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Hyperosmolar Mannitol Stimulates Expression of Aquaporins 4 and 9 through a p38 Mitogen-activated Protein Kinase-dependent Pathway in Rat Astrocytes

Hyperosmolar Mannitol Stimulates Expression of Aquaporins 4 and 9 through a p38 Mitogen-activated... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 45, Issue of November 7, pp. 44525–44534, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Hyperosmolar Mannitol Stimulates Expression of Aquaporins 4 and 9 through a p38 Mitogen-activated Protein Kinase-dependent Pathway in Rat Astrocytes* Received for publication, April 25, 2003, and in revised form, August 14, 2003 Published, JBC Papers in Press, August 27, 2003, DOI 10.1074/jbc.M304368200 Hajime Arima‡§ , Naoki Yamamoto , Kazuya Sobue§, Fuminori Umenishi**, Toyohiro Tada‡‡, Hirotada Katsuya§, and Kiyofumi Asai From the ‡Department of Anesthesia and Critical Care, Okazaki City Hospital, 3-1 Goshoai, Koryuji-cho, Okazaki 444-8553, Japan, the §Department of Anesthesiology and Medical Crisis Management, Nagoya City University, Graduate School of Medical Sciences, Mizuho-ku, Nagoya 467-8601, Japan, the Department of Molecular Neurobiology, Nagoya City University, Graduate School of Medical Sciences, Mizuho-ku, Nagoya 467-8601, Japan, the **Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, and the ‡‡Department of Pathology, Nagoya City University School of Nursing, Mizuho-ku, Nagoya 467-8601, Japan life forms, including archaea, eubacteria, fungi, plants, and all The membrane pore proteins, aquaporins (AQPs), fa- cilitate the osmotically driven passage of water and, in animal phyla (1–3). AQPs are widely distributed, and more some instances, small solutes. Under hyperosmotic con- than one AQP may be present in the same cell. All AQPs seem ditions, the expression of some AQPs changes, and some to have six transmembrane domains with five connecting loops studies have shown that the expression of AQP1 and and their amino and carboxyl termini in the cytoplasm (4 – 6). AQP5 is regulated by MAPKs. However, the mechanisms AQPs are synthesized as monomers, but there is evidence that regulating the expression of AQP4 and AQP9 induced by they exist as tetramers in the membrane and have four water hyperosmotic stress are poorly understood. In this pores (7). The expression of some AQPs is affected by various study, we observed that hyperosmotic stress induced by factors, including vasopressin (8, 9), hypoxia and reoxygenation mannitol increased the expression of AQP4 and AQP9 in (10), growth factors (11), and retinoic acid (12). In addition, cultured rat astrocytes, and intraperitoneal infusion of hyperosmotic stress increases the expression of AQPs 1, 2, 3, mannitol increased AQP4 and AQP9 in the rat brain and 5 (13–19). cortex. In addition, a p38 MAPK inhibitor, but not ERK Many types of cells respond to changes in osmotic pressure and JNK inhibitors, suppressed their expression in cul- with adaptive mechanisms that allow them to re-establish ho- tured astrocytes. AQPs play important roles in main- meostasis of those aspects of cell structure and function that taining brain homeostasis. The expression of AQP4 and have been disturbed osmotically (20). Osmotic stress may dam- AQP9 in astrocytes changes after brain ischemia or age DNA and proteins, resulting in impairment of cell function traumatic injury, and some studies have shown that p38 and the induction of repair processes and protection systems MAPK in astrocytes is activated under similar condi- (21). These relatively nonspecific responses to cell damage may tions. Since mannitol is commonly used to reduce brain be an important aspect of cellular adaptation to osmotic stress. edema, understanding the regulation of AQPs and p38 Mitogen-activated protein kinases (MAPKs), specifically extra- MAPK in astrocytes under hyperosmotic conditions in- duced with mannitol may lead to a control of water cellular signal-regulated kinase (ERK), c-Jun N-terminal pro- movements and a new treatment for brain edema. tein kinase (JNK), and p38 MAPK, are important intracellular signal transduction pathways that are activated in response to changes in osmolality (22–25). Thus, osmoregulation of AQPs Water passes across cell membranes substantially faster might be mediated by MAPKs. There are some reports that the than can be explained by simple diffusion. This is necessary for expression of AQP1 and AQP5 under hyperosmotic conditions the active regulation of water homeostasis under conditions of is regulated by MAPKs (19, 26), but the role of MAPKs in the osmotic stress in the kidneys, lungs, brain, etc., and to meet expression of AQP4 and AQP9 under hyperosmotic stress is these needs, a family of membrane channel proteins evolved. unclear. These proteins, termed “aquaporins” (AQPs), are found in all Regulation of tissue water content and brain volume is crit- ical for normal functioning of the central nervous system, which is highly sensitive to any increase in intracranial pres- * This work was supported by a grant-in-aid for Scientific Research sure. Brain edema may rapidly become life-threatening be- (C), a grant-in-aid for Exploratory Research, and a grant-in-aid for cause of the rigid encasement of the brain. AQPs were sug- Young Scientists (A and B) from the Ministry of Education, Culture, gested to have a role in maintaining the homeostasis of the Sports, Science and Technology of Japan and a Research Grant from the brain (27–29). Among them, numerous studies indicated that Japan Brain Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must the expression of AQP1 (30, 31), AQP4 (32–34), and AQP9 (35) therefore be hereby marked “advertisement” in accordance with 18 is sensitive to brain injury, swelling, and other experimental U.S.C. Section 1734 solely to indicate this fact. interventions. AQP4 in astrocytes of the brain cortex decreased To whom correspondence should be addressed: Dept. of Anesthesia in the acute phase of brain injury (32, 33) and increased in the and Critical Care, Okazaki City Hospital, 3-1 Goshoai, Koryuji-cho, Okazaki 444-8553, Japan. Tel.: 81-564-21-8111; Fax: 81-564-25-2913; E-mail: [email protected]. The abbreviations used are: AQP, aquaporin; MAPK, mitogen-acti- DMEM, low glucose Dulbecco’s modified Eagle’s medium; RT, reverse vated protein kinase; ERK, extracellular signal-regulated kinase; JNK, transcriptase; TK, thymidine kinase; MEK, mitogen-activated protein c-Jun NH2-terminal kinase; GFAP, glial fibrillary acidic protein; l- kinase/extracellular signal-regulated kinase kinase. This paper is available on line at http://www.jbc.org 44525 This is an Open Access article under the CC BY license. 44526 p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions source with excitation at 488 nm and emission at 520 nm. The chro- later phase (32, 34). AQP4-deficient mice survive much longer matograms were visualized and analyzed automatically using GOLD and have a better neurological outcome from experimentally Software (Beckman). The peaks were expressed in relative fluorescence induced brain edema than wild-type mice (36). AQP4 in astro- units, and the retention time was expressed in minutes. AQP4, AQP9, cytes is tightly associated with the blood-brain barrier (37). In and -actin PCR products were compared by integrating each peak the brain cortex, astrocyte-specific AQP9 appears in the infarct area. border zone after transient ischemic stroke (35). Western Blot Analysis—Cells cultured under the specified conditions 2 2 were harvested with Ca - and Mg -free phosphate-buffered saline Hyperosmotic solutions of mannitol are used commonly to (PBS (-)) and centrifuged for 10 min at 800  g at 4 °C. For quantitation reduce brain edema, so it is important to elucidate the rela- of AQP4 and AQP9 protein, cell pellets were suspended in 100 lof tionship between the expression of AQPs and hyperosmolarity Tris-buffered saline (TBS) containing 200 mM phenylmethanesulfonyl induced with mannitol. This study was designed to assess the fluoride, 10 M pepstatin A, 10 M leupeptin, 2 mM EDTA, and 0.5% effects of hyperosmotic stress induced with mannitol on the Nonidet P-40 and then sonicated on ice at 8-watt output for 10 s. expression of AQP4 and AQP9 and to identify the signal trans- Samples were kept at room temperature for 15 min with 5 lofSDS sample buffer. For quantitation of MAPKs and phosphorylated MAPKs, duction pathways regulating the response in rat astrocytes in the cell pellets were suspended in 150 l of buffer (50 mM Tris-HCl, pH which AQP4 and AQP9 are localized (10). We found that the 7.4, 1 mM phenylmethanesulfonyl fluoride, 10 M pepstatin A, 10 M expression of AQP4 and AQP9 is induced in cultured rat astro- leupeptin) and sonicated on ice at 8-watt output for 10 s. The cell lysates cytes and in rat brain cortex by hyperosmotic stress. In addi- were centrifuged for 30 min at 15,000  g at 4 °C, and the supernatants tion, we demonstrated that this induction requires selective were collected and boiled with 5 l of SDS sample buffer for 5 min. For activation and involvement of the p38 MAPK pathway in cul- GFAP, cell pellets were suspended and sonicated as for AQPs and boiled with 5 l of SDS sample buffer for 5 min. Total protein was measured tured rat astrocytes. We believe this study provides the first using a BCA protein assay reagent kit (Pierce). 3 g of protein for AQP4 example of osmotic regulation of AQP4 and AQP9 and the first and GFAP, 20 g for AQP9, and 30 g for MAPKs and phosphorylated evidence that the expression of AQP4 and AQP9 is regulated by MAPKs were separated by 12.5% SDS-polyacrylamide gel electrophore- the p38 MAPK pathway. sis (ATTO, Tokyo, Japan) and transferred to a Clear Blot Membrane-P (ATTO), which was then blocked for 1 h with 5% skim milk in TBS-T (20 EXPERIMENTAL PROCEDURES mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween 20). The membranes were Cell Culture—Cultures of cortical astrocytes were prepared from rat incubated overnight with anti-AQP4 antibody (Chemicon International, postnatal cortices (P2) according to previously described methods (38). Inc., Temecula, CA) diluted 1:750, anti-AQP9 antibody (Chemicon) di- Trypsinized and dissociated cortical cells were cultured in 75-cm cul- luted 1:500, anti-p38 MAPK antibody (Santa Cruz Biotechnology, Santa ture flasks (Corning Glass) containing low glucose (1000 mg/liter) Dul- Cruz, CA) diluted 1:500, anti-phosphorylated p38 MAPK antibody (Cell becco’s modified Eagle’s medium (l-DMEM; Invitrogen) supplemented Signaling Technology, Inc., Beverly, MA) diluted 1:1000, anti-ERK an- with 10% fetal bovine serum (Sigma). After incubation for 5–7 days, the tibody (Promega) diluted 1:5000, anti-phosphorylated ERK antibody cells were trypsinized and subcultured in 60-mm diameter culture (Cell Signaling Technology) diluted 1:1000, anti-JNK antibody (Santa dishes (Falcon; PGC Scientific, Frederick, MD). The cell population Cruz Biotechnology) diluted 1:500, anti-phosphorylated JNK antibody consisted of over 95% astrocytes as determined by immunocytochemical (Cell Signaling Technology) diluted 1:1000, or anti-GFAP antibody staining with anti-glial fibrillary acidic protein (GFAP) antibody. In (Chemicon) diluted 1:1000 all in TBS-T containing 5% skim milk. After neural cells, AQP4 and AQP9 are expressed in astrocytes but not in washing the membranes four times for 15 min each with TBS-T, they neurons, oligodendrocytes, and microglia (10). were incubated for 1 h with 1:1000 diluted (for AQP4, MAPKs, and Hyperosmotic Stress of Cultured Rat Astrocytes—When astrocyte GFAP) or 1:500 diluted (for AQP9) horseradish peroxidase-conjugated cultures became confluent, equal volumes of medium with or without secondary antibody in TBS-T buffer containing 5% skim milk. Protein various concentrations of mannitol (Sigma) were added, and the cul- was visualized using an ECL detection system (Amersham Biosciences). tures were incubated for the indicated times. Osmolality of the medium Relative band intensities were determined by densitometry using TM was measured by the freezing point depression method (Osmostat Kodak Digital Science 1D version 2.0 (Eastman Kodak Co.). OM-6040; ARKREY, Kyoto, Japan). All cultures were maintained at Isolation of the 5-Flanking Regions of Human AQP4 and AQP9 37 °C in an atmosphere of 5% CO , 95% air. Genes—The 5-flanking region of the human AQP4 gene was isolated as Reverse Transcription (RT)-PCR—At the indicated times, total RNA described previously (44). The AQP4 gene is regulated by alternative was isolated using Trizol reagent (Invitrogen). cDNAs were generated promoters upstream of exons 0 and 1. The 5-flanking regions of the from 1 g of total RNA by Superscript II RNase H reverse tran- exon 0 promoter (bp 1930 to 29) (nucleotide 1 corresponds to the scriptase (Invitrogen) primed with oligo(dT) (Invitrogen). They were initiation codon of exon 0) and the exon 1 promoter (bp 297 to 113) amplified with primers designed according to the published sequences. (nucleotide 1 corresponds to the initiation codon of exon 1) were The PCR protocols and primers for AQP4, AQP9, and -actin have been subcloned into the vector pCR2.1 (Invitrogen), and 5 deletion mutants described previously (10). The oligonucleotide primers were as follows: were constructed. Deletion mutants corresponding to bp 1930 to 29 AQP4 (39), 5-TTGGACCAATCATAGGCGC-3 (forward) and 5-GGT- in the exon 0 promoter (eight constructs) and from bp 297 to 133 in CAATGTCGATCACATGC-3 (reverse); AQP9 (40), 5-GATGCCTTCT- the exon 1 promoter were fused to the promoterless vector PGV-B GAGAAGGACG-3 (forward) and 5-AGAGAGCCATCACGACTGC-3 (Toyo-ink, Tokyo, Japan) containing the luciferase reporter gene. (reverse); -actin (41), 5-GACCTGACTGACTACCTCAT-3 (forward) The promoter region of AQP9 (bp 1125 to 55) (nucleotide 1 and 5-TCGTCATACTCCTGCTTGCT-3 (reverse). Multitarget PCRs corresponds to the initiation codon) was also isolated by a similar were performed by coamplifying -actin as the internal standard. The method. Briefly, the region was isolated using a Promoter Finder DNA reaction mixture for AQP4 contained 2 l of the RT reaction product, Walking kit (Clontech, Palo Alto, CA), which contains adapter-linked and the reaction was carried out for 20 cycles, using a 95 °C, 30-s human genomic DNA digested with EcoRV, ScaI, DraI, PvuII, and SspI. denaturing step; a 57 °C, 30-s annealing step; and a 72 °C, 1-min A two-step PCR was performed according to the manufacturer’s instruc- extension step. The reaction mixture for AQP9 contained 2 l of the RT tions. The first PCR consisted of 94 °C for 1 min followed by 30 cycles of reaction product, and the reaction was carried out for 27 cycles, using a 94 °C for 15 s, 60 °C for 30 s, and 68 °C for 4 min and was done in a final 95 °C, 30-s denaturing step; a 58 °C, 30-s annealing step; and a 72 °C, volume of 50 l with 10 pmol of a specific primer, 5-GCTCTTCAAGAC- 1-min extension step. CAGTCTCTGCTTGA-3 (nucleotides 60 to 46). The second PCR was Semiquantification of mRNA—The PCR products were visualized by done with the same protocol using 1 l of the first PCR product and a ethidium bromide staining following separation on 1.5% agarose gels specific primer, 5-CTCTGAGGACTCCTGTTTCTACCAAT-3 (nucleo- and quantified by laser-induced fluorescence-linked capillary gel elec- tides 11 to 35). The PCR products were subcloned into pCR2.1, and trophoresis. Details of laser-induced fluorescence-linked capillary gel both strands were sequenced completely. BLAST searches were per- TM electrophoresis have been reported elsewhere (10, 42, 43). Briefly, 40 l formed on the NCBI sites. The sequence was identical to GenBank of PCR products was applied to a P/ACE system 5010 (Beckman Instru- accession number AC025431. Deletion mutants corresponding to bp ments). Samples were injected at a pressure of 0.5 p.s.i.g. for 20 s. 1125 to 55 in the AQP9 promoter (four constructs) were fused to the Separation was carried out at 9.4 kV for 35 min in a capillary (47 cm promoterless vector PGV-B. long, 100-m diameter) filled with electrophoresis gel buffer containing Cell Transfection and Luciferase Reporter Gene Assay—Reporter as- DNA fluorescence stain (LIFluor dsDNA 1000 kit; Beckman). Laser- says were done on AQP4 and AQP9 gene fragments fused upstream of induced fluorescence detection was preformed using an argon-ion laser the firefly luciferase reporter in PGV-B using the Dual-Luciferase re- p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions 44527 porter assay system (Promega), with which the pRL-thymidine kinase (TK) plasmid (Promega) containing the Renilla luciferase gene under control of the TK promoter was co-transfected as an internal control. For transient transfection, astrocytes in l-DMEM supplemented with 10% fetal bovine serum were plated at 1  10 cells/well in a 48-well plate, incubated for 18 h, and transfected in serum-free medium with 0.4 g of the test plasmid and 0.01 g of pRL-TK using 1 l of Lipofect- AMINE 2000 (Invitrogen). After 4 h, 150 l of l-DMEM containing 20% fetal bovine serum was added. 48 h after transfection, equal volumes of l-DMEM or 3% mannitol in l-DMEM were added. After 24 h, the astrocytes were washed and harvested. The activities of control Renilla luciferase and firefly luciferase were measured in triplicate. Induction of Hyperosmolarity in Rats—All animal studies were un- dertaken after the protocols were approved by the Animals Care and Use Committee of Nagoya City University Graduate School of Medical Sciences. Male Wistar rats (150 g) were used throughout. Animals were anesthetized with pentobarbital (Schering-Plough Animal Health, Kenil- worth, NJ) (3.2 mg/100 g, body weight) and placed under a heating lamp. Intraperitoneal infusion was performed using a modification of a technique described previously (45). A polyurethane catheter (20 gauge) was inserted into the peritoneum by puncture and tightly fixed to avoid leakage of the experimental solution. The animals were then allowed to recover under a heating lamp. An isosmotic solution (121 mmol of NaCl, 5 mmol of KCl, 1 mmol of MgCl , 1 mmol of NaH PO , 2 mmol of CaCl , 2 2 4 2 18 of mmol NaHCO , 22 mmol of glucose) or a hyperosmotic mannitol solution (1.1 mol of mannitol added to the isosmotic solution) was administered by intraperitoneal infusion using an infusion pump. The infusion rate was 1.9 ml/h, and the duration of the infusion was 1, 3, or 6 h. Control animals were anesthetized and cannulated but were not administered any solution. At the conclusion of the experiments, the animals were disconnected from the infusion pump and rapidly decap- itated. Samples for Western blot analyses, done as described above, were obtained from the superficial region of the brain cortex, and cerebral sections (5 mm thick) were obtained for immunohistochemical staining. Blood samples were removed, and plasma osmotic pressure was measured by the freezing point depression method as described above. Immunohistochemical Staining—Coronal sections from the brain were embedded in paraffin by the AMeX method (46). Briefly, tissues were fixed in acetone at 4 °C for 3 days, cleared in methyl benzoate and xylene, and then embedded in paraffin. Sections 3 m thick were prepared and deparaffinized with xylene. Immunoreactivity was visu- alized using the streptavidin/biotin method (Histofine SAB-PO Kit, Nichirei, Tokyo, Japan). After washing the sections with PBS (), they were treated with 0.3% (v/v) hydrogen peroxide in methanol for 30 min to inactivate endogenous peroxidase. They were then immersed in a 1:20 dilution of nonimmune goat serum for 10 min to block nonspecific binding, and after blotting to remove excess serum they were incubated FIG.1. Effect of mannitol on expression of AQP4 and AQP9 mRNAs and proteins. A, rat astrocytes were incubated for6hin at room temperature for 1 h with anti-AQP4 antibody (Chemicon) isosmotic control medium or hyperosmotic medium containing the des- diluted 1:400, anti-AQP9 antibody (Chemicon) diluted 1:100, or anti- ignated concentrations of mannitol. Cells were processed for RT-PCR GFAP antibody (Chemicon) diluted 1:50 in PBS () containing 1% and semiquantification of AQP4 or AQP9 mRNA. The expression of bovine serum albumin. Control sections were treated with nonimmune mRNA (normalized to -actin) (n  8 for each group) is presented as a rabbit immunoglobulins (MBL, Nagoya, Japan). The sections were percentage of the control (mean  S.E.; *, p  0.05 versus control). B, rinsed three times with PBS (), incubated for 30 min each with the rat astrocytes were treated as described in A and processed for immu- secondary antibody, rinsed three times, and then incubated with a noblotting with anti-AQP4 or anti-AQP9 antibody. C, immunoblots (n streptavidin-biotin-peroxidase complex for 15 min. After washing the 8 for each group) were analyzed by densitometry, and band densities sections with PBS (), the peroxidase reaction was developed by incu- are presented as a percentage of the control (mean  S.E.; *, p  0.05 bating them in 0.02% (w/v) 3,3-diaminobenzidine tetrahydrochloride versus control). (Sigma) solution containing 0.003% (v/v) hydrogen peroxide and 10 mM sodium azide. The sections were counterstained with hematoxylin. for RT-PCR or harvesting for immunoblot analysis. Both AQP4 Data Analysis—Data are expressed as means  S.E. of at least four and AQP9 mRNAs (Fig. 1A) and proteins (Fig. 1, B and C) independent experiments. Statistical analysis was performed by one- increased significantly after treatment with 1, 3, and 10% way factorial analysis of variance combined with Scheffe’s test for all mannitol. AQP4 and AQP9 mRNAs and AQP4 protein in- comparison pairs. Reporter gene assays were carried out three times, and the results were analyzed by Student’s t test for statistical signif- creased maximally after treatment with 3% mannitol, whereas icance. Differences with p values  0.05 were considered significant. AQP9 protein peaked after treatment with 1% mannitol. AQP4 and AQP9 mRNAs and proteins were induced to a lesser extent RESULTS with 10% mannitol. Induction of AQP4 and AQP9 in Cultured Rat Astrocytes To analyze the time course of the mannitol-mediated induc- Subjected to Hyperosmotic Stress—To examine the effects of tion of AQP4 and AQP9 expression, astrocytes were treated hyperosmotic stress on the expression of AQP4 and AQP9 with hyperosmotic medium containing 3% mannitol for various mRNAs and proteins, astrocytes were incubated in isosmotic periods of time. AQP4 mRNA increased significantly after 1, 3, control medium (final osmotic pressure 311  1.4 mOsM (n  6, and 12 h of treatment with mannitol, and AQP9 mRNA 4)) or hyperosmotic medium supplemented with 0.3% (324  increased significantly after 3, 6, and 12 h of treatment (Fig. 1.6 mOsM), 1% (363  2.7 mOsM), 3% (491  4.7 mOsM), or 10% 2A). After 24 h, both AQP mRNAs returned to base-line levels. mannitol (854  8.9 mOsM) for 6 h before isolation of total RNA AQP4 and AQP9 proteins increased after6hof treatment with 44528 p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions FIG.3. Effect of cycloheximide on hyperosmotic induction of AQP4 and AQP9 mRNAs. Rat astrocytes were incubated for 6 h with or without 3% mannitol or cycloheximide (25 g/ml). Cells were proc- essed for RT-PCR and semiquantification of AQP4 or AQP9 mRNA. The expression of mRNA (normalized to -actin) (n  4 for each group) is presented as a percentage of the control (mean  S.E.; *, p  0.05 versus control). AQP9 is solute-specific, astrocytes were incubated in isosmotic medium or hyperosmotic medium supplemented with 0.4% NaCl (502  0.7 mOsM), 1% urea (501  2.5 mOsM), 1.5% glycerol (491  4.5 mOsM), 3% sorbitol (485  9.2 mOsM), or 3% mannitol (491  4.7 mOsM), for 6 h. NaCl and glycerol had little effect on the expression of AQP4 and AQP9 mRNAs and pro- teins, whereas they were decreased by urea and increased by mannitol and sorbitol (Fig. 4, A–C). Except for NaCl, the effects of specific solutes on hyperosmotic induction of AQP4 and AQP9 were similar to those reported for AQP1, -3, and -5 (17, 19, 26). Although6hof exposure to hyperosmotic medium supplemented with NaCl did not increase the expression of AQP4 and AQP9, other studies have demonstrated that over 12 h of treatment with NaCl is required to increase the expres- sion of AQP1 and AQP5 (19, 26). To examine the time course of NaCl-, urea-, glycerol-, or sorbitol-mediated induction of AQP4 and AQP9 expression, astrocytes were treated with hyperos- motic medium supplemented with the solutes for various peri- ods of time before harvesting for immunoblot analysis. AQP4 and AQP9 proteins increased after 12 h of treatment with NaCl (Fig. 4D). Although AQP4 protein was at the same high level even after 24 h of treatment, AQP9 protein decreased but was FIG.2. Time course of hyperosmotic induction of AQP4 and AQP9 mRNAs and proteins. A, rat astrocytes were incubated in still higher than in the control. After 12 or 24 h of treatment, hyperosmotic medium containing 3% mannitol. At the indicated times, urea and glycerol did not affect the levels of AQP4 and AQP9 cells were processed for RT-PCR and semiquantification of AQP4 or proteins. The effect of sorbitol was the same as that of mannitol AQP9 mRNA. The expression of mRNA (normalized to -actin) (n  8 (data not shown). for each group) is presented as a percentage of the control at time 0 The Role of MAPKs in the Expression of AQP4 and AQP9 in (mean  S.E.; *, p  0.05 versus time 0). B, rat astrocytes were treated as described in A and processed for immunoblotting with anti AQP4 or Cultured Rat Astrocytes Subjected to Hyperosmotic Stress— anti-AQP9 antibody. C, immunoblots (n  8 for each group) were Hyperosmotic stress can activate signaling through three analyzed by densitometry, and band densities are presented as a per- MAPK pathways, p38 MAPK, ERK, and JNK, all three of centage of the control at time 0 (mean  S.E.; *, p  0.05 versus time 0). which are activated in mouse astrocytes by sorbitol (47). To investigate whether these three MAPKs are activated in rat mannitol (Fig. 2, B and C). Although AQP4 mRNA returned to astrocytes by mannitol, we performed immunoblot analyses of the base-line level after 24 h of treatment with mannitol, AQP4 cells incubated in hyperosmotic medium using antibodies that protein remained at the same high level, suggesting that this react with either the phosphorylated or total amounts of each of gene is regulated at both the transcriptional and post-tran- the three MAPKs. Whereas the total amount of each MAPK scriptional levels. AQP9 protein peaked at 12 h but returned to remained constant, p38 MAPK was activated by hyperosmotic the base-line level after 24 h of treatment with mannitol. stress after 30 min, and activation persisted even at 120 min To see whether hyperosmotic induction of AQP4 and AQP9 (Fig. 5A). ERK (Fig. 5B) and JNK (Fig. 5C) activation both mRNAs requires de novo protein synthesis, astrocytes incu- peaked at 30 min after hyperosmotic exposure. bated in hyperosmotic medium containing 3% mannitol were To investigate the possible involvement of the three MAPK treated with or without the protein synthesis inhibitor, cyclo- cascades in the increased expression of AQP4 in response to heximide (25 g/ml), for 6 h. The increased expression of both mannitol, the effects of the p38 MAPK inhibitor SB203580, the AQP mRNAs induced by mannitol was not affected by cyclo- mitogen-activated extracellular signal-regulated kinases heximide (Fig. 3), suggesting that de novo protein synthesis is (MEK) 1/2 inhibitor PD98059, and the JNK inhibitor SP600125 not involved. were examined. MEK 1/2 are the upstream kinases that acti- To determine whether hyperosmotic induction of AQP4 and vate ERK. The hyperosmotic induction of AQP4 mRNA and p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions 44529 FIG.5. Phosphorylation of MAPKs induced by hyperosmotic stress. Rat astrocytes were incubated in hyperosmotic medium con- taining 3% mannitol. At the indicated times, cells were harvested and processed for immunoblotting using anti-p38 MAPK antibody or anti- phosphorylated p38 MAPK (p-p38) antibody (A), anti-ERK antibody or anti-phosphorylated ERK (p-ERK) antibody (B), or anti JNK antibody or anti-phosphorylated JNK (p-JNK) antibody (C). inhibitor was not a result of nonspecific effects. Thus, it ap- pears that the increased expression of AQP4 in response to mannitol requires signaling through p38 MAPK. RT-PCR re- vealed that SB203580 reduced the expression of AQP4 mRNA in a concentration-dependent manner (Fig. 6C). The same re- sults were obtained with AQP9 (Fig. 7). FIG.4. Hyperosmotic induction of AQP4 and AQP9 mRNAs To determine whether just p38 MAPK activation is sufficient and proteins by different solutes. A, rat astrocytes were incubated for6hin control isosmotic medium (final osmotic pressure, 311  1.4 for inducing AQP4 and AQP9, astrocytes were incubated with mOsm (n  4 for each group)) or medium supplemented with 0.4% NaCl the p38 MAPK activators, hydrogen peroxide (2 mM) or aniso- (502  0.7 mOsm), 1% urea (501  2.5 mOsm), 1.5% glycerol (491  4.5 mycin (50 M), under isosmotic conditions. The total amount of mOsm), 3% sorbitol (485  9.2 mOsm), or 3% mannitol (491  4.7 p38 MAPK remained constant, and both activators caused its mOsm) and then processed for RT-PCR and semiquantification of AQP4 or AQP9 mRNA. The expression of mRNA (normalized to -actin) (n  phosphorylation (Fig. 8, A and B). Activation of p38 MAPK by 4 for each group) is presented as a percentage of that in control isos- hydrogen peroxide increased 15 min after exposure and peaked motic medium (mean  S.E.; *, p  0.05 versus control). B, rat astro- at 60 min (Fig. 8A), whereas its activation by exposure to cytes were treated as described in A and processed for immunoblotting anisomycin peaked at 30 min and was observed only weakly at with anti-AQP4 or anti-AQP9 antibody. C, immunoblots (n  4 for each 60 min (Fig. 8B). Hydrogen peroxide increased AQP4 and group) were analyzed by densitometry, and band densities are pre- sented as a percentage of that in control isosmotic medium (mean AQP9 mRNAs, whereas they were decreased by anisomycin S.E.; *, p  0.05 versus control). D, rat astrocytes were incubated in (Fig. 8C). AQP4 and AQP9 proteins were increased by hydro- hyperosmotic medium supplemented with 0.4% NaCl. At the indicated gen peroxide but were not affected by anisomycin (Fig. 8D). times, cells were processed for immunoblotting with anti-AQP4 or anti- Transcriptional Regulation of AQP4 and AQP9 Genes in AQP9 antibody. The immunoblots (n  4 for each group) were analyzed by densitometry, and band densities are presented as a percentage of Cultured Rat Astrocytes under Hyperosmotic Conditions—The the control at time 0 (mean  S.E.; *, p  0.05 versus time 0). promoter activity of the AQP4 gene has been demonstrated in SF-126 (glioblastoma) and Madin-Darby canine kidney cells protein was inhibited by SB203580 (10 M) but not by PD98059 (44), but that of the AQP9 gene has not been investigated. (10 M) or SP600125 (10 M) (Fig. 6, A and B), and all three Luciferase promoter gene assays were performed to determine inhibitors had no effect on the basal expression of AQP4. To the transcriptional activity of the AQP4 and AQP9 genes in determine whether the p38 MAPK inhibitor specifically pre- astrocytes under isosmotic and hyperosmotic conditions. Eight vented AQP4 induction, astrocytes were incubated with luciferase promoter constructs of AQP4 exon 0, one of AQP4 Me SO. It had no effect on AQP4 expression, suggesting that exon 1, or four of AQP9 were transfected into astrocytes, and the reduction of AQP4 expression caused by the p38 MAPK luciferase activity was measured under isosmotic conditions 44530 p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions FIG.6. Effect of MAPK inhibitors on hyperosmotic induction FIG.7. Effect of MAPK inhibitors on hyperosmotic induction of AQP4 mRNA and protein. A, rat astrocytes were incubated for 6 h of AQP9 mRNA and protein. Rat astrocytes were incubated and with or without 3% mannitol and MAPK inhibitors (the p38 MAPK treated as described in the legend for Fig. 6. Using AQP9 primer and inhibitor SB203580 (10 M), the MEK 1/2 inhibitor PD98059 (10 M), or anti-AQP9 antibody, RT-PCR (A and C) and immunoblotting (B) were the JNK inhibitor SP600125 (10 M)). To determine whether the reduc- carried out as described in the legend for Fig. 6. DMSO,Me SO. tion of AQPs induced by MAPK inhibitors was a result of nonspecific effects, astrocytes were incubated with Me SO (DMSO;5 M). Cells the AQP4 promoter is up-regulated by hyperosmotic stress and were processed for RT-PCR and semiquantification of AQP4 mRNA. The expression of mRNA (normalized to -actin) (n  4 for each group) that a critical cis element involved is located between 345 and is presented as a percentage of the control (mean  S.E.; *, p  0.05 428. On the other hand, the 765/55 and 486/55 AQP9 versus control). B, rat astrocytes were treated as described in A and constructs showed significant induction of luciferase activity, processed for immunoblotting with anti-AQP4 antibody. C, rat astro- but other AQP9 constructs were not affected by hyperosmolar- cytes were incubated for 6 h with or without 3% mannitol and the p38 MAPK inhibitor SB203580 at the indicated concentrations. Cells were ity (Fig. 9B). These results indicate that the AQP9 promoter is processed for RT-PCR and semiquantification of mRNA. The expression also up-regulated by hyperosmotic stress and that a critical cis of mRNA is presented as described in A (mean  S.E.; n  4 for each element is located between 486 and 176. group; *, p  0.05 versus control). Induction of AQP4 and AQP9 Expression in Rat Brain Cortex by Intraperitoneal Infusion of Hyperosmotic Solution—To in- (Fig. 9, A and B). The 297/113 exon 1 construct had the vestigate whether AQP4 and AQP9 expression is induced in rat greatest activity among the AQP4 constructs. All eight con- brain cortex under hyperosmotic conditions, an isosmotic solu- structs derived from the exon 0 promoter had weak luciferase tion (osmotic pressure was 298  5.9 mOsM (n  4)) or a activity. There was an increase in luciferase activity of the exon hyperosmotic solution of mannitol (1368  24.8 mOsM) was 0 830/29 construct compared with the longer isoform, indi- infused intraperitoneally for 1, 3, and 6 h, after which samples cating the presence of a suppressor element just upstream of bp from the superficial region of brain cortex were taken for West- 831. Luciferase activity of the 175/55 AQP9 construct was ern blot analyses, and cerebral sections were taken for immu- greater than that of the other longer AQP9 constructs, indicat- nohistochemical staining. Animals recovered from anesthesia ing the presence of a suppressor element just upstream of bp within 1 h after intraperitoneal cannulation. The position of 174. the intraperitoneal catheter tip was confirmed after decapita- Under hyperosmotic conditions achieved with 3% mannitol, tion, at which time blood samples were obtained from the right the luciferase activities of the 1625/29, 830/29, 515/ atrium. The plasma osmotic pressure of control animals was 29, and 428/29 AQP4 exon 0 constructs were induced 297  3.9 mOsM, and it did not increase after infusion of the significantly (Fig. 9A). Luciferase activity of the 428/29 isosmotic solution (data not shown). Infusion of the hyperos- construct was increased the most. These results indicate that motic solution resulted in an increase of the plasma osmotic p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions 44531 FIG.9. Promoter activities of the 5-flanking regions of the AQP4 and AQP9 genes. A, AQP4 promoter luciferase plasmid con- structs were transfected into rat astrocytes. The transfected cells were incubated for 24 h with or without 3% mannitol, and promoter activity was analyzed. The expression of promoter activities (normalized to Renilla luciferase activity) (n  3 for each group) is presented as a FIG.8. Effect of p38 MAPK activators on the induction of AQP4 percentage of that of the AQP4 exon 0 construct (bp 1930 to 29) and AQP9 mRNAs and proteins. A, rat astrocytes were incubated in activity under isosmotic conditions (mean  S.E.; *, p  0.05, with isosmotic medium containing hydrogen peroxide (2 mM). At the indi- versus without 3% mannitol with each construct). B, AQP9 promoter cated times, cells were harvested and processed for immunoblotting activity was analyzed as described in A. The results (normalized to the using anti-p38 MAPK antibody or anti-phosphorylated p38 MAPK (p- Renilla luciferase activity) are expressed as percentages of the AQP9 p38). B, rat astrocytes were incubated in isosmotic medium containing construct (bp 1125 to 55) activity under isosmotic conditions anisomycin (50 M). The blots were processed as described in A. C, rat (mean  S.E.; n  3 for each group; *, p  0.05, with versus without 3% astrocytes were incubated for 6 h with or without hydrogen peroxide (2 mannitol with each construct). mM) or anisomycin (50 M) under isosmotic conditions. To determine whether the reduction of AQPs expression by MAPK activators was a under the pial surface (28, 29), and we found a normal immu- result of nonspecific effects, astrocytes were also incubated with Me SO nostaining pattern of AQP4 under isosmotic conditions (Fig. (DMSO;5 M). Cells were processed for RT-PCR and semiquantification of AQP4 or AQP9 mRNA. The expression of mRNA (normalized to 10B, a). Positive staining was observed under the pial surface -actin) (n  4 for each group) is presented as a percentage of the and around microvessels, with the area along the blood vessels control (mean  S.E.; *, p  0.05 versus control). D, rat astrocytes were stained weekly. Intraperitoneal infusion of mannitol solution incubated for 6 h with or without hydrogen peroxide (2 mM) or aniso- for 6 h significantly increased the positive staining of AQP4 mycin (50 M) under isosmotic conditions and processed for immuno- under the pial surface and the area along the blood vessels (Fig. blotting with anti-AQP4 or anti-AQP9 antibody. 10B, b). AQP9 was stained weakly in normal brain cortex and was difficult to find. The staining pattern of AQP9 under isos- pressure (316  3.9 mOsM after1hof infusion, 354  6.6 mOsM motic conditions is shown in Fig. 10B, c, with positive staining after 3 h, and 369  6.8 mOsM after 6 h). under the pial surface. Infusion of the hyperosmotic solution for Western blotting (Fig. 10A) showed the brain cortices of rats 6 h increased the positive staining of AQP9 under the pial administered the mannitol solution had increased expression of surface and around the blood vessels (Fig. 10B, d). GFAP was AQP4 and AQP9. AQP4 protein increased after3hof infusion stained weakly and did not change with infusion of an isos- of the mannitol solution and was expressed even when the motic or hyperosmotic solution (data not shown). solution was infused for 6 h. AQP9 protein increased after 1 h DISCUSSION of infusion of the mannitol solution and peaked at 3 h. After 6 h, AQP9 protein decreased but was still higher than in the con- Although hyperosmotic stress often causes a decrease of total trol. GFAP remained constant whether the solution infused DNA synthesis in mammalian cells, the up-regulation of the was isosmotic or hyperosmotic. In normal brain parenchyma, expression of a limited number of genes has been described. AQP4 is distributed in astrocyte foot processes, and AQP4 Recent studies have shown that the expression of AQPs is immunoreactivity was seen mainly around the vessels and induced in mammalian cells by hyperosmotic stress. AQP1 in 44532 p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions sion of AQP4 and AQP9 mRNAs induced by hyperosmotic stress, indicating that the induction of AQP4 and AQP9 does not require de novo protein synthesis and is due to direct stimulation of an intracellular signaling pathway. This is sup- ported by the results of luciferase promoter gene assays. Since AQP4 and AQP9 of astrocytes were induced by hyperosmotic mannitol solution, which is commonly used to reduce brain edema, it is suggested that AQP4 and AQP9 play important roles in the therapy of brain edema. Mannitol decreases intracranial pressure and brain water content and increases the expression of AQP4 and AQP9. How- ever, it is possible that up-regulation of AQP4 in astrocytes contributes to brain edema. AQP4 expression in astrocytes occurs at sites where the blood-brain barrier is disrupted (28). In brain cortex astrocytes, AQP4 decreases within 48 h in the acute phase of brain injury (32, 33), and overexpression of AQP4 is observed in the peri-contusional area after 3 days and later (32). The decrease of AQP4 in the acute phase is described as an endogenous protective mechanism to reduced glial water accumulation and cell swelling (32). The increase of AQP4 in the late phase is explained as the loss of its polarity and its redistribution throughout the astrocyte. AQP4 up-regulation is suggested to be a maladaptation reaction (28). On the other hand, astrocyte-specific AQP9 of the brain cortex appears in the infarct border zone 48 h after transient ischemic stroke (35). AQP9 might be involved in reperfusion edema associated with lactic acidosis, since it is permeable to water and lactic acid (28). Therapy with hyperosmotic mannitol solution might have an influence on the expression of AQP4 and AQP9 in astrocytes after brain injury or ischemia. When cells were treated in hyperosmotic medium containing the various concentrations of mannitol for 6 h, AQP4 protein increased maximally after treatment with 3% mannitol, whereas AQP9 protein peaked with 1% mannitol. When cells were treated with 3% mannitol, AQP4 protein increased after 6 h and remained at the same high level after 24 h, whereas AQP9 protein peaked at 12 h and returned to the base-line level after 24 h. Differences between AQP4 and AQP9 expres- sion were also detected in rat brain cortex by Western blot FIG. 10. Effect of intraperitoneal administration of mannitol analysis and immunohistochemical staining after intraperito- solution on the expression of AQP4 and AQP9 proteins in rat neal infusion of a hyperosmotic solution. These differences brain cortex. A, rats were anesthetized and intraperitoneally cannu- might reflect different roles for AQP4 and AQP9 when astro- lated, and an isosmotic solution or hyperosmotic solution was infused. cytes are exposed to a hyperosmotic environment. After the indicated times, samples were removed from the superficial region of the brain cortex and processed for immunoblotting with anti- AQP1 is increased in mIMCD-3 cells by 12-h treatments with AQP4, anti-AQP9, or anti-GFAP antibody. B, rats were manipulated as hyperosmotic raffinose, glucose, sucrose, sorbitol, and NaCl but described in A. After 6 h, cerebral sections were obtained, and immu- not with urea (26). In keratinocytes, AQP3 is increased by 8-h nohistochemical staining was performed with anti-AQP4 or anti-AQP9 treatments with hyperosmotic mannitol, sorbitol, glucose, su- antibody. a, photomicrograph showing the immunostaining pattern of AQP4 under isosmotic conditions. Positive staining is observed under crose, and NaCl, whereas glycerol has little effect, and the the pial surface and around the microvessels. The area along the blood expression of AQP3 is decreased by urea (17). AQP5 is in- vessel is stained weakly (arrowheads). b, positive staining of AQP4 creased in MLE-15 cells by 16 –20-h treatments with hyperos- increased under the pial surface and the area along the blood vessels (arrowheads) under hyperosmotic conditions. c, photomicrograph show- motic mannitol, sorbitol, and NaCl, whereas urea has no effect ing the immunostaining pattern of AQP9 under isosmotic conditions. (19). In our study, hyperosmotic mannitol, sorbitol, and NaCl Positive staining is observed under the pial surface. There is no staining increased the expression of AQP4 and AQP9, whereas glycerol around the blood vessel (arrowheads). d, positive staining of AQP9 had no effect, and urea decreased them. Although mannitol and increased under the pial surface and was observed around the blood sorbitol increased AQP4 and AQP9 within 6 h, treatment with vessel (arrowheads) under hyperosmotic conditions. Original magnifi- cation was 50. NaCl required 12 h to increase them. The solute specificities of the hyperosmotic induction of AQP4 and AQP9 are similar to those reported in the above studies on AQP1, -3, and -5. A mouse inner medullary cells (13), BALB/c fibroblasts (14), and hyperosmotic gradient is required to increase the expression of mIMCD-3 cells (15); AQP2 in outer medullary collecting duct AQPs. Since urea and glycerol permeate cell membranes rela- (OMCD) cells (16); AQP3 in human keratinocytes (17) and tively freely, they do not create osmotic gradients and do not Madin-Darby canine kidney epithelial cells (18); and AQP5 in mouse lung epithelial (MLE-15) cells (19) have been reported to increase the expression of AQPs (17, 26). Mannitol solution increased the expression of AQP4 and AQP9 in rat brain cortex. increase. Here, we show that hyperosmotic stress induced with mannitol solution increased AQP4 and AQP9 expression in Mannitol crosses the normal blood-brain barrier and concen- cultured rat astrocytes and in rat brain cortex. The protein trates in extracellular spaces of the brain following repeated synthesis inhibitor, cycloheximide, did not suppress the expres- injections (48, 49). It seems reasonable that the increase of p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions 44533 AQP4 and AQP9 in our in vivo experiments is associated with decreases in the expression of AQP4 and AQP9 in a time- and the osmotic gradient of mannitol across the blood-brain barrier concentration-dependent manner, prolonged treatment pre- and/or a direct effect of mannitol. vented subsequent decreases (43, 52). We present a new finding MAPKs are important intracellular signal transduction that the expression of AQP4 and AQP9 is regulated by p38 MAPK. pathways that are involved in the protective response of cells to hyperosmotic stress (22–25). Under hyperosmotic conditions, Because AQP4 and AQP9 in astrocytes play important roles maintaining brain homeostasis, elucidating the intracellular AQP1 is regulated by all three MAPKs (26), and AQP5 is signal pathways that regulate their expression is important. regulated by ERK (19). In our study, all three MAPKs were Among them, p38 MAPK is activated in astrocytes after brain activated, as indicated by Western blotting using a phos- ischemia (53) or injury (54, 55). Other studies demonstrated phospecific antibody. In addition, the effects of inhibitors of p38 that the expression of AQP4 and AQP9 in astrocytes changes MAPK, ERK, and JNK were examined for their effects on under similar conditions (32–35). The increase of AQP4 and AQP4 and AQP9 induction by hyperosmotic stress. Only the AQP9 due to hyperosmotic mannitol, which is commonly ad- p38 MAPK inhibitor suppressed AQP4 and AQP9 expression, ministered to reduce brain edema, was regulated by p38 indicating that AQP4 and AQP9 expression in rat astrocytes MAPK. Clarification of the detailed relationship between AQPs under hyperosmotic conditions can be regulated by the p38 and p38 MAPK in astrocytes may lead to the control of water MAPK pathway. On the other hand, hydrogen peroxide, but not movements and new treatments for brain edema. anisomycin, increased AQP4 and AQP9 expression. Although both agents are potent activators of p38 MAPK, oxidative Acknowledgment—We thank Manami Yamamoto for technical stress like that produced by hydrogen peroxide activates intra- assistance. cellular pathways other than MAPKs, namely the phospho- REFERENCES inositide 3 kinase pathway, phospholipase C signaling, protein 1. 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Hyperosmolar Mannitol Stimulates Expression of Aquaporins 4 and 9 through a p38 Mitogen-activated Protein Kinase-dependent Pathway in Rat Astrocytes

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 45, Issue of November 7, pp. 44525–44534, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Hyperosmolar Mannitol Stimulates Expression of Aquaporins 4 and 9 through a p38 Mitogen-activated Protein Kinase-dependent Pathway in Rat Astrocytes* Received for publication, April 25, 2003, and in revised form, August 14, 2003 Published, JBC Papers in Press, August 27, 2003, DOI 10.1074/jbc.M304368200 Hajime Arima‡§ , Naoki Yamamoto , Kazuya Sobue§, Fuminori Umenishi**, Toyohiro Tada‡‡, Hirotada Katsuya§, and Kiyofumi Asai From the ‡Department of Anesthesia and Critical Care, Okazaki City Hospital, 3-1 Goshoai, Koryuji-cho, Okazaki 444-8553, Japan, the §Department of Anesthesiology and Medical Crisis Management, Nagoya City University, Graduate School of Medical Sciences, Mizuho-ku, Nagoya 467-8601, Japan, the Department of Molecular Neurobiology, Nagoya City University, Graduate School of Medical Sciences, Mizuho-ku, Nagoya 467-8601, Japan, the **Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, and the ‡‡Department of Pathology, Nagoya City University School of Nursing, Mizuho-ku, Nagoya 467-8601, Japan life forms, including archaea, eubacteria, fungi, plants, and all The membrane pore proteins, aquaporins (AQPs), fa- cilitate the osmotically driven passage of water and, in animal phyla (1–3). AQPs are widely distributed, and more some instances, small solutes. Under hyperosmotic con- than one AQP may be present in the same cell. All AQPs seem ditions, the expression of some AQPs changes, and some to have six transmembrane domains with five connecting loops studies have shown that the expression of AQP1 and and their amino and carboxyl termini in the cytoplasm (4 – 6). AQP5 is regulated by MAPKs. However, the mechanisms AQPs are synthesized as monomers, but there is evidence that regulating the expression of AQP4 and AQP9 induced by they exist as tetramers in the membrane and have four water hyperosmotic stress are poorly understood. In this pores (7). The expression of some AQPs is affected by various study, we observed that hyperosmotic stress induced by factors, including vasopressin (8, 9), hypoxia and reoxygenation mannitol increased the expression of AQP4 and AQP9 in (10), growth factors (11), and retinoic acid (12). In addition, cultured rat astrocytes, and intraperitoneal infusion of hyperosmotic stress increases the expression of AQPs 1, 2, 3, mannitol increased AQP4 and AQP9 in the rat brain and 5 (13–19). cortex. In addition, a p38 MAPK inhibitor, but not ERK Many types of cells respond to changes in osmotic pressure and JNK inhibitors, suppressed their expression in cul- with adaptive mechanisms that allow them to re-establish ho- tured astrocytes. AQPs play important roles in main- meostasis of those aspects of cell structure and function that taining brain homeostasis. The expression of AQP4 and have been disturbed osmotically (20). Osmotic stress may dam- AQP9 in astrocytes changes after brain ischemia or age DNA and proteins, resulting in impairment of cell function traumatic injury, and some studies have shown that p38 and the induction of repair processes and protection systems MAPK in astrocytes is activated under similar condi- (21). These relatively nonspecific responses to cell damage may tions. Since mannitol is commonly used to reduce brain be an important aspect of cellular adaptation to osmotic stress. edema, understanding the regulation of AQPs and p38 Mitogen-activated protein kinases (MAPKs), specifically extra- MAPK in astrocytes under hyperosmotic conditions in- duced with mannitol may lead to a control of water cellular signal-regulated kinase (ERK), c-Jun N-terminal pro- movements and a new treatment for brain edema. tein kinase (JNK), and p38 MAPK, are important intracellular signal transduction pathways that are activated in response to changes in osmolality (22–25). Thus, osmoregulation of AQPs Water passes across cell membranes substantially faster might be mediated by MAPKs. There are some reports that the than can be explained by simple diffusion. This is necessary for expression of AQP1 and AQP5 under hyperosmotic conditions the active regulation of water homeostasis under conditions of is regulated by MAPKs (19, 26), but the role of MAPKs in the osmotic stress in the kidneys, lungs, brain, etc., and to meet expression of AQP4 and AQP9 under hyperosmotic stress is these needs, a family of membrane channel proteins evolved. unclear. These proteins, termed “aquaporins” (AQPs), are found in all Regulation of tissue water content and brain volume is crit- ical for normal functioning of the central nervous system, which is highly sensitive to any increase in intracranial pres- * This work was supported by a grant-in-aid for Scientific Research sure. Brain edema may rapidly become life-threatening be- (C), a grant-in-aid for Exploratory Research, and a grant-in-aid for cause of the rigid encasement of the brain. AQPs were sug- Young Scientists (A and B) from the Ministry of Education, Culture, gested to have a role in maintaining the homeostasis of the Sports, Science and Technology of Japan and a Research Grant from the brain (27–29). Among them, numerous studies indicated that Japan Brain Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must the expression of AQP1 (30, 31), AQP4 (32–34), and AQP9 (35) therefore be hereby marked “advertisement” in accordance with 18 is sensitive to brain injury, swelling, and other experimental U.S.C. Section 1734 solely to indicate this fact. interventions. AQP4 in astrocytes of the brain cortex decreased To whom correspondence should be addressed: Dept. of Anesthesia in the acute phase of brain injury (32, 33) and increased in the and Critical Care, Okazaki City Hospital, 3-1 Goshoai, Koryuji-cho, Okazaki 444-8553, Japan. Tel.: 81-564-21-8111; Fax: 81-564-25-2913; E-mail: [email protected]. The abbreviations used are: AQP, aquaporin; MAPK, mitogen-acti- DMEM, low glucose Dulbecco’s modified Eagle’s medium; RT, reverse vated protein kinase; ERK, extracellular signal-regulated kinase; JNK, transcriptase; TK, thymidine kinase; MEK, mitogen-activated protein c-Jun NH2-terminal kinase; GFAP, glial fibrillary acidic protein; l- kinase/extracellular signal-regulated kinase kinase. This paper is available on line at http://www.jbc.org 44525 This is an Open Access article under the CC BY license. 44526 p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions source with excitation at 488 nm and emission at 520 nm. The chro- later phase (32, 34). AQP4-deficient mice survive much longer matograms were visualized and analyzed automatically using GOLD and have a better neurological outcome from experimentally Software (Beckman). The peaks were expressed in relative fluorescence induced brain edema than wild-type mice (36). AQP4 in astro- units, and the retention time was expressed in minutes. AQP4, AQP9, cytes is tightly associated with the blood-brain barrier (37). In and -actin PCR products were compared by integrating each peak the brain cortex, astrocyte-specific AQP9 appears in the infarct area. border zone after transient ischemic stroke (35). Western Blot Analysis—Cells cultured under the specified conditions 2 2 were harvested with Ca - and Mg -free phosphate-buffered saline Hyperosmotic solutions of mannitol are used commonly to (PBS (-)) and centrifuged for 10 min at 800  g at 4 °C. For quantitation reduce brain edema, so it is important to elucidate the rela- of AQP4 and AQP9 protein, cell pellets were suspended in 100 lof tionship between the expression of AQPs and hyperosmolarity Tris-buffered saline (TBS) containing 200 mM phenylmethanesulfonyl induced with mannitol. This study was designed to assess the fluoride, 10 M pepstatin A, 10 M leupeptin, 2 mM EDTA, and 0.5% effects of hyperosmotic stress induced with mannitol on the Nonidet P-40 and then sonicated on ice at 8-watt output for 10 s. expression of AQP4 and AQP9 and to identify the signal trans- Samples were kept at room temperature for 15 min with 5 lofSDS sample buffer. For quantitation of MAPKs and phosphorylated MAPKs, duction pathways regulating the response in rat astrocytes in the cell pellets were suspended in 150 l of buffer (50 mM Tris-HCl, pH which AQP4 and AQP9 are localized (10). We found that the 7.4, 1 mM phenylmethanesulfonyl fluoride, 10 M pepstatin A, 10 M expression of AQP4 and AQP9 is induced in cultured rat astro- leupeptin) and sonicated on ice at 8-watt output for 10 s. The cell lysates cytes and in rat brain cortex by hyperosmotic stress. In addi- were centrifuged for 30 min at 15,000  g at 4 °C, and the supernatants tion, we demonstrated that this induction requires selective were collected and boiled with 5 l of SDS sample buffer for 5 min. For activation and involvement of the p38 MAPK pathway in cul- GFAP, cell pellets were suspended and sonicated as for AQPs and boiled with 5 l of SDS sample buffer for 5 min. Total protein was measured tured rat astrocytes. We believe this study provides the first using a BCA protein assay reagent kit (Pierce). 3 g of protein for AQP4 example of osmotic regulation of AQP4 and AQP9 and the first and GFAP, 20 g for AQP9, and 30 g for MAPKs and phosphorylated evidence that the expression of AQP4 and AQP9 is regulated by MAPKs were separated by 12.5% SDS-polyacrylamide gel electrophore- the p38 MAPK pathway. sis (ATTO, Tokyo, Japan) and transferred to a Clear Blot Membrane-P (ATTO), which was then blocked for 1 h with 5% skim milk in TBS-T (20 EXPERIMENTAL PROCEDURES mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween 20). The membranes were Cell Culture—Cultures of cortical astrocytes were prepared from rat incubated overnight with anti-AQP4 antibody (Chemicon International, postnatal cortices (P2) according to previously described methods (38). Inc., Temecula, CA) diluted 1:750, anti-AQP9 antibody (Chemicon) di- Trypsinized and dissociated cortical cells were cultured in 75-cm cul- luted 1:500, anti-p38 MAPK antibody (Santa Cruz Biotechnology, Santa ture flasks (Corning Glass) containing low glucose (1000 mg/liter) Dul- Cruz, CA) diluted 1:500, anti-phosphorylated p38 MAPK antibody (Cell becco’s modified Eagle’s medium (l-DMEM; Invitrogen) supplemented Signaling Technology, Inc., Beverly, MA) diluted 1:1000, anti-ERK an- with 10% fetal bovine serum (Sigma). After incubation for 5–7 days, the tibody (Promega) diluted 1:5000, anti-phosphorylated ERK antibody cells were trypsinized and subcultured in 60-mm diameter culture (Cell Signaling Technology) diluted 1:1000, anti-JNK antibody (Santa dishes (Falcon; PGC Scientific, Frederick, MD). The cell population Cruz Biotechnology) diluted 1:500, anti-phosphorylated JNK antibody consisted of over 95% astrocytes as determined by immunocytochemical (Cell Signaling Technology) diluted 1:1000, or anti-GFAP antibody staining with anti-glial fibrillary acidic protein (GFAP) antibody. In (Chemicon) diluted 1:1000 all in TBS-T containing 5% skim milk. After neural cells, AQP4 and AQP9 are expressed in astrocytes but not in washing the membranes four times for 15 min each with TBS-T, they neurons, oligodendrocytes, and microglia (10). were incubated for 1 h with 1:1000 diluted (for AQP4, MAPKs, and Hyperosmotic Stress of Cultured Rat Astrocytes—When astrocyte GFAP) or 1:500 diluted (for AQP9) horseradish peroxidase-conjugated cultures became confluent, equal volumes of medium with or without secondary antibody in TBS-T buffer containing 5% skim milk. Protein various concentrations of mannitol (Sigma) were added, and the cul- was visualized using an ECL detection system (Amersham Biosciences). tures were incubated for the indicated times. Osmolality of the medium Relative band intensities were determined by densitometry using TM was measured by the freezing point depression method (Osmostat Kodak Digital Science 1D version 2.0 (Eastman Kodak Co.). OM-6040; ARKREY, Kyoto, Japan). All cultures were maintained at Isolation of the 5-Flanking Regions of Human AQP4 and AQP9 37 °C in an atmosphere of 5% CO , 95% air. Genes—The 5-flanking region of the human AQP4 gene was isolated as Reverse Transcription (RT)-PCR—At the indicated times, total RNA described previously (44). The AQP4 gene is regulated by alternative was isolated using Trizol reagent (Invitrogen). cDNAs were generated promoters upstream of exons 0 and 1. The 5-flanking regions of the from 1 g of total RNA by Superscript II RNase H reverse tran- exon 0 promoter (bp 1930 to 29) (nucleotide 1 corresponds to the scriptase (Invitrogen) primed with oligo(dT) (Invitrogen). They were initiation codon of exon 0) and the exon 1 promoter (bp 297 to 113) amplified with primers designed according to the published sequences. (nucleotide 1 corresponds to the initiation codon of exon 1) were The PCR protocols and primers for AQP4, AQP9, and -actin have been subcloned into the vector pCR2.1 (Invitrogen), and 5 deletion mutants described previously (10). The oligonucleotide primers were as follows: were constructed. Deletion mutants corresponding to bp 1930 to 29 AQP4 (39), 5-TTGGACCAATCATAGGCGC-3 (forward) and 5-GGT- in the exon 0 promoter (eight constructs) and from bp 297 to 133 in CAATGTCGATCACATGC-3 (reverse); AQP9 (40), 5-GATGCCTTCT- the exon 1 promoter were fused to the promoterless vector PGV-B GAGAAGGACG-3 (forward) and 5-AGAGAGCCATCACGACTGC-3 (Toyo-ink, Tokyo, Japan) containing the luciferase reporter gene. (reverse); -actin (41), 5-GACCTGACTGACTACCTCAT-3 (forward) The promoter region of AQP9 (bp 1125 to 55) (nucleotide 1 and 5-TCGTCATACTCCTGCTTGCT-3 (reverse). Multitarget PCRs corresponds to the initiation codon) was also isolated by a similar were performed by coamplifying -actin as the internal standard. The method. Briefly, the region was isolated using a Promoter Finder DNA reaction mixture for AQP4 contained 2 l of the RT reaction product, Walking kit (Clontech, Palo Alto, CA), which contains adapter-linked and the reaction was carried out for 20 cycles, using a 95 °C, 30-s human genomic DNA digested with EcoRV, ScaI, DraI, PvuII, and SspI. denaturing step; a 57 °C, 30-s annealing step; and a 72 °C, 1-min A two-step PCR was performed according to the manufacturer’s instruc- extension step. The reaction mixture for AQP9 contained 2 l of the RT tions. The first PCR consisted of 94 °C for 1 min followed by 30 cycles of reaction product, and the reaction was carried out for 27 cycles, using a 94 °C for 15 s, 60 °C for 30 s, and 68 °C for 4 min and was done in a final 95 °C, 30-s denaturing step; a 58 °C, 30-s annealing step; and a 72 °C, volume of 50 l with 10 pmol of a specific primer, 5-GCTCTTCAAGAC- 1-min extension step. CAGTCTCTGCTTGA-3 (nucleotides 60 to 46). The second PCR was Semiquantification of mRNA—The PCR products were visualized by done with the same protocol using 1 l of the first PCR product and a ethidium bromide staining following separation on 1.5% agarose gels specific primer, 5-CTCTGAGGACTCCTGTTTCTACCAAT-3 (nucleo- and quantified by laser-induced fluorescence-linked capillary gel elec- tides 11 to 35). The PCR products were subcloned into pCR2.1, and trophoresis. Details of laser-induced fluorescence-linked capillary gel both strands were sequenced completely. BLAST searches were per- TM electrophoresis have been reported elsewhere (10, 42, 43). Briefly, 40 l formed on the NCBI sites. The sequence was identical to GenBank of PCR products was applied to a P/ACE system 5010 (Beckman Instru- accession number AC025431. Deletion mutants corresponding to bp ments). Samples were injected at a pressure of 0.5 p.s.i.g. for 20 s. 1125 to 55 in the AQP9 promoter (four constructs) were fused to the Separation was carried out at 9.4 kV for 35 min in a capillary (47 cm promoterless vector PGV-B. long, 100-m diameter) filled with electrophoresis gel buffer containing Cell Transfection and Luciferase Reporter Gene Assay—Reporter as- DNA fluorescence stain (LIFluor dsDNA 1000 kit; Beckman). Laser- says were done on AQP4 and AQP9 gene fragments fused upstream of induced fluorescence detection was preformed using an argon-ion laser the firefly luciferase reporter in PGV-B using the Dual-Luciferase re- p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions 44527 porter assay system (Promega), with which the pRL-thymidine kinase (TK) plasmid (Promega) containing the Renilla luciferase gene under control of the TK promoter was co-transfected as an internal control. For transient transfection, astrocytes in l-DMEM supplemented with 10% fetal bovine serum were plated at 1  10 cells/well in a 48-well plate, incubated for 18 h, and transfected in serum-free medium with 0.4 g of the test plasmid and 0.01 g of pRL-TK using 1 l of Lipofect- AMINE 2000 (Invitrogen). After 4 h, 150 l of l-DMEM containing 20% fetal bovine serum was added. 48 h after transfection, equal volumes of l-DMEM or 3% mannitol in l-DMEM were added. After 24 h, the astrocytes were washed and harvested. The activities of control Renilla luciferase and firefly luciferase were measured in triplicate. Induction of Hyperosmolarity in Rats—All animal studies were un- dertaken after the protocols were approved by the Animals Care and Use Committee of Nagoya City University Graduate School of Medical Sciences. Male Wistar rats (150 g) were used throughout. Animals were anesthetized with pentobarbital (Schering-Plough Animal Health, Kenil- worth, NJ) (3.2 mg/100 g, body weight) and placed under a heating lamp. Intraperitoneal infusion was performed using a modification of a technique described previously (45). A polyurethane catheter (20 gauge) was inserted into the peritoneum by puncture and tightly fixed to avoid leakage of the experimental solution. The animals were then allowed to recover under a heating lamp. An isosmotic solution (121 mmol of NaCl, 5 mmol of KCl, 1 mmol of MgCl , 1 mmol of NaH PO , 2 mmol of CaCl , 2 2 4 2 18 of mmol NaHCO , 22 mmol of glucose) or a hyperosmotic mannitol solution (1.1 mol of mannitol added to the isosmotic solution) was administered by intraperitoneal infusion using an infusion pump. The infusion rate was 1.9 ml/h, and the duration of the infusion was 1, 3, or 6 h. Control animals were anesthetized and cannulated but were not administered any solution. At the conclusion of the experiments, the animals were disconnected from the infusion pump and rapidly decap- itated. Samples for Western blot analyses, done as described above, were obtained from the superficial region of the brain cortex, and cerebral sections (5 mm thick) were obtained for immunohistochemical staining. Blood samples were removed, and plasma osmotic pressure was measured by the freezing point depression method as described above. Immunohistochemical Staining—Coronal sections from the brain were embedded in paraffin by the AMeX method (46). Briefly, tissues were fixed in acetone at 4 °C for 3 days, cleared in methyl benzoate and xylene, and then embedded in paraffin. Sections 3 m thick were prepared and deparaffinized with xylene. Immunoreactivity was visu- alized using the streptavidin/biotin method (Histofine SAB-PO Kit, Nichirei, Tokyo, Japan). After washing the sections with PBS (), they were treated with 0.3% (v/v) hydrogen peroxide in methanol for 30 min to inactivate endogenous peroxidase. They were then immersed in a 1:20 dilution of nonimmune goat serum for 10 min to block nonspecific binding, and after blotting to remove excess serum they were incubated FIG.1. Effect of mannitol on expression of AQP4 and AQP9 mRNAs and proteins. A, rat astrocytes were incubated for6hin at room temperature for 1 h with anti-AQP4 antibody (Chemicon) isosmotic control medium or hyperosmotic medium containing the des- diluted 1:400, anti-AQP9 antibody (Chemicon) diluted 1:100, or anti- ignated concentrations of mannitol. Cells were processed for RT-PCR GFAP antibody (Chemicon) diluted 1:50 in PBS () containing 1% and semiquantification of AQP4 or AQP9 mRNA. The expression of bovine serum albumin. Control sections were treated with nonimmune mRNA (normalized to -actin) (n  8 for each group) is presented as a rabbit immunoglobulins (MBL, Nagoya, Japan). The sections were percentage of the control (mean  S.E.; *, p  0.05 versus control). B, rinsed three times with PBS (), incubated for 30 min each with the rat astrocytes were treated as described in A and processed for immu- secondary antibody, rinsed three times, and then incubated with a noblotting with anti-AQP4 or anti-AQP9 antibody. C, immunoblots (n streptavidin-biotin-peroxidase complex for 15 min. After washing the 8 for each group) were analyzed by densitometry, and band densities sections with PBS (), the peroxidase reaction was developed by incu- are presented as a percentage of the control (mean  S.E.; *, p  0.05 bating them in 0.02% (w/v) 3,3-diaminobenzidine tetrahydrochloride versus control). (Sigma) solution containing 0.003% (v/v) hydrogen peroxide and 10 mM sodium azide. The sections were counterstained with hematoxylin. for RT-PCR or harvesting for immunoblot analysis. Both AQP4 Data Analysis—Data are expressed as means  S.E. of at least four and AQP9 mRNAs (Fig. 1A) and proteins (Fig. 1, B and C) independent experiments. Statistical analysis was performed by one- increased significantly after treatment with 1, 3, and 10% way factorial analysis of variance combined with Scheffe’s test for all mannitol. AQP4 and AQP9 mRNAs and AQP4 protein in- comparison pairs. Reporter gene assays were carried out three times, and the results were analyzed by Student’s t test for statistical signif- creased maximally after treatment with 3% mannitol, whereas icance. Differences with p values  0.05 were considered significant. AQP9 protein peaked after treatment with 1% mannitol. AQP4 and AQP9 mRNAs and proteins were induced to a lesser extent RESULTS with 10% mannitol. Induction of AQP4 and AQP9 in Cultured Rat Astrocytes To analyze the time course of the mannitol-mediated induc- Subjected to Hyperosmotic Stress—To examine the effects of tion of AQP4 and AQP9 expression, astrocytes were treated hyperosmotic stress on the expression of AQP4 and AQP9 with hyperosmotic medium containing 3% mannitol for various mRNAs and proteins, astrocytes were incubated in isosmotic periods of time. AQP4 mRNA increased significantly after 1, 3, control medium (final osmotic pressure 311  1.4 mOsM (n  6, and 12 h of treatment with mannitol, and AQP9 mRNA 4)) or hyperosmotic medium supplemented with 0.3% (324  increased significantly after 3, 6, and 12 h of treatment (Fig. 1.6 mOsM), 1% (363  2.7 mOsM), 3% (491  4.7 mOsM), or 10% 2A). After 24 h, both AQP mRNAs returned to base-line levels. mannitol (854  8.9 mOsM) for 6 h before isolation of total RNA AQP4 and AQP9 proteins increased after6hof treatment with 44528 p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions FIG.3. Effect of cycloheximide on hyperosmotic induction of AQP4 and AQP9 mRNAs. Rat astrocytes were incubated for 6 h with or without 3% mannitol or cycloheximide (25 g/ml). Cells were proc- essed for RT-PCR and semiquantification of AQP4 or AQP9 mRNA. The expression of mRNA (normalized to -actin) (n  4 for each group) is presented as a percentage of the control (mean  S.E.; *, p  0.05 versus control). AQP9 is solute-specific, astrocytes were incubated in isosmotic medium or hyperosmotic medium supplemented with 0.4% NaCl (502  0.7 mOsM), 1% urea (501  2.5 mOsM), 1.5% glycerol (491  4.5 mOsM), 3% sorbitol (485  9.2 mOsM), or 3% mannitol (491  4.7 mOsM), for 6 h. NaCl and glycerol had little effect on the expression of AQP4 and AQP9 mRNAs and pro- teins, whereas they were decreased by urea and increased by mannitol and sorbitol (Fig. 4, A–C). Except for NaCl, the effects of specific solutes on hyperosmotic induction of AQP4 and AQP9 were similar to those reported for AQP1, -3, and -5 (17, 19, 26). Although6hof exposure to hyperosmotic medium supplemented with NaCl did not increase the expression of AQP4 and AQP9, other studies have demonstrated that over 12 h of treatment with NaCl is required to increase the expres- sion of AQP1 and AQP5 (19, 26). To examine the time course of NaCl-, urea-, glycerol-, or sorbitol-mediated induction of AQP4 and AQP9 expression, astrocytes were treated with hyperos- motic medium supplemented with the solutes for various peri- ods of time before harvesting for immunoblot analysis. AQP4 and AQP9 proteins increased after 12 h of treatment with NaCl (Fig. 4D). Although AQP4 protein was at the same high level even after 24 h of treatment, AQP9 protein decreased but was FIG.2. Time course of hyperosmotic induction of AQP4 and AQP9 mRNAs and proteins. A, rat astrocytes were incubated in still higher than in the control. After 12 or 24 h of treatment, hyperosmotic medium containing 3% mannitol. At the indicated times, urea and glycerol did not affect the levels of AQP4 and AQP9 cells were processed for RT-PCR and semiquantification of AQP4 or proteins. The effect of sorbitol was the same as that of mannitol AQP9 mRNA. The expression of mRNA (normalized to -actin) (n  8 (data not shown). for each group) is presented as a percentage of the control at time 0 The Role of MAPKs in the Expression of AQP4 and AQP9 in (mean  S.E.; *, p  0.05 versus time 0). B, rat astrocytes were treated as described in A and processed for immunoblotting with anti AQP4 or Cultured Rat Astrocytes Subjected to Hyperosmotic Stress— anti-AQP9 antibody. C, immunoblots (n  8 for each group) were Hyperosmotic stress can activate signaling through three analyzed by densitometry, and band densities are presented as a per- MAPK pathways, p38 MAPK, ERK, and JNK, all three of centage of the control at time 0 (mean  S.E.; *, p  0.05 versus time 0). which are activated in mouse astrocytes by sorbitol (47). To investigate whether these three MAPKs are activated in rat mannitol (Fig. 2, B and C). Although AQP4 mRNA returned to astrocytes by mannitol, we performed immunoblot analyses of the base-line level after 24 h of treatment with mannitol, AQP4 cells incubated in hyperosmotic medium using antibodies that protein remained at the same high level, suggesting that this react with either the phosphorylated or total amounts of each of gene is regulated at both the transcriptional and post-tran- the three MAPKs. Whereas the total amount of each MAPK scriptional levels. AQP9 protein peaked at 12 h but returned to remained constant, p38 MAPK was activated by hyperosmotic the base-line level after 24 h of treatment with mannitol. stress after 30 min, and activation persisted even at 120 min To see whether hyperosmotic induction of AQP4 and AQP9 (Fig. 5A). ERK (Fig. 5B) and JNK (Fig. 5C) activation both mRNAs requires de novo protein synthesis, astrocytes incu- peaked at 30 min after hyperosmotic exposure. bated in hyperosmotic medium containing 3% mannitol were To investigate the possible involvement of the three MAPK treated with or without the protein synthesis inhibitor, cyclo- cascades in the increased expression of AQP4 in response to heximide (25 g/ml), for 6 h. The increased expression of both mannitol, the effects of the p38 MAPK inhibitor SB203580, the AQP mRNAs induced by mannitol was not affected by cyclo- mitogen-activated extracellular signal-regulated kinases heximide (Fig. 3), suggesting that de novo protein synthesis is (MEK) 1/2 inhibitor PD98059, and the JNK inhibitor SP600125 not involved. were examined. MEK 1/2 are the upstream kinases that acti- To determine whether hyperosmotic induction of AQP4 and vate ERK. The hyperosmotic induction of AQP4 mRNA and p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions 44529 FIG.5. Phosphorylation of MAPKs induced by hyperosmotic stress. Rat astrocytes were incubated in hyperosmotic medium con- taining 3% mannitol. At the indicated times, cells were harvested and processed for immunoblotting using anti-p38 MAPK antibody or anti- phosphorylated p38 MAPK (p-p38) antibody (A), anti-ERK antibody or anti-phosphorylated ERK (p-ERK) antibody (B), or anti JNK antibody or anti-phosphorylated JNK (p-JNK) antibody (C). inhibitor was not a result of nonspecific effects. Thus, it ap- pears that the increased expression of AQP4 in response to mannitol requires signaling through p38 MAPK. RT-PCR re- vealed that SB203580 reduced the expression of AQP4 mRNA in a concentration-dependent manner (Fig. 6C). The same re- sults were obtained with AQP9 (Fig. 7). FIG.4. Hyperosmotic induction of AQP4 and AQP9 mRNAs To determine whether just p38 MAPK activation is sufficient and proteins by different solutes. A, rat astrocytes were incubated for6hin control isosmotic medium (final osmotic pressure, 311  1.4 for inducing AQP4 and AQP9, astrocytes were incubated with mOsm (n  4 for each group)) or medium supplemented with 0.4% NaCl the p38 MAPK activators, hydrogen peroxide (2 mM) or aniso- (502  0.7 mOsm), 1% urea (501  2.5 mOsm), 1.5% glycerol (491  4.5 mycin (50 M), under isosmotic conditions. The total amount of mOsm), 3% sorbitol (485  9.2 mOsm), or 3% mannitol (491  4.7 p38 MAPK remained constant, and both activators caused its mOsm) and then processed for RT-PCR and semiquantification of AQP4 or AQP9 mRNA. The expression of mRNA (normalized to -actin) (n  phosphorylation (Fig. 8, A and B). Activation of p38 MAPK by 4 for each group) is presented as a percentage of that in control isos- hydrogen peroxide increased 15 min after exposure and peaked motic medium (mean  S.E.; *, p  0.05 versus control). B, rat astro- at 60 min (Fig. 8A), whereas its activation by exposure to cytes were treated as described in A and processed for immunoblotting anisomycin peaked at 30 min and was observed only weakly at with anti-AQP4 or anti-AQP9 antibody. C, immunoblots (n  4 for each 60 min (Fig. 8B). Hydrogen peroxide increased AQP4 and group) were analyzed by densitometry, and band densities are pre- sented as a percentage of that in control isosmotic medium (mean AQP9 mRNAs, whereas they were decreased by anisomycin S.E.; *, p  0.05 versus control). D, rat astrocytes were incubated in (Fig. 8C). AQP4 and AQP9 proteins were increased by hydro- hyperosmotic medium supplemented with 0.4% NaCl. At the indicated gen peroxide but were not affected by anisomycin (Fig. 8D). times, cells were processed for immunoblotting with anti-AQP4 or anti- Transcriptional Regulation of AQP4 and AQP9 Genes in AQP9 antibody. The immunoblots (n  4 for each group) were analyzed by densitometry, and band densities are presented as a percentage of Cultured Rat Astrocytes under Hyperosmotic Conditions—The the control at time 0 (mean  S.E.; *, p  0.05 versus time 0). promoter activity of the AQP4 gene has been demonstrated in SF-126 (glioblastoma) and Madin-Darby canine kidney cells protein was inhibited by SB203580 (10 M) but not by PD98059 (44), but that of the AQP9 gene has not been investigated. (10 M) or SP600125 (10 M) (Fig. 6, A and B), and all three Luciferase promoter gene assays were performed to determine inhibitors had no effect on the basal expression of AQP4. To the transcriptional activity of the AQP4 and AQP9 genes in determine whether the p38 MAPK inhibitor specifically pre- astrocytes under isosmotic and hyperosmotic conditions. Eight vented AQP4 induction, astrocytes were incubated with luciferase promoter constructs of AQP4 exon 0, one of AQP4 Me SO. It had no effect on AQP4 expression, suggesting that exon 1, or four of AQP9 were transfected into astrocytes, and the reduction of AQP4 expression caused by the p38 MAPK luciferase activity was measured under isosmotic conditions 44530 p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions FIG.6. Effect of MAPK inhibitors on hyperosmotic induction FIG.7. Effect of MAPK inhibitors on hyperosmotic induction of AQP4 mRNA and protein. A, rat astrocytes were incubated for 6 h of AQP9 mRNA and protein. Rat astrocytes were incubated and with or without 3% mannitol and MAPK inhibitors (the p38 MAPK treated as described in the legend for Fig. 6. Using AQP9 primer and inhibitor SB203580 (10 M), the MEK 1/2 inhibitor PD98059 (10 M), or anti-AQP9 antibody, RT-PCR (A and C) and immunoblotting (B) were the JNK inhibitor SP600125 (10 M)). To determine whether the reduc- carried out as described in the legend for Fig. 6. DMSO,Me SO. tion of AQPs induced by MAPK inhibitors was a result of nonspecific effects, astrocytes were incubated with Me SO (DMSO;5 M). Cells the AQP4 promoter is up-regulated by hyperosmotic stress and were processed for RT-PCR and semiquantification of AQP4 mRNA. The expression of mRNA (normalized to -actin) (n  4 for each group) that a critical cis element involved is located between 345 and is presented as a percentage of the control (mean  S.E.; *, p  0.05 428. On the other hand, the 765/55 and 486/55 AQP9 versus control). B, rat astrocytes were treated as described in A and constructs showed significant induction of luciferase activity, processed for immunoblotting with anti-AQP4 antibody. C, rat astro- but other AQP9 constructs were not affected by hyperosmolar- cytes were incubated for 6 h with or without 3% mannitol and the p38 MAPK inhibitor SB203580 at the indicated concentrations. Cells were ity (Fig. 9B). These results indicate that the AQP9 promoter is processed for RT-PCR and semiquantification of mRNA. The expression also up-regulated by hyperosmotic stress and that a critical cis of mRNA is presented as described in A (mean  S.E.; n  4 for each element is located between 486 and 176. group; *, p  0.05 versus control). Induction of AQP4 and AQP9 Expression in Rat Brain Cortex by Intraperitoneal Infusion of Hyperosmotic Solution—To in- (Fig. 9, A and B). The 297/113 exon 1 construct had the vestigate whether AQP4 and AQP9 expression is induced in rat greatest activity among the AQP4 constructs. All eight con- brain cortex under hyperosmotic conditions, an isosmotic solu- structs derived from the exon 0 promoter had weak luciferase tion (osmotic pressure was 298  5.9 mOsM (n  4)) or a activity. There was an increase in luciferase activity of the exon hyperosmotic solution of mannitol (1368  24.8 mOsM) was 0 830/29 construct compared with the longer isoform, indi- infused intraperitoneally for 1, 3, and 6 h, after which samples cating the presence of a suppressor element just upstream of bp from the superficial region of brain cortex were taken for West- 831. Luciferase activity of the 175/55 AQP9 construct was ern blot analyses, and cerebral sections were taken for immu- greater than that of the other longer AQP9 constructs, indicat- nohistochemical staining. Animals recovered from anesthesia ing the presence of a suppressor element just upstream of bp within 1 h after intraperitoneal cannulation. The position of 174. the intraperitoneal catheter tip was confirmed after decapita- Under hyperosmotic conditions achieved with 3% mannitol, tion, at which time blood samples were obtained from the right the luciferase activities of the 1625/29, 830/29, 515/ atrium. The plasma osmotic pressure of control animals was 29, and 428/29 AQP4 exon 0 constructs were induced 297  3.9 mOsM, and it did not increase after infusion of the significantly (Fig. 9A). Luciferase activity of the 428/29 isosmotic solution (data not shown). Infusion of the hyperos- construct was increased the most. These results indicate that motic solution resulted in an increase of the plasma osmotic p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions 44531 FIG.9. Promoter activities of the 5-flanking regions of the AQP4 and AQP9 genes. A, AQP4 promoter luciferase plasmid con- structs were transfected into rat astrocytes. The transfected cells were incubated for 24 h with or without 3% mannitol, and promoter activity was analyzed. The expression of promoter activities (normalized to Renilla luciferase activity) (n  3 for each group) is presented as a FIG.8. Effect of p38 MAPK activators on the induction of AQP4 percentage of that of the AQP4 exon 0 construct (bp 1930 to 29) and AQP9 mRNAs and proteins. A, rat astrocytes were incubated in activity under isosmotic conditions (mean  S.E.; *, p  0.05, with isosmotic medium containing hydrogen peroxide (2 mM). At the indi- versus without 3% mannitol with each construct). B, AQP9 promoter cated times, cells were harvested and processed for immunoblotting activity was analyzed as described in A. The results (normalized to the using anti-p38 MAPK antibody or anti-phosphorylated p38 MAPK (p- Renilla luciferase activity) are expressed as percentages of the AQP9 p38). B, rat astrocytes were incubated in isosmotic medium containing construct (bp 1125 to 55) activity under isosmotic conditions anisomycin (50 M). The blots were processed as described in A. C, rat (mean  S.E.; n  3 for each group; *, p  0.05, with versus without 3% astrocytes were incubated for 6 h with or without hydrogen peroxide (2 mannitol with each construct). mM) or anisomycin (50 M) under isosmotic conditions. To determine whether the reduction of AQPs expression by MAPK activators was a under the pial surface (28, 29), and we found a normal immu- result of nonspecific effects, astrocytes were also incubated with Me SO nostaining pattern of AQP4 under isosmotic conditions (Fig. (DMSO;5 M). Cells were processed for RT-PCR and semiquantification of AQP4 or AQP9 mRNA. The expression of mRNA (normalized to 10B, a). Positive staining was observed under the pial surface -actin) (n  4 for each group) is presented as a percentage of the and around microvessels, with the area along the blood vessels control (mean  S.E.; *, p  0.05 versus control). D, rat astrocytes were stained weekly. Intraperitoneal infusion of mannitol solution incubated for 6 h with or without hydrogen peroxide (2 mM) or aniso- for 6 h significantly increased the positive staining of AQP4 mycin (50 M) under isosmotic conditions and processed for immuno- under the pial surface and the area along the blood vessels (Fig. blotting with anti-AQP4 or anti-AQP9 antibody. 10B, b). AQP9 was stained weakly in normal brain cortex and was difficult to find. The staining pattern of AQP9 under isos- pressure (316  3.9 mOsM after1hof infusion, 354  6.6 mOsM motic conditions is shown in Fig. 10B, c, with positive staining after 3 h, and 369  6.8 mOsM after 6 h). under the pial surface. Infusion of the hyperosmotic solution for Western blotting (Fig. 10A) showed the brain cortices of rats 6 h increased the positive staining of AQP9 under the pial administered the mannitol solution had increased expression of surface and around the blood vessels (Fig. 10B, d). GFAP was AQP4 and AQP9. AQP4 protein increased after3hof infusion stained weakly and did not change with infusion of an isos- of the mannitol solution and was expressed even when the motic or hyperosmotic solution (data not shown). solution was infused for 6 h. AQP9 protein increased after 1 h DISCUSSION of infusion of the mannitol solution and peaked at 3 h. After 6 h, AQP9 protein decreased but was still higher than in the con- Although hyperosmotic stress often causes a decrease of total trol. GFAP remained constant whether the solution infused DNA synthesis in mammalian cells, the up-regulation of the was isosmotic or hyperosmotic. In normal brain parenchyma, expression of a limited number of genes has been described. AQP4 is distributed in astrocyte foot processes, and AQP4 Recent studies have shown that the expression of AQPs is immunoreactivity was seen mainly around the vessels and induced in mammalian cells by hyperosmotic stress. AQP1 in 44532 p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions sion of AQP4 and AQP9 mRNAs induced by hyperosmotic stress, indicating that the induction of AQP4 and AQP9 does not require de novo protein synthesis and is due to direct stimulation of an intracellular signaling pathway. This is sup- ported by the results of luciferase promoter gene assays. Since AQP4 and AQP9 of astrocytes were induced by hyperosmotic mannitol solution, which is commonly used to reduce brain edema, it is suggested that AQP4 and AQP9 play important roles in the therapy of brain edema. Mannitol decreases intracranial pressure and brain water content and increases the expression of AQP4 and AQP9. How- ever, it is possible that up-regulation of AQP4 in astrocytes contributes to brain edema. AQP4 expression in astrocytes occurs at sites where the blood-brain barrier is disrupted (28). In brain cortex astrocytes, AQP4 decreases within 48 h in the acute phase of brain injury (32, 33), and overexpression of AQP4 is observed in the peri-contusional area after 3 days and later (32). The decrease of AQP4 in the acute phase is described as an endogenous protective mechanism to reduced glial water accumulation and cell swelling (32). The increase of AQP4 in the late phase is explained as the loss of its polarity and its redistribution throughout the astrocyte. AQP4 up-regulation is suggested to be a maladaptation reaction (28). On the other hand, astrocyte-specific AQP9 of the brain cortex appears in the infarct border zone 48 h after transient ischemic stroke (35). AQP9 might be involved in reperfusion edema associated with lactic acidosis, since it is permeable to water and lactic acid (28). Therapy with hyperosmotic mannitol solution might have an influence on the expression of AQP4 and AQP9 in astrocytes after brain injury or ischemia. When cells were treated in hyperosmotic medium containing the various concentrations of mannitol for 6 h, AQP4 protein increased maximally after treatment with 3% mannitol, whereas AQP9 protein peaked with 1% mannitol. When cells were treated with 3% mannitol, AQP4 protein increased after 6 h and remained at the same high level after 24 h, whereas AQP9 protein peaked at 12 h and returned to the base-line level after 24 h. Differences between AQP4 and AQP9 expres- sion were also detected in rat brain cortex by Western blot FIG. 10. Effect of intraperitoneal administration of mannitol analysis and immunohistochemical staining after intraperito- solution on the expression of AQP4 and AQP9 proteins in rat neal infusion of a hyperosmotic solution. These differences brain cortex. A, rats were anesthetized and intraperitoneally cannu- might reflect different roles for AQP4 and AQP9 when astro- lated, and an isosmotic solution or hyperosmotic solution was infused. cytes are exposed to a hyperosmotic environment. After the indicated times, samples were removed from the superficial region of the brain cortex and processed for immunoblotting with anti- AQP1 is increased in mIMCD-3 cells by 12-h treatments with AQP4, anti-AQP9, or anti-GFAP antibody. B, rats were manipulated as hyperosmotic raffinose, glucose, sucrose, sorbitol, and NaCl but described in A. After 6 h, cerebral sections were obtained, and immu- not with urea (26). In keratinocytes, AQP3 is increased by 8-h nohistochemical staining was performed with anti-AQP4 or anti-AQP9 treatments with hyperosmotic mannitol, sorbitol, glucose, su- antibody. a, photomicrograph showing the immunostaining pattern of AQP4 under isosmotic conditions. Positive staining is observed under crose, and NaCl, whereas glycerol has little effect, and the the pial surface and around the microvessels. The area along the blood expression of AQP3 is decreased by urea (17). AQP5 is in- vessel is stained weakly (arrowheads). b, positive staining of AQP4 creased in MLE-15 cells by 16 –20-h treatments with hyperos- increased under the pial surface and the area along the blood vessels (arrowheads) under hyperosmotic conditions. c, photomicrograph show- motic mannitol, sorbitol, and NaCl, whereas urea has no effect ing the immunostaining pattern of AQP9 under isosmotic conditions. (19). In our study, hyperosmotic mannitol, sorbitol, and NaCl Positive staining is observed under the pial surface. There is no staining increased the expression of AQP4 and AQP9, whereas glycerol around the blood vessel (arrowheads). d, positive staining of AQP9 had no effect, and urea decreased them. Although mannitol and increased under the pial surface and was observed around the blood sorbitol increased AQP4 and AQP9 within 6 h, treatment with vessel (arrowheads) under hyperosmotic conditions. Original magnifi- cation was 50. NaCl required 12 h to increase them. The solute specificities of the hyperosmotic induction of AQP4 and AQP9 are similar to those reported in the above studies on AQP1, -3, and -5. A mouse inner medullary cells (13), BALB/c fibroblasts (14), and hyperosmotic gradient is required to increase the expression of mIMCD-3 cells (15); AQP2 in outer medullary collecting duct AQPs. Since urea and glycerol permeate cell membranes rela- (OMCD) cells (16); AQP3 in human keratinocytes (17) and tively freely, they do not create osmotic gradients and do not Madin-Darby canine kidney epithelial cells (18); and AQP5 in mouse lung epithelial (MLE-15) cells (19) have been reported to increase the expression of AQPs (17, 26). Mannitol solution increased the expression of AQP4 and AQP9 in rat brain cortex. increase. Here, we show that hyperosmotic stress induced with mannitol solution increased AQP4 and AQP9 expression in Mannitol crosses the normal blood-brain barrier and concen- cultured rat astrocytes and in rat brain cortex. The protein trates in extracellular spaces of the brain following repeated synthesis inhibitor, cycloheximide, did not suppress the expres- injections (48, 49). It seems reasonable that the increase of p38 MAPK Regulates AQP4 and -9 under Hyperosmotic Conditions 44533 AQP4 and AQP9 in our in vivo experiments is associated with decreases in the expression of AQP4 and AQP9 in a time- and the osmotic gradient of mannitol across the blood-brain barrier concentration-dependent manner, prolonged treatment pre- and/or a direct effect of mannitol. vented subsequent decreases (43, 52). We present a new finding MAPKs are important intracellular signal transduction that the expression of AQP4 and AQP9 is regulated by p38 MAPK. pathways that are involved in the protective response of cells to hyperosmotic stress (22–25). Under hyperosmotic conditions, Because AQP4 and AQP9 in astrocytes play important roles maintaining brain homeostasis, elucidating the intracellular AQP1 is regulated by all three MAPKs (26), and AQP5 is signal pathways that regulate their expression is important. regulated by ERK (19). In our study, all three MAPKs were Among them, p38 MAPK is activated in astrocytes after brain activated, as indicated by Western blotting using a phos- ischemia (53) or injury (54, 55). Other studies demonstrated phospecific antibody. In addition, the effects of inhibitors of p38 that the expression of AQP4 and AQP9 in astrocytes changes MAPK, ERK, and JNK were examined for their effects on under similar conditions (32–35). The increase of AQP4 and AQP4 and AQP9 induction by hyperosmotic stress. Only the AQP9 due to hyperosmotic mannitol, which is commonly ad- p38 MAPK inhibitor suppressed AQP4 and AQP9 expression, ministered to reduce brain edema, was regulated by p38 indicating that AQP4 and AQP9 expression in rat astrocytes MAPK. Clarification of the detailed relationship between AQPs under hyperosmotic conditions can be regulated by the p38 and p38 MAPK in astrocytes may lead to the control of water MAPK pathway. On the other hand, hydrogen peroxide, but not movements and new treatments for brain edema. anisomycin, increased AQP4 and AQP9 expression. Although both agents are potent activators of p38 MAPK, oxidative Acknowledgment—We thank Manami Yamamoto for technical stress like that produced by hydrogen peroxide activates intra- assistance. cellular pathways other than MAPKs, namely the phospho- REFERENCES inositide 3 kinase pathway, phospholipase C signaling, protein 1. 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Published: Nov 1, 2003

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