Hypertonicity-induced Aquaporin-1 (AQP1) Expression Is Mediated by the Activation of MAPK Pathways and Hypertonicity-responsive Element in the AQP1 Gene

Aquaporin-1 (AQP1) is a water channel that is induced by hypertonicity. The present study was undertaken to clarify the osmoregulation mechanism of AQP1 in renal medullary cells. In cultured mouse medullary (mIMCD-3) cells, AQP1 expression was significantly induced by hypertonic treatment with impermeable solutes, whereas urea had no effect on AQP1 expression. This result indicates the requirement of a hypertonic gradient. Hypertonicity activated ERK, p38 kinase, and JNK in mIMCD-3 cells. Furthermore, all three MAPKs were phosphorylated by the upstream activation of MEK1/2, MKK3/6, and MKK4, respectively. The treatments with MEK inhibitor U0126, p38 kinase inhibitor SB203580, and JNK inhibitor SP600125 significantly attenuated hypertonicity-induced AQP1 expression in mIMCD-3 cells. In addition, hypertonicity-induced AQP1 expression was significantly reduced by both the dominant-negative mutants of JNK1- and JNK2-expressing mIMCD-3 cells. NaCl-inducible activity of AQP1 promoter, which contains a hypertonicity response element, was attenuated in the presence of U0126, SB203580, and SP600125 in a dose-dependent manner and was also significantly reduced by the dominant-negative mutants of JNK1 and JNK2. These data demonstrate that the activation of ERK, p38 kinase, and JNK pathways and the hypertonicity response element in the AQP1 promoter are involved in hypertonicity-induced AQP1 expression in mIMCD-3 cells. Aquaporin-1 (AQP1) is a water channel that is induced by hypertonicity. The present study was undertaken to clarify the osmoregulation mechanism of AQP1 in renal medullary cells. In cultured mouse medullary (mIMCD-3) cells, AQP1 expression was significantly induced by hypertonic treatment with impermeable solutes, whereas urea had no effect on AQP1 expression. This result indicates the requirement of a hypertonic gradient. Hypertonicity activated ERK, p38 kinase, and JNK in mIMCD-3 cells. Furthermore, all three MAPKs were phosphorylated by the upstream activation of MEK1/2, MKK3/6, and MKK4, respectively. The treatments with MEK inhibitor U0126, p38 kinase inhibitor SB203580, and JNK inhibitor SP600125 significantly attenuated hypertonicity-induced AQP1 expression in mIMCD-3 cells. In addition, hypertonicity-induced AQP1 expression was significantly reduced by both the dominant-negative mutants of JNK1- and JNK2-expressing mIMCD-3 cells. NaCl-inducible activity of AQP1 promoter, which contains a hypertonicity response element, was attenuated in the presence of U0126, SB203580, and SP600125 in a dose-dependent manner and was also significantly reduced by the dominant-negative mutants of JNK1 and JNK2. These data demonstrate that the activation of ERK, p38 kinase, and JNK pathways and the hypertonicity response element in the AQP1 promoter are involved in hypertonicity-induced AQP1 expression in mIMCD-3 cells. aquaporin-1 extracellular signal-regulated kinase c-Jun NH2-terminal kinase mitogen-activated protein kinase mitogen-activated extracellular signal-regulated kinase kinase mitogen-activated protein kinase kinase inner medullary collecting duct mouse hypertonicity response element tonicity-responsive enhancer dominant negative Aquaporins (AQPs),1 a family of water channels, function as a water-selective transporting protein in cell membranes (1Agre P. Preston G.M. Smith B.L. Jung J.S. Raina S. Moon C. Guggino W.B. Nielsen S. Am. J. Physiol. 1993; 265: F463-F476Crossref PubMed Google Scholar). Aquaporin-1 (AQP1) was first discovered in human erythrocytes as a water channel for high osmotic water permeability (2Preston G.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11110-11114Crossref PubMed Scopus (727) Google Scholar, 3Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1705) Google Scholar). In addition to erythrocytes, AQP1 is abundantly present in the epithelium of kidney proximal tubules and descending thin limbs and endothelium of the descending vasa recta (4Nielsen S. Smith B.L. Christensen E.I. Knepper M.A. Agre P. J. Cell Biol. 1993; 120: 371-383Crossref PubMed Scopus (455) Google Scholar). AQP1 has been suggested to be important in constitutive water reabsorption, especially in the epithelial cells of the renal medulla. The AQP1-expressing vasa recta of the renal medulla are critical in generating and maintaining an axial osmotic gradient through the medulla (5Nielsen S. Knepper M.A. Kwon T. Fr℘kiaer J. Diseases of the Kidney and Urinary Tract. Lippincott Williams and Wilkins, Philadelphia, PA2001: 109-134Google Scholar). Sodium chloride (NaCl), urea, and water transporters in the inner medullary collecting duct (IMCD) all play an important role in the regulation of solute-free water excretion in the kidney. Although most cells in mammals are not normally stressed by hypertonicity, epithelial cells of the renal medulla are constantly subjected to a hypertonic condition. Specifically, as a consequence of the urinary concentrating mechanism, cells in the renal inner medulla are normally exposed to a variety of high concentrations of NaCl and urea. Hypertonicity, which results from a high concentration of salt and urea, provides a mechanical stress to shrink medullary cells. However, medullary cells adapt to hypertonicity by a variety of responses through an acute influx of NaCl and water (6Hoffmann E.K. Simonsen L.O. Physiol. Rev. 1989; 69: 315-382Crossref PubMed Scopus (741) Google Scholar), chronic accumulation of organic osmolytes (7Yancey P.H. Clarl M.E. Hand S.C. Bowlus R.D. Somero G.N. Science. 1982; 217: 1214-1222Crossref PubMed Scopus (3042) Google Scholar), and acute activation of immediate early and heat shock genes (8Cohen D. Wasserman J. Gullans S. Am. J. Physiol. 1991; 261: C594-C601Crossref PubMed Google Scholar, 9Dasgupta S. Hohman T.C. Carper D. Exp. Eye Res. 1992; 54: 461-470Crossref PubMed Scopus (179) Google Scholar). Although the amounts of total RNA transcription, DNA synthesis, and protein synthesis are significantly decreased by hypertonicity, a limited number of genes are up-regulated by hypertonicity-induced transcription (10Burg M.B. Kwon E.D. Kultz D. Annu. Rev. Physiol. 1997; 59: 437-455Crossref PubMed Scopus (331) Google Scholar, 11Burg M.B. Kwon E.D. Kultz D. FASEB J. 1996; 10: 1598-1606Crossref PubMed Scopus (159) Google Scholar). The cellular and molecular mechanisms of aquaporin-2 (AQP2) regulation via vasopressin in the collecting duct of the kidney are well understood (12Fushimi K. Uchida S. Hara Y. Hirata Y. Marumo F. Sasaki S. Nature. 1993; 361: 549-552Crossref PubMed Scopus (876) Google Scholar, 13Nielsen S. DiGiovanni S.R. Christensen E.I. Knepper M.A. Harris H.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11663-11667Crossref PubMed Scopus (668) Google Scholar, 14Umenishi F. Verbavatz J.M. Verkman A.S. Biophys. J. 2000; 78: 1024-1035Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). However, the AQP1 regulation in the kidney is still unknown. Studies performed in the human primary proximal tubule epithelial cell (15Jenq W. Cooper D.R. Bittle P. Ramirez G. Biochem. Biophys. Res. Commun. 1999; 256: 240-248Crossref PubMed Scopus (34) Google Scholar) and mouse medullary cell line (mIMCD-3) (16Jenq W. Mathieson I.M. Ihara W. Ramirez G. Biochem. Biophys. Res. Commun. 1998; 245: 804-809Crossref PubMed Scopus (50) Google Scholar) demonstrate that AQP1 mRNA and protein expressions were up-regulated by hypertonicity. It was also reported that the AQP1 transcript was up-regulated by a hypertonicity response element (HRE) in the promoter region of AQP1 gene that was located at −54 to −46, which responded to hypertonic stimulation (17Umenishi F. Schrier R.W. Biochem. Biophys. Res. Commun. 2002; 292: 771-775Crossref PubMed Scopus (43) Google Scholar). This HRE sequence is different from previously reported tonicity-responsive enhancer (TonE) consensus sequence, which is also responsible for hypertonicity. Additional studies in mIMCD-3 cells showed that all three MAPKs (ERK, p38 kinase, and JNK) were activated by hypertonicity (18Zhang Z. Cohen D.M. Am. J. Physiol. 1996; 271: F1234-F1238PubMed Google Scholar, 19Berl T. Siriwardana G. Ao L. Butterfield L.M. Heasley L.E. Am. J. Physiol. 1997; 272: F305-F311PubMed Google Scholar). Thus, it was possible that the osmoregulation of AQP1 may be mediated by MAPK pathways, which are induced by hypertonicity. However, the role of MAPKs in hypertonicity-induced AQP1 expression remains unclear. The objective of the present study was to elicit the cellular and molecular mechanisms of hypertonicity-induced AQP1 expression in renal medullary cells. We show here that hypertonicity-induced AQP1 expression is mediated by the activation of the ERK, p38 kinase, and JNK pathways in mIMCD-3 cells. Functional analysis of the AQP1 promoter containing the HRE sequence also demonstrated that the AQP1 promoter activity was stimulated by hypertonicity and regulated by MAPK pathways. mIMCD-3 cells used in this study were obtained from the American Type Culture Collection. Cells were cultured at 37 °C and 5% CO2 in Dulbecco's modified Eagle's/F-12 medium supplemented with 10% fetal bovine serum. Total RNA was isolated from mIMCD-3 cells using a TRIzol reagent (Invitrogen). Two micrograms of total RNA was reverse-transcribed and then directly amplified by PCR using two sets of primers: AQP1 sense primer, 5′-CGGGCTGTCATGTACATCATCGCCCA-3′ (nucleotides 276–301); AQP1 antisense primer, 5′-CCCAATGAACGGCCCCACCCAGAAA-3′ (nucleotides 632–656); glyceraldehyde-3-phosphate dehydrogenase sense primer, 5′-ATGGGAAGCTTGTCATCAACGGGAA-3′ (nucleotides 184–208); glyceraldehyde-3-phosphate dehydrogenase antisense primer, 5′-TGGCAGGTTTCTCCAGGCGGCACGT-3′ (nucleotides 729–753). The PCR amplification was performed for 30 cycles as follows: 94 °C for 30 s, 60 °C for 60 s, and 72 °C for 60 s. The PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining. mIMCD-3 cells grown on 6-cm dishes were washed with ice-cold phosphate-buffered saline and suspended with 10 mm Tris-HCl, pH 7.5, containing 200 mm sucrose and homogenized by 10 passages through a 27-gauge needle. The homogenate was centrifuged at 3000 × g for 10 min at 4 °C. The supernatant was collected, and protein concentration was measured using the Bradford protein assay method (Bio-Rad protein assay kit). Protein (10 μg) from the cell extract was resolved on a 12% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. The membrane was incubated with a rabbit polyclonal anti-human AQP1 antibody for 1 h. As a protein loading control, the membrane was also incubated with anti-β-actin antibody (Sigma). For the analysis of MAPK signaling pathways, cells were lysed with ice-cold lysis buffer (50 mm β-glycerophosphate, pH 7.2, 0.5% Triton X-100, 0.1 mm sodium vanadate, 2 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol). The lysates were resolved on a 12% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. The membranes were incubated with antibodies to phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, JNK, phospho-mitogen-activated ERK kinase (MEK) 1/2, MEK1/2, phospho-MAPK kinase (MKK) 3/6, MKK3, phospho-MKK4 (Cell Signaling Technology Inc., Beverly, MA), or MKK4 (StressGen, Victoria, British Columbia, Canada). After washing, the membrane was incubated with anti-rabbit IgG horseradish peroxidase secondary antibody (Amersham Biosciences). The immunoreactive bands were visualized by enhanced chemiluminescence method (PerkinElmer Life Sciences). The plasmid pCAT-basic (Promega, Madison, WI) was used to examine the promoter activity of the 5′-flanking region in the human AQP1 gene (17Umenishi F. Schrier R.W. Biochem. Biophys. Res. Commun. 2002; 292: 771-775Crossref PubMed Scopus (43) Google Scholar, 20Umenishi F. Verkman A.S. Genomics. 1998; 47: 341-349Crossref PubMed Scopus (39) Google Scholar, 21Umenishi F. Schrier R.W. Biochem. Biophys. Res. Commun. 2002; 293: 913-917Crossref PubMed Scopus (25) Google Scholar). The AQP1 promoter construct (−54/+23; CP-54), which contains the HRE sequence, was generated by PCR and ligated into Hind III andXbaI sites of pCAT-basic vector. The sense primer corresponded to nucleotides −54 to −33 and contained an engineeredHind III restriction site. The antisense primer for amplification corresponded to nucleotides +23 to +4 and contained an engineered XbaI restriction site. The CAT construct was confirmed by sequence analysis. The transient transfection experiment was performed by the modification of previous reports (17Umenishi F. Schrier R.W. Biochem. Biophys. Res. Commun. 2002; 292: 771-775Crossref PubMed Scopus (43) Google Scholar, 20Umenishi F. Verkman A.S. Genomics. 1998; 47: 341-349Crossref PubMed Scopus (39) Google Scholar, 21Umenishi F. Schrier R.W. Biochem. Biophys. Res. Commun. 2002; 293: 913-917Crossref PubMed Scopus (25) Google Scholar, 22Umenishi F. Verkman A.S. Genomics. 1998; 50: 373-377Crossref PubMed Scopus (58) Google Scholar). mIMCD-3 cells were resuspended at 2 × 107 cells/ml in Dulbecco's modified Eagle's/F-12 medium without serum and transfected by electroporation under 300 V at 500 microfarads in a 0.4-mm cuvette. Each transfection was performed with 10 μg of the AQP1 promoter-CAT construct (CP-54) and 5 μg of the plasmid pSV-β-galactosidase (Promega) on a total volume of 300 μl. After overnight incubation, cells were incubated with or without 100 mm NaCl. After a 48-h incubation, the cells were harvested, and cell extracts were isolated using the reporter lysis buffer (Promega). β-Galactosidase activity was measured in every experiment for normalization. CAT activity was measured (Amersham and the previous study demonstrated that AQP1 expression was induced by hypertonic medium supplemented with NaCl in mouse renal medullary (mIMCD-3) cells (16Jenq W. Mathieson I.M. Ihara W. Ramirez G. Biochem. Biophys. Res. Commun. 1998; 245: 804-809Crossref PubMed Scopus (50) Google Scholar). examine the hypertonic of AQP1 in mIMCD-3 cells, cells were incubated in medium supplemented with and and impermeable osmolytes and After the treatment with cell lysates were by using AQP1 protein expression was by and not urea These data that AQP1 by hypertonicity a hypertonic gradient. to the for hypertonic of AQP1 mIMCD-3 cells were incubated in hypertonic medium by the addition of 100 mm NaCl. were at for analysis. As in 1 AQP1 protein significantly by to hypertonic medium and at h. the AQP1 in medium cells were incubated in medium by the addition of or with different addition of mm 100 mm or 100 mm to the medium was to AQP1 protein expression in mIMCD-3 cells, and of AQP1 was obtained by the to medium However, the addition of 200 mm to the medium showed AQP1 expression that of 100 and mm the cell significantly decreased at that condition. data that AQP1 expression in mIMCD-3 cells is significantly in hypertonic in a and dose-dependent AQP1 expression may be by mRNA or or protein the mechanism, cells were with the RNA synthesis inhibitor or protein synthesis inhibitor and then incubated under or hypertonic condition. The expression of AQP1 mRNA and protein were by and respectively. As in with or AQP1 mRNA and protein These results that both and are for AQP1 by hypertonicity. show that all three MAPKs (ERK, p38 kinase, and JNK) be activated by hypertonicity (18Zhang Z. Cohen D.M. Am. J. Physiol. 1996; 271: F1234-F1238PubMed Google Scholar, 19Berl T. Siriwardana G. Ao L. Butterfield L.M. Heasley L.E. Am. J. Physiol. 1997; 272: F305-F311PubMed Google Scholar). signaling was involved in hypertonic of AQP1 in mIMCD-3 cells, antibodies for the phosphorylated or total of of three MAPKs were Cells were with containing 100 or 200 mm urea for 10 and then the of all three MAPKs were by analysis. NaCl significantly activated the ERK, p38 kinase, and JNK pathways in mIMCD-3 cells, whereas urea activated the ERK These data confirmed the results reported previously (18Zhang Z. Cohen D.M. Am. J. Physiol. 1996; 271: F1234-F1238PubMed Google Scholar, Z. Cohen D.M. Am. J. Physiol. 1999; PubMed Google Scholar). activation of MAPKs is mediated by MEK or ERK is activated by and whereas p38 kinase is activated by and and JNK is activated by MKK4 J. T. J. Science. PubMed Scopus Google Scholar, K. J. J.M. Nature. PubMed Scopus Google Scholar, J. Y. S. 1997; PubMed Scopus Google Scholar, J. Y. Z. L. J. Biol. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, J.S. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the activation of MAPKs in mIMCD-3 cells in response to hypertonicity, the for the upstream activation of MAPKs was using antibodies for the phosphorylated or total of MEK1/2, MKK3/6, or As in NaCl activated the MEK1/2, MKK3/6, and MKK4 pathways, whereas urea activated the Thus, NaCl significantly stimulated the of all three of MAPKs in mIMCD-3 cells, urea the ERK the activation of ERK, p38 kinase, and JNK was involved in the regulation of hypertonicity-induced AQP1 a MEK U0126, a p38 kinase SB203580, and a JNK SP600125 B.L. Sasaki W. A. S. Y. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Z. L. L. J. PubMed Scopus Google Scholar), were the of MAPK mIMCD-3 cells were with the different concentrations of U0126, SB203580, or and then ERK, p38 kinase, and JNK were analyzed by As in all three MAPK the for MAPKs the of a the of the result in to the effect of MAPK on AQP1 protein expression. Cells were with MAPK inhibitor and stimulated with NaCl. Cell lysates were analyzed by were to expression of by of AQP1 expression was significantly by and SP600125 The treatment of a concentration with MAPK inhibitor AQP1 expression not this result provides that AQP1 by hypertonicity the activation of ERK, p38 kinase, and JNK pathways. As an to the of JNK on hypertonicity-induced AQP1 mIMCD-3 cells were with the dominant-negative mutants of JNK1 or and pSV-β-galactosidase After of without or with 100 mm AQP1 protein expression was by analysis. β-Galactosidase activity was measured in experiment for of transfection were to expression of by of As in AQP1 expression was significantly reduced by both the and mIMCD-3 cells. the activation of ERK, p38 kinase, and JNK pathways is for AQP1 in mIMCD-3 cells that is responsible for hypertonicity. results demonstrated that both NaCl and urea activated the ERK the ERK activation is to AQP1 or which are as of ERK were As in all three activated the ERK However, not AQP1 protein expression under or hypertonic the ERK activation is not to AQP1 expression by hypertonicity. previous study that a HRE is present in the AQP1 promoter which is located in the region from −54 to (17Umenishi F. Schrier R.W. Biochem. Biophys. Res. Commun. 2002; 292: 771-775Crossref PubMed Scopus (43) Google Scholar). This critical element to hypertonicity and is for and hypertonicity-induced expression of the AQP1 the of ERK, p38 kinase, and JNK pathways in hypertonicity-induced AQP1 expression at the transient transfection were performed using the AQP1 construct which contains the HRE These results are showed in cells were transfected with the the treatment with NaCl to an in the promoter whereas the treatment with urea promoter activity was attenuated in the presence of U0126, SB203580, and SP600125 in a dose-dependent In promoter activity was significantly by the dominant-negative mutants of JNK1 or These results that the HRE in AQP1 promoter is for the hypertonicity-induced by ERK, p38 kinase, and JNK pathways on AQP1 expression. In the to Smith that the to solute-free water excretion by a urinary concentrating mechanism was an important in the from water to mammals H.W. to The of and Scholar). has been the mechanism, the osmotic and of the vasopressin R.W. T. Am. J. Physiol. Google Scholar), the concentrating and mechanisms T. Schrier R.W. and Philadelphia, Scholar), the of the collecting duct vasopressin A. S. C. A. P. W. Nature. 1992; PubMed Scopus Google Scholar), and most the of the renal water channels, M.A. J. D. Nielsen S. Kidney 1996; Full Text PDF PubMed Scopus Google Scholar). Although the and to the collecting membrane of is a critical in the mechanism S. D. Christensen E.I. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), is an important role of AQP1 in the proximal descending thin of and cells of the vasa recta (4Nielsen S. Smith B.L. Christensen E.I. Knepper M.A. Agre P. J. Cell Biol. 1993; 120: 371-383Crossref PubMed Scopus (455) Google Scholar). of the AQP1 gene both in T. A. Verkman A.S. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and Agre P. J. PubMed Scopus Google Scholar) been to a in urinary concentrating study that a hypertonic NaCl in mouse medullary cells is with an of AQP1 (16Jenq W. Mathieson I.M. Ihara W. Ramirez G. Biochem. Biophys. Res. Commun. 1998; 245: 804-809Crossref PubMed Scopus (50) Google Scholar). The mechanism for this effect not been an of AQP1 protein expression the urinary concentrating mechanism, of AQP1 urinary The present study was undertaken to examine the cellular and molecular mechanisms on the effect of hypertonic stress to AQP1 protein expression. The study was performed with mouse medullary (mIMCD-3) cells incubated in medium supplemented with NaCl or urea as well as with medium containing impermeable osmolytes and AQP1 protein expression was in medium containing all of urea. urea cell membrane not an osmotic results a hypertonic gradient in this of AQP1 protein expression. studies demonstrated the and dose-dependent of the hypertonic gradient using or to AQP1 protein expression. The and treatment suggested the of and in hypertonicity-induced AQP1 expression. study demonstrated that AQP1 protein was in AQP1 by hypertonicity in mouse which was mediated by the Agre P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that AQP1 by hypertonicity at both the and Hypertonicity activated all three MAPK signaling pathways (ERK, p38 kinase, and JNK) in mIMCD-3 cells. These signaling pathways were to hypertonicity-induced AQP1 expression. Specifically, it was demonstrated that the of of ERK, p38 kinase, or JNK signaling by a inhibitor significantly reduced hypertonicity-induced AQP1 expression. U0126, SB203580, or SP600125 the ERK, p38 kinase, or JNK in a dose-dependent respectively. the ERK by was at 5 not not The used in the present study was The was previously reported by Z. Cohen D.M. Am. J. Physiol. 1999; PubMed Google Scholar) and suggested a ERK and p38 kinase Although which was as a JNK the JNK activation in a dose-dependent it has been demonstrated that this inhibitor also had a p38 at a concentration the used in the present study B.L. Sasaki W. A. S. Y. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). It be that mIMCD-3 cells are to of MAPK signaling Furthermore, showed that the of JNK by dominant-negative mutants of JNK1 and as an significantly reduced hypertonicity-induced AQP1 expression. that the activation of all three MAPK is for AQP1 by hypertonicity. In mIMCD-3 cells, NaCl activated the ERK, p38 kinase, and JNK, urea activated the Although both NaCl and urea activated the ERK, NaCl induced AQP1 expression. Furthermore, ERK as and not AQP1 expression under or hypertonic condition. These results that the ERK activation is not to AQP1 expression by hypertonicity. study also that hypertonicity-induced expression in mouse epithelial cells was mediated by the the ERK activation was not for by hypertonicity Agre P. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism via MAPK pathways in hypertonicity-induced AQP1 expression is still unclear. data that the three MAPKs may be to that to in the AQP1 The of AQP1 gene by hypertonicity results from a that directly the promoter region of AQP1 studies demonstrate that the genes of the J.S. S.C. J.S. Kwon J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), S.C. J.S. Kwon Am. J. Physiol. 1998; Google Scholar), and Jung M.B. A. J. Biol. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) are up-regulated in response to hypertonicity. The enhancer (TonE) consensus sequence present in genes the It that AQP1 by hypertonicity is mediated by an osmotic response element as However, no consensus sequence is present in a AQP1 promoter region F. Verkman A.S. Genomics. 1998; 47: 341-349Crossref PubMed Scopus (39) Google Scholar). We a HRE in the AQP1 gene that is different from consensus sequence (17Umenishi F. Schrier R.W. Biochem. Biophys. Res. Commun. 2002; 292: 771-775Crossref PubMed Scopus (43) Google Scholar). the AQP1 construct that contains the HRE sequence, showed that of three MAPK and dominant-negative mutants of JNK1 or significantly reduced AQP1 promoter activity in a In urea not the promoter In addition, the promoter activity was not by U0126, SB203580, or SP600125 under and W. Thus, the which to hypertonicity, is for the regulation of hypertonicity-induced AQP1 expression. of a protein that to the HRE the regulation of hypertonicity-induced AQP1 expression. In the present results demonstrate that hypertonicity-induced AQP1 expression is regulated by ERK, p38 kinase, and JNK activation and the HRE in the AQP1 of MAPK results in of hypertonicity-induced AQP1 that all three MAPK signaling pathways are for AQP1 We for and Heasley for the dominant-negative mutants of JNK1 and

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