Induced MBP Kinase Activation
As shown in Fig 2, both pretreatment with 100 nmol/L PMA for 24 hours and pretreatment with 1 µmol/L calphostin C for 20 minutes inhibited only to a limited extent (not significantly) hypoxia-induced as well as hypoxia/reoxygenation–induced MBP kinase activation. This suggests that PKC may be only partially involved, if at all, in both hypoxia-induced and hypoxia/reoxygenation–induced MBP kinase activation. We also performed control experiments to examine whether calphostin C itself could activate MAP kinases. However, calphostin C treatment had no significant effect on MBP kinase activity (data not shown).

View larger version (40K): [in this window] [in a new window]   Figure 2. Effect of PKC downregulation on hypoxia (hypox)– and hypoxia/reoxygenation (reoxy)–induced MBP kinase activation. Serum-starved cardiac myocytes were pretreated with 100 nmol/L PMA for 24 hours or 1 µmol/L calphostin C for 20 minutes and then subjected to hypox and reoxy for the indicated time periods and lysed in buffer A. Cell extracts were incubated with a substrate (MBP) in kinase buffer, and then aliquots of the supernatants were spotted onto P81 paper (Whatman), washed, dried, and counted by the Cerenkov technique. The results shown represent the mean±SEM from four experiments. The control activity (hypox 0 or hypox 60) is designated as 1.0.

Hypoxia and Hypoxia/Reoxygenation Stimulate S6 Kinase Activity
MAP kinases are known to phosphorylate one of the S6 kinases (p90^rsk) and regulate its activity.^{31 32} p90^rsk is one of the growth factor–induced protein kinases, localized in the cytoplasm and the nucleus, and may participate in transcriptional regulation of immediate-early genes, such as c-fos gene expression, and their target genes by phosphorylating serum response factors.^{33 34} To examine whether hypoxia or hypoxia/reoxygenation also activates S6 kinase (p90^rsk), we measured S6 peptide kinase activity in the immunoprecipitates with control rabbit IgG or anti–S6 kinase II antibody from cardiac myocytes. Fig 3A shows the time course of S6 kinase activation by hypoxia and hypoxia/reoxygenation. Hypoxia and hypoxia/reoxygenation increased S6 kinase activity by 1.7- and 1.4-fold, respectively. S6 kinase activity was led to a maximum level at 10 minutes by hypoxia and at 15 minutes by reoxygenation after 60 minutes of hypoxia. Control rabbit IgG did not immunoprecipitate S6 peptide kinase activity either by hypoxia or hypoxia/reoxygenation (data not shown). We confirmed that almost equal amounts of S6 kinase protein were immunoprecipitated in each reaction by Western analysis using the anti–S6 kinase II antibody (Fig 3B).

View larger version (36K): [in this window] [in a new window]   Figure 3. Hypoxia (Hypox) and hypoxia/reoxygenation (Reoxy) activate S6 kinase (p90rsk). Serum-starved cardiac myocytes were subjected to Hypox and Reoxy for the indicated time periods and lysed in buffer A. A, Cell extracts were immunoprecipitated with anti–S6 (rsk) kinase antibody and incubated with a substrate (S6 peptide) in kinase buffer, and then aliquots of the supernatants were spotted onto P81 paper (Whatman), washed, dried, and counted by the Cerenkov technique. The results shown represent the mean±SEM from three experiments. The control activity (Hypox 0 or Hypox 60) is designated as 1.0. *P<.001 vs control (Hypox 0); **P<.001 vs control (Hypox 60). B, To confirm that equal amounts of S6 kinase protein were immunoprecipitated in each reaction, aliquots of the cell extracts were also immunoprecipitated with anti–S6 kinase antibody and subjected to Western analysis using the anti–S6 kinase antibody.

Hypoxia and Hypoxia/Reoxygenation Induce Hyperphosphorylation of Raf-1
Raf-1 is a serine/threonine kinase known to play an important role in the signaling process in cell growth and differentiation.^{35 36 37} Recent studies showed that Raf-1 phosphorylates and activates the MAP kinase pathway and is also phosphorylated by MAP kinases.^{38 39 40 41 42 43 44 45} Therefore, first, we examined whether or not hypoxia or hypoxia/reoxygenation phosphorylates and activates Raf-1. The phosphorylation state of Raf-1 was investigated by examining its electrophoretic mobility.⁴⁶ As shown in Fig 4, hypoxia resulted in retarded electrophoretic mobility of Raf-1 within 15 minutes after the start of stimulation. The maximum retardation of electrophoretic mobility was observed at 30 minutes of hypoxia. We could detect only a slight, but significant, retardation of electrophoretic mobility at 30 minutes of reoxygenation. Although the observed mobility retardation was not great, especially in reoxygenation, we confirmed that the same pattern of mobility retardation was induced by these stimuli in at least five experiments. Therefore, we concluded that the mobility retardation induced by hypoxia/reoxygenation as well as by hypoxia was significant.

View larger version (16K): [in this window] [in a new window]   Figure 4. Hypoxia (Hypox) and hypoxia/reoxygenation (Reoxy) hyperphosphorylate Raf-1. Serum-starved cardiac myocytes were subjected to Hypox and Reoxy for the indicated time periods and lysed in buffer A. Cell extracts were electrophoresed on SDS–polyacrylamide gels, transferred to a membrane, and Western-blotted with an anti–Raf-1 antibody. The antibody-antigen complexes were visualized by alkaline phosphatase reaction.

Activation of MAPKK by Raf-1 Induced by Hypoxia and Hypoxia/Reoxygenation
Raf-1 is known to be an immediate upstream activator of MAPKK as well as a downstream substrate for MAP kinases.^{27 38 39 40 42} Therefore, next, we examined the MAPKKK activity of Raf-1 induced by hypoxia or hypoxia/reoxygenation. As shown in Fig 5A, hypoxia and hypoxia/reoxygenation increased the MAPKKK activity of Raf-1 by 2.2- and 1.7-fold, respectively. MAPKKK activity was led to a maximum level at 5 minutes by hypoxia and at 5 minutes by reoxygenation after 60 minutes of hypoxia. We confirmed that almost equal amounts of Raf-1 protein were immunoprecipitated in each reaction by Western analysis using the anti–Raf-1 antibody (Fig 5A, Raf-1 protein).

View larger version (33K): [in this window] [in a new window]   Figure 5. Hypoxia (Hypox) and hypoxia/reoxygenation (Reoxy) stimulate MAPKKK activity of Raf-1 and MAPKK. A, Activation of the MAPKKK activity of Raf-1. Serum-starved cardiac myocytes were subjected to Hypox and Reoxy for the indicated time periods and lysed in buffer A. Cell extracts were immunoprecipitated with an anti–Raf-1 antibody and subjected to the MAPKKK assay by using a recombinant MAPKK as a substrate (described in "Materials and Methods"). The control activity (Hypox 0 or Hypox 60) is designated as 1.0. *P<.001 vs control (Hypox 0); **P<.01 vs control (Hypox 60). To confirm that equal amounts of Raf-1 protein were immunoprecipitated in each reaction, aliquots of the samples were also immunoprecipitated and subjected to Western analysis using the anti–Raf-1 antibody. B, Activation of MAPKK. Serum-starved cardiac myocytes were subjected to Hypox and Reoxy for the indicated time periods and lysed in buffer A. Cell extracts were immunoprecipitated with an anti-MAPKK antibody and subjected to the MAPKK assay using a recombinant MAP kinase as a substrate (described in "Materials and Methods"). Fold induction values represent the averages of three experiments in which the kinase activity at the 73-kD or 84-kD bands was measured by densitometric scanning of the phosphoimage of MAPKKK or MAPKK assay, respectively. The control activity at Hypox 0 or Hypox 60 is designated as 1.0 for Hypox or Reoxy, respectively. Each autoradiogram shows the results of one typical experiment. The control activity (Hypox 0 or Hypox 60) is designated as 1.0. P<.005 vs control (Hypox 0); P<.05 vs control (Hypox 60). To confirm that equal amounts of MAPKK protein were immunoprecipitated in each reaction, aliquots of the samples were also immunoprecipitated and subjected to Western analysis using the anti-MAPKK antibody.

Activation of MAP Kinase by MAPKK Induced by Hypoxia and Hypoxia/Reoxygenation
MAPKK (or MEK) is a protein kinase specific for both tyrosine and serine/threonine. Recent studies have shown that MAPKK is activated by MAPKKK (such as Raf-1) as well as an immediate upstream activator of MAP kinases and functions as a key intermediate in the MAP kinase cascade.^{27 28 38 39 40 42 47 48 49} To confirm that MAP kinase cascade is really activated sequentially by the stimuli, we examined the MAPKK activity stimulated by hypoxia or hypoxia/reoxygenation. As shown in Fig 5B, hypoxia and hypoxia/reoxygenation increased MAPKK activity by 2.8- and 1.8-fold, respectively. MAPKK activity was led to a maximum level at 5 minutes by hypoxia and at 10 minutes by reoxygenation after 60 minutes of hypoxia. We confirmed that almost equal amounts of MAPKK protein were immunoprecipitated in each reaction by Western analysis using the anti-MAPKK antibody (Fig 5B, MAPKK protein).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, first, we showed that kinase activity toward MBP is increased by both hypoxia and hypoxia/reoxygenation (Fig 1A). Then, we demonstrated by using an in-gel kinase assay that 42-kD and 44-kD proteins were responsible for the increased MBP kinase activity (Fig 1B and 1C), indicating that 42-kD and 44-kD MAP kinases were activated by these stimuli. It was shown that MAP kinases can phosphorylate and enhance the transcriptional activity of c-jun protein (c-Jun).⁵⁰ Recently, MAP kinases were shown to phosphorylate transcription factor p62^TCF, which is known to form a ternary complex with p67^SRF at the c-fos promotor serum response element and to enhance the ternary complex formation, leading to c-fos induction.⁵¹ Therefore, we speculate that possible induction of c-fos and phosphorylation and activation of c-Jun by MAP kinases in response to hypoxia and hypoxia/reoxygenation may synergistically activate the function of the AP-1 complex. Previous studies⁵² showing that both antioxidants and H₂O₂ induced activation of AP-1 in vitro strongly support this idea. Next, we showed that both hypoxia and hypoxia/reoxygenation activated S6 kinase (Fig 3). Because MAP kinases phosphorylate one of the S6 kinases (p90^rsk), the activation of S6 kinase by hypoxia and hypoxia/reoxygenation was thought to be through phosphorylation by MAP kinases. p90^rsk is known to participate in the transcriptional regulation of c-fos through phosphorylation of serum response factors as well as in ribosomal protein synthesis through phosphorylation of 40S ribosomal subunit protein S6.^{32 33 34} p90^rsk was also shown to act in the nucleus as a potentially important kinase of nuclear protein lamin C.⁵³ Thus, p90^rsk may act as a mediator between the intracellular second messenger pathways and the intranuclear events and may lead to specific gene expression.

It has been reported that Raf-1 kinase has MAPKKK activity and lies upstream from MAPKK and MAP kinases in various cells and that Raf-1 and MAPKK can be also phosphorylated by MAP kinases directly or indirectly as downstream substrates.^{27 38 39 40 42} Recently, it has been shown that maximal hyperphosphorylation of Raf-1 and MAPKK was substantially achieved after the maximal activation of MAPKKK of Raf-1, MAPKK, and MAP kinases and that maximal hyperphosphorylation of Raf-1 was accompanied by a significant decrease in MAPKKK activity.²⁷ This strongly suggested that the MAPKKK activity of Raf-1 can be downregulated by a feedback mechanism through hyperphosphorylation by MAP kinases. In the present study, by examining the electrophoretic mobility, we found that maximal hyperphosphorylation of Raf-1 was induced at 30 minutes of hypoxia (Fig 4). Using recombinant MAPKK as a substrate, we also examined the MAPKKK activity of Raf-1 stimulated by hypoxia or hypoxia/reoxygenation. We found that maximal MAPKKK activity of Raf-1 was induced at 3 to 5 minutes of hypoxia and at 5 minutes of hypoxia/reoxygenation, respectively, and that the MAPKKK activity significantly decreased at 10 minutes of hypoxia (Fig 5A). Our data support the feedback mechanism of Raf-1 kinase activity. However, we could not exclude the possibility that downregulation of the MAPKKK activity of Raf-1 was mediated by an unknown mechanism (other than MAP kinase) and that the signals upstream from Raf-1 were transient. Furthermore, using recombinant MAP kinase as a substrate, we examined the MAPKK (or MEK) activity. We found that maximal MAPKK activity was induced at 10 minutes of hypoxia and at 10 to 15 minutes of hypoxia/reoxygenation, respectively (Fig 5B). These data confirmed the sequential activation of MAPKKK of Raf-1, MAPKK, MAP kinases, and p90^rsk induced by both hypoxia and hypoxia/reoxygenation. This was followed by the hyperphosphorylation of Raf-1, which is associated with the downregulation of the MAPKKK activity of Raf-1. The time courses of the activation of these kinases induced by hypoxia and hypoxia/reoxygenation are summarized in Fig 6. We speculate that the activation of these kinases revealed in the present study was not so remarkable because serum starvation for 24 hours could not completely downregulate these kinases in cardiac myocytes. In the present study, we balanced the hypoxic medium with 0.1% O₂ and 21% CO₂. CO₂ (21%) lowered the pH of the medium from 7.42 (normoxic) to 7.07 (hypoxic, 0.1% O₂ and 21% CO₂). Therefore, we did control experiments to determine whether the pH change (7.42 to 7.07) itself could induce the activation of MBP kinase activity, and we found that 20% of the effect of hypoxia was due to the alteration of pH (data not shown). The pH of the hypoxic medium incubated with cardiac myocytes for 60 minutes was between 7.4 and 7.5. Therefore, we thought that the effect of pH change was minimal and could be ignored, as for reoxygenation. In accordance with the results of previous studies on the redox control mechanism of transcription factors,⁵² the signals generated by these second messengers seem to converge into activation of AP-1 or NF-B in response to hypoxia or hypoxia/reoxygenation, respectively. PKC appeared to be only partially activated, if at all, by these stimuli in the present study.

View larger version (14K): [in this window] [in a new window]   Figure 6. Time courses of the activation of the MAPKKK activity of Raf-1, MAPKK, MAP kinases, and S6 kinase induced by hypoxia (Hypox) and hypoxia/reoxygenation (Reoxy) were estimated from the patterns of kinase activation rather than having been determined by precise measurement.

p21^ras and Raf-1 are known to operate downstream from cell surface–associated tyrosine kinases⁵⁴ and upstream from MAP kinases^{41 55} and to serve as important intermediates in the pathway leading to the induction of transcription factors. p21^ras was shown to interact directly with Raf-1⁵⁶ and to be upstream from Raf-1 by the observations that transformation by oncogenic ras can be suppressed by the expression of antisense RNA for c-raf-1 or the kinase-defective Raf-1 mutant.^{35 55} Therefore, we are currently investigating whether or not both hypoxia and hypoxia/reoxygenation activate p21^ras.

	Selected Abbreviations and Acronyms

 

AP-1 = activator protein-1 DTT = dithiothreitol GST = glutathione S-transferase MAP = mitogen-activated protein MAPKK = MAP kinase kinase MAPKKK = MAP kinase kinase kinase MBP = myelin basic protein PCR = polymerase chain reaction PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate

	Acknowledgments

 
This study was supported by a grant for cardiomyopathy and a grant for intractable vasculitis from the Ministry of Health and Welfare, Japan; a grant for scientific research from the Ministry of Education, Science, and Culture, Japan; a grant from the Kowa Life Science Foundation; a grant from the Ichiro Kanehara Foundation; a grant from the Kanae Foundation of Research for New Medicine; and a grant from the Japan Foundation of Cardiovascular Research. We thank Kaori Takahashi for excellent technical assistance.

Received December 27, 1994; accepted September 12, 1995.

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