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【摘要】 Vasopressin V 1a and V 2 receptors (V 1a R and V 2 R, respectively) distribute in the collecting duct of the kidney. Although the function of V 2 R mediating the antidiuretic effect of AVP has been investigated in detail, the role of V 1a R in the collecting ducts has not been elucidated. In the present study, we have investigated the role of the V 1a R pathway in V 2 R promoter activity. We cloned the 5'-flanking region of rat V 2 R (rV 2 R) and investigated rV 2 R promoter activity in the LLC-PK 1 cell line transfected to express rat V 1a R (rV 1a R) dominantly (LLC-PK 1 /rV 1a R). AVP induced a transient increase, followed by a sustained decrease, of rV 2 R promoter activity in these cells. This AVP-induced decrease of rV 2 R promoter activity was inhibited by V 1a R, but not V 2 R, antagonist. PMA mimicked this decrease of rV 2 R promoter activity. On the contrary, 8-(4-chlorophenylthio)-cAMP increased rV 2 R promoter activity. These PMA- and 8-(4-chlorophenylthio)-cAMP-induced effects were not observed on the deletion segment of the 5'-flanking region lacking CAAT and SP1 sites. In conclusion, 1 ) expression of the V 2 R is downregulated via the V 1a R pathway in LLC-PK 1 /rV 1a R cells, and 2 ) expression of the V 2 R is downregulated by the PMA-induced PKC pathway and upregulated by the cAMP-PKA pathway. These opposite effects of PKC and PKA appear to be regulated by the same promoter region of CAAT and SP1.
【关键词】 transcription regulation collecting ducts
THE MAIN ROLE OF THE KIDNEY is to maintain body fluid homeostasis by producing concentrated or diluted urine. Among many hormones, AVP plays a major role in the urine-concentrating mechanism. There are two types of AVP receptors in the kidney: vasopressin V 1a and V 2 receptors (V 1a R and V 2 R, respectively).
The V 2 R is distributed at the basolateral membrane of the collecting ducts and mediates the antidiuretic effect of AVP ( 22, 25 ). The V 2 R is coupled to G s proteins and increases cAMP in cytoplasm as a second messenger. Subsequently, PKA is activated. As a consequence of V 2 R stimulation from the basolateral membrane, excretion and reabsorption of water, electrolytes, and urea are induced through transporters such as aquaporin-2 (AQP2), Na-K-Cl cotransporter type 1, and renal urea transporter type A 1 in the collecting ducts ( 16, 34, 37 ).
The V 1a R is localized mainly in the vascular system and glomeruli and exerts a vasopressor effect ( 24 ). The V 1a R is coupled to G q/11 proteins and stimulates phospholipase C. Activation of phospholipase C releases Ca 2+ from the endoplasmic reticulum, and subsequently PKC is activated. In the kidney, V 1a R mRNA is expressed not only in the glomeruli but also in the collecting ducts ( 28, 30 ). Although the function of the V 2 R has been investigated in detail, the physiological role of the V 1a R in the collecting ducts remains unclear.
Previous studies have suggested that the V 1a R is present at the luminal membrane of the collecting ducts. Ando et al. ( 1 ) demonstrated that luminal AVP induces sustained hyperpolarization of transepithelial voltage. Naruse et al. ( 21 ) demonstrated that luminal AVP induces changes in luminal conductance. Our previous immunohistochemical study ( 22, 28 ) revealed that the V 1a R was mainly localized at the luminal membrane and in the cytoplasm, whereas the V 2 R is localized at the basolateral membrane in the rat collecting duct.
Bankir ( 2 ) proposed a role for urinary AVP via the V 1a R and the interaction between V 1a R- and V 2 R-mediated effects. We previously demonstrated that fractional extraction of AVP is closely related to fractional excretion of Na + in patients with chronic renal failure ( 23 ). Our previous study also showed that luminal AVP decreases osmotic water permeability in the presence of basolateral AVP in the inner medullary collecting ducts ( 22 ). In addition, the increase of V 1a R mRNA and decrease of V 2 R mRNA were simultaneously observed during metabolic acidosis ( 28 ). These studies strongly indicate that body fluid homeostasis is maintained not only by basolateral AVP, but also by luminal AVP, presumably via the V 1a R pathway.
On the basis of previous reports, we hypothesized that V 1a R stimulation by luminal AVP might modulate the basolateral AVP-induced activity by regulating V 2 R expression at the transcriptional level. To address this hypothesis, we characterized the rat V 2 R (rV 2 R) promoter region and investigated the regulation of rV 2 R promoter activity in LLC-PK 1 cells that were transfected to express rat V 1a R (rV 1a R) dominantly. Furthermore, we analyzed the effects of the PKC and PKA pathways on V 2 R promoter activity and investigated the cross talk between V 1a R and V 2 R signaling pathways in LLC-PK 1 cells.
MATERIALS AND METHODS
Reagents. [Arg 8 ]-vasopressin (AVP) was obtained from Sigma-Aldrich (St. Louis, MO). RO 31-8220 and 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) were obtained from Sigma-Aldrich (Steinheim, Germany). PMA and 4 -PMA were purchased from Promega (Madison, WI). OPC-21268 and OPC-31260 were kind gifts from Otsuka Pharmaceutical (Tokushima, Japan).
Cloning of the 5'-flanking region of the rV 2 R gene. The 1,092-bp 5'-flanking region of the rV 2 R gene was cloned by PCR amplification, with rat genomic DNA used as a template. An Expand High Fidelity PLUS PCR System (Roche Applied Science, Penzberg, Germany) was used for PCR amplification. Gene-specific sense and antisense primers were designed on the basis of the published cDNA sequence of the rV 2 R gene ( 17 ).
Reporter plasmid construction. For analysis of rV 2 R promoter activity, a series of deletion mutants were synthesized by PCR using sense primers designed to downstream (-664, -345, -245, -225, -165, -155, -124, -92, and -46 bp) and antisense primers on exon 1 (+57 bp). A deletion mutant, lacking -164 to 47 bp from the 5'-flanking region, was synthesized by the in vitro overlap-extension PCR method, as described previously ( 20 ). PCR-amplified products were subcloned into a pGL3-basic luciferase reporter vector (Promega).
Cell culture, transfection, and determination of promoter activity. LLC-PK 1 cells (catalog no. JCRB0060, Health Science Research Resources Bank, Tokyo, Japan) were maintained in DMEM supplemented with 100 IU/ml penicillin-streptomycin and 10% fetal bovine serum (Invitrogen, Tokyo, Japan).
For transfection studies, cells were seeded in 12-well clusters (Corning) and grown to reach 50% confluence. FuGENE6 transfection reagent (Roche Applied Science, Indianapolis, IN) was used to cotransfect 0.314 pmol of pGL3-rV 2 R promoter constructs and 3.75 fmol of pRL-TK constructs into the cells. Promoter activity was examined after treatment with AVP, cpt-cAMP, PMA, and vehicle (DMSO).
For determination of reporter activity, firefly luciferase activity from the pGL3 reporter vector and Renilla luciferese activity from the pRL-TK vector were measured by the Dual-Luciferase Assay System (Promega) on a luminometer (model TD-20/20, Turner Design).
Establishment of a stable LLC-PK 1 cell line expressing rV 1a R. The rV 1a R gene (GenBank accession no. BC088095 ) was synthesized by PCR amplification from a pExpress1 vector containing rV 1a R cDNA (Open Biosystems) and subcloned into a pcDNA5/FRT vector. Gene-specific sense and antisense primers were designed on the basis of the published cDNA sequence of the rV 1 R gene ( 19 ).
LLC-PK 1 stably expressing the rV 1a R was constructed using the Flp-In System (Invitrogen Life Technologies, Carlsbad, CA), which is able to create isogenic cell lines with one or more Flp recombination targets (FRT). To prepare a new LLC-PK 1 /FRT cell line, LLC-PK 1 cells were transfected with pFRT/lacZeo and selected in Zeocin (250 µg/ml) for 2 wk. Cells were confirmed to have the FRT site by -galactosidase staining assay (Invitrogen Life Technologies). Positive cells were named LLC-PK 1 /FRT.
The LLC-PK 1 /FRT cell line was cotransfected with pOG44, a vector for transient expression of Flp recombinase, and pcDNA5/FRT/rV 1a R, an expression vector containing the coding region for the rV 1a R protein, and possesses the FRT site for homologous recombination. Since the pcDNA5/FRT vector also contained the hygromycin B-resistant gene, LLC-PK 1 /FRT cells stably transfected with rV 1a R (LLC-PK 1 /rV 1a R) were selected in 800 µg/ml hygromycin B. Single-cell colonies were used to analyze the interaction between rV 1a R stimulation and rV 2 R promoter activity.
mRNA isolation and cDNA synthesis. MgNA Pure LC mRNA Isolation Kit II (Roche Diagnostics, Tokyo, Japan) was used for mRNA extraction. The cell pellets were diluted with 300 µl of lysis buffer and dissolved in a buffer containing a chaotropic salt and an RNase inactivator. The 3'-poly(A + ) from the released mRNA hybridizes to the added biotin-labeled oligo(dT). This complex is immobilized onto the surface of streptavidin-coated magnetic beads. After a DNase digestion step, unbound substances were removed by three washing steps, and purified mRNA was eluted with a low-salt buffer. cDNA was synthesized using the High-Capacity cDNA Archive Kit (Applied Biosystems).
Intracellular Ca 2+ measurement. LLC-PK 1 cells expressing the rV 1a R were cultured in 96-well black plates to reach 100% confluence. According to the protocol for the Calcium Kit Fluo 3 (Dojindo Laboratories, Kumamoto, Japan), cells were preincubated in 100 µl of loading buffer containing 5 mg/l fluo 3-AM, 1.25 mM probenecid, and 0.04% Pluronic F-127 at 37°C for 1 h. After preincubation, loading buffer was replaced with 100 µl of recording medium containing 1.25 mM probenecid. Fluorescence intensity was measured as the Ca 2+ concentration for 5 min after AVP treatment with use of a plate reader (Mulch Micro MTP-800Lab, Corona, Niigata, Japan).
Data analysis. Values are means ± SE. Statistical analysis was performed by ANOVA and multiple comparison (Bonferroni's or Sheffé's test) or by Student's t -test. P < 0.05 was considered significant.
RESULTS
Analysis and characterization of the 1,092-bp segment from the 5'-flanking region of the rV 2 R gene. The sequence up to 1,092 bp upstream from the transcriptional initiation site of the rV 2 R was identified by Mandon et al. ( 17 ) ( Fig. 1 ). We confirmed the whole sequence using our PCR product. This region contains multiple motif sequences that may play an important role in transcriptional regulation. This gene has no TATA box, but it does have a CAAT box at -82 bp from the transcription initiation site. There are seven binding sites for PEA3 and one each for AP1, AP3, and SP1. As is the case for other genes, these consensus sequences are located close to the transcription initiation site.
Fig. 1. Nucleotide sequence of the 5'-flanking region of the rat vasopressin V 2 receptor (rV 2 R) gene. Inverse PCR was used to clone the 5'-flanking region. Possible promoter regulatory elements are shown in boxes.
Basal promoter activity of the rV 2 R is shown in Fig. 2. Deletion of the 1,092-bp 5'-flanking region to 345 bp significantly increased promoter activity: 25- to 30-fold greater in the 345-bp promoter segment than in control pGL3-basic. However, deletion of the segment further downstream decreased promoter activity. By deletion to 46 bp, the promoter activity was completely abolished.
Fig. 2. Promoter activity of the rV 2 R in LLC-PK 1 cells with deletion analysis. LLC-PK 1 cells were cotransfected with pGL3-rV 2 R promoter constructs and pRL-TK constructs. Luciferase activity was measured 42 h after transfection. The 345-bp 5'-flanking region of rV 2 R showed the highest promoter activity among deletion segments. Values are means ± SE ( n = 6). * P < 0.05 vs. pGL3 basic.
Effect of AVP on intracellular Ca 2+ concentration in LLC-PK1 cells transfected with rV 1a R. RT-PCR was used to confirm mRNA expression of the rV 1a R in LLC-PK 1 cells transfected with rV 1a R by the Flp-In System. cDNA (2 µg) obtained by reverse transcription from cell lysate was amplified for 35 cycles by PCR. Agarose gel electrophoresis showed the PCR products at the appropriate molecular weight, as well as the rV 1a R-positive control ( Fig. 3 ). These findings indicate that the rV 1a R is expressed in LLC-PK 1 /rV 1a R cells at the mRNA level. To demonstrate the function of the rV 1a R in this cell line, we investigated the accumulation of intracellular Ca 2+ by measuring the change of fluo 3 fluorescence intensity ( Fig. 4 ). In LLC-PK 1 /rV 1a R cells, treatment with 10 -9 M AVP increased fluorescence intensity, indicating an increase of the intracellular Ca 2+ concentration. Maximal intensity was achieved <30 s after addition of AVP ( Fig. 4 A ). AVP dose dependently increased intracellular Ca 2+ ( Fig. 4 B ). These results indicate that rV 1a R protein is present on the cell membrane and is functionally active in LLC-PK 1 /rV 1a R cells. In contrast, in non-rV 1a R-transfected LLC-PK 1 (LLC-PK 1 /FRT) cells, only a high dose (10 -7 M) AVP increased Ca 2+ concentration.
Fig. 3. Agarose gel electrophoresis for detection of rat vasopressin V 1a receptor (rV 1 R) mRNA in LLC-PK 1 /rV 1a R cells. The 1.5-kbp bands indicate rV 1a R. cDNA (2 µg) obtained by RT with cell as template was amplified for 35 cycles by PCR. Lane 2, amplification by cDNA in LLC-PK 1 /rV 1a R cells (transfected with rV 1a R using Flp-In System); lane 3, negative control amplified by purified mRNA in LLC-PK 1 /rV 1a R cells; lane 4, positive control amplified by pcDNA5 plasmid with rV 1a R as template; lane 5, negative control amplified by cDNA in LLC-PK 1 /FRT cells; lane 6, negative control amplified by purified mRNA in LLC-PK 1 /FRT cells; lanes 1 and 7, molecular markers.
Fig. 4. Alteration of relative fluorescence intensity for accumulation of intracellular Ca 2+. Fluorescence intensity for fluo 3, indicating accumulation of intracellular Ca 2+, was measured in LLC-PK 1 /FRT and LLC-PK 1 /rV 1a R cells at 1-s intervals for 5 min after treatment with AVP or DMSO (vehicle). A : alteration of fluorescence intensity relative to mean basic intensity (before AVP stimulation). AVP at 10 -9 M increased intracellular Ca 2+ in LLC-PK 1 /rV 1a R, but not LLC-PK 1 /FRT, cells. B : dose-response effect of AVP on intracellular Ca 2+ in LLC-PK 1 /rV 1a R and LLC-PK 1 /FRT cells shown as mean fluorescence intensity 30 s after treatment with AVP or vehicle. AVP dose dependently increased intracellular Ca 2+ in LLC-PK 1 /rV 1a R cells. Values are means ± SE ( n = 6). * P < 0.05 vs. vehicle. P < 0.05 vs. LLC-PK 1 /FRT.
Downregulation of rV 2 R promoter activity via rV 1a R stimulation. Deletion study of the promoter element demonstrated the greatest activity at a 345-bp segment of the rV 2 R promoter ( Fig. 2 ). Therefore, we transfected the 345-bp 5'-flanking region of the rV 2 R in LLC-PK 1 /rV 1a R cells to investigate the interaction between V 2 R promoter activity and V 1a R stimulation. First, we examined the time course of the effect of 10 -8 M AVP on rV 2 R promoter activity ( Fig. 5 A ). AVP transiently increased rV 2 R promoter activity 2 h after AVP treatment. Sustained suppression of rV 2 R promoter activity was observed 12-24 h after AVP treatment. Although rV 2 R promoter activity was transiently increased 2-4 h after AVP treatment in LLC-PK1/FRT cells, a significant decrease of rV 2 R promoter activity was not observed at 12-24 h ( Fig. 5 B ). In addition, rV 2 R promoter activity was decreased over a wide range of AVP concentrations in LLC-PK 1 /rV1aR cells ( Fig. 6 ). The decrease of rV 2 R promoter activity by 10 -10 M AVP was inhibited by a V 1a R-specific antagonist (OPC-21268) in LLC-PK 1 /rV 1a R cells ( Fig. 7 A ). This restoration of rV 2 R promoter activity by a V 1a R-specific antagonist was more distinct when the cells were treated with high-dose (10 -8 M) AVP ( Fig. 7 C ). In contrast, a V 2 R-specific antagonist (OPC-31260) did not restore V 2 R promoter activity. A PKC inhibitor (RO 31-8220), as well as the V 1a R-specific antagonist, inhibited the decrease of rV 2 R promoter activity. Although AVP slightly decreased rV 2 R promoter activity in LLC-PK 1 /FRT cells ( Fig. 7 B ), the change was not statistically significant, and neither V 1a R nor V 2 R antagonists restored V 2 R promoter activity.
Fig. 5. Time course of effect of AVP on rV 2 R promoter activity in LLC-PK 1 /rV 1a R and LLC-PK 1 /FRT cells. Each cell was cotransfected with the 5'-flanking region (-345 to +57 bp) of rV 2 R. At 18 h after transfection, cells were incubated with 10 -8 M AVP. Luciferase assay was performed 0, 2, 4, 12, and 24 h after AVP stimulation. A : luciferase activity in LLC-PK 1 /rV 1a R cells. AVP transiently increased rV 2 R promoter activity 2 h after AVP treatment. Sustained decrease of activity was observed at 12-24 h. B : luciferase activity in LLC-PK 1 /FRT cells. AVP transiently increased rV 2 R promoter activity. Values are means ± SE ( n = 6). * P < 0.05 vs. vehicle.
Fig. 6. Dose-dependent effect of AVP on rV 2 R promoter activity in LLC-PK1/rV 1a R cells. LLC-PK 1 /rV 1a R cells were cotransfected with the 5'-flanking region (-345 to +57 bp) of rV 2 R and incubated with 10 -11 -10 -7 M AVP for 24 h. rV 2 R promoter activity was decreased over a wide range of AVP concentrations. Values are means ± SE ( n = 6). * P < 0.05 vs. vehicle.
Fig. 7. Effect of AVP on rV 2 R promoter activity in LLC-PK 1 /rV 1a R and LLC-PK 1 /FRT cells. Each cell was cotransfected with the 5'-flanking region (-345 to +57 bp) of rV 2 R and incubated with AVP, antagonists, and PKC inhibitor for 12 or 24 h. Final concentration of each solution is as follows: 10 -10 or 10 -8 M AVP, 10 -6 M OPC-21268 (a V 1a R-specific antagonist), 10 -7 M OPC-31260 (a V 2 R-specific antagonist), and 10 -7 M RO 31-8220 (a PKC inhibitor). A : luciferase activity in LLC-PK 1 /rV 1a R cells treated with 10 -10 M AVP for 12 h. B : luciferase activity in LLC-PK 1 /FRT cells treated with 10 -10 M AVP for 12 h. AVP significantly decreased rV 2 R promoter activity in LLC-PK 1 /rV 1a R, but not LLC-PK 1 /FRT, cells. AVP effect was abolished by OPC-21268 and RO 31-8220, but not by OPC-31260. C : luciferase activity in LLC-PK 1 /rV 1a R cells treated with 10 -8 M AVP for 24 h. Note distinct restoration of rV 2 R promoter activity by OPC-21268. Values are means ± SE ( n = 6). * P < 0.05 vs. AVP.
Identification of PKC and PKA response element in the rV 2 R promoter region. To analyze signal transduction, we treated LLC-PK 1 cells with PMA, a PKC activator. First, we used the 1,092-bp 5'-flanking region of the rV 2 R to examine the time course of the effect of PMA on rV 2 R promoter activity ( Fig. 8 A ). rV 2 R promoter activity was decreased 6-12 h after treatment with PMA. rV 2 R promoter activity was suppressed by PMA in a dose-dependent manner ( Fig. 8 B ). On the basis of this finding, we performed additional experiments on the cells 12 h after they were treated with 10 -6 M PMA. In contrast to PMA, 4 -PMA, a PMA analog, did not change rV 2 R promoter activity ( Fig. 9 ). rV 2 R promoter activity was downregulated by PMA, even in short segments ( Fig. 10 ). However, this downregulation was abolished by deletion of the promoter region to 46 bp.
Fig. 8. Dose- and time-dependent effect of PMA on rV 2 R promoter activity. A : time-dependent effect of PMA and 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) on rV 2 R promoter activity in LLC-PK 1 cells. LLC-PK 1 cells were cotransfected with the 5'-flanking region (-1,092 to +57 bp) of rV 2 R. At 18 h after transfection with promoter construct, cells were exposed to 10 -6 M PMA or 4 x 10 -4 M cpt-cAMP. Luciferase activity was assayed 0, 2, 4, 6, and 12 h after PMA or cpt-cAMP stimulation. rV 2 R promoter activity was increased 2-12 h after cpt-cAMP and decreased 6-12 h after PMA. B : dose-dependent effect of PMA on rV 2 R promoter activity in LLC-PK 1 cells. LLC-PK 1 cells were cotransfected with the 5'-flanking region (-1,092 to +57 bp) of rV 2 R and exposed to 10 -8 -10 -6 M PMA for 12 h. PMA dose dependently decreased rV 2 R promoter activity. Values are means ± SE ( n = 6). * P < 0.05 vs. vehicle.
Fig. 9. Effect of PMA, cpt-cAMP, and 4 -PMA on rV 2 R promoter activity in LLC-PK 1 cells. LLC-PK 1 cells were cotransfected with the 5'-flanking region (-1,092 to +57 bp) of rV 2 R and exposed to DMSO, 10 -6 M PMA, 4 x 10 -4 M cpt-cAMP, or 10 -6 M 4 -PMA for 12 h. rV 2 R promoter activity was significantly decreased by PMA and decreased by cpt-cAMP. 4 -PMA did not change rV 2 R promoter activity. Values are means ± SE ( n = 6). * P < 0.05 vs. vehicle.
Fig. 10. Deletion series of rV 2 R promoter element and promoter activity. LLC-PK 1 cells were cotransfected with a deletion series of the 5'-flanking region from -1,092 to -46 bp of rV 2 R and exposed to 10 -6 M PMA for 12 h. PMA suppressed rV 2 R promoter activity, even in short segments. Effect of PMA was abolished in the deletion mutant lacking CAAT and SP1 elements (-345, -165/-46, and +57). Values are means ± SE ( n = 6). * P < 0.05.
To examine the role of the PKA pathway in V 2 R signal transduction, we added cpt-cAMP to LLC-PK 1 cells. rV 2 R promoter activity was significantly increased 2-12 h after treatment with cpt-cAMP ( Fig. 8 A ). These opposite effects caused by PMA and cpt-cAMP were abolished by deletion at -164 to -47 bp on the 345-bp 5'-flanking region of rV 2 R ( Fig. 11 B ). This mutant lacks consensus sequences of CAAT and Sp1 from the original 5'-flanking region.
Fig. 11. Effect of PMA and cpt-cAMP on rV 2 R promoter activity of the deletion mutant in LLC-PK 1 cells. LLC-PK 1 cells were cotransfected with the deletion mutant lacking CAAT and SP1 elements (from -164 to -47 bp) and exposed to 10 -6 M PMA or 4 x 10 -4 M cpt-cAMP or DMSO for 12 h. A : effect of PMA and cpt-cAMP on the 5'-flanking region (-345 to +57 bp). B : effect of PMA and cpt-cAMP on the deletion mutant (-345, -165/-46, and +57). Opposite effects of PMA and cpt-cAMP on rV 2 R promoter activity were abolished on the deletion mutant. Values are means ± SE ( n = 6). * P < 0.05 vs. vehicle.
DISCUSSION
In the present study, we used an LLC-PK 1 cell line to investigate the interaction between V 2 R promoter activity and the V 1a R pathway. The LLC-PK 1 cell line, which is derived from the proximal tubule of porcine kidney, has been widely used to investigate the kinetics of the V 2 R and AQP2 because of its response to vasopressin stimulation ( 12, 13 ). Moreover, the promoter analysis, trafficking, and recycling of AQP2 have been investigated in LLC-PK 1 cells ( 5, 9, 13 ). Preliminarily, we also measured rV 2 R promoter activity in Madin-Darby canine kidney, COS-7, and HEK-293 cells, but the greatest increase of V 2 R promoter activity was observed in LLC-PK 1 cells. On the basis of these reports and our preliminarily data, we considered the LLC-PK 1 cell line to be suitable for our purpose.
In previous studies, stimulation of LLC-PK 1 cells with 10 -10 -10 -7 M AVP increased cAMP, indicating expression of the V 2 R in LLC-PK 1 cells ( 11 ). In contrast, the increase of intracellular Ca 2+ in response to V 1a R stimulation required the high dose (10 -7 -5 x 10 -6 M) of AVP in LLC-PK 1 cells ( 3, 7, 27 ). Our present results also showed an increase of intracellular Ca 2+ in LLC-PK 1 /FRT cells in response to only high-dose AVP ( Fig. 4 B ). It is considered that LLC-PK 1 cells might express endogenous V 1a R; however, this functional role could be small at physiological AVP concentration.
In the present study, using the Flp-In System, we have established an LLC-PK 1 cell line stably expressing the rV 1a R. This cell line expresses rV 1a R mRNA and shows an increase of intracellular Ca 2+ in response to 10 -10 -10 -7 M AVP, suggesting functional expression of the rV 1a R ( Figs. 3 and 4 ). Since the host cell line (LLC-PK 1 /FRT) can be transfected with any other genes of interest in FRT sites, this cell line could be useful for further study. It should be possible to compare the kinetics of plural genes or proteins in isogenic cell conditions. For example, it might be interesting to analyze a functional interaction of the V 1a R with the oxytocin receptor, which has been demonstrated to cross-react with vasopressin ( 6, 10, 32, 33 ).
AVP caused a sustained decrease of rV 2 R promoter activity in LLC-PK 1 /rV 1a R cells ( Fig. 5 ). In addition, suppression of the AVP-induced V 2 R was inhibited by a V 1a R-specific antagonist and a PKC inhibitor ( Fig. 7, A and C ). These findings strongly indicate that V 2 R promoter activity could be downregulated via the V 1a R-PKC pathway. It is known that the physiological concentration of urinary AVP usually ranges from 10 -11 to 10 -9 M ( 18 ). Our results were obtained within this range of AVP concentrations, indicating that V 1a R stimulation affects V 2 R promoter activity under physiological conditions.
However, some issues remain unresolved. In contrast to the dose-dependent increase of intracellular Ca 2+ by AVP, rV 2 R promoter activity showed the two-phased decrease over a wide range of AVP concentrations ( Fig. 6 ). In addition, the V 1a R antagonist and the PKC inhibitor did not completely restore V 2 R promoter activity. Perhaps a pathway other than the rV 1a R-PKC pathway decreases V 2 R promoter activity in LLC-PK1 cells.
rV 2 R promoter activity was increased in the early phase by addition of AVP to LLC-PK 1 /rV 1a R and LLC-PK 1 /FRT cells ( Fig. 5 ). It is suggested that AVP increased rV 2 R promoter activity via the V 2 R pathway. In the late phase, rV 2 R promoter activity was slightly decreased, even in LLC-PK 1 /FRT cells ( Fig. 7 B ). However, this slight decrease in LLC-PK 1 /FRT cells was also observed after treatment with V 1a R antagonist, V 2 R antagonist, and PKC inhibitor (not shown) and was not statistically significant.
Wong and Tsui ( 35 ) showed that V 2 R mRNA is upregulated by PKA stimulation and downregulated by PKC stimulation in the rat inner medullary collecting duct. As shown in Fig. 9, rV 2 R promoter activity was decreased by PMA and increased by cpt-cAMP. These results indicate that rV 2 R promoter activity is modulated by PKC and PKA pathways.
The time-dependent effects of AVP, PMA, and cpt-cAMP on rV 2 R promoter activity demonstrate that differences in time course between V 1a R and V 2 R stimulation affect rV 2 R promoter activity. rV 2 R promoter activity was increased in the early phase and decreased in the late phase by AVP ( Fig. 5 A ). The increase of rV 2 R promoter activity by cpt-cAMP was observed 2 h after AVP treatment, whereas the decrease of rV2R promoter activity appeared 6 h after addition of AVP ( Fig. 8 A ). Although intracellular Ca 2+ was rapidly increased to maximal concentration via V 1a R stimulation ( Fig. 4 ), the effect of cAMP on V 2 R promoter activity via V 2 R stimulation could be more potent and could occur before the effect of V 1a R stimulation. It is suggested that the early AVP-induced increase of rV 2 R promoter activity is caused by V 2 R stimulation and the delayed AVP-induced decrease is mediated by V 1a R stimulation. As discussed in previous studies, a 10- to 100-fold higher dose of AVP seems to be required for V 1a R than for V 2 R activation ( 2, 26 ). The response of the V 1a R to AVP might be slower than the response of the V 2 R.
By the deletion mutant study of the rV 2 R promoter, consensus sequences of CAAT and SP1 were speculated to be the PKC-responsive element. Interestingly, our results showed that the same element was also crucial for cAMP-induced V 2 R promoter activity. It is suggested that the opposite effects of PKC and PKA on regulation could affect the same consensus sequences, such as CAAT and/or SP1, in this gene. Several reports have demonstrated that CAAT and/or SP1 are related to cAMP-inducible promoter activity ( 8, 9, 14 ). However, a direct correlation between these consensus sequences and PKC has not been reported. An indirect mechanism for suppression of V 2 R expression via the PKC pathway at the transcriptional level could be supposed.
Klingler et al. ( 15 ) demonstrated that long-term (5 h) exposure to PMA decreased cAMP accumulation in Chinese hamster ovary cells stably transfected with the V 1a R and V 2 R, suggesting an interaction between PMA stimulation and cAMP accumulation. Previous reports have indicated a connection between PKC and cAMP in cultured collecting tubular cells ( 4, 29 ). These studies have suggested that PKC caused phosphorylation of adenylyl cyclase or, alternatively, phosphorylation of G i protein. We confirmed the PMA-induced decrease of cAMP accumulation in LLC-PK 1 cells (not shown). The interaction between PMA-induced PKC and AVP-induced cAMP could support our hypothesis of cross talk between the V 1a R and V 2 R signaling pathways.
It is well known that expression of V 2 R mRNA and protein is decreased when plasma AVP increases in vivo ( 31, 36 ). We have demonstrated that inhibition of V 1a R increased V 2 R mRNA in the rat outer medullary collecting duct ( 28 ). However, downregulation of V 2 R expression has not been investigated in detail. Our present study could support this downregulation of the V 2 R at the transcriptional level.
Our results can be summarized as follows. 1 ) Expression of the V 2 R is downregulated via the V 1a R pathway and upregulated via the V 2 R pathway. 2 ) Expression of the V 2 R is downregulated by the PMA-induced PKC signaling pathway and upregulated by the cAMP-PKA signaling pathway. These opposite effects of PKC and PKA stimulation could be exerted on the same consensus sequences in the V 2 R promoter region, such as CAAT and SP1. 3 ) Intracellular cross talk between the V 1a R and V 2 R pathways in the collecting duct cells appears to strictly and sensitively maintain body fluid homeostasis.
GRANTS
This study was supported by Ministry of Education, Culture, Sports, Science, and Technology of Japan Grants-in-Aid for Scientific Research 18590895, 17590833, 167904660, 16590791, 16590792, 16390246, 15590852, and 14370321.
ACKNOWLEDGMENTS
We thank Dr. Tetsushi Kagawa for valuable suggestions regarding the luciferase assay and Takanobu Matsuzaki and Noriko Teramoto for technical assistance. We are grateful for support by the staff at the Gene Technology Center at Kumamoto University. We also thank Dr. Mark A. Knepper for critical reading of the manuscript.
【参考文献】
Ando Y, Tabei K, Asano Y. Luminal vasopressin modulates transport in the rabbit cortical collecting duct. J Clin Invest 88: 952-959, 1991.
Bankir L. Antidiuretic action of vasopressin: quantitative aspects and interaction between V 1a and V 2 receptor-mediated effects. Cardiovasc Res 51: 372-390, 2001.
Bonventre JV, Skorecki KL, Kreisberg JI, Cheung JY. Vasopressin increases cytosolic free calcium concentration in glomerular mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 251: F94-F102, 1986.
Dixon BS, Breckon R, Burke C, Anderson RJ. Phorbol esters inhibit adenylate cyclase activity in cultured collecting tubular cells. Am J Physiol Cell Physiol 254: C183-C191, 1988.
Gustafson CE, Levine S, Katsura T, McLaughlin M, Aleixo MD, Tamarappoo BK, Verkman AS, Brown D. Vasopressin regulated trafficking of a green fluorescent protein-aquaporin 2 chimera in LLC-PK 1 cells. Histochem Cell Biol 110: 377-386, 1998.
Han JS, Maeda Y, Knepper MA. Dual actions of vasopressin and oxytocin in regulation of water permeability in terminal collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 265: F26-F34, 1993.
Himpens B, De SH, Casteels R. Intracellular Ca 2+ signaling induced by vasopressin, ATP, and epidermal growth factor in epithelial LLC-PK 1 cells. Am J Physiol Cell Physiol 265: C966-C975, 1993.
Horton CD, Halvorson LM. The cAMP signaling system regulates LH gene expression: roles of early growth response protein-1, SP1 and steroidogenic factor-1. J Mol Endocrinol 32: 291-306, 2004.
Hozawa S, Holtzman EJ, Ausiello DA. cAMP motifs regulating transcription in the aquaporin 2 gene. Am J Physiol Cell Physiol 270: C1695-C1702, 1996.
Inoue T, Naruse M, Nakayama M, Kurokawa K, Sato T. Oxytocin affects apical sodium conductance in rabbit cortical collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 265: F487-F503, 1993.
Jans DA, Peters R, Jans P, Fahrenholz F. Vasopressin V 2 -receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK 1 renal epithelial cells. J Cell Biol 114: 53-60, 1991.
Jans DA, Peters R, Zsigo J, Fahrenholz F. The adenylate cyclase-coupled vasopressin V 2 -receptor is highly laterally mobile in membranes of LLC-PK 1 renal epithelial cells at physiological temperature. EMBO J 8: 2481-2488, 1989.
Katsura T, Verbavatz JM, Farinas J, Ma T, Ausiello DA, Verkman AS, Brown D. Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK 1 epithelial cells. Proc Natl Acad Sci USA 92: 7212-7216, 1995.
Kinane TB, Shang C, Finder JD, Ercolani L. cAMP regulates G-protein i-2 -subunit gene transcription in polarized LLC-PK 1 cells by induction of a CCAAT box nuclear binding factor. J Biol Chem 268: 24669-24676, 1993.
Klingler C, Ancellin N, Barrault MB, Morel A, Corman B. Potentiation of receptor-mediated cAMP production: role in the cross-talk between vasopressin V 1a and V 2 receptor transduction pathways. Biochem J 330: 1023-1028, 1998.
Knepper MA. Molecular physiology of urinary concentrating mechanism: regulation of aquaporin water channels by vasopressin. Am J Physiol Renal Physiol 272: F3-F12, 1997.
Mandon B, Bellanger AC, Elalouf JM. Inverse PCR-mediated cloning of the promoter for the rat vasopressin V 2 receptor gene. Pflügers Arch 430: 12-18, 1995.
Moses AM, Steciak E. Urinary and metabolic clearances of arginine vasopressin in normal subjects. Am J Physiol Regul Integr Comp Physiol 251: R365-R370, 1986.
Murasawa S, Matsubara H, Kijima K, Maruyama K, Mori Y, Inada M. Structure of the rat V 1a vasopressin receptor gene and characterization of its promoter region and complete cDNA sequence of the 3'-end. J Biol Chem 270: 20042-20050, 1995.
Nakayama Y, Nonoguchi H, Kiyama S, Ikebe M, Tashima Y, Shimada K, Tanzawa K, Tomita K. Intranephron distribution and regulation of endothelin-converting enzyme-1 in cyclosporin A-induced acute renal failure in rats. J Am Soc Nephrol 10: 562-571, 1999.
Naruse M, Yoshitomi K, Hanaoka K, Imai M, Kurokawa K. Electrophysiological study of luminal and basolateral vasopressin in rabbit cortical collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 268: F20-F29, 1995.
Nonoguchi H, Owada A, Kobayashi N, Takayama M, Terada Y, Koike J, Ujiie K, Marumo F, Sakai T, Tomita K. Immunohistochemical localization of V 2 vasopressin receptor along the nephron and functional role of luminal V 2 receptor in terminal inner medullary collecting ducts. J Clin Invest 96: 1768-1778, 1995.
Nonoguchi H, Takayama M, Owada A, Ujiie K, Yamada T, Nakashima O, Sakuma Y, Koike J, Terada Y, Marumo F, Tomita K. Role of urinary arginine vasopressin in the sodium excretion in patients with chronic renal failure. Am J Med Sci 312: 195-201, 1996.
Phillips PA, Abrahams JM, Kelly JM, Mooser V, Trinder D, Johnston CI. Localization of vasopressin binding sites in rat tissues using specific V 1 and V 2 selective ligands. Endocrinology 126: 1478-1484, 1990.
Robben JH, Knoers NV, Deen PM. Regulation of the vasopressin V 2 receptor by vasopressin in polarized renal collecting duct cells. Mol Biol Cell 15: 5693-5699, 2004.
Star RA, Nonoguchi H, Balaban R, Knepper MA. Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct. J Clin Invest 81: 1879-1888, 1988.
Tang MJ, Weinberg JM. Vasopressin-induced increases of cytosolic calcium in LLC-PK 1 cells. Am J Physiol Renal Fluid Electrolyte Physiol 251: F1090-F1095, 1986.
Tashima Y, Kohda Y, Nonoguchi H, Ikebe M, Machida K, Star RA, Tomita K. Intranephron localization and regulation of the V 1a vasopressin receptor during chronic metabolic acidosis and dehydration in rats. Pflügers Arch 442: 652-661, 2001.
Teitelbaum I. Protein kinase C inhibits arginine vasopressin-stimulated cAMP accumulation via a G i -dependent mechanism. Am J Physiol Renal Fluid Electrolyte Physiol 264: F216-F220, 1993.
Terada Y, Tomita K, Nonoguchi H, Yang T, Marumo F. Different localization and regulation of two types of vasopressin receptor messenger RNA in microdissected rat nephron segments using reverse transcription polymerase chain reaction. J Clin Invest 92: 2339-2345, 1993.
Terashima Y, Kondo K, Mizuno Y, Iwasaki Y, Oiso Y. Influence of acute elevation of plasma AVP level on rat vasopressin V 2 receptor and aquaporin-2 mRNA expression. J Mol Endocrinol 20: 281-285, 1998.
Terashima Y, Kondo K, Oiso Y. Administration of oxytocin affects vasopressin V 2 receptor and aquaporin-2 gene expression in the rat. Life Sci 64: 1447-1453, 1999.
Terrillon S, Durroux T, Mouillac B, Breit A, Ayoub MA, Taulan M, Jockers R, Barberis C, Bouvier M. Oxytocin and vasopressin V 1a and V 2 receptors form constitutive homo- and heterodimers during biosynthesis. Mol Endocrinol 17: 677-691, 2003.
Tomita K, Pisano JJ, Knepper MA. Control of sodium and potassium transport in the cortical collecting duct of the rat. Effects of bradykinin, vasopressin, and deoxycorticosterone. J Clin Invest 76: 132-136, 1985.
Wong NL, Tsui JK. Angiotensin II upregulates the expression of vasopressin V 2 mRNA in the inner medullary collecting duct of the rat. Metabolism 52: 290-295, 2003.
Yokoi H, Nagasaki H, Tachikawa K, Arima H, Murase T, Miura Y, Hirabayashi M, Oiso Y. Adaptation to sustained high plasma vasopressin in water and electrolyte homeostasis in the rat transgenic for the metallothionein-vasopressin fusion gene. J Endocrinol 173: 23-33, 2002.
Zhang C, Sands JM, Klein JD. Vasopressin rapidly increases phosphorylation of UT-A1 urea transporter in rat IMCDs through PKA. Am J Physiol Renal Physiol 282: F85-F90, 2002.
作者单位：Department of Nephrology, Graduate School of Medical Sciences, Kumamoto University, Kunamoto, Japan