From the
Department of Biological Sciences,
College of Natural Sciences, University of Ulsan, Ulsan 680-749, South Korea,
the ¶Department of Pediatrics, College of
Medicine, University of Ulsan, Ulsan 682-060, South Korea, the
||Department of Biomedical Sciences and
Technologies, University of Udine, 33100 Udine, Italy, and the
Division of Neuroscience, Oregon
National Primate Research Center/Oregon Health and Science University,
Beaverton, Oregon 97006
Received for publication, March 27, 2003 , and in revised form, April 29, 2003.
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ABSTRACT |
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INTRODUCTION |
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Given the importance of AoGen in the pathogenesis of ANG II-dependent hypertension (10), it is critically important to acquire a more complete understanding of the mechanisms regulating central AoGen gene expression. The genomic structure of the AoGen gene, including its 5'-flanking region, has been characterized (1113), and recently AoGen-activating elements in the proximal promoter region of the AoGen gene were found to be essential for the regulation of AoGen gene expression in the liver (14) and for the AoGen-dependent control of blood pressure by both the liver (15) and brain (16). Nevertheless, the identity of the binding protein(s) recognized by these AoGen-activating elements remains unknown.
Here we report that the homeodomain-containing transcription factor, TTF-1, regulates AoGen gene transcription in the SFO. TTF-1 was first found in the thyroid gland (17, 18) and then shown to also be expressed in the embryonic diencephalon and lung (19). Recently, we and others reported that TTF-1 remains expressed in discrete regions of the postnatal rat brain (20, 21). In the hypothalamus, TTF-1 is present in sets of neurons containing luteinizing hormone-releasing hormone, enkephalin, and pituitary adenylate cyclase-activating polypeptide (20, 22), in addition to ependymoglial cells lining the third ventricle and astrocytic tanycytes of the median eminence, where it is coexpressed with the epidermal growth factor-related receptor ErbB2 (20). We also showed that TTF-1 binds to specific recognition motifs present in each one of these promoters to regulate transcriptional activity (20, 22).
In the present report, we identify the SFO as an unexpected site of TTF-1 expression in the brain. Because the SFO is a prominent site of AoGen synthesis, we sought to determine whether there is a functional, hierarchical relationship between TTF-1 and AoGen. The results of this study demonstrate that both mRNAs are indeed expressed by the same cells of the SFO, that TTF-1 regulates AoGen transcription via binding to a specific binding motif in the 5'-flanking region of the AoGen gene, and that inhibition of TTF-1 synthesis by antisense oligodeoxynucleotide (ODN) administration decreases AoGen synthesis and leads to a decrease in water intake and an increase in urine excretion.
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EXPERIMENTAL PROCEDURES |
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Double in Situ HybridizationTo localize TTF-1 and AoGen mRNAs in the SFO, the brains of four rats were fixed by transcardiac perfusion of 4% parformaldehyde-borate buffer, pH 9.5, as recommended (23). After an overnight postfixation in the same fixative containing 10% sucrose, the brains were blocked and frozen at 85 °C until use. Thirty-µm sections were then prepared with a freezing sliding microtome and processed for hybridization as reported (24). The hybridization procedure was that recommended by Simmons et al. (23), as described earlier by us (24), using a [35S]UTP-labeled TTF-1 cRNA probe (20) and a digoxygenin-UTP-labeled AoGen cRNA. The latter was transcribed from a 205-bp AoGen cDNA template generated by PCR amplification (see below for additional details). Following an overnight hybridization at 45 °C, the slides were washed and processed for digoxygenin detection as described (24). After dehydration, the slides were dipped in Ilford K5 emulsion (without defatting) instead of the NTB-2 emulsion used for isotopic hybridization and were exposed to the emulsion for 3 weeks at 4 °C. At this time, the slides were developed, quickly dehydrated, dried, and coverslipped for microscopic examination.
Combined Immunohistochemistry-in Situ HybridizationTo determine if TTF-1 is expressed in neurons or glial cells of the SFO, these cell types were identified by immunohistochemistry, and the presence of TTF-1 mRNA was assessed using the [35S]UTP-labeled TTF-1 mRNA described above. Neurons were identified using a monoclonal antibody against NeuN, a neuron-specific nuclear protein (25). This antibody stains the nuclei of most neurons of the central and peripheral nervous system, with the exception of some subsets, such as cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells (25). The antibody was purchased from Chemicon (Temecula, CA) and used at a 1:200 dilution. Astroglial cells were visualized with rabbit polyclonal antibodies against glial fibrillary acidic protein (GFAP) obtained from Dako (Carpinteria, CA) and used at a 1:500 dilution. The brains were fixed and sectioned as outlined above and then subjected to immunohistochemistry using a procedure described elsewhere (26, 27). A modification to the method was the addition of a ribonuclease inhibitor to the immunoreactions. The ribonuclease inhibitor SUPERaseIn (Ambion, Austin, TX) was added (at 100 units/ml) to the solutions containing both the primary and secondary antibodies. Prior detection of ribonuclease activity in the different solutions used for immunohistochemistry using a fluorometric RNase detection assay (RNaseAlertTM Lab Test kit, Ambion) demonstrated that only these two solutions had detectable ribonuclease levels. Upon completion of the immunohistochemical reaction, the sections were mounted on glass slides and were dried overnight in a vacuum oven. The next day they were hybridized with the TTF-1 cRNA. Control sections were incubated with a sense TTF-1 probe transcribed from the same plasmid but linearized on its 3'-end to transcribe the coding strand of TTF-1. Following posthybridization washes, the sections were exposed to NTB-2 emulsion for 3 weeks. At this time, the reaction was developed, and the sections were counterstained with 1% methyl green before microscopic examination.
RNA Extraction and RNase Protection Assay (RPA)RPA was used to quantitate changes in TTF-1 and AoGen mRNA abundance in the SFO and arginine vasopressin (AVP) mRNA in the supraoptic nucleus (SON). Total RNA was isolated as reported (28) and subjected to RPA using the procedure previously described by Ma et al. (29) with some modifications (30). The preparation of antisense RNA probes for TTF-1, AoGen, AVP, and cyclophilin followed a procedure described in detail elsewhere (20, 31, 32). RNA samples (5 µg/tube) or different amounts of in vitro synthesized TTF-1 and AoGen sense RNA (0.061 pg/tube) were hybridized with 500,000 cpm of 32P-labeled TTF-1, AoGen, and AVP cRNA probes, respectively, for 1820 h at 45 °C. The tissue RNA samples were simultaneously hybridized to a cyclophilin cRNA probe (5000 cpm/tube) to correct for procedural losses. At the completion of hybridization, the samples were treated with ribonucleases A and T1 to digest unhybridized RNA species. The protected cRNA fragments were separated on a polyacrylamide-urea gel (5% acrylamide, 7 M urea), and the hybridization signals were detected by exposure of dried gels to X-Omat x-ray film (Eastman Kodak Co.). The intensity of the signals obtained was quantified as reported (29, 30).
PCR Cloning of an AoGen cDNA Fragment and 5'-Flanking Region of the AoGen GeneA cDNA fragment of AoGen was cloned by reverse transcription-PCR from rat liver RNA. The specific primer sets (sense primer, 5'-CCT GAA GGC CAC CAT CTT CT-3'; antisense primer, 5'-CAG GGT CTT CTC ATC CAC G-3') were designed to generate a 205-bp AoGen cDNA fragment corresponding to nucleotides 74278 in rat AoGen mRNA (NCBI GenBankTM data base, accession number L00091 [GenBank] ). A rat AVP cDNA fragment was also cloned by reverse transcription-PCR from the rat hypothalamic RNA. The specific primer sets (sense primer, 5'-GCA AGG CTT CTG GCC AGA CT-3'; antisense primer, 5'-CGC CAT GAT GCT CAA CAC TA-3') were designed to generate a 283-bp AVP cDNA fragment corresponding to nucleotides 26308 in rat AVP mRNA (NCBI GenBankTM data base, accession number M25646 [GenBank] ).
The proximal promoter of the rat AoGen gene (689 to +49 bp) to be employed for promoter analysis was also cloned by PCR from rat liver DNA using the sequence information deposited in the NCBI GenBankTM data base (accession number M12113 [GenBank] ). The sense primer was 5'-GGA TCC ACC CGT CTC ATT CTC-3', and the antisense primer was 5'-CAA GAG GGC TCT GCT TAC CTT-3'. The resulting DNA fragment was inserted into the luciferase reporter plasmid (pGL-3 basic; Promega, Madison, WI), and its sequence was confirmed by DNA sequencing. Mutant AoGen promoter constructs carrying deletions of the TTF-1 binding motifs were generated using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions; the intended mutations were confirmed by sequencing.
Cell Culture and Assays for Luciferase ActivityAoGen
promoter analysis was performed in C6 glioma cells, because they endogenously
express AoGen (see Supplementary Fig. 1). C6 cells were grown in Dulbecco's
modified Eagle's medium/F-12 containing 10% fetal bovine serum. Twenty-four h
after seeding the cells in six-well plates, they were transiently transfected
with the AoGen promoter-luciferase reporter construct (AoGen-P) using
LipofectAMINE (Invitrogen) along with different concentrations (100500
ng/well) of the expression vector, pcDNA 3.1-zeo (Invitrogen), containing the
TTF-1 coding region (TTF-1-pcDNA)
(20,
22). Transfection efficiency
was normalized by co-transfecting the -galactosidase reporter plasmid
(pCMV-
-gal; Clontech, Palo Alto, CA) at 20 ng/ml. The transfected cells
were harvested 48 h after transfection for luciferase and
-galactosidase
assays as reported (20).
Electrophoretic Mobility Shift Assay (EMSA)The procedure
for expression and purification of the TTF-1 homeodomain (TTF-1HD) protein has
been described (33).
Double-stranded oligodeoxynucleotides, labeled with 32P at the
5' terminus, were used as probes for the EMSA. Sequences of the
oligodeoxynucleotides used are shown in
Table I. Oligodeoxynucleotides
C and C were used as a positive and negative control, respectively
(34). EMSA was performed as
previously described (22,
33,
34). Electrophoretically
separated signals corresponding to the protein-bound and free DNA were
quantitated with Multi-analyst software. Binding of TTF-1HD to
oligodeoxynucleotides representing different regions of the AoGen 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
(17).
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To determine endogenous binding activity of nuclear extracts from the SFO to the TTF-1 binding domains in the 5'-flanking region of AoGen gene, nuclear protein fractions from the rat SFO were prepared according to the method of Andrews and Faller (35), utilizing the mixture of protease inhibitors recommended by Kuhn et al. (36). The binding assay was performed as previously described (20), using 15 µg of protein and 20,000 cpm of probe. 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.
Intracerebral Administration of Antisense (AS) TTF-1 ODN and Assessment of Water Intake and Urine OutputTo determine whether TTF-1 controls AoGen synthesis in vivo and hence has a physiological role in the regulation of water intake and urine excretion, a phosphorothioate AS TTF-1 ODN (GenoTech Corp., Daejeon, Korea) was delivered in the vicinity of the SFO. 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 previously reported (22). 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 intracerebral injection, the ODNs were diluted to a final concentration of 100 ng/µl artificial cerebrospinal fluid (22). Under pentobarbital (7.5 mg/kg body weight) and ketamine hydrochloride (25 mg/kg body weight) anesthesia, a polyethylene cannula (outer diameter, 1.05 mm; inner diameter, 0.35 mm) was stereotaxically implanted into the brain with its opening protruding onto the top of the SFO (coordinates: AP = 1.3 mm caudal to the bregma; V = 4.3 mm from the dura mater; L = 0.0 mm from the midsagittal line) as recommended (37). After a week of recovery, the ODNs (2 nmol in 4 µl of artificial cerebrospinal fluid) were injected with a Hamilton syringe, and water intake, urine excretion, and body weight were measured. One day following a single ODN administration, total RNA or nuclear proteins were extracted from the SFO to determine AoGen mRNA and TTF-1 protein content. To determine the effect of AS TTF-1 ODN on renin-induced water intake, renin (1 milliunit/2 µl) was injected 4 h after AS ODN injection through the same cannula, and water intake was determined for 1 h, beginning 1 h after the renin injection.
Western Blot Analysis of TTF-1 ProteinNuclear protein
extracts were prepared according to the method of Andrews and Faller
(35), and Western blots using
TTF-1 antiserum were performed as reported
(22). The proteins were
detected with an ECL kit (Amersham Biosciences) according to the protocol
provided by the manufacturer; membranes were exposed to x-ray film for 3 min.
Standardization of applied protein concentration was done by Western blotting
of -tubulin with specific antibody (Sigma).
Determination of Plasma AVP and ANG IIPlasma AVP and ANG II levels were determined by radioimmunoassay using radioimmunoassay kits (AVP kit from Diasorin Inc., Stillwater, MN; ANG II kit from Mitsubishi Kagaku Bio-Clinical Laboratories Inc., Tokyo, Japan) and the procedure provided by each manufacturer. Blood samples were collected in ice-cold tubes containing EDTA 13 h after ODN injection, and the plasma was separated by centrifugation before the assays. The resulting values are expressed as pg/ml.
StatisticsChanges in water intake and urine excretion were analyzed by a two-way analysis of variance followed by the Student Neuman-Keuls multiple comparison test. Changes observed between two groups were analyzed by Student's t test.
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RESULTS |
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TTF-1 mRNA Is Present in Neurons and Nonneuronal, GFAP-negative Cells of the SFOTo determine whether the TTF-1 gene is expressed in neurons or glial cells of the SFO, we performed combined immunohistochemistry-in situ hybridization experiments. As shown in Fig. 2, most (if not all) GFAP-positive cells detected with the polyclonal antibodies used were confined to the internal portion of the SFO in a region that separates the SFO from the ventral hippocampal commissure and had no detectable TTF-1 signal (Fig. 2, AC). In contrast, neurons identified by the presence of NeuN immunoreactivity were mostly located in the central portion of the SFO (Fig. 2D), and some of them were TTF-1 mRNA-positive (Fig. 2, F and G). Surprisingly, most TTF-1 mRNA-positive cells contained neither NeuN nor GFAP immunoreactivity (Fig. 2, CG), suggesting that they are tanycytes and/or astrocytic tanycytes known to make up the majority of cells present in circumventricular organs such as the SFO (38), the median eminence of the hypothalamus (39), and the subcommissural organ (40). Although these cells are considered to be glial, astrocytic tanycytes (39) are for the most part GFAP-negative (40, 41). This cellular distribution is shown at a higher magnification in Fig. 2G. Sections incubated with the sense TTF-1 RNA probe did not show specific hybridization signals (Fig. 2, H and I).
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Both TTF-1 and AoGen mRNA Content in the SFO Increased after Water DeprivationIn agreement with the hybridization histochemistry results, both TTF-1 and AoGen mRNAs, simultaneously detected by RPA (Fig. 3A), were present in the SFO (Fig. 3B). Water deprivation significantly increased both mRNA levels compared with the euhydrated control group (Fig. 3C), as assessed after 48 h of water deprivation. In contrast to the relatively small (but statistically significant) change found in TTF-1 mRNA levels, the content of TTF-1 protein was greatly increased by water deprivation (Fig. 3D). In harmony with these changes, water-deprived rats showed a significant increase in plasma osmolality and a decrease in body weight compared with control animals (Table II).
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Identification of Putative TTF-1 Binding Motifs in the Rat AoGen PromoterTo find potential TTF-1 binding motifs in the 5'-flanking region of the rat AoGen gene, the promoter sequence generated by PCR amplification (see above) was analyzed using a search program provided on the World Wide Web at molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html. Several putative consensus motifs for transcription factors were detected (Fig. 4), including binding sites for CCAAT-box/enhancer-binding protein (C/EBP), cAMP-response element binding protein, GATA-1, and glucocorticoid receptor. Based on the sequence identity with reported conserved motifs of TTF-1 binding domains (42), seven putative TTF-1 binding domains were detected (Fig. 4, double underline).
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TTF-1 Transactivates AoGen Promoter ActivityFunctional analysis of the AoGen promoter demonstrated that this promoter is transcriptionally active over a wide range of concentrations in C6 cells (Fig. 5A). Cotransfection with different concentrations of a TTF-1 expression vector resulted in a dose-dependent increase in AoGen promoter activity (Fig. 5B). Endogenous expression of AoGen mRNA in C6 cells was also increased by expressing TTF-1 (Supplementary Fig. 1).
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TTF-1 Binds to TTF-1 Putative Binding Domains in the 5'-Flanking Region of AoGen GeneEMSAs were performed to determine the ability of TTF-1HD to recognize the putative TTF-1 binding domains present in the AoGen promoter. Double-stranded oligodeoxynucleotide probes (Table I), containing the presumptive TTF-1 binding motifs and their flanking sequences shown in Fig. 4, were employed. Of the seven putative binding motifs, four were recognized by TTF-1HD (Fig. 6A). The site at 643 showed the strongest signal, whereas the sites at 446, 125, and 14 had relatively weak signals. The sites at 591, 329, and 135 showed no binding activity and thus cannot be considered as TTF-1 binding domains.
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Nuclear proteins from the rat SFO strongly bound the oligonucleotide probes
(446, 125, and 14)
(Fig. 6B) that
contained the binding motifs used by TTF-1 to transactivate the AoGen promoter
(see below) (Fig. 7). The
interaction of the labeled probes with SFO nuclear proteins was reduced by the
addition of a 50-fold excess of unlabeled oligonucleotide C. In contrast, an
oligonucleotide carrying a mutated core TTF-1-binding sequence (C) was
ineffective. Preincubation of SFO nuclear proteins with a TTF-1 antibody
delayed the migration of the protein-DNA complex, indicating that TTF-1 is
indeed part of this complex.
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Loss of AoGen Transactivation by TTF-1 after Deletion of the TTF-1 Binding Core Motif at 125To determine whether the sites at 643, 446, 125, and 14, recognized by TTF-1HD in EMSA assays, are required for TTF-1 to transactivate the AoGen promoter, we deleted each of them by site-directed mutagenesis and examined the ability of TTF-1 to transactivate the mutated promoters. As shown in Fig. 7, TTF-1-dependent transactivation of the AoGen promoter appeared to mostly require the core motif at 125, since deletion of this site resulted in an almost complete loss of TTF-1-dependent transactivation. Whereas deletion of either the 446 or 14 sites partially diminished TTF-1-induced AoGen promoter activity, no decrease was found after deletion of the 643 site. A triple deletion, including the 125 site, decreased TTF-1 transactivational activity to a level similar to that caused by the deletion of the 125 site alone. Deletion of all four TTF-1 binding domains completely abolished TTF-1-dependent activation of the AoGen promoter.
Effect of AS TTF-1 ODN on AoGen mRNA Content in the SFOTo
determine whether an in vivo decrease in TTF-1 availability results
in a diminished steady-state level of AoGen mRNA, a single injection of AS
TTF-1 ODN or its corresponding scrambled DNA sequence was administered
intracerebrally, targeting the SFO region of 2-month-old male rats. One day
after the injection, SFOs were collected for measurement of TTF-1 protein and
AoGen mRNA. As shown in Fig.
8A, the TTF-1 antibody detected a 40-kDa band
similar in size to TTF-1, as previously reported for the rat hypothalamus
(22). TTF-1 AS ODN-treated
animals showed a significant decrease in TTF-1 protein content as compared
with both sham-operated controls and SCR ODN-injected animals
(Fig. 8, A and
B). Importantly, AoGen mRNA levels were significantly
decreased in the SFO of AS TTF-1 ODN-injected rats, as determined by RPA
(Fig. 8C). However, no
change in AoGen mRNA level was observed in the PVA
(Fig. 8D). Thus, in
keeping with its transactivational activity in vitro, TTF-1 is
required for maintaining AoGen expression specifically in the SFO in
vivo.
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Effect of AS TTF-1 ODN on Water Intake and Urinary ExcretionTo determine whether the decrease in AoGen gene expression, a result of TTF-1 synthesis inhibition in the SFO, has physiological consequences, we measured water intake and urinary excretion as indices of body fluid homeostasis. Daily water intake was measured every day for 4 days, beginning 2 days before the ODN injection. As shown in Fig. 9, water consumption remained steady until the time of injection. Twenty-four h after the AS TTF-1 ODN injection, there was a dramatic reduction in daily water intake (Fig. 9A). To further characterize this effect, a detailed profile of water intake after ODN injection, assessed as cumulative water intake, was obtained at 3-h intervals for 24 h. In keeping with the reduction in daily water intake, the AS TTF-1 ODN injection decreased cumulative water intake compared with controls (Fig. 9B). Significant differences between the AS ODN-injected and two control groups began 10 h after ODN injection and persisted for the remainder of the 24-h period studied.
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Renin, an enzyme that converts AoGen to ANG I, is a potent dipsogen that increases water intake within 1 h of its systemic administration. The renin-induced increase in water intake was partially suppressed by the administration of AS TTF-1 ODN 4 h before renin injection (Fig. 9C).
Urinary volume, determined in a metabolic cage at daily intervals, was significantly increased 1 day after the injection of AS TTF-1 ODN (Fig. 10A). In contrast, no change in body weight occurred during this period (Fig. 10B).
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Effect of AS TTF-1 ODN on Plasma AVP and ANG II LevelsBlood samples were collected 13 h after the ODN injection (i.e. at the time when cumulative water intake begins to decrease in response to the AS ODN against TTF-1 mRNA) (Fig. 9B, open arrow). Whereas plasma AVP levels declined after the AS ODN injection (Fig. 11A), plasma ANG II titers increased (Fig. 11B). The AS ODN treatment also induced a significant decrease in AVP mRNA content in the SON (Fig. 11, C and D).
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DISCUSSION |
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The existence of a hierarchical relationship between TTF-1 and AoGen was initially suggested by our identification of consensus TTF-1 binding motifs in the 5'-flanking region of the AoGen gene. These binding motifs are intermingled with canonical binding sites for transcription factors already known to control AoGen gene expression such as glucocorticoid receptor, cAMP-response element-binding protein, and C/EBP. Perhaps the most important regulators of AoGen synthesis are C/EBP-related transcription factors. Several members of the C/EBP family have been shown to regulate AoGen gene expression in liver (4345). Some of them are important for the liver-specific expression of AoGen (46, 47), and especially DBP, a liver-enriched transcription factor, binds to the proximal AoGen promoter and increases its activity (46). A recent report that shows that a subpopulation of African-Americans at high risk for hypertension have a point mutation of the C/EBP binding domain in the AoGen promoter provides genetic evidence for its critical role in the regulation of AoGen synthesis (48).
Although the 5'-flanking region of the AoGen gene has seven putative TTF-1 binding domains, identified as such because of their similarity to the canonical TTF-1 binding motif present in TTF-1 target genes (33, 42, 49), only four of them recognize the TTF-1 homeodomain protein. Surprisingly, the binding affinity of these sites for TTF-1HD does not correlate well with their biological activity. Whereas the site at 643 showed the strongest binding activity, deletion of the site at 125, which had only 23% TTF-1HD binding activity as compared with the canonical TTF-1 binding site in the thyroglobulin promoter (oligomer C), was sufficient to prevent TTF-1 transactivation of the AoGen promoter. In contrast, mutation of the 643 site had no effect. This could be due to differences in binding and transcription activity in an in vivo three-dimensional DNA structure and the in vitro binding between DNA and protein in an EMSA and/or to the requirement, for full binding activity, of protein domains outside the homeodomain. Regardless of the explanation for this discrepancy, the results are not without precedent, since domains with relatively weaker binding activities have been reported to be important in the in vivo action of homeodomain-containing proteins (50). Our results also show that in addition to the site at 125, binding domains at 446 and 14 are important but not critical for the transactivation of AoGen by TTF-1. Deletions of the core motifs at 446 and 14 resulted in less AoGen promoter transactivation by TTF-1, but significant activity still remained. Of interest in this context is the presence of putative GATA-1 binding sites near those of TTF-1 at 446 and 125. Recent data showed that GATA-6 (51, 52) and GATA-4 (51) directly interact with TTF-1 to cooperatively transactivate TTF-1 target genes. It is possible that a similar interaction may contribute to the transcriptional regulation of AoGen expression by TTF-1 in the SFO. Further study will be necessary to clarify this potential interaction.
Cells containing TTF-1 mRNA were not observed in the PVA, whereas many AoGen-positive cells were detected in this region. The absence of TTF-1 mRNA in these cells suggests that TTF-1-dependent regulation of AoGen expression is limited to the SFO. The present histochemical data show that most TTF-1 mRNA-containing cells are located in the central portion of the SFO, a region that has few, if any, GFAP-positive cells but is composed by a majority of cells immunonegative for both GFAP and the neuronal marker NeuN. Because this region of the SFO is mostly composed of modified ependymoglial cells (38), which in other CVOs are not recognizable by GFAP staining (40, 41), it is likely that they represent a major site of TTF-1 expression. The median eminence, another ventricular organ, also contains numerous ependymoglial cells of the astrocytic tanycyte type (39), which for the most part are GFAP-negative (41). Thus, the present results indicate that TTF-1 and its target gene AoGen are mainly expressed in ependymoglial cells and neurons of the SFO.
Water deprivation leads to an up-regulation of ANG II receptor binding and AT1A receptor gene expression (5, 53) as well as an increase in steady state AoGen mRNA levels in the rat SFO (5). The simultaneous increase in TTF-1 mRNA and protein and AoGen mRNA observed in the SFO after 2 days of water deprivation suggests that dehydration up-regulates AoGen synthesis in the SFO via activation of TTF-1 production. This interpretation is supported by the results of blocking TTF-1 synthesis via in vivo administration of antisense oligodeoxynucleotides. This treatment resulted in two key alterations in body fluid homeostasis: inhibition of water intake and increased urine excretion (the latter reflecting a decrease in renal water reabsorption). These two changes represent the major homeostatic responses to hypervolemia and hypoosmolality and are regulated by ANG II secreted from both the SFO and peripheral sources (1).
The SFO seems to be mainly responsible for increasing water ingestion in response to circulating ANG II and other dipsogenic inputs, since destruction of the SFO results in the loss of drinking in response to intravenous ANG II (54, 55). Destruction of the SFO or infusion of an ANG II antagonist into the SFO also prevented water consumption in response to adrenergic activation (56). Because some angiotensin-sensitive structures involved in water drinking behavior lie inside the blood-brain barrier and cannot be directly reached by circulating ANG II (1), SFO-intrinsic angiotensins appear to play an important role in the regulation of these structures. In fact, inhibition of brain ANG II synthesis via an intracerebral injection of AS AoGen ODN (57) results in a decreased water intake, as predicted by the concept that brain ANG II is important for the regulation of water consumption. In addition to showing that blockade of TTF-1 synthesis targeted to the SFO decreases water intake, our results also show that AS TTF-1 ODN significantly decreased the renin (a potent dipsogen)-induced increase of water intake. Thus, AoGen synthesized in the SFO under the facilitatory control of TTF-1 may serve as a renin substrate and play an important role in the central regulation of water drinking behavior.
AVP-secreting magnocellular neurons terminate in the posterior pituitary, where AVP, the major hormone required for water conservation through its antidiuretic effect, is stored and released into the bloodstream in response to increases in osmolality or hypovolemia (58). Peripheral administration of ANG II can stimulate AVP release by acting on CVOs such as the SFO or directly on the pituitary; intracerebral injection of ANG II can also cause AVP release (59). Ablation of the SFO abolished the stimulatory effect of peripherally administered ANG II on AVP release (60, 61), indicating that the central mechanism used by ANG II to stimulate AVP release requires the SFO. By showing that TTF-1 synthesis blockade by AS ODN injection reduces AoGen expression in the SFO, decreases the AVP mRNA content of the SON, and reduces circulating plasma AVP levels, while increasing urine excretion, the present results suggest that AoGen production is required for the SFO-dependent regulation of AVP synthesis and secretion. Accordingly, AoGen produced in the SFO appears to be an important regulator of AVP-dependent renal water retention.
Surprisingly, in contrast to the decreased AVP level, plasma ANG II level was significantly increased by the AS TTF-1 ODN injection. This increase may be due to a homeostatic response coping with a transient hypovolemia induced by decreases both in drinking and renal reabsorption. Hypovolemia is sensed by vascular stretch receptor and then triggers an increase in circulating ANG II level (1, 62).
In conclusion, our results show that the AoGen gene expressed in cells of the SFO is under the transcriptional regulation of TTF-1, a homeodomain gene previously thought to be required only for the basal forebrain morphogenesis. Our data also demonstrate that this regulatory mechanism plays an important role in the control of body fluid homeostasis. We anticipate that identification of this novel control system will provide new insights into the understanding of the central processes that, operating within the brain, regulate body fluid homeostasis in normalcy and disease.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains an additional figure.
Supported by Neurobiological Research Fund Grant M1-0108-00-0016 through
Korea Institute of Science & Technology Evaluation and Planning.
** Supported by Consiglio Nazionale delle Ricerche (Target Project on
Biotechnology) and Ministero della Università e Ricerca Scientifica e
Tecnologica.
Supported by National Institutes of Health Grants HD-25123, U-54 HD-18185,
and RR00163 for the operation of the Oregon National Primate Research
Center.
¶¶ To whom correspondence should be addressed. Tel.: 82-52-259-2351; Fax: 82-52-259-1694; E-mail: bjlee{at}mail.ulsan.ac.kr.
1 The abbreviations used are: RAS, renin-angiotensin system; ANG,
angiotensin; AoGen, angiotensinogen; AS, antisense; AVP, arginine vasopressin;
C/EBP, CCAAT-box/enhancer-binding protein; CVO, circumventricular organ; ODN,
oligodeoxynucleotide; PVA, anterior thalamic paraventricular nucleus; SCR,
scrambled; SFO, subfornical organ; TTF-1HD, TTF-1 homeodomain; GFAP, glial
fibrillary acidic protein; RPA, RNase protection assay; SON, supraoptic
nucleus; EMSA, electrophoretic mobility shift assay.
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ACKNOWLEDGMENTS |
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REFERENCES |
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