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• W2049226316 abstract "In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5′-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation. In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5′-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation. Cerebrospinal fluid (CSF), 6The abbreviations used are: CSF, cerebrospinal fluid; AQP-1, aquaporin-1; TTF-1, thyroid transcription factor-1; AS, antisense; ODN, oligodeoxynucleotide; FISH, fluorescence in situ hybridization; RT, reverse transcription; DIG, digoxygenin; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; TTF-1 HD, TTF-1 homeodomain; EMSA, electrophoretic mobility shift assay; SCR, scrambled; FITC, fluorescence isothiocyanate; ICP, intracranial pressure; dDAVP, 1-deamino-8-d-arginine vasopressin; AS, antisense. a major constituent of the extracellular fluid in the central nervous system, fills the ventricles of the brain, spinal canal, and subarachnoid space. Most CSF in the cerebral ventricular system is formed in discrete sites, including the choroid plexus and circumventricular organs of the lateral, third, and fourth ventricles. Altogether, these sites produce nearly 90% of all ventricular CSF (1Brown P.D. Davies S.L. Speake T. Millar I.D. Neuroscience. 2004; 129: 957-970Crossref PubMed Scopus (320) Google Scholar). CSF has several important functions. Foremost, it provides a mechanical cushion to protect the brain from impact with the bony calvarium when the head moves (2Segal M.B. J. Inherit. Metab. Dis. 1993; 16: 617-638Crossref PubMed Scopus (108) Google Scholar) and also serves as a flexible physical support for the brain. In addition, it facilitates the transport of nutrients, peptides, and hormones into the brain. Because CSF communicates with the interstitial fluid of the brain, it plays an important role in maintaining a constant external environment for neurons and glia (3Johanson C.E. Preston J.E. Chodobski A. Stopa E.G. Szmydynger-Chodobska J. McMillan P.N. Am. J. Physiol. 1999; 276: C82-C90Crossref PubMed Google Scholar). Regulation of brain water content and brain volume is critical for normal functioning of the central nervous system, which is highly sensitive to any change in fluid osmolality. Aquaporins (AQPs), a family of small integral membrane proteins, have been implicated in the regulation of water homeostasis in the brain (4Venero J.L. Vizuete M.L. Machado A. Cano J. Prog. Neurobiol. 2001; 63: 321-336Crossref PubMed Scopus (180) Google Scholar). Numerous studies have shown that the expression of AQP-1, -4, and -9 is sensitive to brain injury, swelling, and other experimental interventions that affect the homeostatic regulation of brain water balance (5Arima H. Yamamoto N. Sobue K. Umenishi F. Tada T. Katsuya H. Asai K. J. Biol. Chem. 2003; 278: 44525-44534Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 6Gunnarson E. Zelenina M. Aperia A. Neuroscience. 2004; 129: 947-955Crossref PubMed Scopus (112) Google Scholar, 7Papadopoulos M.C. Verkman A.S. J. Biol. Chem. 2005; 280: 13906-13912Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). AQP-1, a water channel for high osmotic water permeability, is abundantly present in the epithelium of kidney, lung, eye, liver, and brain (8Umenishi F. Verkman A.S. Gropper M.A. DNA Cell Biol. 1996; 15: 475-480Crossref PubMed Scopus (87) Google Scholar). AQP-1 was first discovered in human erythrocytes (9Preston G.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11110-11114Crossref PubMed Scopus (723) Google Scholar). In the brain, AQP-1 is mainly expressed in the apical (CSF-facing) membrane of the ventricular choroid plexus epithelium and regulates the formation of CSF (10Speake T. Freeman L.J. Brown P.D. Biochim. Biophys. Acta. 2003; 1609: 80-86Crossref PubMed Scopus (117) Google Scholar, 11Oshio K. Watanabe H. Song Y. Verkman A.S. Manley G.T. FASEB J. 2005; 19: 76-78Crossref PubMed Scopus (340) Google Scholar). The mechanisms underlying the transcriptional control of AQP-1 gene expression in the brain, however, are poorly understood. We report that thyroid transcription factor-1 (TTF-1), a homeodomain-containing transcription factor, regulates AQP-1 gene transcription in the choroid plexus. TTF-1 was first identified in the thyroid gland (12Civitareale D. Lonigro R. Sinclair A.J. DiLauro R. EMBO J. 1989; 8: 2537-2542Crossref PubMed Scopus (325) Google Scholar), and its cDNA was cloned and characterized as having sequence homology with the Drosophila NKx-2 homeodomain transcription factor (13Guazzi S. Price M. De Felice M. Damante G. Mattei M.G. Di Lauro R. EMBO J. 1990; 9: 3631-3639Crossref PubMed Scopus (470) Google Scholar). A different study identified the lung and fetal diencephalon as additional sites of TTF-1 expression (14Lazzaro D. Price M. de Felice M. Di Lauro R. Development (Camb.). 1991; 113: 1093-1104Crossref PubMed Google Scholar). We and others have reported that TTF-1 remains expressed in the discrete regions of the postnatal rat brain (15Lee B.J. Cho G.J. Norgren Jr., R.B. Junier M.P. Hill D.F. Tapia V. Costa M.E. Ojeda S.R. Mol. Cell. Neurosci. 2001; 17: 107-126Crossref PubMed Scopus (105) Google Scholar, 16Nakamura K. Kimura S. Yamazaki M. Kawaguchi A. Inoue K. Sakai T. Brain Res. Dev. Brain Res. 2001; 130: 159-166Crossref PubMed Scopus (41) Google Scholar). More recently, we showed that TTF-1 is expressed in the subfornical organ where it regulates angiotensinogen synthesis (17Son Y.J. Hur M.K. Ryu B.J. Park S.K. Damante G. D'Elia A.V. Costa M.E. Ojeda S.R. Lee B.J. J. Biol. Chem. 2003; 278: 27043-27052Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). During the course of these experiments, we noticed the presence of TTF-1 mRNA in the rat choroid plexus, and thus we examined the 5′-flanking region of the AQP-1 gene; we found several conserved core TTF-1-binding motifs. Promoter analysis performed in the BAS8.1 (astrocyte) and C6 (glioblastoma) cell lines showed that TTF-1 activated AQP-1 transcription in a dose-dependent manner. Electrophoretic mobility shift assays showed that TTF-1 bound specific subsets of putative DNA recognition sites in the AQP-1 promoter. In vivo experiments in which inhibition of TTF-1 synthesis was achieved by administration of antisense (AS) oligodeoxynucleotide (ODN) into the lateral ventricle decreased AQP-1 synthesis and, in turn, led to a decrease in CSF formation. Animals and Tissue Preparation—Two-month-old male Sprague-Dawley rats (Daehan Animal Breeding Co., Chungwon, Korea) were used in this study. Following arrival, rats were housed in a room with a conditioned photoperiod (12-h light/12-h darkness, lights on from 600 to 1800 h) and temperature (23–25 °C) and allowed ad libitum access to tap water and pelleted rat chow. Animals were sacrifice, and the choroid plexus was collected from their brains. The tissues were quickly frozen on dry ice and stored at -80 °C until RNA or protein isolation was performed. Some brain samples were placed in embedding medium (Tissue-Tek, Torrance, CA) and frozen by placing them in 2-methylbutane (isopentane) immersed in liquid nitrogen for 2 min and then stored in a -80 °C freezer for cryosectioning at a later time. Twelve-μm cryostat sections were prepared, maintaining the cryostat temperature at -20 °C, and were mounted onto SuperFrost slides (Fisher). The slides were kept at -80 °C until used for fluorescence in situ hybridization (FISH). PCR Cloning of TTF-1 and AQP-1 cDNA Fragments and 5′-Flanking Region of the AQP-1 Gene—A cDNA fragment derived from TTF-1 mRNA was cloned by reverse transcription (RT)-PCR from rat hypothalamic RNA. The specific primer sets (sense primer, 5′-AAC AGT CAA GCA AAT CCA AC-3′; antisense primer, 5′-AAT ACC AAA CCG TGG AGT AA-3′) were designed to generate a 552-bp TTF-1 cDNA fragment corresponding to nucleotides 1669–2220 in rat TTF-1 mRNA (NCBI GenBank™ accession number X53858). A rat AQP-1 cDNA fragment was also cloned by RT-PCR from rat choroid plexus RNA. The specific primer set (sense primer, 5′-AGC GAA ATC AAG AAG AAG C-3′; antisense primer, 5′-ATA TCA TCA GCA TCC AGG TC-3′) generates a 773-bp AQP-1 cDNA fragment corresponding to nucleotides 7–779 in rat AQP-1 mRNA (NCBI GenBank™ data base, accession number NM_012778). The cDNA fragments were cloned into a pEZ-T vector (RNA Corp., Seoul, Korea); constructs were confirmed by DNA sequencing. The proximal promoter of the human AQP-1 gene (-1779 to +22 bp) that was to be employed for promoter analysis was also cloned by PCR from human genomic DNA using the sequence information deposited in the NCBI GenBank™ data base (accession number AF026479). The sense primer was 5′-ACT TAT GAC CTT CGG CCA CC-3′, and the antisense primer was 5′-GGT GCT CAA TTC CCT CTG AG-3′. The 1802-nucleotide-long PCR product was inserted into a luciferase reporter plasmid (pGL-3 basic, Promega, Madison, WI), and its sequence was confirmed by DNA sequencing. Fourteen AQP-1 promoter constructs containing individually mutated core TTF-1-binding motifs were generated using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions; the intended mutations were confirmed by DNA sequencing. Fluorescence in Situ Hybridization (FISH)—The TTF-1 cDNA template described above was linearized by SpeI and NcoI digestion for the production of either sense or antisense RNA probes, respectively, and the AQP-1 cDNA was linearized using either NotI or SalI. In vitro transcription of digoxygenin (DIG)-labeled or fluorescein-labeled cRNA probes was performed using an RNA labeling mix (Roche Applied Science) and T3 and T7 RNA polymerases (Promega). Tissue sections were fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, for 10 min. Following two 5-min washes with PBS, the slides were incubated in a 0.25% acetylation solution for 10 min to reduce ionic background. After acetylation, the sections were washed three times in 2× SSPE (pH 7.4, 0.3 m NaCl, 20 mm NaH2PO4, 2 mm EDTA) for 5 min and dehydrated in ascending ethanol concentrations (75, 90, and 100%, 2 min each). To reduce nonspecific background, prehybridization was performed in hybridization buffer (50% formamide, 2× SSPE, 1× Denhardt’s solution, 1 mg/ml yeast tRNA, 100 μg/ml polyadenylic acid, 500 μg/ml salmon sperm DNA, and 4 units/ml RNase inhibitor) at 60 °C for 3 h. The hybridization procedure was performed overnight at 60 °C using 200 μl of hybridization solution containing 300 ng of probe (a DIG-labeled cRNA probe to detect TTF-1 mRNA or a fluorescein-labeled cRNA probe to detect AQP-1 mRNA). The next morning the sections were incubated in 50% formamide and 2× SSPE at 62 °C for 1 h and washed with 0.2× SSPE and 0.1× SSPE (30 min each) at 62 °C. After the last washing step, to reduce endogenous peroxidase activity, the slides were incubated with 1% H2O2 in TNT buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl, 0.3% Triton X-100) for 30 min and washed three times with TNT buffer for 5 min each. A blocking step with TNB blocking buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl, 0.5% Blocking Reagent (PerkinElmer Life Sciences)) for 1 h at room temperature was followed by 1 h of incubation with horseradish peroxidase (HRP)-conjugated anti-DIG antibody (a final concentration of 1.5 milliunits/ml; Roche Applied Science) or with HRP-conjugated anti-fluorescein antibody (0.75 milliunits/ml; Roche Applied Science) in TNB blocking buffer. After washing three times in TNT buffer for 5 min each, mRNA signals of TTF-1 and AQP-1 were detected by using a Cy3-labeled and FITC-labeled tyramide signal amplification system, respectively (PerkinElmer Life Sciences). Simultaneous Detection of TTF-1 and AQP-1 Transcripts Using Double FISH—To visualize mRNA expression of these two genes, sections were hybridized simultaneously with a DIG-labeled TTF-1 cRNA probe and a fluorescein-labeled AQP-1 cRNA probe. The detailed procedure for detection of each probe is described above. Briefly, the DIG-labeled TTF-1 probe was detected by incubating with HRP-conjugated DIG antibody (anti-DIG-HRP). After incubation with Cy3-coupled tyramide to detect the DIG-labeled TTF-1 probe, the sections were washed three times in TNT buffer for 5 min each. To quench the peroxidase activity of anti-DIG-HRP, the sections were incubated with 2% H2O2 in TNT buffer for 2 h and washed three times in TNT buffer for 5 min each. To detect fluorescein-labeled AQP-1 probe, sections were then placed in TNB blocking buffer for 1 h at room temperature and incubated with HRP-conjugated anti-fluorescein antibody for 1 h. After washing three times in TNT buffer for 5 min each, signals were detected by incubating with FITC-coupled tyramide. Finally, the slides were dipped in Hoechst 33258 (Sigma) solution for visualization of cell nuclei. Microscopy and Imaging—FISH images were captured with an InfinityX CCD camera (Lumenera Corp., Ontario, Canada) attached to an Axioskop2 Plus fluorescence microscope (Zeiss, Thornwood, NY). Merged images were analyzed using i-solution (iMTechnology, Seoul, Korea) image processing software. The Cy3 fluorescence image was visualized with a 546-nm green excitation filter set. FITC signal was observed with a 450–490-nm blue excitation filter set. Hoechst 33258 fluorescence emission from cell nuclei was observed with a 365-nm excitation filter. In addition, confocal microscope images were acquired using a Fluoview FV500 confocal microscope (Olympus, Tokyo, Japan). Further image processing was carried out using Adobe Photoshop 7.0 software. No selective visual enhancement of specific area or cells was applied to the images. Cell Culture and Assays for Luciferase Activity—BAS8.1 and C6 cells were grown in Dulbecco’s modified Eagle’s medium/F-12 medium containing 10% fetal bovine serum, and the cultures were maintained at either 34 °C (BAS8.1 cells) or 37 °C (C6 cells) in an atmosphere of 5% CO2, 95% air. Twenty four h after seeding the cells in 12-well plates, they were transiently transfected with the AQP-1 promoter-luciferase reporter construct (AQP-1-P) using Lipofectamine (Invitrogen) along with different concentrations (100–400 ng/well) of the expression vector pcDNA 3.1-zeo (Invitrogen) containing the TTF-1 coding region (TTF-1-pcDNA) (15Lee B.J. Cho G.J. Norgren Jr., R.B. Junier M.P. Hill D.F. Tapia V. Costa M.E. Ojeda S.R. Mol. Cell. Neurosci. 2001; 17: 107-126Crossref PubMed Scopus (105) Google Scholar). Transfection efficiency was normalized by cotransfecting the β-galactosidase reporter plasmid (pCMV-β-gal; Clontech) at 20 ng/ml. Cells were harvested 24 h after transfection and used for both luciferase and β-galactosidase assays as described previously (18Kim M.S. Hur M.K. Son Y.J. Park J.I. Chun S.Y. D'Elia A.V. Damante G. Cho S. Kim K. Lee B.J. J. Biol. Chem. 2002; 277: 36863-36871Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Cells containing different combination of vectors were simultaneously cultured and assayed for each experiment. In each case, we used 6 wells per condition, and each experiment was repeated 2–3 times. Electrophoretic Mobility Shift Assay (EMSA)—The procedure for expression and purification of the TTF-1 homeodomain (TTF-1HD) protein has been described previously (19Damante G. Pellizzari L. Esposito G. Fogolari F. Viglino P. Fabbro D. Tell G. Formisano S. Di Lauro R. EMBO J. 1996; 15: 4992-5000Crossref PubMed Scopus (90) Google Scholar). Double-stranded oligodeoxynucleotides 5′-labeled with 32P were used as probes. Sequences of the oligodeoxynucleotides employed are shown in Table 1. Oligodeoxynucleotides C and Cβ were used as a positive and negative control, respectively (20Pellizzari L. Tell G. Damante G. Biochem. J. 1999; 337: 253-262Crossref PubMed Google Scholar). EMSA was performed as described previously (17Son Y.J. Hur M.K. Ryu B.J. Park S.K. Damante G. D'Elia A.V. Costa M.E. Ojeda S.R. Lee B.J. J. Biol. Chem. 2003; 278: 27043-27052Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 18Kim M.S. Hur M.K. Son Y.J. Park J.I. Chun S.Y. D'Elia A.V. Damante G. Cho S. Kim K. Lee B.J. J. Biol. Chem. 2002; 277: 36863-36871Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), using the TTF-1 HD and oligonucleotides at final concentrations of 150 and 5 mm, respectively. Electrophoretically separated signals corresponding to the protein-bound and free DNA were quantitated with Multianalyst software. Binding of TTF-1HD to the oligodeoxynucleotides representing different regions of the AQP-1 promoter was expressed as a percentage of TTF-1HD binding to oligodeoxynucleotide C, which contains the core TTF-1 binding domain and flanking region of the thyroglobulin gene promoter (12Civitareale D. Lonigro R. Sinclair A.J. DiLauro R. EMBO J. 1989; 8: 2537-2542Crossref PubMed Scopus (325) Google Scholar).TABLE 1EMSA oligonucleotide probes Each sequence represents the sense strand of probes; core TTF-1 binding motifs are underlined. * and + denote positive and negative control probes, respectively (see text).Location in the 5′-flanking region of AQP-1 geneProbe sequencesC*5′-CACTGCCCATCAAGTGTTCTTGA-3′Cβ+5′-CACTGCCCAGTCACGCGTTCTTGA-3′-17325′-CAGGCAGGGCCCTTGGGCCACCAC-3′-16355′-GCAATTCACCCCAAGAGCCAGGCC-3′-15605′-GCCCCTGGACAAGAGGCATAAG-3′-15335′-CCCACTCATCCTTGCCCTGCCCC-3′-13245′-CATGCAGCAGGCAAGAGGGCCAGG-3′-12915′-CCTGACAGCTTGCTGGCCCTG-3′-12675′-GTCCAGATTCTTGTTTGTTGGCTG-3′-11125′-CTTTCTGCTTCTTGGCTCTGGGTC-3′-10275′-CTCAGCCCTGCTTGTCTCACTGTC-3′-9435′-CTTTCTCCTTGTCTGTCTCTTCTC-3′-7935′-GTGTGACTGCCTTGAGAGGAAAG-3′-7245′-CAAATCGCTCTTGAGAAGTTTGGG-3′-5165′-CGGGAGGACTTGACTGCCCCTCTG-3′-2735′-GTGGGGCCAGCTTGGAAGAATTTC-3′> Open table in a new tab To determine endogenous binding activity of nuclear extracts from the choroid plexus to the TTF-1 binding domains in the 5′-flanking region of the AQP-1 gene, nuclear protein fractions from the rat choroid plexus were prepared according to the method of Andrews and Faller (21Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar), utilizing the mixture of protease inhibitors recommended by Kuhn et al. (22Kuhn R. Monuki E.S. Lemke G. Mol. Cell. Biol. 1991; 11: 4642-4650Crossref PubMed Scopus (51) Google Scholar). The binding assay was performed as described previously (17Son Y.J. Hur M.K. Ryu B.J. Park S.K. Damante G. D'Elia A.V. Costa M.E. Ojeda S.R. Lee B.J. J. Biol. Chem. 2003; 278: 27043-27052Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), using 15 μg of protein and 20,000 cpm of probe (-724 in Table 1). To further confirm the presence of immunoreactive TTF-1 in the nuclear extracts, the proteins were incubated with 1 μl of undiluted TTF-1 antiserum (NeoMarkers, Fremont, CA) or preimmune serum for 30 min at room temperature before initiating the binding reactions. Intracerebroventricular Administration of AS TTF-1 ODN—To determine the effect of blocking TTF-1 expression on AQP-1 synthesis and formation of CSF, a phosphorothioate AS TTF-1 ODN (GenoTech Corp., Daejeon, Korea) was delivered into the lateral ventricle of adult male rats. The AS TTF-1 ODN used to disrupt TTF-1 synthesis (5′-GAC TCA TCG ACA TGA TTC GGC GTC-3′) was directed against the sequence surrounding the first ATG codon of TTF-1 mRNA as reported previously (17Son Y.J. Hur M.K. Ryu B.J. Park S.K. Damante G. D'Elia A.V. Costa M.E. Ojeda S.R. Lee B.J. J. Biol. Chem. 2003; 278: 27043-27052Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 18Kim M.S. Hur M.K. Son Y.J. Park J.I. Chun S.Y. D'Elia A.V. Damante G. Cho S. Kim K. Lee B.J. J. Biol. Chem. 2002; 277: 36863-36871Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). As a control, a scrambled (SCR) sequence of identical base composition was used (5′-AGT CCT ACT CGG TAC GTA TGC AGC-3′). For the intracerebroventricular injection, ODNs were diluted to a final concentration of 1 nmol/μl in artificial cerebrospinal fluid (in mm, 126 NaCl, 2.5 KCl, 1.24 NaH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3, 10 d-glucose, pH 7.3, and bubbled with 95% air, 5% CO2). Injections were performed under pentobarbital (7.5 mg/kg body weight) and ketamine hydrochloride (25 mg/kg body weight) anesthesia. A polyethylene cannula (outer diameter of 1.05 mm and inner diameter of 0.35 mm) containing an inner stylet was stereotaxically implanted into the brain with its opening protruding from the roof of the lateral ventricle (coordinates were as follows: antero-posterior = 1.0 mm caudal to the bregma; ventral = 3.6 mm from the dura mater; lateral = 0.16 mm from the mid-line). After a week of recovery, the inner stylet was removed, and the ODNs (4 μl) were injected twice with a Hamilton syringe at a 12-h interval. The animals were sacrificed 12 h after the second ODN administration, and total RNA and proteins were extracted from the choroid plexus of the lateral ventricle. Changes in CSF secretion were also measured in other animals 12 h after the second ODN injection. Western Blots—Choroid plexus tissue from SCR or AS ODN-treated animals was homogenized in lysis buffer (T-PER tissue protein extract reagent; Pierce) containing a protease inhibitor mixture (1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 3 mm aprotinin) and 1 mm sodium orthovanadate, pH 6.8. Electrophoretically separated polypeptides were transferred onto a nitrocellulose membrane at 20 mA for 16 h using transfer buffer (25 mm Tris, 192 mm glycine, and 20% methanol, pH 8.3). The nitrocellulose membrane was blocked with 1% bovine serum albumin in PBS. After incubation with a monoclonal mouse TTF-1 antibody (1:2000) (NeoMarkers) or a polyclonal goat AQP-1 antibody (1:5000) (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h, bound antibody was detected with an ECL detection kit (Amersham Biosciences), according to the manufacturer’s recommended protocol. The membranes were exposed to x-ray film for 5 min in a dark room. Real Time PCR—Total RNA was isolated from the rat choroid plexus using TRIzol reagent (Sigma). Total RNA (2 μg) was reverse-transcribed and amplified by a real time PCR using two sets of primers as follows: AQP-1 sense primer, 5′-CATGTATATCATCGCGGAGT-3′, and antisense primer, 5′-CACAGCCAGTGTAGTCAATG-3′; glyceraldehyde-3-phosphate dehydrogenase sense primer, 5′-TGTGAACGGATTTGGCCGTA-3′, and antisense primer, 5′-ACTTGCCGTGGGTAGAGTCA-3′. Real time PCRs (20-μl total volume, containing 5 pmol of primer, 10 μl of SYBR green dye (Qiagen, Valencia, CA), and 2 μl of cDNA) were carried out in capillaries of a DNA Engine Opticon continuous fluorescence detection system (MJ Research Inc., Waltham, MA) for ∼40 cycles. Assays for CSF Production—CSF production was measured by the ventriculocisternal perfusion method as described previously (23Davson H. Hollingsworth J.G. Carey M.B. Fenstermacher J.D. J. Neurobiol. 1982; 13: 293-318Crossref PubMed Scopus (50) Google Scholar). Animals who were intracerebroventricular-injected with ODNs were anesthetized with pentobarbital (7.5 mg/kg body weight) and ketamine hydrochloride (25 mg/kg body weight), placed on a stereotaxic device, and subjected to catheterization of the lateral ventricle and the cisterna magna. Lateral ventricles of the brain were perfused with artificial cerebrospinal fluid containing 1 mg/ml fluorescence isothiocyanate (FITC)-dextran (Mr 70,000) as a volume marker (7Papadopoulos M.C. Verkman A.S. J. Biol. Chem. 2005; 280: 13906-13912Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar) at 4 μl min-1 using a slow drive syringe pump (KDS 100, kdScientific, Holliston, MA). CSF samples from the cisterna magna were collected at 10-min intervals for 90 min. Fluorescence from the collected CSF samples was measured in a fluorescence spectrophotometer (Wallac Victor 1420 multilabel counter; EG & G Wallac, Turku, Finland). The rate of CSF secretion was calculated as described previously (11Oshio K. Watanabe H. Song Y. Verkman A.S. Manley G.T. FASEB J. 2005; 19: 76-78Crossref PubMed Scopus (340) Google Scholar): CSF secretion rate (μl min-1) = Fin(Din - Dout)/Dout. Din is the fluorescence of FITC-dextran in the in flow solution and Dout is fluorescence of FITC-dextran in the outflow solution. Fin is the rate of artificial cerebrospinal fluid perfusion (4 μl min-1). Measurements of Survival Rate of Animals and Brain Water Content after Acute Water Intoxication—To determine the effect of TTF-1 AS ODN on the survival rate of animals subjected to acute water intoxication, rats were given distilled water containing 1-deamino-8-d-arginine vasopressin (dDAVP, 0.4 μg/kg body weight; Sigma) equal to a 25% of body weight by intraperitoneal injection, as reported previously (24Manley G.T. Fujimura M. Ma T. Noshita N. Filiz F. Bollen A.W. Chan P. Verkman A.S. Nat. Med. 2000; 6: 159-163Crossref PubMed Scopus (1303) Google Scholar), 12 h after the second ODN injection. Beginning immediately after water injection, the survival rate of animals was monitored for 24 h. To measure brain water content (11Oshio K. Watanabe H. Song Y. Verkman A.S. Manley G.T. FASEB J. 2005; 19: 76-78Crossref PubMed Scopus (340) Google Scholar), some animals were sacrificed 1 h after water injection, and their brains were weighed immediately and dried in a vacuum oven at 105 °C for 24 h. The dried brains were weighed again, and % brain water content was calculated as (wet weight - dry weight) × 100/wet weight. Statistics—The differences between several groups were analyzed by analysis of variance with Dunnett’s multiple comparison test. Student’s t test was used for the comparison of two groups. TTF-1 and AQP-1 mRNA Are Coexpressed in the Rat Choroid Plexus—The choroid plexus is a tissue in the ventricle regions of the brain with branched structures made up of numerous villi that are composed of a single layer of epithelial cells overlying a core of connective tissue and blood capillaries (1Brown P.D. Davies S.L. Speake T. Millar I.D. Neuroscience. 2004; 129: 957-970Crossref PubMed Scopus (320) Google Scholar). Previous reports have shown that AQP-1 is exclusively expressed in the apical membrane of the choroid plexus epithelium (25Nielsen S. Smith B.L. Christensen E.I. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7275-7279Crossref PubMed Scopus (654) Google Scholar). We used FISH to determine the localization of TTF-1 and AQP-1 mRNAs in the choroid plexus. FISH showed that TTF-1 mRNA was expressed in epithelial cells of the lateral ventricular choroid plexus (Fig. 1, A and C, red), and as expected, AQP-1 was exclusively expressed in choroid plexus epithelial cells (Fig. 1, B and D, green). Double FISH demonstrated that TTF-1 mRNA was present in all AQP-1 mRNA-containing cells of the choroid plexus (Fig. 1, E and F, yellow). The donut-shaped fluorescence staining strongly revealed that both mRNA signals were present only in the cytoplasm surrounding cellular nuclei stained with Hoechst 33258 (Fig. 1, C and D). We also confirmed the colocalization of TTF-1 and AQP-1 mRNAs in the choroid plexus by classical double in situ hybridization using DIG-labeled TTF-1 cRNA and 35S-UTP-labeled AQP-1 cRNA probes (data not shown). In control experiments, hybridization with DIG-labeled TTF-1 sense RNA or fluorescein-labeled AQP-1 sense RNA probes revealed absence of specific signals detected in the choroid plexus (data not shown). TTF-1 Transactivates the AQP-1 Promoter—The coexpression of TTF-1 and AQP-1 suggested that the former may control transcription of the AQP-1 gene. Thus, we evaluated whether TTF-1 was able to activate the AQP-1 promoter. In this study, we used the 5′-flanking region of the human AQP-1 gene, as no confirmed promoter sequence for mouse or rat AQP-1 has been reported to date. We also used a rat TTF-1 expression vector to transfect either mouse BAS8.1 glial progenitor cells or rat glioma C6 cells, both of which express AQP-1 mRNA detected by RT-PCR (data not shown). Rat TTF-1 is expected" @default.
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• W2049226316 title "Thyroid Transcription Factor-1 Facilitates Cerebrospinal Fluid Formation by Regulating Aquaporin-1 Synthesis in the Brain" @default.
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