Nucleoporin 88 (Nup88) Is Regulated by Hypertonic Stress in Kidney Cells to Retain the Transcription Factor Tonicity Enhancer-binding Protein (TonEBP) in the Nucleus
2008; Elsevier BV; Volume: 283; Issue: 36 Linguagem: Inglês
10.1074/jbc.m802381200
ISSN1083-351X
AutoresAna Andres‐Hernando, Miguel A. Lanaspa, Christopher J. Rivard, Tomás Berl,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoAntibody microarray technology identified Nup88 (nucleoporin 88) as a highly up-regulated protein in response to osmotic stress in inner medullary collecting duct (IMCD3) cells. Changes in expression were verified by Western blot and quantitative PCR for protein and message expression. In mouse and human kidney, Nup88 expression was substantial in the papilla, whereas it was nearly absent in the cortex. Furthermore, the expression of Nup88 increased 410.4 ± 22% in the papilla of mice after 36 h of thirsting. Nup88 protein expression in IMCD3 cells was significantly up-regulated in the first 8 h following exposure to acute osmotic stress, indicating that Nup88 is an early response protein. To define the function of Nup88 in the osmotic stress response, the transcription factor associated with hypertonicity, tonicity enhancer-binding protein (TonEBP), was cloned upstream of the green fluorescent protein. Employing this construct, we demonstrate that silencing Nup88 in IMCD3 cells acutely stressed to hypertonic conditions reduces nuclear retention of TonEBP, resulting in a substantial blunting in transcription of important osmotic stress response target genes and reduced cell viability. Finally, we show that in IMCD3 cells, nuclear export of TonEBP under isotonic conditions involves CRM-1 but under hypertonic stress is CRM1-independent. Our data, therefore, suggest that Nup88 is up-regulated in response to hypertonic stress and acts to retain TonEBP in the nucleus, activating transcription of critical osmoprotective genes. Antibody microarray technology identified Nup88 (nucleoporin 88) as a highly up-regulated protein in response to osmotic stress in inner medullary collecting duct (IMCD3) cells. Changes in expression were verified by Western blot and quantitative PCR for protein and message expression. In mouse and human kidney, Nup88 expression was substantial in the papilla, whereas it was nearly absent in the cortex. Furthermore, the expression of Nup88 increased 410.4 ± 22% in the papilla of mice after 36 h of thirsting. Nup88 protein expression in IMCD3 cells was significantly up-regulated in the first 8 h following exposure to acute osmotic stress, indicating that Nup88 is an early response protein. To define the function of Nup88 in the osmotic stress response, the transcription factor associated with hypertonicity, tonicity enhancer-binding protein (TonEBP), was cloned upstream of the green fluorescent protein. Employing this construct, we demonstrate that silencing Nup88 in IMCD3 cells acutely stressed to hypertonic conditions reduces nuclear retention of TonEBP, resulting in a substantial blunting in transcription of important osmotic stress response target genes and reduced cell viability. Finally, we show that in IMCD3 cells, nuclear export of TonEBP under isotonic conditions involves CRM-1 but under hypertonic stress is CRM1-independent. Our data, therefore, suggest that Nup88 is up-regulated in response to hypertonic stress and acts to retain TonEBP in the nucleus, activating transcription of critical osmoprotective genes. The cells that inhabit the hypertonic environment of the inner medulla of the kidney possess a number of adaptative mechanisms that allow them to survive in this environment (1Burg M.B. Kwon E.D. Kultz D. Annu. Rev. Physiol. 1997; 59: 437-455Crossref PubMed Scopus (330) Google Scholar, 2Burg M.B. Kwon E.D. Kültz D. FASEB J. 1996; 10: 1598-1606Crossref PubMed Scopus (159) Google Scholar, 3Ferraris J.D. Burg M.B. Contrib. Nephrol. 2006; 152: 125-141Crossref PubMed Scopus (30) Google Scholar, 4Handler J.S. Kwon H.M. Kidney Int. 1996; 49: 1682-1683Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 5Handler J.S. Kwon H.M. Nephron. 2001; 87: 106-110Crossref PubMed Scopus (36) Google Scholar). The classical osmotic stress response involves the prompt transcription of several target genes by the tonicity enhancer-binding protein (TonEBP), 2The abbreviations used are:TonEBPtonicity enhancer-binding proteinARaldose reductaseTauTtaurine transporterQPCRquantitative PCRDAPI4′,6-diamidino-2-phenylindoleGFPgreen fluorescent proteinLMBleptomycin B. also known as NFAT5 (6Handler J.S. Kwon H.M. Kidney Int. 2001; 60: 408-411Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 7Jeon U.S. Kim J.A. Sheen M.R. Kwon H.M. Acta Physiol. (Oxf.). 2006; 187: 241-247Crossref PubMed Scopus (85) Google Scholar, 8Woo S.K. Kwon H.M. Int. Rev. Cytol. 2002; 215: 189-202Crossref PubMed Scopus (45) Google Scholar, 9Woo S.K. Lee S.D. Kwon H.M. Pflugers Arch. 2002; 444: 579-585Crossref PubMed Scopus (117) Google Scholar). Under isotonic conditions (300 mosmol/kg H2O), TonEBP is mainly present in the cytosol with only minor localization in the nucleus. However, under hypertonic stress, TonEBP is translocated to the nucleus, where it enhances the transcription of genes that are important in the early osmotic stress response. These genes includes aldose reductase (AR) (10Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (473) Google Scholar), the sodium-myoinositol transporter (10Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (473) Google Scholar), the BGT1 (betaine/GABA transporter 1) (11Miyakawa H. Woo S.K. Chen C.P. Dahl S.C. Handler J.S. Kwon H.M. Am. J. Physiol. 1998; 274: F753-F761PubMed Google Scholar), the taurine transporter (TauT) (12Ito T. Fujio Y. Hirata M. Takatani T. Matsuda T. Muraoka S. Takahashi K. Azuma J. Biochem. J. 2004; 382: 177-182Crossref PubMed Scopus (108) Google Scholar), and heat shock protein 70 (Hsp70) (13Woo S.K. Lee S.D. Na K.Y. Park W.K. Kwon H.M. Mol. Cell. Biol. 2002; 22: 5753-5760Crossref PubMed Scopus (177) Google Scholar) among others. Expression of these target genes results in the accumulation of a number of compatible organic osmolytes (mainly sorbitol, myoinositol, betaine, and taurine) that allow the cell to compensate the extracellular osmotic gradient and, hence, adapt to hypertonic stress (see Refs. 3Ferraris J.D. Burg M.B. Contrib. Nephrol. 2006; 152: 125-141Crossref PubMed Scopus (30) Google Scholar and 6Handler J.S. Kwon H.M. Kidney Int. 2001; 60: 408-411Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 7Jeon U.S. Kim J.A. Sheen M.R. Kwon H.M. Acta Physiol. (Oxf.). 2006; 187: 241-247Crossref PubMed Scopus (85) Google Scholar, 8Woo S.K. Kwon H.M. Int. Rev. Cytol. 2002; 215: 189-202Crossref PubMed Scopus (45) Google Scholar, 9Woo S.K. Lee S.D. Kwon H.M. Pflugers Arch. 2002; 444: 579-585Crossref PubMed Scopus (117) Google Scholar for excellent reviews). One of the main mechanisms involved in the regulation of TonEBP activity under hypertonic stress is nucleocytoplasmic trafficking (7Jeon U.S. Kim J.A. Sheen M.R. Kwon H.M. Acta Physiol. (Oxf.). 2006; 187: 241-247Crossref PubMed Scopus (85) Google Scholar, 9Woo S.K. Lee S.D. Kwon H.M. Pflugers Arch. 2002; 444: 579-585Crossref PubMed Scopus (117) Google Scholar, 14Cha J.H. Woo S.K. Han K.H. Kim Y.H. Handler J.S. Kim J. Kwon H.M. J. Am. Soc. Nephrol. 2001; 12: 2221-2230Crossref PubMed Google Scholar, 15Tong E.H. Guo J.J. Huang A.L. Liu H. Hu C.D. Chung S.S. Ko B.C. J. Biol. Chem. 2006; 281: 23870-23879Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). tonicity enhancer-binding protein aldose reductase taurine transporter quantitative PCR 4′,6-diamidino-2-phenylindole green fluorescent protein leptomycin B. Our laboratory has employed several proteomic approaches, including two-dimensional difference gel electrophoresis (16Rivard C.J. Brown L.M. Almeida N.E. Maunsbach A.B. Pihakaski-Maunsbach K. Andres-Hernando A. Capasso J.M. Berl T. J. Biol. Chem. 2007; 282: 6644-6652Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and antibody microarray (17Lanaspa M.A. Almeida N.E. Andres-Hernando A. Rivard C.J. Capasso J.M. Berl T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 13672-13677Crossref PubMed Scopus (40) Google Scholar) technologies to evaluate hypertonicity-induced up-regulation of important proteins in inner medullary collecting duct (IMCD3) cells. Here, we describe the marked up-regulation of Nup88 (nucleoporin 88) under hypertonic stress. Nup88 was first identified as an interacting partner of Nup214 (nucleoporin 214) (18Fornerod M. van Deursen J. van Baal S. Reynolds A. Davis D. Murti K.G. Fransen J. Grosveld G. EMBO J. 1997; 16: 807-816Crossref PubMed Scopus (400) Google Scholar). These two proteins are components of a nuclear pore complex in the nuclear membrane and are involved in the nucleocytoplasmic trafficking of different molecules, including transcription factors (19Hutten S. Kehlenbach R.H. Mol. Cell. Biol. 2006; 26: 6772-6785Crossref PubMed Scopus (117) Google Scholar, 20Roth P. Xylourgidis N. Sabri N. Uv A. Fornerod M. Samakovlis C. J. Cell Biol. 2003; 163: 701-706Crossref PubMed Scopus (69) Google Scholar, 21Uv A.E. Roth P. Xylourgidis N. Wickberg A. Cantera R. Samakovlis C. Genes Dev. 2000; 14: 1945-1957PubMed Google Scholar, 22Xylourgidis N. Roth P. Sabri N. Tsarouhas V. Samakovlis C. J. Cell Sci. 2006; 119: 4409-4419Crossref PubMed Scopus (52) Google Scholar). It was previously demonstrated in Drosophila that Nup88 does not interfere in the nuclear import of NF-κB and instead acts as an inhibitor of CRM1-mediated protein export (20Roth P. Xylourgidis N. Sabri N. Uv A. Fornerod M. Samakovlis C. J. Cell Biol. 2003; 163: 701-706Crossref PubMed Scopus (69) Google Scholar, 22Xylourgidis N. Roth P. Sabri N. Tsarouhas V. Samakovlis C. J. Cell Sci. 2006; 119: 4409-4419Crossref PubMed Scopus (52) Google Scholar). In experiments with mutated (inactive) Nup88, CRM1 expression is mislocalized in the nucleus and is not present in the nuclear membrane. Interestingly, this mislocalization resulted in a continuous shuttling of the NF-κB Rel-like transcription factor Dorsal in and out of the nucleus (22Xylourgidis N. Roth P. Sabri N. Tsarouhas V. Samakovlis C. J. Cell Sci. 2006; 119: 4409-4419Crossref PubMed Scopus (52) Google Scholar). These data suggest that CRM1 and, therefore, Nup88 expression are necessary for regulating Dorsal nuclear export. The mechanism by which Nup88 functions is by sequestering CRM1 at the nuclear pore and thereby avoiding its recycling back to the nucleus for another round of export. Similarly, another Rel-like transcription factor, Dif, seems to be regulated by Nup88 expression in Drosophila (20Roth P. Xylourgidis N. Sabri N. Uv A. Fornerod M. Samakovlis C. J. Cell Biol. 2003; 163: 701-706Crossref PubMed Scopus (69) Google Scholar). Likewise, other members of the Rel-like transcription factor class, including TonEBP (10Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (473) Google Scholar), may be controlled by the expression of Nup88. The up-regulation of Nup88 under hypertonic stress in the kidney has not been previously described. The present work was undertaken to confirm the observations made by antibody microarray analysis and further characterize the osmotic regulation of expression, half-life, cellular distribution, and in vivo expression. Furthermore, we identify a potential role for Nup88 in the nuclear retention of TonEBP in hypertonically stressed cells. Materials—Cell culture medium, FCS, and antibiotics were from Invitrogen. Antibodies to Nup88 and GS15 were obtained from Clontech, anti-AR antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), anti-Hsp70 was from Stressgen (Ann Arbor, MI), and anti-β-actin was from Cell Signaling (Danvers, MA). All other chemicals were purchased from Sigma. Antibody Microarray—Antibody microarrays and reagents were purchased from Clontech. Microarrays were processed as per the manufacturer's protocol and as previously described (17Lanaspa M.A. Almeida N.E. Andres-Hernando A. Rivard C.J. Capasso J.M. Berl T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 13672-13677Crossref PubMed Scopus (40) Google Scholar). Antibody microarrays were analyzed using a ProScanArray HT scanner (PerkinElmer Life Sciences). Cell Culture—The established IMCD3 cell line originally developed by Rauchman et al. (23Rauchman M.I. Nigam S.K. Delpire E. Gullans S.R. Am. J. Physiol. 1993; 265: F416-F424Crossref PubMed Google Scholar) was provided by Steve Gullans (Rx Gen, Hamden, CT). IMCD3 cells chronically adapted to 600 and 900 mosmol/kg H2O were previously established in our laboratory (24Capasso J.M. Rivard C.J. Berl T. Am. J. Physiol. 2001; 280: F768-F776Crossref PubMed Google Scholar, 25Capasso J.M. Rivard C.J. Enomoto L.M. Berl T. Ann. N. Y. Acad. Sci. 2003; 986: 410-415Crossref PubMed Scopus (17) Google Scholar) and were compared with cells grown at isotonic conditions. In experiments involving hypertonic stress, the media in culture dishes were exchanged for that with added NaCl to the specified osmolality, depending on the experiment. Osmolality was determined with a microosmometer (model 3300; Advanced Instruments, Norwood, MA). Cell Viability and Growth Experiments—Cell viability in tissue culture stress experiments was determined by cell counts following exposure to sublethal osmotic stress. Experiments were initiated once cells reached confluence at isotonic conditions in 24-well flat bottom tissue culture plates (35-3047; Falcon BD Labware, Franklin Lakes, NJ), with each experimental time point after exposure to the sublethal osmotic stress performed in triplicate. In cell growth studies, cell counts were initiated when cultures had reached ∼90% confluence and followed for an additional 80-h period with counts at 24 and 48 h. Growth curves were fit to linear regression using the Prism 4.0 software, and the slopes of the curves were compared for relative differences in growth rate. Stable Cell Lines Silenced for Nup88 Expression—IMCD3 cultures were transfected with the pSM2 empty vector (v2MM_173601) or the short hairpin RNA vector pSM2-Nup88 (v2HS_152407; Open Biosystems) using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Stable transfectants (clones) were selected from colonies growing in plates from a 10-fold dilution series in media prepared with 10 μg/ml puromycin antibiotic (Sigma). The absence of Nup88 expression in silenced clones was verified by Western blot of cell lysates obtained from these clones grown in media adjusted to 500 mosmol/kg H2O for 48 h. Cloning and Transfection of the Construct TonEBP-GFP—The first 800 bp of mouse TonEBP, including the nuclear export signal, the auxiliary export domain, and the nuclear localization signal, was obtained by PCR using the following primers, which contained, respectively, XhoI and EcoRI restriction sites (both underlined) for directional cloning into pAcGFP-N1 vector (Clontech): sense, 5′-GAC CTC GAG ATG CCC TCG GAC TTC ATC TCA TTG CTC-3′; antisense, 5′-GAC GAA TTC GAG GTG CTT TGG CAC TGT CGG CAT CAA-3′. Transfection into IMCD3 cells or into the stable cell lines expressing pSM2-Nup88 vector or pSM2-Empty vector control was performed using Lipofectamine 2000. Mouse Kidney Tissues—C57/B6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were subjected to food and ad libitum water or deprived for water for 36 h. Urine samples were collected from the bladder for osmolality analysis. Mice were harvested by cervical dislocation, and kidneys were removed. Papilla and cortex tissues were dissected and homogenized with a glass tissue grinder on ice with mitogen-activated protein kinase lysis buffer for protein and analyzed as described (24Capasso J.M. Rivard C.J. Berl T. Am. J. Physiol. 2001; 280: F768-F776Crossref PubMed Google Scholar). Human Kidney Tissues—Human kidney tissues from cortex and papilla were obtained under the Colorado Multiple Institutional Review Board and National Institutes of Health Grant U19A10636030 from a kidney that was not suitable for transplantation. Tissues were processed for protein as described above. RNA Extraction, Analysis, and Message Quantification—Cytosolic RNA was isolated from confluent cell cultures using the RNeasy kit (Qiagen, Valencia, CA) as per the manufacturer's protocol. Before quantitative PCR (QPCR), sample RNA concentration and integrity was assessed by UV spectrometry (absorbance at 260 nm) and by capillary electrophoresis using a bioanalyzer (model 210, Agilent, Foster City, CA; using the 28 S to 18 S rRNA ratio), respectively. RNA was converted to cDNA using the iScript cDNA synthesis kit (Bio-Rad). QPCR was performed using primer pairs identified in supplemental Table 3, designed using Beacon Designer 7.0 (Premier Biosoft, Palo Alto, CA). QPCR runs were performed using the SYBR green JumpStart Taq Readymix QPCR kit (Sigma) on an I-Cycler (Bio-Rad). QPCR runs were analyzed by agarose gel electrophoresis and melt curve to verify that the correct amplicon was produced. β-Actin RNA was used as internal control in all QPCRs, and the amount of RNA was calculated by the comparative CT method. Protein Extraction and Western Blotting—Cell protein lysates were obtained from confluent cell culture dishes as previously described (24Capasso J.M. Rivard C.J. Berl T. Am. J. Physiol. 2001; 280: F768-F776Crossref PubMed Google Scholar). Protein concentration was determined by the BCA protein assay (Pierce). Seventy micrograms of total protein was loaded per lane for SDS-PAGE (7.5%, w/v) analysis and then transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibody and visualized by using an alkaline phosphatase secondary antibody and Lumi-Phos reagent (Pierce) as described by the manufacturer. Chemiluminescence was recorded with an Image Station 440CF (Kodak Digital Science), and results were analyzed with 1D Image Software (Kodak Digital Science). Blots were also analyzed for β-actin as a loading control. Confocal Fluorescence Microscopy—IMCD3 cells were seeded in 8-well chambers (Nunc) and stained using an anti-Nup88 antibody (1:50 dilution in phosphate-buffered saline; Clontech) or anti-TonEBP antibody (1:400 dilution in PBS) kindly provided by Dr. H. Moo Kwon (University of Maryland). Inmunofluorescence was carried out as previously described (17Lanaspa M.A. Almeida N.E. Andres-Hernando A. Rivard C.J. Capasso J.M. Berl T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 13672-13677Crossref PubMed Scopus (40) Google Scholar). Preparations and TonEBP-GFP fusion protein transfections were imaged with a ×40 water immersion objective using a laser-scanning confocal microscope (model LSM510; Zeiss, Thornwood, NY). Data were analyzed with LSM Image analyzer postacquisition software (Zeiss). Statistics and Data Analysis—All data are presented as the mean ± S.E. Data graphics and statistical analysis were performed using Instat (version 3.0) and Prism 4 (both from GraphPad, San Diego, CA). Independent replicates for each data point (n) are identified in figure legends. p < 0.05 was recognized as statistically significant. Nup88 Expression Is Up-regulated under Hypertonic Stress—Employing antibody microarray proteomics (Clontech, Mountain View, CA), we found that 5% of the 512 proteins analyzed were up-regulated (17Lanaspa M.A. Almeida N.E. Andres-Hernando A. Rivard C.J. Capasso J.M. Berl T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 13672-13677Crossref PubMed Scopus (40) Google Scholar) in IMCD3 cells chronically adapted to hypertonic stress (900 mosmol/kg H2O) as compared with cells grown under isotonic conditions. As shown in Fig. 1A, the Golgi vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (GS15) is representative of the greater than 90% of the proteins whose expression did not change under hypertonic conditions. In contrast, Nup88 demonstrated a substantial increase in expression in hypertonically adapted cells. A statistical analysis of four spots for each protein (from two arrays; i.e. dye swap (Fig. 1B)) showed an internal normalization ratio for Nup88 signal intensity of 1.65 (p < 0.001) as compared with 1.05 for GS15. Validation of antibody microarray data was performed by QPCR and Western blot. Data shown in Fig. 1C indicate a substantial increase in Nup88 message levels (7-fold; p < 0.001) in cells adapted to 900 mosmol/kg H2O as compared with isotonic conditions (300 mosmol/kg H2O). This up-regulation was also substantial for protein expression (12.9-fold; p < 0.001) under the same conditions. For comparison, Western blots prepared with anti-GS15 demonstrated equal protein expression in IMCD3 cells at isotonic and hypertonic conditions (Fig. 1D). Expression of Nup88 in Renal Cortex and Medulla from Rodents and Human Kidney—To assess whether the changes seen in cultured cells are also observed in vivo, we examined the renal tissues of mice and a human kidney. Western blot data shown in Fig. 2A reveal a near absence of Nup88 protein expression in the cortex of both species. In contrast, substantial Nup88 protein expression was determined in the papilla for both mice and human. These determinations were made in mice on ad libitum water intake. The difference between cortex and papilla is quantitatively depicted in Fig. 2B. We also examined the kidney tissues of mice subjected to 36 h of thirsting (urine osmolality increased from 1,424 ± 211 to 3,105 ± 524 mosmol/kg H2O; n = 6). As shown in Fig. 2B, thirsting increased Nup88 expression by 2-fold (107 ± 12%, p < 0.01) in the cortex and 5-fold (410 ± 22%, p < 0.01) in the papilla tissues. Immunocytochemical Localization of Nup88 in Cells Adapted to Hypertonicity—Immunofluorescence microscopy studies were undertaken to assess the presence and localization of Nup88 protein in IMCD3 cells at isotonic conditions and chronically adapted to hypertonicity (900 mosmol/kg H2O). Fig. 3 shows that Nup88 expression (green) is substantial in IMCD3 cells chronically adapted to 900 mosmol/kg H2Oas compared with isotonic conditions where no Nup88 signal is found. The presence of Nup88 in the nuclear membrane is confirmed by colocalization with the nuclear marker DAPI. Kinetics of Nup88 Message and Protein Expression with Acute Exposure to Hypertonicity—Nup88 protein expression in IMCD3 cells subjected to acute sublethal osmotic stress was evaluated to determine the timing of expression. To this end, we performed Western blot analysis for protein at numerous time points after exposure to acute sublethal hypertonicity (550 mosmol/kg H2O). Data shown in Fig. 4 demonstrate a significant increase in Nup88 protein expression after 4-6 h of hypertonic stress (p < 0.01). To assess the half-life for Nup88 message and protein, IMCD3 cells were returned to isotonic conditions. Data shown in Fig. 5 were curve-fit for exponential decay, and the half-life was calculated to be 2.8 and 18.4 h for message and protein, respectively. Separately, cells were treated with actinomycin D or cyclohexamide to evaluate potential changes in the stability of Nup88 message and protein. These data were essentially identical to the data shown in Fig. 5 indicating no significant changes in the stability of Nup88 message or protein (data not shown).FIGURE 5Estimation of half-life for Nup88 mRNA and protein in IMCD3 cells. A, QPCR and Western blot analysis were from three independent experiments performed in duplicate (n = 6). B, a representative Western blot including a β-actin loading control is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Studies on the Osmotic and Ionic Mediators of the Up-Regulation of Nup88 Expression to Hypertonic Stress—To assess whether the effects observed with increasing NaCl levels were unique to this solute, Nup88 protein expression was measured after exposure to other mediators added to the medium to reach the same osmolality (550 mosmol/kg H2O). As depicted in Fig. 6, replacement of sodium by choline or chloride by acetate does not affect the response (p < 0.01 versus isotonic). Other solutes, including sucrose, mannitol, and, interestingly, urea, also caused a marked increase in Nup88 protein expression (p < 0.01 versus isotonic). Development and Use of a TonEBP-GFP Construct to Study Trafficking in IMCD3 Cells under Changing Osmotic Stress—Primers for the N terminus of human TonEBP (amino acids 1-267) that include the nuclear export signal, auxiliary export domain, and nuclear localization signal were designed to amplify an 800-bp fragment from cDNA of murine IMCD3 cells chronically adapted at 900 mosmol/kg H2O. Sequence data for the amplicon and its translation are shown as supplemental data (supplemental Tables 1 and 2, respectively). The amplicon was cloned into pAcGFP-N1 (Clontech) upstream of the green fluorescent protein (GFP). To validate the utility of the TonEBP-GFP construct, a comparison was made with antibody labeling of fixed cells. Fig. 7 shows the results obtained from IMCD3 cells transfected with the TonEBP-GFP construct (columns 2 and 4) as compared with immunohistochemistry analysis (columns 1 and 3). In both cases, cells maintained at isotonic conditions (300 mosmol/kg H2O) demonstrated that the majority of the TonEBP signal is present in the cytosol (with some nuclear staining), whereas in cells acutely stressed to 550 mosmol/kg H2O for 30 min, TonEBP is rapidly shifted to the nucleus. Analysis of confocal images reveals that the TonEBP-GFP construct cells demonstrate a more profound and quantifiable image as compared with immunostained cells. The application of the TonEBP-GFP construct also, and more importantly, allows for live imaging of TonEBP trafficking in IMCD3 cells in response to changes in acute osmotic stress. We therefore employed the TonEBP-GFP construct in further experiments. TonEBP Is Exported from the Nucleus under Hypertonic Stress in Cells Silenced for Nup88 Expression—IMCD3 cells were silenced for expression of Nup88 employing a commercial vector pSM2-Nup88 (Open Biosystems). BLAST and alignment analysis were performed to ascertain the selectivity of the silencing sequence with other genes that may induce off-target responses. Analysis reveals a <70% homology of the silencing sequence with well known nucleoporins and exportins. In addition, message levels for nucleoporins and exportins reported to be involved with Nup88, including Nup153 (26Kodiha M. Tran D. Qian C. Morogan A. Presley J.F. Brown C.M. Stochaj U. Biochim. Biophys. Acta. 2008; 1783: 405-418Crossref PubMed Scopus (45) Google Scholar), Nup358 (27Bernad R. van der Velde H. Fornerod M. Pickersgill H. Mol. Cell. Biol. 2004; 24: 2373-2384Crossref PubMed Scopus (133) Google Scholar), Nup214 (28Bernad R. Engelsma D. Sanderson H. Pickersgill H. Fornerod M. J. Biol. Chem. 2006; 281: 19378-19386Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and CRM1 (18Fornerod M. van Deursen J. van Baal S. Reynolds A. Davis D. Murti K.G. Fransen J. Grosveld G. EMBO J. 1997; 16: 807-816Crossref PubMed Scopus (400) Google Scholar), did not change significantly in Nup88-silenced cells as compared with empty vector controls (data not shown). Stable silenced clones using puromycin (10 μg/ml) were validated as depicted in supplemental Fig. 1A. Two clones (clones 3 and 6) were selected for further experiments, since they showed nearly complete silencing of Nup88 expression under hypertonic stress. Fig. 8A depicts a stable control cell line expressing pSM2-Empty Vector (left) or pSM2-Nup88 clone 3 (right) and transiently transfected with the TonEBP-GFP construct. In cells expressing the empty vector control, TonEBP (green) is present in the nucleus after 8 h of acute sublethal stress (550 mosmol/kg H2O), as demonstrated by its colocalization with DAPI (blue). In contrast, in Nup88-silenced cells (right), the TonEBP is mainly present in the cytosol under hypertonic conditions. Comparative fluorescence measurement of the TonEBP-GFP signal is shown in Fig. 8B and reveals that 80 ± 4% of the TonEBP signal remains in the nucleus of empty vector control cells, whereas only 25 ± 2% of TonEBP remains in nucleus of cells silenced for Nup88 expression (p < 0.001, n > 35 cells/condition). This finding is supported by a significant decrease in message levels of TonEBP target genes (p < 0.001). As shown in Fig. 9A, levels of AR, sodium-myoinositol transporter, BGT1, TauT, and Hsp70 are lower in Nup88 silenced cells (solid bars) as compared with empty vector control cells (open bars) (p < 0.001). Fig. 9B depicts a representative Western blot for AR and Hsp70 from these cells in which levels of AR protein expression are significantly blunted in cells silenced for Nup88 as compared with empty vector control cells. Interestingly, there is a complete absence of Hsp70 protein expression in cells silenced for Nup88, corroborating the data obtained by QPCR. In contrast, mRNA levels of non-TonEBP target genes, such as the α1 subunit of the Na/K-ATPase (ATP1a1) (29Capasso J.M. Rivard C. Berl T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13414-13419Crossref PubMed Scopus (43) Google Scholar), did not change in Nup88-silenced cells as compared with the empty vector control cells (Fig. 9A). We also examined the expression of AR and Hsp70 in a clone that was partially silenced, clone 4, and a clone that had almost full Nup88 expression, clone 5 (see supplemental Fig. 1A). As shown in supplemental Fig. 1, B, C, and D, an intermediate phenotype was found for AR, and Hsp70 mRNA (Fig. 1C) and protein (Fig. 1D) expression were found in the partially silenced clone 4. In contrast, no significant difference was found between empty vector control cells and the nonsilenced clone 5. Silencing Nup88 Expression in IMCD3 Cells Increases Cellular Osmosensitivity to Acute Hyperton
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