Differential regulation of aquaporin expression in astrocytes by protein kinase C

Molecular Brain Research 95 (2001) 110–116 www.elsevier.com / locate / bres

Research report

Differential regulation of aquaporin expression in astrocytes by protein kinase C a, b c b a Naoki Yamamoto *, Kazuya Sobue , Taishi Miyachi , Masaaki Inagaki , Yutaka Miura , b a Hirotada Katsuya , Kiyofumi Asai b

a Department of Bioregulation Research, Nagoya City University Medical School, Mizuho-ku, Nagoya 467 -8601, Japan Department of Anesthesiology and Resuscitology, Nagoya City University Medical School, Mizuho-ku, Nagoya 467 -8601, Japan c Department of Pediatrics, Nagoya City University Medical School, Mizuho-ku, Nagoya 467 -8601, Japan

Accepted 28 August 2001

Abstract Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [6], that increase plasma membrane water permeability in secretory and absorptive cells. In astrocytes of the central nervous system (CNS), using the reverse transcription-polymerase chain reaction (RT-PCR), we previously detected AQP3, 5 and 8 mRNAs in addition to the reported AQP4 and 9. However the mechanisms regulating the expression of these AQPs are not known. In this study, we investigated the effects of a protein kinase C (PKC) activator on the expression of AQP4, 5 and 9 in cultured rat astrocytes. Treatment of the cells with TPA caused decreases in AQP4 and 9 mRNAs and proteins in time- and concentration-dependent manners. The TPA-induced decreases in AQP4 and 9 mRNAs were inhibited by PKC inhibitors. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decreases in AQP4 and 9 mRNAs caused by TPA. Pretreatment of cells with an inhibitor of protein synthesis, cycloheximide, did not inhibit the decreases in AQP4 and 9 mRNAs induced by TPA. These results suggest that signal transduction via PKC may play important roles in regulating the expression of AQP4 and 9.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Uptake and transporters Keywords: Astrocyte; Aquaporin; Protein kinase C; TPA

1. Introduction Aquaporins (AQPs) are water-selective channels that function to increase plasma membrane water permeability in secretory and adsorptive cells that require rapid or regulated water movement. Ten subtypes of aquaporins have been identified in mammals, aquaporin1-9 and the intrinsic protein of lens (also referred as AQP0). Structurally, AQPs may be divided into two groups depending on

Abbreviations: AQP, aquaporin; CNS, central nervous system; DMEM, Dulbecco’s modified Eagle’s medium; RT-PCR, reverse transcriptionpolymerase chain reaction; LIF–CGE, laser-induced fluorescence linked capillary-gel electrophoresis; TPA, 12-O-tetradecanoylphorbol,13-acetate *Corresponding author. Tel.: 181-52-853-8200; fax: 181-52-8423316. E-mail address: [email protected] (N. Yamamoto).

their sequence homology with the bacterial glycerol facilitator (GlpF) and the bacterial water channel (AQP-Z). (AQP3, 7 and 9 are closely related to the GlpF, whereas the other aquaporins (AQP0, 1, 2, 4, 5, 6 and 8) have more homology with AQP-Z. Functionally, with the exception of AQP 0 and 6, AQPs have high water permeability. In addition to having of water permeability, some AQP subtypes are also permeable to glycerol (AQP3, 7 and 7L) and / or urea (AQP3, 7, 7L, 8 and 9) [32]. The predicted six-transmembrane-spanning topology and deduced amino acid sequences of distinct aquaporin genes are highly conserved between species and family members [1,26]. In the central nervous system (CNS), membrane water transport is important for brain volume homeostasis, cerebrospinal fluid (CSF) production, and the pathogenesis of brain edema. Control of water flux and cell volume is critical in the brain where swelling may be fatal. More-

0169-328X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00254-6

N. Yamamoto et al. / Molecular Brain Research 95 (2001) 110 – 116

over, the regulation of water transport is critical because osmolarity changes in extracellular fluids can affect neuronal cell functions. Furthermore, AQP4 is localized in multiple sites corresponding to membranes where orthogonally aggregated 6–7 nm particles (OAPs) have been observed by freeze-fracture electron microscopy [5], suggesting that OAPs consist of AQP4 protein. This hypothesis was confirmed with AQP4-transfected Chinese hamster ovary (CHO) cells [35] and transgenic knockout AQP4 mice [33] and by direct immunogold labeling [29]. Recently, Manley et al. [17] reported that deletion of AQP4 in knockout mice reduced cerebral edema in response to water intoxication and stroke, and improved the clinical indices of survival and neurological status. Modification of the membrane density of these OAPs has been reported for several pathological states, including brain ischemia [22,30], epilepsy [9], and muscular dystrophy [37]. Recently, we defined the expression profile of the AQP family in neural cells and reported that AQP4 and 9 mRNAs were downregulated in hypoxic astrocytes [34]. However, there are no reports about the mechanism of regulation of AQPs in the CNS, except for one showing that the expression of AQP4 mRNA was attenuated by treating astrocytes with 12-O-tetradecanoylphorbol,13-acetate (TPA) [21]. In this study, we examined the effect of TPA on the expression of AQP4, 5 and 9 mRNAs and proteins in cultured rat astrocytes.

2. Materials and methods

2.1. Astrocyte culture Cultures of cortical astrocytes were prepared from rat postnatal cortex (P2) according to the methods described by Kato et al. [13]. Trypsinized and dissociated cortical cells were cultured in 75-cm 2 culture flasks (Costar) containing low glucose (1000 mg / l) Dulbecco’s modified Eagle’s medium (l-DMEM; Gibco BRL) supplemented with 10% fetal bovine serum (FBS; Sigma). After incubation for 5–7 days, the cells were trypsinized and subcultured in [ 60-mm culture dishes (Falcon). The cell

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population consisted of over 95% astrocytes as determined by immunocytochemical examination with antiglial fibrillary acidic protein (GFAP).

2.2. TPA treatment and PKC blockage in astrocytes When astrocytes became confluent in [ 60-mm culture dishes l-DMEM containing 10% FBS was replaced with serum-free l-DMEM. To examine the effect of TPA (Sigma) on the expression of AQP4, 5 and 9 mRNAs, astrocytes were treated with TPA or the vehicle. Total RNA was isolated with Trizol reagent (Gibco BRL) at the indicated times. To investigate whether the TPA-induced effect is mediated by the activation of PKC, astrocytes were pretreated with H-7 (Tocris) or chelerythrine (Calbiochem) for 1 h or with TPA for 24 h. After the cells were incubated with TPA for another 6 h, total RNA was isolated with Trizol reagent. To determine whether the TPA-induced decrease in AQP4 and 9 mRNAs requires de novo protein synthesis, astrocytes were pretreated with 20 mg / ml cycloheximide for 1 h and total RNA was isolated 6 h after the TPA treatment.

2.3. Semiquantification of mRNA The cDNAs were generated from 1 mg of total RNA by Superscript II RNase H 2 (GibcoBRL) reverse transcriptase primed with oligo (dT) 18 . They were amplified with primers designed according to the published sequences. The PCR protocols and primers for AQP 4, 5, 9 and b-actin are shown in Table 1. Multitarget PCRs were performed coamplifying b-actin as an internal standard. The reactions for AQP4 contained 1 ml of RT reaction product as template DNA and were carried out for 20 cycles, using a 958C, 30 s denaturing step; a 578C, 30 s annealing step; and a 728C, 1 min extension step. The reactions for AQP5 contained 2 ml of RT reaction product as template DNA and were carried out for 30 cycles, using a 958C, 30 s denaturing step; a 608C, 30 s annealing step; and a 728C, 1 min extension step. The reactions for AQP9 contained 1 ml of RT reaction product as template DNA and were carried out for 27 cycles, using a 958C, 30 s

Table 1 RT-PCR primers Gene

Primer

Products size (bp)

Reference

AQP4

59-TTGGACCAATCATAGGCGC-39 59-GGTCAATGTCGATCACATGC-39

214

Jung et al. [11]

AQP5

59-TGGCCATAGGTACCTTAGCC-39 59-ACAGCCGGTGAAGTAGATCC-39

395

Raina et al. [28]

AQP9

59-GATGCCTTCTGAGAAGGACG-39 59-AGAGAGCCATCACGACTGC-39

218

Ko et al. [14]

b-Actin

59-GACCTGACTGACTACCTCAT-39 59-TCGTCATACTCCTGCTTGCT-39

542

Nudel et al. [24]

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denaturing step; a 588C, 30 s annealing step; and a 728C, 1 min extension step. PCR products were quantified by laser-induced fluorescence linked capillary-gel electrophoresis (LIF–CGE). Details of the method are reported elsewhere [12,20,34]. The peaks are expressed in relative fluorescence units and the retention time in min. PCR products of AQP4, AQP5, AQP9, and b-actin were compared by integrating each peak area.

2.4. Western blot analysis TPA treated astrocytes were washed with Ca 21 - and Mg 21 -free phosphate buffered saline (PBS (2)) at appropriate times and collected by centrifugation for 10 min at 800 g. Cell pellets were suspended in 100 ml Tris-buffered saline (TBS) containing 200 mM phenylmethanesulfonyl fluoride (PMSF), 10 mM pepstatin A, 10 mM leupeptin and 5 mM EDTA and sonicated at 8-W output for 150 s on ice. The protein contents were determined with a BCA protein assay reagent kit (Pierce). Samples were separated by 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and then transferred to an Immobilon-P transfer membrane (Millipore). Blotted membranes were blocked with 5% skim milk and then incubated with primary antibodies (Chemicon) against AQP4, 5 or 9 which were visualized by reaction with horseradish peroxidase (HRP)-linked second antibodies (Amersham) and an ECL or ECL plus detection system (Amersham).

elsewhere) [34]. Therefore, PCR was terminated at 20, 30 and 27 cycles for AQP4, 5 and 9, respectively. With these conditions the amplification curves of AQP4, 5 and 9 and b-actin were linear when plotted against the amount of added RT reaction products (reported elsewhere) [34]. The sequences of PCR products were confirmed by a direct sequencing. To evaluate the effects of TPA on the expression AQP4, 5 and 9 mRNAs, astrocytes were incubated for up to 24 h with 100 nM TPA. The maximal decreases in AQP4 and 9 mRNAs were observed 6 h after TPA treatment, and were sustained for at least 24 h. In contrast, AQP5 mRNA was not changed by the treatment with TPA (Fig. 1). Western blot analysis showed expression patterns similar to the results of RT-PCR (Fig. 2). The concentration-dependence of the effect of TPA on AQP4 and 9 mRNAs expression was investigated and as shown in Fig. 3, the TPA-induced decreases in AQP4 and 9 mRNAs were observed even

2.5. Statistical analysis All data were entered into a computer and analyzed using the STATVIEW program (Abacus Concepts). The effect of drug stimulation on the expression of AQP4 and 9 mRNAs was compared with control values using multivariate analysis of variance (MANOVA), where there were two variables. When statistical significance was observed, one-way ANOVA with Scheffe’s posthoc test was performed for each set of variables. P values #0.05 were considered significant.

3. Results

3.1. Effect of TPA on expression of AQP4, 5 and 9 mRNAs We examined the regulation of AQP expression by TPA. As a preliminary experiment, 1 mg of total astrocyte RNA was amplified by RT-PCR and the amount of PCR products for AQP4, 5 and 9 or b-actin was measured every two cycles by LIF–CGE. The amplification rate of b-actin mRNA paralleled that of AQP4, 5 or 9 between 18 and 24, 28 and 32, or 24 and 28 cycles, respectively (reported

Fig. 1. Time course of AQP4, 5 and 9 mRNA expression in cultured astrocytes treated with TPA. Astrocytes were treated with TPA (100 nM) or the vehicle at time 0. Total RNA was isolated at the indicated times. Levels of AQP4, 5 and 9 mRNAs are expressed as relative PCR product ratios (AQP4:b-actin, AQP5:b-actin and AQP9:b-actin). Each point is the mean6S.D. of eight determinations. *, P,0.05 compared with the timematched controls.

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Fig. 2. Time course of appearance of AQP4, 5 and 9 protein in cultured astrocytes treated with TPA. Astrocytes were treated with TPA (100 nM) or the vehicle at time 0. Samples, 3 mg for AQP4, 40 mg for AQP5 and 40 mg for AQP9, were separated by SDS–PAGE and transferred to Immobilon-P membranes. Immunodetection was performed as described in the text. Blots shown are representative of three experiments.

when astrocytes were treated with TPA at a concentration as low as 10 nM.

3.2. Role of PKC in AQP4 and 9 mRNA decrease

Fig. 3. Concentration-dependent effect of TPA on expression of AQP4, 5 and 9 mRNAs. Total RNA was isolated from astrocytes 6 h after TPA treatment (10, 100 or 1000 nM). Levels of AQP4, 5 and 9 mRNAs are expressed as relative PCR product ratios (AQP4:b-actin, AQP5:b-actin and AQP9:b-actin). Each point is the mean6S.D. of eight determinations. *, P,0.05 compared with the control (vehicle treated).

To ascertain whether the TPA-induced decreases in AQP4 and 9 mRNAs are mediated by PKC activation, we examined the effects of H-7 or chelerythrine, a relatively specific PKC inhibitor. Pretreatment of astrocytes with H-7 abolished the TPA-induced decreases in AQP4 and 9 mRNAs in a concentration-dependent manner (Fig. 4A). The effects of TPA were completely abolished when the cells were pretreated with 100 mM H-7, and similar results were obtained by pretreatment with 3 mM chelerythrine (Fig. 4B). To test whether the effects of TPA are mediated by PKC activation, PKC-depletion was achieved by a prolonged exposure to TPA before the TPA-treatment. After a 24-h pretreatment of astrocytes with 1 mM TPA, the expression of AQP4 and 9 mRNAs was not affected by a subsequent TPA stimulation and showed almost the same levels as the control (Fig. 5). These results may indicate that the TPA-induced decrease in AQP4 and 9 mRNAs are mediated by PKC activation. To examine whether the TPA-induced decreases in AQP4 and 9 mRNAs requires de novo protein synthesis, astrocytes were pretreated with cycloheximide (20 mg / ml), a protein synthesis inhibitor, 1 h prior to TPA treatment. As shown in Fig. 6, the effect of TPA was the same in cycloheximide-pretreated and non-treated astrocytes. From these results, it seems that TPA decreases the expression of

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Fig. 4. Effect of PKC inhibitors on expression of AQP4 and 9 mRNAs. (A) Effect of H-7 (10, 30 or 100 mM) on TPA-induced decreases in AQP4 and 9 mRNAs. (B) Effect of chelerythrine (1 or 3 mM) on TPA-induced decreases in AQP4 and 9 mRNAs. Astrocytes were pretreated with H-7, chelerythrine or the vehicle for 1 h. Total RNA was isolated 6 h after TPA treatment. Values are the means6S.D. of six determinations. *, P,0.05 compared with the control (vehicle treated).

Fig. 5. Effect of pretreatment with TPA for 24 h on TPA-induced decreases in AQP4 and 9 mRNAs. Astrocytes were pretreated with 1 mM of TPA (first stimulation) or the vehicle for 24 h. Then, they were treated with 100 nM of TPA (second stimulation), and total RNAs were isolated 6 h after the second stimulation. Values are the means6S.D. of six determinations. *, P,0.05 compared with the control (vehicle treated).

Fig. 6. Effect of cycloheximide (CHX) on TPA-induced decrease in AQP4 and 9 mRNA. Astrocytes were pretreated with cycloheximide (20 mg / ml) or the vehicle for 1 h. Total RNA was isolated 6 h after TPA treatment. Values are the means6S.D. of six determinations. *, P,0.05 compared with the control (vehicle treated).

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AQP4 and 9 mRNAs at the transcriptional level without involvement of de novo protein synthesis.

4. Discussion The control of the water flux in the CNS is critical and the role of AQPs are receiving attention recently. In fact, AQP4 was reported to play a major role in the cause of brain edema [16]. We reported that the expression of AQPs in astrocytes is upregulated by hypoxia and reoxygenation [34]. However, there is only one report showing that the expression of AQP4 mRNA in astrocytes is attenuated by TPA which has many kinds of biological effects via the activation of PKC [21], and the mechanism of regulation of AQP expression in the CNS remains unclear. The PKC pathway is one of the major signal transduction pathways that regulates cell growth, mRNA expression, and enzyme activation in astrocytes [2,3,10,19,23,25,27,31,36]. Therefore, in this study, we focused on the PKC pathway and examined the effect of TPA on the expression of AQP4, 5, and 9 in cultured astrocytes. Previously we demonstrated that five subtypes of AQPs (3, 4, 5, 8, and 9) are expressed in neural cells [4,5,34]. It might be important to investigate the different mechanisms regulating the expression of the various AQPs. In this study, we found that the expression of AQP4 and 9 mRNAs and proteins are attenuated by TPA treatment, but AQP5 was unaffected by treatment of the cells with TPA (Figs. 1–3). From these results, the expression of AQPs may be regulated by a complex system which changes water movement. The effects of TPA were inhibited by pretreatment of the cells with PKC inhibitors and by PKC depletion (Figs. 4 and 5). These results indicate that the effects of TPA on the cells are closely associated with the activation of PKC. Pretreatment of the cells with cycloheximide (20 mg / ml) did not inhibit the effects of TPA (Fig. 6). Thus, our results suggest that de novo protein synthesis is not required for the TPA-induced decrease in AQP4 and 9 mRNAs. In the ischemic brain PKC-a, -b, and -g isoforms are downregulated [8] and PKC-delta is upregulated [15]. PKC stimulates AQP4 phosphorylation and reduces water influx via AQP4 [7]. There are two possibilities that in the ischemic brain downregulated PKC isoforms increase water flux through AQPs or upregulated PKC isoforms decrease water flux through AQPs. Potentially, AQPs play important roles in the rate of progression of brain edema from ischemia or other brain injury or in the reversal of brain edema. Additional in vivo studies are needed to reveal the mechanism of regulation of water flux via AQPs in relation to PKC activity in the CNS. In conclusion, we examined the effect of TPA on the expression of AQP4, 5 and 9 mRNAs in primary rat astrocyte cultures. We found that the regulation of expression of AQP9 mRNA and protein is similar to that of

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AQP4 and involves TPA-sensitive PKC in astrocytes. Since it is known that PKC activity increases in inflamed tissue [18], our data contribute to understanding the mechanisms of brain dysfunction. Previously we reported that AQP5 expression in astrocytes were downregulated after hypoxia and transiently upregulated after hypoxia and reoxygenation [34]. However, AQP5 was unaffected by treatment of the cells with TPA. This must have a different signal pathway. To elucidate the regulation of AQP5 in astrocytes, further studies need to be undertaken.

Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research on General Research (B) and (C) in Priority Areas from the Ministry of Education, Science, Sports and Culture, Japan; a Grant for Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan, and a Health Sciences Research Grants for Research on Environmental Health from the Ministry of Health and Welfare, Japan.

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