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H+ exchanger NHE3 in the kidney
Kidney International, Vol. 67 (2005), pp. 1410–1419
ION CHANNELS – MEMBRANE TRANSPORT – INTEGRATIVE PHYSIOLOGY
Circadian clock genes directly regulate expression of the Na+/H+ exchanger NHE3 in the kidney MOHAMMAD SAIFUR ROHMAN,1 NORIAKI EMOTO, HIDEMI NONAKA, RYUSUKE OKURA, MASATAKA NISHIMURA, KAZUHIRO YAGITA, GIJSBERTUS T.J. VAN DER HORST, MASAFUMI MATSUO, HITOSHI OKAMURA, and MITSUHIRO YOKOYAMA Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School, Kobe, Japan; Division of Molecular Brain Science, Department of Brain Sciences, Kobe University Graduate School, Kobe, Japan; Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan; and MGC, Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands
Circadian clock genes directly regulate expression of the Na+ /H+ exchanger NHE3 in the kidney. Background. Daily rhythms in mammalian physiology are generated by a transcription/translation feedback loop orchestrated by a set of clock genes. However, little is known about the molecular cascade from the clock gene oscillators to cellular function. Methods. The mRNA expression profiles of NHE3 and clock genes were examined in mice and rat kidneys. First, luciferase assays followed by a site directed mutagenesis of an E-box sequence were used to assess the CLOCK:BMAL1-transactivated NHE3 promoter activity. A direct binding of CLOCK:BMAL1 heterodimers to an E-box sequences of NHE3 promoter was confirmed by electrophoretic mobility shift assay (EMSA). Results. We present evidence that renal tubular NHE3, the Na+ /H+ exchanger critical for systemic electrolyte and acidbase homeostasis, is a clock-controlled gene regulated directly by CLOCK:BMAL1 heterodimers in kidneys. NHE3 mRNA level in rat kidney displayed circadian kinetics, and this circadian expression was severely blunted in homozygous CRY1/2 double-deficient mice, suggesting that the transcriptional machinery of peripheral clocks in renal tubular cells directly regulates the circadian expression of NHE3. By analyzing the 5 upstream region of the NHE3 gene, we found an E box critical for the transcription of NHE3 via the CLOCK:BMAL1-driven circadian oscillator. The circadian expression of NHE3 mRNA was reflected by oscillating protein levels in the proximal tubules of the rat kidney. Conclusion. NHE3 should represent an output gene of the peripheral oscillators in kidney, which is regulated directly by CLOCK:BMAL1 heterodimers. 1 The current address for Dr. Rohman is Faculty of Medicine, Brawijaya University, Malang, Indonesia.
Key words: NHE3, clock controlled gene, peripheral clock, circadian, acid-base homeostasis. Received for publication July 22, 2004 and in revised form October 9, 2004 Accepted for publication October 25, 2004 C
2005 by the International Society of Nephrology
Circadian rhythmicity is observed in many aspects of cellular function, including membrane excitation, energy metabolism, and cell division [1–3]. In mammals, the clock system is composed of a central clock in the suprachiasmatic nucleus which generates the standard time of the body at systemic levels, and peripheral clocks which perform the effector processes of the circadian rhythms with the aid of systemic suprachiasmatic nucleus signals in the variety of organs [4, 5]. In both central and peripheral clock systems, circadian rhythmicity is generated at the cellular level by the circadian core oscillator composed of an autoregulatory transcription-(post)translation-based feedback loop involving a set of clock genes. In this molecular loop, two transcriptional activator genes, CLOCK and BMAL1, regulate gene expression by binding to specific enhancer elements, termed E boxes (CTCGTG). Target genes of these activators include several repressor proteins, including PER1, PER2, PER3, CRY1, and CRY2, which function to inhibit the CLOCK:BMAL1 complex, thus generating a circadian oscillation in their own transcription [6–8]. In the kidney, plasma Na+ concentration and renal Na+ excretion are known to display a significant diurnal variation in both animals and humans [9–13]. The mechanisms involving these diurnal changes are related to the regulation of renal blood flow and renal cellular functions, though the underlying molecular mechanism remains unclear. Since a number of genes in the kidney show diurnal variation, we searched for genes likely involved in these processes. NHE3 is one of the Na+ /H+ exchangers (NHEs) that catalyze the electroneutral exchange of one extracellular Na+ for one intracellular H+ across the plasma membrane, to mediate bulk reabsorption of filtered Na+ in the proximal convoluted tubule [14–16]. Gene knockout experiments in mice have been suggested that NHE3 is required for maintenance of
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normal set point for Na+ fluid volume balance. In addition, NHE3 knockout mice shown a decreased blood pressure, an elevated plasma aldosterone and renin mRNA in the kidney [17], thus supporting the view that the major renal transporter mediating Na+ reabsorption plays a central role in long-term control of arterial blood pressure [18, 19]. In the present study, we investigated whether the expression of NHE3 has diurnal variations, and whether it is regulated by the circadian clock system. We demonstrate here that NHE3 mRNA and protein display circadian expression in the rodent kidney. Circadian expression of NHE3 is mediated by the direct control of CLOCK:BMAL1 heterodimers through an E box in the promoter of the NHE3 gene. This conclusion is further supported by genetic studies. In homozygous CRY1/2 double-deficient mice, circadian expression of NHE3 mRNA is significantly reduced. Immunohistochemistry experiments clearly showed the colocalization of NHE3 and PER2 in the proximal tubule cells in kidney. Taken together, these results strongly suggest that the transcriptional regulation of NHE3 is controlled by the core oscillator, and that NHE3 represents a clock-controlled gene (Ccg) in the kidney. METHODS Materials Enzymes used in molecular cloning were obtained from Roche Molecular Biochemicals or from New England Biolabs (Beverly, MA, USA). Animals and procedures Sprague-Dawley rats purchased 5 weeks postpartum were exposed to 2 weeks of 12-hour light (fluorescent light, 300 lux)/dark cycles and then kept in complete darkness (dark/dark) for 2 days as a continuation of the dark phase of the last cycle. Three- to 5-week-old Balb-c and mCry1−/− mCry2−/− mice [20] were housed under the same conditions as Sprague-Dawley rats. The mRNA expression profiles of NHE3 and clock genes were examined in the second dark-dark cycle every 4 hours, starting at the beginning of the light cycle. The care and use of the animals strictly followed the guidelines of the Animal Research Committee of Kobe University Graduate School of Medicine. Northern blot analysis Ten micrograms of total RNA was electrophoresed in a 1.2% denaturing agarose gel containing formaldehyde and transferred onto nylon membranes. The blots were hybridized in QuickHyb hybridization solution (Stratagene, La Jolla, CA, USA) at 68◦ C or in a 50% formamide-containing hybridization solution at 42◦ C. To obtain a specific probe for rat NHE3 (rNHE3), a fragment of the rNHE3 cDNA was amplified by reverse
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transcriptase-polymerase chain reaction (RT-PCR) using primers 5 -GGTCAATGTGGACTTCAGCAC3 and 5 -GGGGAGAACACAGGATTATCAAT-3 (GeneBank, accession number M.85300.1). The clock gene probes were generated as previously described [21]. The probes were labeled with [a 32 P] deoxycytidimine triphosphate (dCTP) using random priming after sequence confirmations, and were exposed to the imaging plates of a Fuji-Bio Imaging Analyzer BAS 2000 (Fuji Photo Film, Kanagawa, Japan). Construction of plasmids The promoter of rNHE3 was constructed by amplification of a 1.36 kb fragment of the 5 flanking region of rNHE3 gene from rat genomic DNA using the primers 5 -TCCAGTTCCTTACCCAGTCAGTCTC-3 and 5 GCTCCAGGAGCCGACACGCATAC-3 . The amplified fragment was digested with BamHI followed by blunt end generation at one end and cut with KpnI at the other. This fragment was directionally cloned into SmaI-KpnI digested picagene basic vector (PGV-B) luciferase reporter (Tokyo, Japan), resulting in a −1360/+58 bp (relative to the transcriptional initiation sites) promoter construct. The constructs spanning −605/+58 bp, −484/+58 bp, −315/+58 bp, and −17/+58 bp were generated by digesting the 1.36 kb fragment of BamHI-KpnI 5 flanking region of rNHE3 with EcoRI, NcoI, SalI, and SmaI, respectively. These fragments were subcloned into the SmaI/KpnI sites of PGV-B and used after confirmation by enzymatic digestion. Deletion of −141/−18 bp fragment was done with BstXI and SmaI to remove the putative promoter region, whereas removal of the −17/+58 bp fragment was accomplished by digestion of the −1360/+58 bp promoter construct with SmaI and KpnI. Clock gene expression constructs were obtained by RT-PCR from the coding regions of mPer2 (AF035830), mCry1 (AB000777), hBMAL1 (AB000813), and hClock (AB002332), as previously described [22, 23]. Mutagenesis of E box Mutant construct was made by site directed mutagenesis [24] using the Muta-Gene Phagemid In Vitro Mutagenesis Kit, version 2 (Bio-Rad Laboratories, Hercules, CA, USA) as described by the manufacturer. All constructs were verified by sequencing the final plasmids. Transcriptional assay Opossum kidney (OK) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 2 mmol/L L-glutamine, Earle’s balanced salt solution (BSS), and 0.1 mmol/L nonessential amino acids supplemented with 10% fetal bovine serum (FBS), 1.5 g/L sodium bicarbonate, and 1.0 mmol/L sodium pyruvate (Sigma Chemical Co., St. Louis, MO, USA). OK cells passages 4 to 10 were plated at 70% to 80% confluency in 60 mm dishes 24 hours before transfection. The cells were transfected with
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3 lg (total) of expression plasmids with the indicated insert in pcDNA3 (Invitrogen, Carlsbad, CA, USA) and 1.7 lg of reporter plasmid using Lipofectamine Plus (Invitrogen) according to the manufacturer’s instructions. To correct for variation in transfection efficiency, pcDNA3 vector was adjusted to the total amount of DNA per dish. Cell extracts were prepared 23 to 24 hours after transfection by a lysis buffer. Twenty microliters of the extract was taken for luciferase assay using a luminometer as described by the manufacturer (Pikkajin). For statistical analysis, a two sample t test was applied. Gel shift assay Nuclear extracts were prepared as described [25]. Briefly, CLOCK:BMAL1-transfected or untransfected OK cells were washed with 5 mL 1 × Tris-buffered saline (TBS) and pelleted by centrifugation at 1500g for 5 minutes. The pellet was resuspended in 1 mL TBS, transferred into an Eppendorf tube, and followed by spinning for 5 seconds. The supernatant was removed and the pellet was dissolved in 40 lL cold buffer A [10 mmol/L Hepes, pH 7.9; 10 mmol/L KCl; 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA); 0.1 mmol/L etheylenglycol tetraacetate (EGTA); 1 mmol/L dithiothreitol (DTT); and 0.5 mnol/L phenylmethylsulfonyl fluoride (PMSF)]. The cells were allowed to swell on ice for 15 minutes, after which 25 lL of a 10% NP-40 was added. The tubes were vigorously vortexed for 10 seconds and centrifuged for 30 seconds at 15,000 rpm. The nuclear pellet was dissolved in 50 lL ice- cold buffer containing 20 mmol/L Hepes, pH 7.9; 0.4 mol/L NaCl; 1 mmol/L EDTA; 1 mmol/L EGTA; 1 mmol/L DTT; and 1 mmol/L PMSF. The nuclear extract was centrifuged at 3000 rpm for 5 minutes, and 7 lL of the supernatant was used for the gel shift assay. Electrophoretic mobility shift assay (EMSA) was carried out according to the manufacturer’s protocol (Promega, Madison, WI, USA) using a doublestranded oligonucleotide: 5 -ACGCAGCAGACTGTTT GCATTCACGTGCGC-3 , including the E-box binding site derived from the rNHE3 promoter. The oligonucleotide was labeled with c adenosine triphosphate (ATP) using T4 polynucleotide kinase. For the competition experiment, a 50-fold excess of double stranded unlabeled nucleotide 5 -ACGCAGCAGACTGTTTGCATTCAC GTGCGC-3 or probe containing a mutated E-box site 5 -ACGCAGCAGACTGTTTGCATTGGATCCCGC3 was used. Immunoblotting Kidneys were homogenized in homogenization buffer (50 mmol/L Tris, pH 7.5; 1 mmol/L EGTA; 250 mmol/L sucrose; 1 mmol/L DTT; 0.1 mmol/L leupeptin; 1 lmol/L pepstatin A; and 0.25 mmol/L PMSF), followed by centrifugation at 1000 × g for 10 minutes at 4◦ C. The super-
natants were centrifuged at 100,000 × g for 30 minutes at 4◦ C, and the pellets containing membrane proteins were resuspended in homogenization buffer. The protein was assayed by a Multiskan JX Thermo Labsystem. Membrane proteins were solubilized in sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% polyacrylamide gels. Immunoblots were incubated in primary antiNHE3 monoclonal antibody (Chemicon Int., Temecula, CA, USA) (MAB3134) at a 1:750 dilution in the same buffer for overnight at 4◦ C. The blots were then washed in washing buffer and incubated in secondary antimouse IgG antibody for 1 hour. The signals were visualized as described [24]. Immunohistochemistry Sections of rat kidney were analyzed at CT8 and CT20 by immunohistochemistry (N = 3 at each time point) for expression of NHE3 protein. Rats were deeply anesthetized with ether and intracardially perfused with ice-cold saline for 2 minutes, followed by a fixative containing 4% paraformaldehyde in 0.1 mol/L phosphate buffer for 5 minutes. The kidneys were removed, postfixed, embedded in paraffin, and cut into 5 lm thick sections. Sections were incubated in anti-NHE-3 antibody (Chemicon Int.) (mouse monoclonal, 1:1000) for 2 days at 4◦ C, then treated with fluorescein isothiocyanate (FITC)conjugated antimouse IgG (1:100) (Vector Laboratories, Burlingame, CA, USA), and observed with confocal fluorescent microscopy (Carl Zeiss AG, Oberkochen, Germany). For double labeling, sections were treated with rabbit anti-PER2 antibody and mouse anti-NHE3 antibody followed by cyanine-3 (Cy3)-conjugated donkey antirabbit IgG and FITC-conjugated donkey antimouse IgG. Slices were washed, mounted with glycerol on a cover glass, and observed under a confocal fluorescence microscope. Data analysis and statistics The significance of differences between groups and among time points was determined by two-way or oneway analysis of variance (ANOVA). Values are considered significantly different if P < 0.05. A test was calculated using Statview software. RESULTS NHE3 transcripts display a circadian rhythmic expression in kidney We observed rhythmic expression of the rat NHE3 (rNHE3) gene, a predominant Na+ /H+ exchanger in the rat kidney. As shown in Figure 1, the amount of rNHE3 mRNA in the rat kidney began to increase at CT4 (circadian time used for assessing biologic time in a complete
Rohman et al: Circadian regulation of NHE3 by peripheral clocks
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darkness cycle, as described in experimental procedures), increasing steadily to the point of highest accumulation at CT16. rNHE3 mRNA levels subsequently decreased and stayed low until CT4. The maximum rNHE3 mRNA level was 2.5-fold higher than the minimal level. Circadian expression of clock gene and NHE3 mRNA is severely blunted in Cry1/Cry2-double mutant mice Next, we examined whether clock genes may control the rhythmic pattern of NHE3 transcription. First, we checked the circadian expression profiles of clock genes in the kidney of wild-type and arrhythmic mCry mutant mice (mCry1−/− and mCry2−/− ). Northern blot analysis revealed that mPer2, the main oscillator gene in the mPer family, was rhythmic at the mRNA level in the kidney of wild-type mice with a peak at CT12-16 and a trough at CT0-4. As expected, mPer2 mRNA no longer cycles in Cry1/Cry2-deficient mice and expression levels are constantly high (Fig. 2A). mBMAL1 mRNA showed an opposite rhythmic expression profile to mPer2 in wild-type mice, and was constantly low in the kidney of Cry1/Cry2deficient mice (Fig. 2B). Next, we performed Northern blot analysis to determine whether the daily rhythm of mouse NHE3 (mNHE3) is abolished in the absence of the circadian pacemaker. In wild-type kidney, the accumulation of mNHE3 fluctuated during the day. In marked contrast to wild-type mice, Cry1/Cry2 double-mutant mice failed to show significant mNHE3 mRNA cycling in the kid-
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ney (Fig. 2C). These results suggest that the circadian rhythmic expression of the mNHE3 gene is blunted in the absence of clock gene rhythmicity in Cry1/Cry2-double mutant mice, and thus must depend on the presence of an intact circadian oscillator. The rNHE3 promoter contains an E box To dissect the molecular mechanism behind the rhythmic expression of NHE3 at the transcriptional level, we first performed a series of assays to determine the important regions of the rNHE3 promoter for transcriptional activity. Constructs containing various lengths of the rNHE3 5 flanking regions inserted upstream of the firely luciferase gene were transiently transfected into OK cells as described in the Methods section. As shown in Figure 3A, a −315/+58 bp construct showed comparable promoter activity to those containing up to 1360 bp of 5 sequence. By contrast, the −17/+58 bp SmaI-KpnI construct displayed substantially decreased promoter activity exhibiting only a threefold activation of luciferase activity compared to the promoterless PGV-B vector. These results suggested that the fragment from −315 to −18 bp is important for basal transcription of rNHE3. To confirm that this promoter region was directly involved in rNHE3 gene expression, the full-length (−1360/+58) reporter construct was digested with BstXI and SmaI to delete bp −141 to −18. Deletion of this region resulted in an 87% decrease in promoter activity as compared to that of the −315/+58 bp construct, suggesting that sequences between bp −141 to –18 are essential for promoter activity of rNHE3 in OK cells. We then examined whether regions further downstream might play an important role in rNHE3 promoter activity by removing the −17/+58 bp fragment. The transcriptional assay showed that deletion of this fragment significantly decreased promoter activity, indicating that the −17 to +58 fragment was required for optimal rNHE3 basal activity even if it was not sufficient to show high levels of promoter activity when expressed alone. Taken together, the transcriptional assay suggested that the region encompassing –141/+58 bp is important for rNHE3 promoter activity in OK cells. We next analyzed the sequence of the −141/+58 bp promoter region of rNHE3 and found that an E-box element (CACGTG), which is the consensus binding site of CLOCK and BMAL1, is located at bp −86 to −81 (Fig. 3B). The rat and mouse NHE3 genes are 92.7% identical in the –107/+58 bp regions with the E-box elements of rat and mouse NHE3 genes being completely conserved. CLOCK and BMAL1 transactivate the rNHE3 promoter through the E-box site The presence of an E box at the promoter region, and the resemblance of the rNHE3 gene expression profile to that of previously reported CLOCK/BMAL1-mediated
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Fig. 2. Circadian rhythms of clock genes and NHE3 mRNA expression are blunted in mCry1−/− mCry2−/− . Northern blot analysis of mPer2 (A), mBMAL1 (B), and mNHE3 (C) in wild-type mice (left panel) and mCry1−/− mCry2/− (right panel). The peak values observed in wild-type mice were adjusted to 100 after normalization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Values are expressed as means ± SE (N = 3). The mice were housed under 12hour light ()/12-hour dark (), Light/dark cycles lasted for at least 2 weeks. CT is circadian time used for assessing biologic time in a dark/dark cycle; CT0 is lights on and CT12 is lights off.
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+1 +58 Fig. 3. The NHE3 promoter contains an E box. (A) The rat NHE3 promoter activity in opossum kidney (OK) cells. Luciferase reporter plasmids containing various lengths of the rat NHE3 5 flanking region were generated and transiently transfected into OK cells as described in the Methods section. Deletion of BstXI-SmaI and SmaI-KpnI fragments, corresponding to bp −141 to −18 and −17 to +58, respectively, resulted in an 87% decrease in promoter activity compared to that of the −314/+58 bp construct, suggesting the –141 to +58 bp fragment is essential for promoter activity of rat NHE3 in OK cells. Luciferase activity is represented as the ratio (mean ± SEM) of the indicated rat NHE3 5 flanking region value to the promoterless picagene basic vector (PGV-B) value. (B) The rat NHE3 promoter region contains an E-box element (CACGTG), the consensus binding site of CLOCK and BMAL1. The E box located at bp −86 to −81 of the rat NHE3 promoter. The rat and mouse NHE3 genes are 92.7% identical in the –107/+58 bp regions with the E-box elements of rat and mouse NHE3 are completely conserved. +1 represents the transcriptional initiation site of the rat NHE3 promoter sequences.
E box-regulated genes (e.g., dbp, mPer1, and vasopression) suggest the possibility that the circadian oscillation of the rNHE3 gene is regulated through this E box. To investigate this possibility, we performed a transient transfection experiment using a 1360 bp fragment of the 5 flanking region of the rNHE3 promoter/luciferase reporter plasmids. Transient transfection of BMAL1 alone resulted in decreased rNHE3 promoter by 35% of basal activity. However, transfection of CLOCK alone did not significantly change rNHE3 promoter activity. Cotransfection of CLOCK and BMAL1 increased rNHE3 promoter activity by five- to sixfold (Fig. 4). Deleting bp −141 to –18 containing an E box of rNHE3 promoter (−1360 −141 ∼−18) markedly diminished this induction by CLOCK and BMAL1 (data not shown). We then generated a point mutation of this E box to determine whether it is important for the transactivation of the rNHE3 promoter by CLOCK and BMAL1. Mutation of the E box completely abolished the CLOCK and BMAL1 mediated transactivation (Fig. 4). Thus, the E-box ele-
ment is important for CLOCK:BMAL1 transactivation of the rNHE3 promoter. To assess whether PER and CRY, the components of the negative limb of the circadian feedback loop, could inhibit CLOCK:BMAL1-driven rNHE3 expression, we cotransfected PER2 and CRY1 with CLOCK-BMAL1 in OK cells. We observed that PER2 alone partially inhibited CLOCK:BMAL1 activation of rNHE3, whereas cotransfection of CRY1 alone abolished the induction of the rNHE3 promoter activity (Fig. 5). These data suggest that PER2 and CRY1 can suppress CLOCK:BMAL1 mediated transactivation of rNHE3 gene expression.
The E box in the rNHE3 promoter directly interacts with the CLOCK:BMAL1 heterodimer To examine whether transcriptional activation of NHE3 by CLOCK:BMAL1 is due to direct binding of CLOCK:BMAL1 to the rNHE3 promoter, we performed a gel shift assay (Fig. 6). When a radiolabeled 30 bp probe,
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Fig. 4. CLOCK and BMAL1 transactivates the rat NHE3 promoter through an E-box site. Transfection of 0.5 lg hCLOCK or hBMAL1 into opossum kidney (OK) cells with the rat NHE3 promoter reporter construct did not increase NHE3 transcriptional activity. Cotransfection of hCLOCK and hBMAL1 increased promoter activity by five ∼sixfold. Mutation of the E box completely abolished the hCLOCK and hBMAL1-mediated transactivation. The E box at bp –86 to –81 was mutated from CACGTG to GGATCC. The results are the mean of three independent experiments. Fold Induction represents the ratio of luciferase activity in cells transfected with expression plasmids to that of cells transfected with empty vector (pcDNA3) (mean ± SEM). The NHE3 construct is 1.36 kb of the rat NHE3 promoter/luciferase plasmids. is the rat NHE3 promoter reporter construct containing an E-box mutation. ∗ P < 0.01 vs. NHE3 construct only: # P < 0.01 vs. NHE3 construct cotransfected with hCLOCK and hBMAL1 into OK cells.
encompassing the putative E box binding site, was incubated with a nuclear extract from untransfected OK cells, only a weak band was observed, which is believed to be due to binding to endogenous proteins. In contrast, nuclear extracts from OK cells transfected with CLOCK and BMAL1 expression vector gave a clear single band. This intense band was abolished by addition of a 50-fold excess of an unlabeled competitor probe containing the E-box consensus sequence, whereas addition of a 50-fold excess of competitor probe containing a mutated E-box sequence had no effect. Taken together, these results suggest that the intense band represents a specific CLOCK:BMAL1-E-box complex, and that CLOCK:BMAL1 transactivates NHE3 through direct binding of CLOCK:BMAL1 to the E box of the NHE3 promoter. Circadian expression of NHE3 protein in the kidney To investigate whether the circadian oscillation of NHE3 is observed at the protein level, we performed immunoblot analysis on the samples obtained from rat kidney (Fig. 7). NHE3 protein levels reached a peak at CT20-24 (0) and a trough at CT8, 8-hour delayed of rNHE3 mRNA levels. These findings demonstrate that
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Fig. 5. PER2 or CRY1 inhibits transactivation of the NHE3 promoter by CLOCK:BMAL1. Cotransfection of 0.3 lg mPER2 or mCRY1 with hCLOCK and hBMAL1 into opossum kidney (OK) cells abolished the induction of the NHE3 promoter. Fold induction represents the ratio (mean ± SE) (N = 3) of luciferase activity in cells transfected with the expression plasmid to that of cells transfected with empty vector (pcDNA3) (mean ± SEM). ∗ P < 0.01 vs. NHE3 construct only; # P < 0.01 vs. NHE3 construct cotransfected with hCLOCk and hBMAL1 into OK cells.
the circadian expression of NHE3 is observed at the protein level as well as the transcriptional level. NHE3 protein colocalizes with PER2 in the kidney NHE3 protein expression was examined in the kidney by immunohistochemistry (Fig. 8A). Similar to previous reports [26, 27], the NHE3 protein was expressed in the epithelial brush border of the proximal convoluted tubules in the renal cortex, and the thick ascending limbs of Henle in the renal medulla. We found that the immunoreactivity in the brush border was higher at CT20 than at CT8, although the NHE3 immunoreactivity in the renal medulla was not altered. To test whether NHE3 and the core clock protein PER2 coexpressed in the same cells, we performed double-labeling histochemical experiments. As shown by Figure 8B, NHE3 staining on the tubular surface and PER2 staining in the nucleus are present in the same cells of the proximal tubule of the kidney. This in vivo study therefore complements our in vitro results that NHE3 is under the direct control of peripheral clock genes, where the CLOCK:BMAL1 heterodimer transactivates the promoter and PER2 inhibits this transactivation. DISCUSSION In the present study, we have provided several lines of evidence showing that NHE3, a well-characterized
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Fig. 6. CLOCK and BMAL1 directly binds the E box of the rat NHE3 promoter. A gel shift assay with a probe encompassing the putative CLOCK:BMAL1 heterodimer E-box binding site, 5 -ACGCAGCAGACTGTTTGCATTCACGTGCGC-3 , were performed as described in the Methods section. For the competition experiments, a 50-fold excess of unlabeled double-stranded oligonucleotide probe or the double-stranded oligonucleotide probe containing a mutated E box (5 -ACGCAGCAGACTGTTTGCATTGGATCCCGC3 ) was added. An arrow indicates the band representing the CLOCK:BMAL1 and probe complex. Arrowheads point to the free probes.
gene with both physiologic and pathologic functions, is a clock controlled gene (Ccg) directly regulated by the CLOCK:BMAL1-driven circadian oscillator in the kidney. First, NHE3 transcripts exhibit an apparent circadian oscillation in the kidney. Second, the rhythmic expression of NHE3 mRNA was blunted in the Cry double mutant mice that are known to have lost the biological clock [20, 28]. Third, luciferase-based transcription assays demonstrated that the NHE3 gene is activated by CLOCK:BMAL1 heterodimers and repressed by PER2 and CRY1 proteins. Fourth, the gel shift assay revealed that CLOCK:BMAL1 heterodimers transactivate NHE3 through direct binding to the E box in the promoter region. Fifth, immunohistochemical analysis showed that NHE3 and PER2 proteins are coexpressed in the same cells within the renal cortex. Finally, the rhythmic expression of NHE3 was observed at the protein level. Considering the fact that NHE3 is a membrane transporter, NHE3 should qualify as an output gene that can be directly linked to the peripheral clockwork in the kidney. It is already known that circadian gene expression is tissue specific and participates in the principal function of individual organs. However, is it also possible that differential regulation within a particular organ might occur to reflect regionally specific functions? In this respect, it is interesting to notice that in contrast to renal cortex, NHE3 immunoreactivity did not significantly change between CT8 and CT20 in the renal medulla. Since PER2
Fig. 7. Circadian oscillation of rNHE3 protein levels. Peak protein expression was set at 100. Representative Western blots (upper panel). Normalized protein levels (mean ± SEM) (N = 6) (lower panel). Peak protein expression was set at 100. One-way analysis of variance (ANOVA) (P = 0.003). Light phase (). Dark phase (). CT is circadian time used for assessing biologic time in a darl/dark cycle; CT0 is lights on and CT12 is lights off.
protein expression is rhythmic in the renal cortex, but arrhythmic in the renal medulla (data not shown), circadian changes of NHE3 protein in the renal cortex may be under the direct control of the negative limbs of the clock genes. Since the amount of NHE3 in the thick ascending limb of Henle is very small as compared to that in the proximal tubule [26, 27], the changes of NHE3 protein expression in whole kidney observed by Western blot likely reflect NHE3 expression in the proximal tubule in the renal cortex. Based on a recent study with a DNA array demonstrating that only a small subset of Ccgs are directly controlled by the core feedback loop in peripheral tissues, NHE3 represents a rare example of Ccg [29]. In mammals, the most established mode of regulation of Ccgs involves the direct control by the core feedback loop of the circadian oscillators through CLOCK:BMAL1 heterodimers (so-called first-order Ccgs) [30]. Examples of such Ccgs include arginine vasopressin (AVP), which encodes a neuropeptide that modulates the firing-rate rhythm from the suprachiasmatic nucleus. The rhythmic expression of AVP mRNA in the suprachiasmatic nucleus appeared to be under the direct control of CLOCK:BMAL1 heterodimers through an E box in its promoter region [31]. It is important to note that the expression of this gene in other parts of the hypothalamus (i.e., a subpopulation of neurons of the paraventricular and supraoptic nuclei) is not regulated in a circadian manner, indicating AVP is a Ccg in the suprachiasmatic nucleus neurons but not in cells in other regions. To our knowledge, NHE3 is the first membrane protein established as a first order Ccg in peripheral tissues.
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A
B
Fig. 8. Expression of NHE3 and PER2 protein in the kidney. (A) Immunohistochemistry of NHE3 at CT8 and CT20. NHE3 protein was expressed in the epithelial brush border of the proximal convoluted tubules (pct), but not in the glomerulus (gl). The immunoreactivity in the brush border was highest at CT20, although the NHE3 immunoreactivity in renal medulla was not altered. Immunohistochemistry was performed as described in the Methods section. Bar indicates 10 lm. (B) Colocalization of NHE3 and PER2. Imunohistochemical localization of NHE3 and PER2 protein in a section of paraformaldehyde-fixed rat kidney embedded in paraffin. Sections were double labeled with antibodies against NHE3 (green) and PER2 (red) as described in the Methods section. Bar indicates 10 lm. Note that nuclear PER2-positive cells also show NHE3 immunoreactivity. CT is circadian time used for assessing biologic time in a dark/dark cycle; CT8 is lights on and CT20 is lights off.
Cloning and characterization of the rat NHE3 gene revealed a variety of modes in its transcriptional regulation. Sequence analysis of the 5 flanking promoter region demonstrated the presence of putative cis-acting elements recognized by various transcription factors, including activating protein (AP)-1, AP-2, C/EBP, glucocorticoid, and thyroid hormone receptors. [32]. In this regard, in the present study, we cannot rule out the possibility that the apparent circadian change of NHE3 expression in vivo may be due to changes in these factors. In fact, diurnal expression of glucocorticoid appears to be controlled by the internal pacemaker, and glucocorticoid is known to regulate the expression of NHE3. However, based on the fact that the regulation of NHE3 expression by glucocorticoid is a rather chronic process [32], it is unlikely that in vivo changes in NHE3 expression is mediated by glucocorticoid. Nevertheless, the transcriptional regulation of NHE3 expression has been extensively studied. However, little attention has been paid to the diurnal variations of its expression. Our study exposes a novel dynamic system of transcriptional regulation of the NHE3 gene in the kidney. In addition to altered expression of NHE3 mRNA and protein, the various factors act in union to achieve the fine regulation of NHE3 activity by controlling phosphorylation of the protein and/or accessory factors, alterations in the surface expression of the exchangers, and changes mediated through the cytoskeleton. For instance, NHE3, in addition to being present at the surface, is detectable in intracellular vesicles [33–35]. A fast and effective way to control the function of cell surface protein,
such as NHE3, is by altering the number of available molecules at the plasma membrane where NHE3 functionally active [32]. In the present study, we have shown that the amount of plasma membrane NHE3 was circadian changed throughout the day. These results were confirmed by immunohistochemistry. Given that hundreds of genes are circadian regulated in the rate-limiting steps for the principal functions of an organ, including components of oxidative phosphorylation and intracellular trafficking, it is possible that post-translational processes in the regulation of NHE3 activity may also be controlled by the circadian clock system [29]. Whether NHE3 activity exhibits a circadian variation remains to be investigated. What is the physiologic relevance for the direct control of NHE3 by the circadian clock system, and hence by the peripheral core oscillator system? The circadian clock system is thought to provide a selective advantage for “prediction” or “anticipation” enabling the organ to prepare for environmental changes, eventually optimizing the appropriate response [36]. Since NHE3 plays an important role in the basic homeostasis of the organism through the regulation of pH, Na+ , and water balance, fine control of the NHE3 activity by the internal clock should allow adaptation to both acute and chronic changes in environment. For instance, peripheral clocks are synchronized mainly by the photoperiod through the suprachiasmatic nucleus clock [29]. However, when feeding is restricted to the light phase in nocturnal mice and rats, peripheral clocks may escape suprachiasmatic nucleus control and be reset by food availability [37]. In such a case, direct control of NHE3 by peripheral
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oscillators has particular value because it enables the local dominant cue (i.e., food availability) to be utilized immediately.
14. 15.
CONCLUSION We have provided evidence demonstrating that NHE3 is a first order Ccg in the peripheral organs. NHE3 appears to be a critical mediator of circadian information since it is regulated in the most direct way through which the core feedback loop could transduce its signal to downstream effectors. Therefore, we predict that NHE3 will be useful as a marker gene for studying temporal regulations of physiology and behavior by clock genes. ACKNOWLEDGMENTS This work was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. G.T.J.van der H. was supported by a VICI grant from The Netherlands Organization for Scientific Research. Reprint requests to Noriaki Emoto, M.D., Ph.D., Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7–5-1, Kusunoki, Chuo, Kobe, 650–0017, Japan. E-mail: [email protected]
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ION CHANNELS – MEMBRANE TRANSPORT – INTEGRATIVE PHYSIOLOGY
Circadian clock genes directly regulate expression of the Na+/H+ exchanger NHE3 in the kidney MOHAMMAD SAIFUR ROHMAN,1 NORIAKI EMOTO, HIDEMI NONAKA, RYUSUKE OKURA, MASATAKA NISHIMURA, KAZUHIRO YAGITA, GIJSBERTUS T.J. VAN DER HORST, MASAFUMI MATSUO, HITOSHI OKAMURA, and MITSUHIRO YOKOYAMA Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School, Kobe, Japan; Division of Molecular Brain Science, Department of Brain Sciences, Kobe University Graduate School, Kobe, Japan; Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan; and MGC, Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands
Circadian clock genes directly regulate expression of the Na+ /H+ exchanger NHE3 in the kidney. Background. Daily rhythms in mammalian physiology are generated by a transcription/translation feedback loop orchestrated by a set of clock genes. However, little is known about the molecular cascade from the clock gene oscillators to cellular function. Methods. The mRNA expression profiles of NHE3 and clock genes were examined in mice and rat kidneys. First, luciferase assays followed by a site directed mutagenesis of an E-box sequence were used to assess the CLOCK:BMAL1-transactivated NHE3 promoter activity. A direct binding of CLOCK:BMAL1 heterodimers to an E-box sequences of NHE3 promoter was confirmed by electrophoretic mobility shift assay (EMSA). Results. We present evidence that renal tubular NHE3, the Na+ /H+ exchanger critical for systemic electrolyte and acidbase homeostasis, is a clock-controlled gene regulated directly by CLOCK:BMAL1 heterodimers in kidneys. NHE3 mRNA level in rat kidney displayed circadian kinetics, and this circadian expression was severely blunted in homozygous CRY1/2 double-deficient mice, suggesting that the transcriptional machinery of peripheral clocks in renal tubular cells directly regulates the circadian expression of NHE3. By analyzing the 5 upstream region of the NHE3 gene, we found an E box critical for the transcription of NHE3 via the CLOCK:BMAL1-driven circadian oscillator. The circadian expression of NHE3 mRNA was reflected by oscillating protein levels in the proximal tubules of the rat kidney. Conclusion. NHE3 should represent an output gene of the peripheral oscillators in kidney, which is regulated directly by CLOCK:BMAL1 heterodimers. 1 The current address for Dr. Rohman is Faculty of Medicine, Brawijaya University, Malang, Indonesia.
Key words: NHE3, clock controlled gene, peripheral clock, circadian, acid-base homeostasis. Received for publication July 22, 2004 and in revised form October 9, 2004 Accepted for publication October 25, 2004 C
2005 by the International Society of Nephrology
Circadian rhythmicity is observed in many aspects of cellular function, including membrane excitation, energy metabolism, and cell division [1–3]. In mammals, the clock system is composed of a central clock in the suprachiasmatic nucleus which generates the standard time of the body at systemic levels, and peripheral clocks which perform the effector processes of the circadian rhythms with the aid of systemic suprachiasmatic nucleus signals in the variety of organs [4, 5]. In both central and peripheral clock systems, circadian rhythmicity is generated at the cellular level by the circadian core oscillator composed of an autoregulatory transcription-(post)translation-based feedback loop involving a set of clock genes. In this molecular loop, two transcriptional activator genes, CLOCK and BMAL1, regulate gene expression by binding to specific enhancer elements, termed E boxes (CTCGTG). Target genes of these activators include several repressor proteins, including PER1, PER2, PER3, CRY1, and CRY2, which function to inhibit the CLOCK:BMAL1 complex, thus generating a circadian oscillation in their own transcription [6–8]. In the kidney, plasma Na+ concentration and renal Na+ excretion are known to display a significant diurnal variation in both animals and humans [9–13]. The mechanisms involving these diurnal changes are related to the regulation of renal blood flow and renal cellular functions, though the underlying molecular mechanism remains unclear. Since a number of genes in the kidney show diurnal variation, we searched for genes likely involved in these processes. NHE3 is one of the Na+ /H+ exchangers (NHEs) that catalyze the electroneutral exchange of one extracellular Na+ for one intracellular H+ across the plasma membrane, to mediate bulk reabsorption of filtered Na+ in the proximal convoluted tubule [14–16]. Gene knockout experiments in mice have been suggested that NHE3 is required for maintenance of
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normal set point for Na+ fluid volume balance. In addition, NHE3 knockout mice shown a decreased blood pressure, an elevated plasma aldosterone and renin mRNA in the kidney [17], thus supporting the view that the major renal transporter mediating Na+ reabsorption plays a central role in long-term control of arterial blood pressure [18, 19]. In the present study, we investigated whether the expression of NHE3 has diurnal variations, and whether it is regulated by the circadian clock system. We demonstrate here that NHE3 mRNA and protein display circadian expression in the rodent kidney. Circadian expression of NHE3 is mediated by the direct control of CLOCK:BMAL1 heterodimers through an E box in the promoter of the NHE3 gene. This conclusion is further supported by genetic studies. In homozygous CRY1/2 double-deficient mice, circadian expression of NHE3 mRNA is significantly reduced. Immunohistochemistry experiments clearly showed the colocalization of NHE3 and PER2 in the proximal tubule cells in kidney. Taken together, these results strongly suggest that the transcriptional regulation of NHE3 is controlled by the core oscillator, and that NHE3 represents a clock-controlled gene (Ccg) in the kidney. METHODS Materials Enzymes used in molecular cloning were obtained from Roche Molecular Biochemicals or from New England Biolabs (Beverly, MA, USA). Animals and procedures Sprague-Dawley rats purchased 5 weeks postpartum were exposed to 2 weeks of 12-hour light (fluorescent light, 300 lux)/dark cycles and then kept in complete darkness (dark/dark) for 2 days as a continuation of the dark phase of the last cycle. Three- to 5-week-old Balb-c and mCry1−/− mCry2−/− mice [20] were housed under the same conditions as Sprague-Dawley rats. The mRNA expression profiles of NHE3 and clock genes were examined in the second dark-dark cycle every 4 hours, starting at the beginning of the light cycle. The care and use of the animals strictly followed the guidelines of the Animal Research Committee of Kobe University Graduate School of Medicine. Northern blot analysis Ten micrograms of total RNA was electrophoresed in a 1.2% denaturing agarose gel containing formaldehyde and transferred onto nylon membranes. The blots were hybridized in QuickHyb hybridization solution (Stratagene, La Jolla, CA, USA) at 68◦ C or in a 50% formamide-containing hybridization solution at 42◦ C. To obtain a specific probe for rat NHE3 (rNHE3), a fragment of the rNHE3 cDNA was amplified by reverse
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transcriptase-polymerase chain reaction (RT-PCR) using primers 5 -GGTCAATGTGGACTTCAGCAC3 and 5 -GGGGAGAACACAGGATTATCAAT-3 (GeneBank, accession number M.85300.1). The clock gene probes were generated as previously described [21]. The probes were labeled with [a 32 P] deoxycytidimine triphosphate (dCTP) using random priming after sequence confirmations, and were exposed to the imaging plates of a Fuji-Bio Imaging Analyzer BAS 2000 (Fuji Photo Film, Kanagawa, Japan). Construction of plasmids The promoter of rNHE3 was constructed by amplification of a 1.36 kb fragment of the 5 flanking region of rNHE3 gene from rat genomic DNA using the primers 5 -TCCAGTTCCTTACCCAGTCAGTCTC-3 and 5 GCTCCAGGAGCCGACACGCATAC-3 . The amplified fragment was digested with BamHI followed by blunt end generation at one end and cut with KpnI at the other. This fragment was directionally cloned into SmaI-KpnI digested picagene basic vector (PGV-B) luciferase reporter (Tokyo, Japan), resulting in a −1360/+58 bp (relative to the transcriptional initiation sites) promoter construct. The constructs spanning −605/+58 bp, −484/+58 bp, −315/+58 bp, and −17/+58 bp were generated by digesting the 1.36 kb fragment of BamHI-KpnI 5 flanking region of rNHE3 with EcoRI, NcoI, SalI, and SmaI, respectively. These fragments were subcloned into the SmaI/KpnI sites of PGV-B and used after confirmation by enzymatic digestion. Deletion of −141/−18 bp fragment was done with BstXI and SmaI to remove the putative promoter region, whereas removal of the −17/+58 bp fragment was accomplished by digestion of the −1360/+58 bp promoter construct with SmaI and KpnI. Clock gene expression constructs were obtained by RT-PCR from the coding regions of mPer2 (AF035830), mCry1 (AB000777), hBMAL1 (AB000813), and hClock (AB002332), as previously described [22, 23]. Mutagenesis of E box Mutant construct was made by site directed mutagenesis [24] using the Muta-Gene Phagemid In Vitro Mutagenesis Kit, version 2 (Bio-Rad Laboratories, Hercules, CA, USA) as described by the manufacturer. All constructs were verified by sequencing the final plasmids. Transcriptional assay Opossum kidney (OK) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 2 mmol/L L-glutamine, Earle’s balanced salt solution (BSS), and 0.1 mmol/L nonessential amino acids supplemented with 10% fetal bovine serum (FBS), 1.5 g/L sodium bicarbonate, and 1.0 mmol/L sodium pyruvate (Sigma Chemical Co., St. Louis, MO, USA). OK cells passages 4 to 10 were plated at 70% to 80% confluency in 60 mm dishes 24 hours before transfection. The cells were transfected with
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3 lg (total) of expression plasmids with the indicated insert in pcDNA3 (Invitrogen, Carlsbad, CA, USA) and 1.7 lg of reporter plasmid using Lipofectamine Plus (Invitrogen) according to the manufacturer’s instructions. To correct for variation in transfection efficiency, pcDNA3 vector was adjusted to the total amount of DNA per dish. Cell extracts were prepared 23 to 24 hours after transfection by a lysis buffer. Twenty microliters of the extract was taken for luciferase assay using a luminometer as described by the manufacturer (Pikkajin). For statistical analysis, a two sample t test was applied. Gel shift assay Nuclear extracts were prepared as described [25]. Briefly, CLOCK:BMAL1-transfected or untransfected OK cells were washed with 5 mL 1 × Tris-buffered saline (TBS) and pelleted by centrifugation at 1500g for 5 minutes. The pellet was resuspended in 1 mL TBS, transferred into an Eppendorf tube, and followed by spinning for 5 seconds. The supernatant was removed and the pellet was dissolved in 40 lL cold buffer A [10 mmol/L Hepes, pH 7.9; 10 mmol/L KCl; 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA); 0.1 mmol/L etheylenglycol tetraacetate (EGTA); 1 mmol/L dithiothreitol (DTT); and 0.5 mnol/L phenylmethylsulfonyl fluoride (PMSF)]. The cells were allowed to swell on ice for 15 minutes, after which 25 lL of a 10% NP-40 was added. The tubes were vigorously vortexed for 10 seconds and centrifuged for 30 seconds at 15,000 rpm. The nuclear pellet was dissolved in 50 lL ice- cold buffer containing 20 mmol/L Hepes, pH 7.9; 0.4 mol/L NaCl; 1 mmol/L EDTA; 1 mmol/L EGTA; 1 mmol/L DTT; and 1 mmol/L PMSF. The nuclear extract was centrifuged at 3000 rpm for 5 minutes, and 7 lL of the supernatant was used for the gel shift assay. Electrophoretic mobility shift assay (EMSA) was carried out according to the manufacturer’s protocol (Promega, Madison, WI, USA) using a doublestranded oligonucleotide: 5 -ACGCAGCAGACTGTTT GCATTCACGTGCGC-3 , including the E-box binding site derived from the rNHE3 promoter. The oligonucleotide was labeled with c adenosine triphosphate (ATP) using T4 polynucleotide kinase. For the competition experiment, a 50-fold excess of double stranded unlabeled nucleotide 5 -ACGCAGCAGACTGTTTGCATTCAC GTGCGC-3 or probe containing a mutated E-box site 5 -ACGCAGCAGACTGTTTGCATTGGATCCCGC3 was used. Immunoblotting Kidneys were homogenized in homogenization buffer (50 mmol/L Tris, pH 7.5; 1 mmol/L EGTA; 250 mmol/L sucrose; 1 mmol/L DTT; 0.1 mmol/L leupeptin; 1 lmol/L pepstatin A; and 0.25 mmol/L PMSF), followed by centrifugation at 1000 × g for 10 minutes at 4◦ C. The super-
natants were centrifuged at 100,000 × g for 30 minutes at 4◦ C, and the pellets containing membrane proteins were resuspended in homogenization buffer. The protein was assayed by a Multiskan JX Thermo Labsystem. Membrane proteins were solubilized in sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% polyacrylamide gels. Immunoblots were incubated in primary antiNHE3 monoclonal antibody (Chemicon Int., Temecula, CA, USA) (MAB3134) at a 1:750 dilution in the same buffer for overnight at 4◦ C. The blots were then washed in washing buffer and incubated in secondary antimouse IgG antibody for 1 hour. The signals were visualized as described [24]. Immunohistochemistry Sections of rat kidney were analyzed at CT8 and CT20 by immunohistochemistry (N = 3 at each time point) for expression of NHE3 protein. Rats were deeply anesthetized with ether and intracardially perfused with ice-cold saline for 2 minutes, followed by a fixative containing 4% paraformaldehyde in 0.1 mol/L phosphate buffer for 5 minutes. The kidneys were removed, postfixed, embedded in paraffin, and cut into 5 lm thick sections. Sections were incubated in anti-NHE-3 antibody (Chemicon Int.) (mouse monoclonal, 1:1000) for 2 days at 4◦ C, then treated with fluorescein isothiocyanate (FITC)conjugated antimouse IgG (1:100) (Vector Laboratories, Burlingame, CA, USA), and observed with confocal fluorescent microscopy (Carl Zeiss AG, Oberkochen, Germany). For double labeling, sections were treated with rabbit anti-PER2 antibody and mouse anti-NHE3 antibody followed by cyanine-3 (Cy3)-conjugated donkey antirabbit IgG and FITC-conjugated donkey antimouse IgG. Slices were washed, mounted with glycerol on a cover glass, and observed under a confocal fluorescence microscope. Data analysis and statistics The significance of differences between groups and among time points was determined by two-way or oneway analysis of variance (ANOVA). Values are considered significantly different if P < 0.05. A test was calculated using Statview software. RESULTS NHE3 transcripts display a circadian rhythmic expression in kidney We observed rhythmic expression of the rat NHE3 (rNHE3) gene, a predominant Na+ /H+ exchanger in the rat kidney. As shown in Figure 1, the amount of rNHE3 mRNA in the rat kidney began to increase at CT4 (circadian time used for assessing biologic time in a complete
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CT rNHE3 GAPDH
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CT Fig. 1. Circadian oscillation of rNHE3 mRNA. Rats were kept in light/dark cycles as described in the Methods section. Total RNA (10 lg) from kidney was used for Northern blot analysis, and the signals were quantified and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative Northern blots (upper panel). Normalized mRNA levels (mean ± SEM) (N = 6) (lower panel). Peak mRNA expression was set at 100. One-way analysis of variance (ANOVA) (P < 0.0001). CT is circadian time used for assessing biologic time in a dark/dark cycle; CT0 is lights on and CT12 is lights off.
darkness cycle, as described in experimental procedures), increasing steadily to the point of highest accumulation at CT16. rNHE3 mRNA levels subsequently decreased and stayed low until CT4. The maximum rNHE3 mRNA level was 2.5-fold higher than the minimal level. Circadian expression of clock gene and NHE3 mRNA is severely blunted in Cry1/Cry2-double mutant mice Next, we examined whether clock genes may control the rhythmic pattern of NHE3 transcription. First, we checked the circadian expression profiles of clock genes in the kidney of wild-type and arrhythmic mCry mutant mice (mCry1−/− and mCry2−/− ). Northern blot analysis revealed that mPer2, the main oscillator gene in the mPer family, was rhythmic at the mRNA level in the kidney of wild-type mice with a peak at CT12-16 and a trough at CT0-4. As expected, mPer2 mRNA no longer cycles in Cry1/Cry2-deficient mice and expression levels are constantly high (Fig. 2A). mBMAL1 mRNA showed an opposite rhythmic expression profile to mPer2 in wild-type mice, and was constantly low in the kidney of Cry1/Cry2deficient mice (Fig. 2B). Next, we performed Northern blot analysis to determine whether the daily rhythm of mouse NHE3 (mNHE3) is abolished in the absence of the circadian pacemaker. In wild-type kidney, the accumulation of mNHE3 fluctuated during the day. In marked contrast to wild-type mice, Cry1/Cry2 double-mutant mice failed to show significant mNHE3 mRNA cycling in the kid-
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ney (Fig. 2C). These results suggest that the circadian rhythmic expression of the mNHE3 gene is blunted in the absence of clock gene rhythmicity in Cry1/Cry2-double mutant mice, and thus must depend on the presence of an intact circadian oscillator. The rNHE3 promoter contains an E box To dissect the molecular mechanism behind the rhythmic expression of NHE3 at the transcriptional level, we first performed a series of assays to determine the important regions of the rNHE3 promoter for transcriptional activity. Constructs containing various lengths of the rNHE3 5 flanking regions inserted upstream of the firely luciferase gene were transiently transfected into OK cells as described in the Methods section. As shown in Figure 3A, a −315/+58 bp construct showed comparable promoter activity to those containing up to 1360 bp of 5 sequence. By contrast, the −17/+58 bp SmaI-KpnI construct displayed substantially decreased promoter activity exhibiting only a threefold activation of luciferase activity compared to the promoterless PGV-B vector. These results suggested that the fragment from −315 to −18 bp is important for basal transcription of rNHE3. To confirm that this promoter region was directly involved in rNHE3 gene expression, the full-length (−1360/+58) reporter construct was digested with BstXI and SmaI to delete bp −141 to −18. Deletion of this region resulted in an 87% decrease in promoter activity as compared to that of the −315/+58 bp construct, suggesting that sequences between bp −141 to –18 are essential for promoter activity of rNHE3 in OK cells. We then examined whether regions further downstream might play an important role in rNHE3 promoter activity by removing the −17/+58 bp fragment. The transcriptional assay showed that deletion of this fragment significantly decreased promoter activity, indicating that the −17 to +58 fragment was required for optimal rNHE3 basal activity even if it was not sufficient to show high levels of promoter activity when expressed alone. Taken together, the transcriptional assay suggested that the region encompassing –141/+58 bp is important for rNHE3 promoter activity in OK cells. We next analyzed the sequence of the −141/+58 bp promoter region of rNHE3 and found that an E-box element (CACGTG), which is the consensus binding site of CLOCK and BMAL1, is located at bp −86 to −81 (Fig. 3B). The rat and mouse NHE3 genes are 92.7% identical in the –107/+58 bp regions with the E-box elements of rat and mouse NHE3 genes being completely conserved. CLOCK and BMAL1 transactivate the rNHE3 promoter through the E-box site The presence of an E box at the promoter region, and the resemblance of the rNHE3 gene expression profile to that of previously reported CLOCK/BMAL1-mediated
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Fig. 2. Circadian rhythms of clock genes and NHE3 mRNA expression are blunted in mCry1−/− mCry2−/− . Northern blot analysis of mPer2 (A), mBMAL1 (B), and mNHE3 (C) in wild-type mice (left panel) and mCry1−/− mCry2/− (right panel). The peak values observed in wild-type mice were adjusted to 100 after normalization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Values are expressed as means ± SE (N = 3). The mice were housed under 12hour light ()/12-hour dark (), Light/dark cycles lasted for at least 2 weeks. CT is circadian time used for assessing biologic time in a dark/dark cycle; CT0 is lights on and CT12 is lights off.
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+1 +58 Fig. 3. The NHE3 promoter contains an E box. (A) The rat NHE3 promoter activity in opossum kidney (OK) cells. Luciferase reporter plasmids containing various lengths of the rat NHE3 5 flanking region were generated and transiently transfected into OK cells as described in the Methods section. Deletion of BstXI-SmaI and SmaI-KpnI fragments, corresponding to bp −141 to −18 and −17 to +58, respectively, resulted in an 87% decrease in promoter activity compared to that of the −314/+58 bp construct, suggesting the –141 to +58 bp fragment is essential for promoter activity of rat NHE3 in OK cells. Luciferase activity is represented as the ratio (mean ± SEM) of the indicated rat NHE3 5 flanking region value to the promoterless picagene basic vector (PGV-B) value. (B) The rat NHE3 promoter region contains an E-box element (CACGTG), the consensus binding site of CLOCK and BMAL1. The E box located at bp −86 to −81 of the rat NHE3 promoter. The rat and mouse NHE3 genes are 92.7% identical in the –107/+58 bp regions with the E-box elements of rat and mouse NHE3 are completely conserved. +1 represents the transcriptional initiation site of the rat NHE3 promoter sequences.
E box-regulated genes (e.g., dbp, mPer1, and vasopression) suggest the possibility that the circadian oscillation of the rNHE3 gene is regulated through this E box. To investigate this possibility, we performed a transient transfection experiment using a 1360 bp fragment of the 5 flanking region of the rNHE3 promoter/luciferase reporter plasmids. Transient transfection of BMAL1 alone resulted in decreased rNHE3 promoter by 35% of basal activity. However, transfection of CLOCK alone did not significantly change rNHE3 promoter activity. Cotransfection of CLOCK and BMAL1 increased rNHE3 promoter activity by five- to sixfold (Fig. 4). Deleting bp −141 to –18 containing an E box of rNHE3 promoter (−1360 −141 ∼−18) markedly diminished this induction by CLOCK and BMAL1 (data not shown). We then generated a point mutation of this E box to determine whether it is important for the transactivation of the rNHE3 promoter by CLOCK and BMAL1. Mutation of the E box completely abolished the CLOCK and BMAL1 mediated transactivation (Fig. 4). Thus, the E-box ele-
ment is important for CLOCK:BMAL1 transactivation of the rNHE3 promoter. To assess whether PER and CRY, the components of the negative limb of the circadian feedback loop, could inhibit CLOCK:BMAL1-driven rNHE3 expression, we cotransfected PER2 and CRY1 with CLOCK-BMAL1 in OK cells. We observed that PER2 alone partially inhibited CLOCK:BMAL1 activation of rNHE3, whereas cotransfection of CRY1 alone abolished the induction of the rNHE3 promoter activity (Fig. 5). These data suggest that PER2 and CRY1 can suppress CLOCK:BMAL1 mediated transactivation of rNHE3 gene expression.
The E box in the rNHE3 promoter directly interacts with the CLOCK:BMAL1 heterodimer To examine whether transcriptional activation of NHE3 by CLOCK:BMAL1 is due to direct binding of CLOCK:BMAL1 to the rNHE3 promoter, we performed a gel shift assay (Fig. 6). When a radiolabeled 30 bp probe,
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Fig. 4. CLOCK and BMAL1 transactivates the rat NHE3 promoter through an E-box site. Transfection of 0.5 lg hCLOCK or hBMAL1 into opossum kidney (OK) cells with the rat NHE3 promoter reporter construct did not increase NHE3 transcriptional activity. Cotransfection of hCLOCK and hBMAL1 increased promoter activity by five ∼sixfold. Mutation of the E box completely abolished the hCLOCK and hBMAL1-mediated transactivation. The E box at bp –86 to –81 was mutated from CACGTG to GGATCC. The results are the mean of three independent experiments. Fold Induction represents the ratio of luciferase activity in cells transfected with expression plasmids to that of cells transfected with empty vector (pcDNA3) (mean ± SEM). The NHE3 construct is 1.36 kb of the rat NHE3 promoter/luciferase plasmids. is the rat NHE3 promoter reporter construct containing an E-box mutation. ∗ P < 0.01 vs. NHE3 construct only: # P < 0.01 vs. NHE3 construct cotransfected with hCLOCK and hBMAL1 into OK cells.
encompassing the putative E box binding site, was incubated with a nuclear extract from untransfected OK cells, only a weak band was observed, which is believed to be due to binding to endogenous proteins. In contrast, nuclear extracts from OK cells transfected with CLOCK and BMAL1 expression vector gave a clear single band. This intense band was abolished by addition of a 50-fold excess of an unlabeled competitor probe containing the E-box consensus sequence, whereas addition of a 50-fold excess of competitor probe containing a mutated E-box sequence had no effect. Taken together, these results suggest that the intense band represents a specific CLOCK:BMAL1-E-box complex, and that CLOCK:BMAL1 transactivates NHE3 through direct binding of CLOCK:BMAL1 to the E box of the NHE3 promoter. Circadian expression of NHE3 protein in the kidney To investigate whether the circadian oscillation of NHE3 is observed at the protein level, we performed immunoblot analysis on the samples obtained from rat kidney (Fig. 7). NHE3 protein levels reached a peak at CT20-24 (0) and a trough at CT8, 8-hour delayed of rNHE3 mRNA levels. These findings demonstrate that
0 NHE3 construct hClock hBMAL1 mPer2 mCry1
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Fig. 5. PER2 or CRY1 inhibits transactivation of the NHE3 promoter by CLOCK:BMAL1. Cotransfection of 0.3 lg mPER2 or mCRY1 with hCLOCK and hBMAL1 into opossum kidney (OK) cells abolished the induction of the NHE3 promoter. Fold induction represents the ratio (mean ± SE) (N = 3) of luciferase activity in cells transfected with the expression plasmid to that of cells transfected with empty vector (pcDNA3) (mean ± SEM). ∗ P < 0.01 vs. NHE3 construct only; # P < 0.01 vs. NHE3 construct cotransfected with hCLOCk and hBMAL1 into OK cells.
the circadian expression of NHE3 is observed at the protein level as well as the transcriptional level. NHE3 protein colocalizes with PER2 in the kidney NHE3 protein expression was examined in the kidney by immunohistochemistry (Fig. 8A). Similar to previous reports [26, 27], the NHE3 protein was expressed in the epithelial brush border of the proximal convoluted tubules in the renal cortex, and the thick ascending limbs of Henle in the renal medulla. We found that the immunoreactivity in the brush border was higher at CT20 than at CT8, although the NHE3 immunoreactivity in the renal medulla was not altered. To test whether NHE3 and the core clock protein PER2 coexpressed in the same cells, we performed double-labeling histochemical experiments. As shown by Figure 8B, NHE3 staining on the tubular surface and PER2 staining in the nucleus are present in the same cells of the proximal tubule of the kidney. This in vivo study therefore complements our in vitro results that NHE3 is under the direct control of peripheral clock genes, where the CLOCK:BMAL1 heterodimer transactivates the promoter and PER2 inhibits this transactivation. DISCUSSION In the present study, we have provided several lines of evidence showing that NHE3, a well-characterized
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Fig. 6. CLOCK and BMAL1 directly binds the E box of the rat NHE3 promoter. A gel shift assay with a probe encompassing the putative CLOCK:BMAL1 heterodimer E-box binding site, 5 -ACGCAGCAGACTGTTTGCATTCACGTGCGC-3 , were performed as described in the Methods section. For the competition experiments, a 50-fold excess of unlabeled double-stranded oligonucleotide probe or the double-stranded oligonucleotide probe containing a mutated E box (5 -ACGCAGCAGACTGTTTGCATTGGATCCCGC3 ) was added. An arrow indicates the band representing the CLOCK:BMAL1 and probe complex. Arrowheads point to the free probes.
gene with both physiologic and pathologic functions, is a clock controlled gene (Ccg) directly regulated by the CLOCK:BMAL1-driven circadian oscillator in the kidney. First, NHE3 transcripts exhibit an apparent circadian oscillation in the kidney. Second, the rhythmic expression of NHE3 mRNA was blunted in the Cry double mutant mice that are known to have lost the biological clock [20, 28]. Third, luciferase-based transcription assays demonstrated that the NHE3 gene is activated by CLOCK:BMAL1 heterodimers and repressed by PER2 and CRY1 proteins. Fourth, the gel shift assay revealed that CLOCK:BMAL1 heterodimers transactivate NHE3 through direct binding to the E box in the promoter region. Fifth, immunohistochemical analysis showed that NHE3 and PER2 proteins are coexpressed in the same cells within the renal cortex. Finally, the rhythmic expression of NHE3 was observed at the protein level. Considering the fact that NHE3 is a membrane transporter, NHE3 should qualify as an output gene that can be directly linked to the peripheral clockwork in the kidney. It is already known that circadian gene expression is tissue specific and participates in the principal function of individual organs. However, is it also possible that differential regulation within a particular organ might occur to reflect regionally specific functions? In this respect, it is interesting to notice that in contrast to renal cortex, NHE3 immunoreactivity did not significantly change between CT8 and CT20 in the renal medulla. Since PER2
Fig. 7. Circadian oscillation of rNHE3 protein levels. Peak protein expression was set at 100. Representative Western blots (upper panel). Normalized protein levels (mean ± SEM) (N = 6) (lower panel). Peak protein expression was set at 100. One-way analysis of variance (ANOVA) (P = 0.003). Light phase (). Dark phase (). CT is circadian time used for assessing biologic time in a darl/dark cycle; CT0 is lights on and CT12 is lights off.
protein expression is rhythmic in the renal cortex, but arrhythmic in the renal medulla (data not shown), circadian changes of NHE3 protein in the renal cortex may be under the direct control of the negative limbs of the clock genes. Since the amount of NHE3 in the thick ascending limb of Henle is very small as compared to that in the proximal tubule [26, 27], the changes of NHE3 protein expression in whole kidney observed by Western blot likely reflect NHE3 expression in the proximal tubule in the renal cortex. Based on a recent study with a DNA array demonstrating that only a small subset of Ccgs are directly controlled by the core feedback loop in peripheral tissues, NHE3 represents a rare example of Ccg [29]. In mammals, the most established mode of regulation of Ccgs involves the direct control by the core feedback loop of the circadian oscillators through CLOCK:BMAL1 heterodimers (so-called first-order Ccgs) [30]. Examples of such Ccgs include arginine vasopressin (AVP), which encodes a neuropeptide that modulates the firing-rate rhythm from the suprachiasmatic nucleus. The rhythmic expression of AVP mRNA in the suprachiasmatic nucleus appeared to be under the direct control of CLOCK:BMAL1 heterodimers through an E box in its promoter region [31]. It is important to note that the expression of this gene in other parts of the hypothalamus (i.e., a subpopulation of neurons of the paraventricular and supraoptic nuclei) is not regulated in a circadian manner, indicating AVP is a Ccg in the suprachiasmatic nucleus neurons but not in cells in other regions. To our knowledge, NHE3 is the first membrane protein established as a first order Ccg in peripheral tissues.
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A
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Fig. 8. Expression of NHE3 and PER2 protein in the kidney. (A) Immunohistochemistry of NHE3 at CT8 and CT20. NHE3 protein was expressed in the epithelial brush border of the proximal convoluted tubules (pct), but not in the glomerulus (gl). The immunoreactivity in the brush border was highest at CT20, although the NHE3 immunoreactivity in renal medulla was not altered. Immunohistochemistry was performed as described in the Methods section. Bar indicates 10 lm. (B) Colocalization of NHE3 and PER2. Imunohistochemical localization of NHE3 and PER2 protein in a section of paraformaldehyde-fixed rat kidney embedded in paraffin. Sections were double labeled with antibodies against NHE3 (green) and PER2 (red) as described in the Methods section. Bar indicates 10 lm. Note that nuclear PER2-positive cells also show NHE3 immunoreactivity. CT is circadian time used for assessing biologic time in a dark/dark cycle; CT8 is lights on and CT20 is lights off.
Cloning and characterization of the rat NHE3 gene revealed a variety of modes in its transcriptional regulation. Sequence analysis of the 5 flanking promoter region demonstrated the presence of putative cis-acting elements recognized by various transcription factors, including activating protein (AP)-1, AP-2, C/EBP, glucocorticoid, and thyroid hormone receptors. [32]. In this regard, in the present study, we cannot rule out the possibility that the apparent circadian change of NHE3 expression in vivo may be due to changes in these factors. In fact, diurnal expression of glucocorticoid appears to be controlled by the internal pacemaker, and glucocorticoid is known to regulate the expression of NHE3. However, based on the fact that the regulation of NHE3 expression by glucocorticoid is a rather chronic process [32], it is unlikely that in vivo changes in NHE3 expression is mediated by glucocorticoid. Nevertheless, the transcriptional regulation of NHE3 expression has been extensively studied. However, little attention has been paid to the diurnal variations of its expression. Our study exposes a novel dynamic system of transcriptional regulation of the NHE3 gene in the kidney. In addition to altered expression of NHE3 mRNA and protein, the various factors act in union to achieve the fine regulation of NHE3 activity by controlling phosphorylation of the protein and/or accessory factors, alterations in the surface expression of the exchangers, and changes mediated through the cytoskeleton. For instance, NHE3, in addition to being present at the surface, is detectable in intracellular vesicles [33–35]. A fast and effective way to control the function of cell surface protein,
such as NHE3, is by altering the number of available molecules at the plasma membrane where NHE3 functionally active [32]. In the present study, we have shown that the amount of plasma membrane NHE3 was circadian changed throughout the day. These results were confirmed by immunohistochemistry. Given that hundreds of genes are circadian regulated in the rate-limiting steps for the principal functions of an organ, including components of oxidative phosphorylation and intracellular trafficking, it is possible that post-translational processes in the regulation of NHE3 activity may also be controlled by the circadian clock system [29]. Whether NHE3 activity exhibits a circadian variation remains to be investigated. What is the physiologic relevance for the direct control of NHE3 by the circadian clock system, and hence by the peripheral core oscillator system? The circadian clock system is thought to provide a selective advantage for “prediction” or “anticipation” enabling the organ to prepare for environmental changes, eventually optimizing the appropriate response [36]. Since NHE3 plays an important role in the basic homeostasis of the organism through the regulation of pH, Na+ , and water balance, fine control of the NHE3 activity by the internal clock should allow adaptation to both acute and chronic changes in environment. For instance, peripheral clocks are synchronized mainly by the photoperiod through the suprachiasmatic nucleus clock [29]. However, when feeding is restricted to the light phase in nocturnal mice and rats, peripheral clocks may escape suprachiasmatic nucleus control and be reset by food availability [37]. In such a case, direct control of NHE3 by peripheral
Rohman et al: Circadian regulation of NHE3 by peripheral clocks
oscillators has particular value because it enables the local dominant cue (i.e., food availability) to be utilized immediately.
14. 15.
CONCLUSION We have provided evidence demonstrating that NHE3 is a first order Ccg in the peripheral organs. NHE3 appears to be a critical mediator of circadian information since it is regulated in the most direct way through which the core feedback loop could transduce its signal to downstream effectors. Therefore, we predict that NHE3 will be useful as a marker gene for studying temporal regulations of physiology and behavior by clock genes. ACKNOWLEDGMENTS This work was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. G.T.J.van der H. was supported by a VICI grant from The Netherlands Organization for Scientific Research. Reprint requests to Noriaki Emoto, M.D., Ph.D., Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7–5-1, Kusunoki, Chuo, Kobe, 650–0017, Japan. E-mail: [email protected]
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