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Originally published In Press as doi:10.1074/jbc.M705495200 on August 9, 2007

J. Biol. Chem., Vol. 282, Issue 41, 29821-29830, October 12, 2007
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1,25-Dihydroxyvitamin D3 Suppresses Renin Gene Transcription by Blocking the Activity of the Cyclic AMP Response Element in the Renin Gene Promoter*

Weihua Yuan{ddagger}, Wei Pan{ddagger}, Juan Kong{ddagger}, Wei Zheng{ddagger}, Frances L. Szeto§, Kari E. Wong§, Ronald Cohen{ddagger}, Anna Klopot{ddagger}, Zhongyi Zhang{ddagger}, and Yan Chun Li{ddagger}1

From the {ddagger}Department of Medicine and §Committee on Molecular Metabolism and Nutrition, Division of Biological Sciences, the University of Chicago, Chicago, Illinois 60637

Received for publication, July 5, 2007 , and in revised form, August 8, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) down-regulates renin expression. To explore the molecular mechanism, we analyzed the mouse Ren-1c gene promoter by luciferase reporter assays. Deletion analysis revealed two DNA fragments from –2725 to –2647 (distal fragment) and from –117 to +6 (proximal fragment) that are sufficient to mediate the repression. Mutation of the cAMP response element (CRE) in the distal fragment blunted forskolin stimulation as well as 1,25(OH)2D3 inhibition of the transcriptional activity, suggesting the involvement of CRE in 1,25(OH)2D3-induced suppression. EMSA revealed that 1,25(OH)2D3 markedly inhibited nuclear protein binding to the CRE in the promoter. ChIP and GST pull-down assays demonstrated that liganded VDR blocked the binding of CREB to the CRE by directly interacting with CREB with the ligand-binding domain, and the VDR-mediated repression can be rescued by CREB, CBP, or p300 overexpression. These data indicate that 1,25(OH)2D3 suppresses renin gene expression at least in part by blocking the formation of CRE-CREB-CBP complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The renin-angiotensin system (RAS)2 plays a central role in the regulation of blood pressure and electrolyte and volume homeostasis (1). Renin, the first and rate-limiting component of the RAS, is a protease synthesized and secreted predominantly by the juxtaglomerular (JG) cells in the kidney. Renin is encoded by one gene in humans. In mice, some strains have one renin gene (e.g. Ren-1c in C57BL/6 strain), and others contain two renin genes (e.g. Ren-1d and Ren-2 in J129 strain), which probably result from a duplication of the 21 kb Ren-1c-like ancestral gene (2). Ren-1 and Ren-2 share 97% amino acid identity (3). The Ren-1 protein is the major source of circulating renin and thus believed to be the major regulator of the renin-angiotensin cascade.

Renin biosynthesis is regulated by a complex network of regulatory proteins (4). Transgenic studies have demonstrated that the cis-DNA elements required for the tissue-specific and developmental stage-specific expression as well as for response to a variety of physiological stimuli are located within 5 kb of the 5'-flanking region of the murine renin gene (5–7). In the 4.1 kb 5'-flanking region of the Ren-1c gene, a proximal minimal promoter (–117 to +6) and a potent 242-bp enhancer (–2866 to –2625) upstream of the transcriptional start site have been shown to be necessary for a high level expression of the renin gene in As4.1 cells (8). Recent studies have revealed the involvement of multiple regulatory proteins in the regulation of renin expression, which include nuclear receptors LXR {alpha} and beta (9, 10), Ear2 (11), and RAR/RXR complex (12), and transcription factors CREB/CREM and USF1/USF2 (13, 14), HOXD10-PBX1b complex and Est-1 (14, 15), Sp1/Sp3 (16), and NF-Y (17).

Cyclic AMP (cAMP) is long known to be a major intracellular signal that stimulates renin production in JG cells. It is well established that cAMP signals through cAMP response elements (CRE) located in target gene promoters, which interacts with members of the ATF/CREB/CREM bZIP transcription factor family in homodimeric or heterodimeric forms. Intracellular cAMP is converted from ATP by adenylate cyclase activated by membrane receptors; cAMP binds to the regulatory subunit of protein kinase A (PKA) to free the catalytic subunit, which enters the nucleus and phosphorylates CREB at serine 133 or CREM at serine 117, resulting in the recruitment of ubiquitous co-activators CBP/p300 to promote gene transcription (18 –20). In fact, a number of CREs have been identified in renin gene promoters that play crucial roles in renin gene transcription. For example, in the human renin gene promoter, a CRE identified at –219 is bound by CREB and is partially responsible for cAMP induction in chorionic cells (21); another study also shows that renin transcription activation by cAMP is partly mediated by a CREB-dependent mechanism via this CRE in Calu-6 cells (22). In the mouse Ren-1d gene promoter, an overlapping cAMP-response element and a negative response element from –619 to –588, known as CNRE, is reported to be bound by LXR{alpha} and respond to cAMP stimulation (10). In the Ren-1c promoter, a CRE at –2688 is bound by CREB/CREM and is essential for the high level of basal renin expression in As4.1 cells (13). cAMP has been reported to stimulate renin gene transcription in As4.1 cells (23).

Our previous studies have demonstrated that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the hormonal form of vitamin D, is a crucial negative regulator of renin biosynthesis (24). The repressive activity of 1,25(OH)2D3 is mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily (25). Mice lacking the VDR or deficient in vitamin D develop hyperreninemia, high blood pressure, cardiac hypertrophy, and other renal abnormalities (24, 26, 27). Analysis of the Ren-1c gene promoter reveals that 1,25(OH)2D3 negatively regulates renin gene transcription (24); however, the molecular mechanism underlying this transcriptional repression remains unclear. In the present study we have addressed this important question by molecular approaches. We found that the transrepression of renin gene expression by 1,25(OH)2D3 is mediated by the CRE within the enhancer in the renin gene promoter. Our study identifies the cAMP signaling pathway, a major regulatory pathway involved in renin biosynthesis, as the target of vitamin D in this negative regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—Construction of the 4.1-kb Ren-1c promoter reporter, pGL4.1k-Luc (see construct A in Fig. 1), has been described previously (24). Deletion constructs of the Ren-1c promoter luciferase reporter were mostly obtained using convenient restriction sites within the DNA sequence of the promoter. Constructs B, C, D, and E were generated by deleting the regions of the Ren-1c 4.1 kb promoter 5' to the restriction sites at –3205 (SpeI), –2673 (BstE II), –1729 (EcoR I) and –1219 (KpnI), respectively. Construct F was made by deleting the fragment from –1729 (EcoRI) to –392 (StuI) from construct B. Constructs G and H were generated by cloning an 803-bp PCR fragment from –3245 to –2442 or a 480-bp PCR fragment from –2922 to –2442, respectively, into the SacI/BglII sites in pGL-117bp plasmid described previously (24). pSBX was generated by deleting the BstXI (–2682)-BglII fragment from construct H; pSBE was generated by deleting the BstEII (–2673)-BglII fragment from construct H; pBXB was generated by deleting the SacI-BstXI (–2682) fragment from construct H. p4-6, p4-7, p5-6, and p5-7 were derived by cloning into the SacI/BglII sites of pGL-117bp the PCR fragments from –2818 to –2647, –2818 to –2671, –2725 to –2647 or –2725 to –2671, respectively, as illustrated in Fig. 1. pSV40E is the pGL3-Control purchased from Promega (Madison, WI). pSV40E-117 was generated by cloning the fragment from –117 to +6 into the Hind III site of pGL3-Enhancer vector (Promega). p3xCRE-117 was generated by inserting a synthetic double-stranded oligonucleotide fragment containing 3 copies of the CRE (5'-TGACATCA-3') of the Ren-1c promoter into the SacI/BglII sites of pGL-117bp, whereas the CRE reporter plasmid p4xCRE-Luc was purchased from Stratagene (La Jolla, CA). p4-6mCRE was generated by mutating the CRE sequence (5'-TGACATCA-3') in p4-6 to 5'-TGt-gATCA-3', and p4-6mRARE generated by mutating one RARE half-site (5'-TGACCT-3') to 5'-TttCCT-3', both using the QuikChange site-directed mutagenesis kit from Stratagene. pcDNA-hVDR(-AF-2) was constructed by cloning PCR-generated hVDR-(1–413) cDNA fragment into pcDNA3.1. pcDNA-hVDRR274L and pcDNA-hVDRR391C were generated by site-directed mutagenesis using pcDNA-hVDR as the template (QuikChange mutagenesis kit). pVP16-CREB was kindly provided by Dr. Richard Goodman (Oregon Health and Science University).

Cell Culture—As4.1, HEK293, and mesangial cells (MC, SV40 MES 13) were purchased from American Type Culture Collection (Manassas, VA). As4.1 cells were JG cells derived from kidney tumors of SV40 T antigen transgenic mice (7). As4.1 cells stably transfected with hVDR, namely As4.1-hVDR cells, has been described previously (24). As4.1 cells stably transfected with hVDR-(1–413), As4.1-hVDR-AF-2, were carried out as described previously (24). As4.1 and HEK293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and penicillin and streptomycin, and MCs were cultured in DMEM/F12 supplemented with 10% heat-inactivated fetal bovine serum and penicillin and streptomycin. No retinoids were present or added in the media. All cells were grown in a 5% CO2 incubator at 37 °C.

Luciferase Reporter Assays—Cells were cultured in 24-well plates to 60–70% confluency and transiently co-transfected with a luciferase reporter plasmid and the pRL-TK plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Six hours after transfection, the cells were treated for 24–36 h with 1,25(OH)2D3 (2 x 10–8 M), forskolin (1 µM), 8-bromo-cAMP (1 µM), H89 (5 µM) or in combinations as indicated in each specific experiment. In some experiments, the cells were co-transfected with expression plasmid for CREB, CBP, or p300 as indicated. Luciferase activity was determined using the Dual-Luciferase Reporter assay system (Promega) and a Luminometer TD-20/20 (Turner Designs). Each transfection was carried out in triplicate and repeated at least three times. Luciferase activity was normalized to the Renilla luciferase activity. In some experiments, pGL3-Basic was included in cell co-transfection for calculation of fold induction. All transfection experiments were repeated at least twice.

Electrophoretic Mobility Shift Assays (EMSA)—As4.1-hVDR cells grown in 10-cm dishes were treated with ethanol or 2 x 10–8 M 1,25(OH)2D3 for 24 h. Nuclear extracts were obtained by following an established method (28). Briefly, cells were scraped and suspended in ice-cold hypotonic solution (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.1 M phenylmethylsulfonyl fluoride) for 10 min on ice and homogenized, and the nuclei were collected by centrifugation. The nuclei were suspended in low salt buffer (20 mM HEPES, pH 7.9, 20% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol). KCl concentration was then adjusted to 0.3 M by addition of 4 M KCl, and the nuclear suspension was rocked at 4 °C for 30 min. The nuclear extract was collected by centrifugation at 25,000 x g. For EMSA, double-stranded oligodeoxynucleotide probes were end-labeled with [{gamma}-32P]ATP using polynucleotide kinase and purified with spin columns. Probe A is 5'-ATGGTGACCTGGCTGTACTCTGACCTCTG-3' (Underlined are the RAR and RXR binding sites); Probe B is 5'-AATCCTCCCAATGACATCACTAACCA-3' (the CRE is underlined). The labeled probe was incubated with nuclear extracts (10 µg) at room temperature for 30 min in the presence of 100 µg/ml polyd(I:C), and the mixture was electrophoresed on a 4% non-denatured polyacrylamide gel (28). Specificity of protein-DNA interaction was confirmed by competition with excess unlabeled probes. Incubation with anti-CREB-1 or anti-CREM-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was also used to confirm the presence of the corresponding protein in the DNA-protein complex. The gel was dried and exposed to x-ray film for autoradiography.

Chromatin Immunoprecipitation (ChIP) Assays—ChIP assays were performed using a commercial kit from Upstate%20Biotechnology">Upstate Biotechnology (Lake Placid, NY) with some modifications. Briefly, As4.1-hVDR cells were treated with ethanol or 2 x 10–8 M 1,25(OH)2D3. After 24 h, the cells were treated with 1% formaldehyde to cross-link the histones and genomic DNA. The cells were lysed and sonicated to shear the chromatin. The sonicated chromatin was incubated with anti-CREB-1, anti-CREM-1, anti-CBP, or anti-VDR antibody (Santa Cruz Biotechnology) or with non-immune IgG as controls. After an overnight incubation, the chromatin-antibody complex was precipitated with protein-A-agarose beads and digested with proteinase K to remove the proteins. The DNA isolated from the complex was subject to PCR amplification using primers flanking the CRE site in the Ren-1c gene promoter: 5'-–2786GAACTTGTAGGTCCTGCCCG-3' (forward) and 5'-–2567CAAACTATAGATCAGGCAGG-3' (reverse), or primers flanking the VDRE in Cyp24a1 gene promoter reported previously (29): 5'-GGTTATCTCCGGGGTGGAGT-3' (forward) and 5'-AGTGGCCAATGAGCACGC-3' (reverse). The PCR products were run on a 2% agarose gel and stained with ethidium bromide.

Northern Blot—Renin mRNA was determined by Northern blot analysis as described previously (24). Briefly, As4.1 cells were treated with different reagents as indicated for 24 h, and total cellular RNAs were extracted using the TRIzol reagent (Invitrogen). The RNAs were separated on agarose gel and transferred onto nylon membranes. The membranes were hybridized with 32P-labeled renin cDNA probe. The membranes were then stripped and rehybridized with 36B4 cDNA probe for internal loading control. The mRNA transcripts were detected by autoradiography.

GST Pull-down Assays—pGEX-hVDR was constructed by cloning a PCR-generated cDNA sequence encoding the hinge and ligand-binding domain (LBD) of hVDR (amino acids 110–427) into pGEX-4T-1 (Amersham Biosciences) as reported previously (30). pGEX-4T-1 and pGEX-hVDR were transformed into Escherichia coli BL21 bacteria and GST and GST-LBD-VDR fusion proteins were purified using glutathione-Sepharose 4B beads (Amersham Biosciences) according to the instruction from the manufacturer, and protein purity was determine by SDS-PAGE, and stained with Coomassie Blue. For the assay, HEK293 cells were transfected with pCMV-CREB using Lipofectamine 2000. After cultured for 24 h, the transfected cells were lysed with phosphate-buffered saline containing 0.5% Triton X-100, 10 mM NaF, 2 mM Na3VO4, and 2 mM phenylmethylsulfonyl fluoride. For binding, GST or GST-LBD-VDR beads were mixed with 500 µg of lysates containing a proteinase inhibitor mixture, and the mixture was rotated at 4 °C for 2 h in the absence or presence of 10–7 M 1,25(OH)2D3 in the binding buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA pH 8.0, and 0.5% Nonidet P-40. The beads were precipitated by centrifugation and washed four times with 0.5 ml of binding buffer. The beads were then suspended in Laemmli buffer, and proteins were resolved by SDS-PAGE (31). After being transfered onto an Immobilon-P membrane, CREB was visualized by Western blot assay using anti-CREB antibody.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Two DNA Fragments in the Ren-1c Promoter Mediate Renin Transrepression by 1,25(OH)2D3—Our previous studies have shown that 1,25(OH)2D3 suppresses renin gene transcription (24). To further explore the molecular mechanism of this transrepression, we systematically analyzed the 4.1-kb 5'-flanking region of the Ren-1c gene by a sequential deletion strategy. The 4.1-kb Ren-1c promoter contains all cis-elements required for tissue-specific and developmental stage-specific expression of the renin gene, as well as for response to a variety of physiological stimuli (5–7), and we showed that its activity was dramatically suppressed by 1,25(OH)2D3 (24) (Fig. 1A, construct A). A series of deletion constructs were generated and used to transfect As4.1-hVDR cells for luciferase reporter assays. As shown in Fig. 1A, all constructs that contain the previously identified 242-bp enhancer region between –2866 and –2625 (8) (i.e. constructs A, B, F, G, and H) maintained a high basal activity, and their activity was markedly suppressed by 1,25(OH)2D3 treatment. In contrast, in constructs that lack this enhancer region (i.e. constructs C, D, and E), the basal activity was decreased drastically as expected, and the activity was barely suppressed by 1,25(OH)2D3 (Fig. 1A).

To narrow down the DNA fragments that mediate the vitamin D transrepression, we subjected construct H, the shortest construct in this series of deletion, to further deletion analyses. As shown in Fig. 1B, once again, luciferase constructs (i.e. pBXB and p5-7) lacking the enhancer region had little activity, while the activity of all enhancer-containing constructs was high and suppressed by 1,25(OH)2D3. These studies revealed that two DNA fragments in the Ren-1c promoter, a 78-bp fragment from –2725 to –2647 that contains a CRE at –2688 (designated as distal fragment, see Fig. 4A for the sequence) and a 121-bp fragment from –117 to +6 that is the minimal promoter (designated as proximal fragment), together were sufficient to mediate 1,25(OH)2D3-induced transrepression (see construct p5-6 in Fig. 1B). When the distal fragment was replaced with SV40 enhancer (pSV40E-117) or three copies of the CRE sequence (p3xCRE-117 in Fig. 1C), the basal activity was dramatically reduced as expected; interestingly, however, in contrast to the pSV40E construct that was made of SV40 enhancer and promoter, 1,25(OH)2D3 was able to inhibit the activity of pSV40E-117 and p3xCRE-117 by 19 and 38%, respectively (Fig. 1C). These data suggest that both the minimal promoter and the CRE are crucial for this negative regulation. We mainly focused on the distal fragment in this study.


Figure 1
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  		FIGURE 1.
Deletion analysis of the renin gene promoter reveals two DNA fragments that are sufficient to mediate vitamin D suppression of renin gene transcription. As4.1-hVDR cells were transfected with each luciferase reporter plasmid construct that contains different regions of the 4.1-kb Ren-1c illustrated schematically on the left. The transfected cells were treated with ethanol (open box) or 1,25(OH)2D3 (2 x 10–8M)(filled box) for 36 h before luciferase activity was determined. Data were representative of at least three independent experiments. In the plasmid constructs, the open box at the right end represents the minimal promoter, and dashed lines represent deleted sequences between two DNA fragments. The nucleotide positions of each DNA fragment in the constructs are indicated by numbers. A, systematic deletion analysis of 4.1-kb Ren-1c gene promoter. Constructs A–H contain different regions of the 4.1-kb promoter as indicated. The fragment from –2866 to –2625, indicated by two vertical dashed lines, is the 242-bp enhancer identified previously to be important for renin gene expression. B, deletion analysis of construct H. The CRE at –2688 is indicated by a vertical dashed line. Note a 78-bp CRE-containing fragment from –2725 to –2647 and the minimal promoter (see construct p5-6) are sufficient to mediate the transcriptional repression induced by 1,25(OH)2D3. C, reporter constructs used to address the role of the 117-bp minimal promoter and the CRE in the transcriptional repression induced by 1,25(OH)2D3.

 
The CRE Is Required for 1,25(OH)2D3-induced Transrepression—Cyclic AMP is long known to be a potent stimulator of renin biosynthesis. The CRE within the distal fragment is known to play a critical role in basal renin expression in As4.1 cells (13). To test whether vitamin D suppresses renin expression by targeting the CRE, we determined the effect of 1,25(OH)2D3 using the p5-6 reporter construct, which contained the CRE in the distal fragment (Fig. 2A). As expected, 1,25(OH)2D3 markedly inhibited the promoter activity in As4.1-hVDR cells (Fig. 2B). Because As4.1 cells did not respond to forskolin (Fig. 2B) because of the constitutively active PKA pathway as reported previously (13), we explored the activity using mesangial cells (MCs) and HEK293 cells. In these cells, the promoter activity was dramatically induced by forskolin, and the induction was markedly attenuated by 1,25(OH)2D3 (Fig. 2, C and D). One difference between these cell lines was that 1,25(OH)2D3 always inhibited the promoter activity much more in As4.1 cells (by 70–80%) than in MCs or HEK293 cells (by 40–50%). As expected, forskolin induction was blocked by H89, a PKA inhibitor (Fig. 2E). Therefore, the CRE in the distal fragment can be activated by the PKA pathway and suppressed by vitamin D. Similar vitamin D regulation was observed using p4xCRE-Luc reporter, a universal CRE reporter that contains 4 copies of consensus CRE sequence 5'-TGACGTCA-3' (data not shown).

To validate that the CRE is directly involved in the repression of renin gene induced by 1,25(OH)2D3, we mutated the CRE site by site-directed mutagenesis in the p4-6 reporter construct to generate p4-6mCRE (Fig. 2F). We chose p4-6 because of its high basal activity (Fig. 1B), so that the activity of the mutant construct would still be high enough for detection. Indeed, mutation of the CRE site in p4-6 reporter reduced the activity by >80%. Interestingly, the CRE mutation markedly diminished, but did not completely eliminate, the repressive effect of 1,25(OH)2D3 in As4.1 cells. 1,25(OH)2D3 inhibited p4-6 activity by 80% and p4-6mCRE by 30% (Fig. 2G, compare p4-6 and p4-6mCRE), suggesting that both the CRE and the proximal fragment are critical for vitamin D inhibition of renin expression. Because the cAMP-PKA signaling pathway is constitutively active in As4.1 cells (13), we used MCs to confirm the data. As expected, forskolin dramatically stimulated the activity of p4-6, and 1,25(OH)2D3 suppressed the basal as well as forskolin-induced p4-6 activity in MCs (Fig. 2H). Most interestingly, when p4-6mCRE was used to transfect MCs, neither forskolin stimulation nor vitamin D inhibition was observed (Fig. 2I), confirming that the CRE is absolutely required to mediate the suppression of renin transcription by 1,25(OH)2D3. Unlike As4.1 cells, mutation of the CRE completely eliminated the vitamin D repression in MCs, suggesting the existence of profound differences in renin gene regulation in As4.1 cells and MCs. In fact, renin mRNA expression in MCs is very low and only detectable by RT-PCR (data not shown).

A previous study identified an unconventional RARE within the 242-bp enhancer that is bound by RAR/RXR heterodimer and plays a critical role in renin gene expression (12). This RARE is composed of two TGACCT half-sites separated by 10 nucleotides and is 16-bp downstream of the CRE (see Fig. 4A for details). To address whether this RARE is involved in the vitamin D-induced renin gene transrepression, we mutated the second half-site of the RARE to generate p4-6mRARE reporter (Fig. 2J), and then determined the mutant reporter activity in response to 1,25(OH)2D3 treatment. A similar mutation has been shown previously to disrupt RAR/RXR binding and reduce renin expression in As4.1 cells (12). Interestingly, although the basal activity of p4-6mRARE was reduced by about 75% compared with that of p4-6 reporter as expected, the RARE mutation has no effect on vitamin D-induced suppression of the reporter activity (Fig. 2K). This result indicates that the RARE is unlikely to mediate the transrepression induced by vitamin D.


Figure 2
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  		FIGURE 2.
The CRE in renin gene promoter is required for 1,25(OH)2D3-induced transrepression of renin gene expression. A–E, 1,25(OH)2D3 suppresses the CRE-containing renin enhancer. The reporter plasmid p5-6 (A) was used to transfect As4.1-hVDR cells (B), MCs (C), and HEK293 cells (D and E). F–I, mutation of the CRE diminishes 1,25(OH)2D3-induced transrepression. Reporter plasmid p4-6 or p4-6mCRE (F) was used to transfect As4.1-hVDR cells (G) or MCs (H and I), respectively, as indicated. J and K, mutation on the RARE site has not effect on 1,25(OH)2D3-induced transrepression. Reporter plasmid p4-6 or p4-6-mRARE (J) was used to transfect As4.1-hVDR cells (K). In all experiments, the transfected cells were treated with ethanol (C), 1,25(OH)2D3 (2 x 10–8 M) (VD), forskolin (1 µM)(F), forskolin and 1,25(OH)2D3 (F+VD), H89 (1 µM), or forskolin and H89 (F+H89) as indicated for 24 h before luciferase activity was determined. Luciferase activity is expressed as percentage of the ethanol-treated control. *, p < 0.01 versus C or F, respectively; **, p < 0.01 versus C.

 


Figure 3
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  		FIGURE 3.
Blocking the cAMP-PKA pathway causes a synergistic effect on vitamin D repression of renin gene expression. A, As4.1-hVDR cells were treated with ethanol (C), 1,25(OH)2D3 (2 x 10–8 M)(VD), forskolin (1 µM)(F), 8-bromo-cAMP (8-Br-cAMP, 5 µM), H89 (1 µM), forskolin, and 1,25(OH)2D3 (F+VD), 8-bromo-cAMP and 1,25(OH)2D3, or H89 and 1,25(OH)2D3 as indicated for 24 h. Total cellular RNAs were isolated, and renin mRNA expression was determined by Northern blotting (15 µg of RNA/lane). B, As4.1-hVDR cells were transfected with p5-6 reporter plasmid, and the transfected cells were treated with ethanol (C), H89, forskolin, 1,25(OH)2D3, forskolin, and 1,25(OH)2D3 or H89 and 1,25(OH)2D3 as indicated. Luciferase activity was determined after 24 h.

 
We further explored the relationship between the vitamin D and the cAMP-PKA signaling pathways in As4.1 cells by measuring renin mRNA expression under a variety of conditions. As shown in Fig. 3, neither forskolin nor 8-bromo-cAMP markedly up-regulated renin mRNA expression; however, when the PKA pathway was blocked by H89, renin mRNA was markedly reduced. Interestingly, combined treatment with 1,25(OH)2D3 and H89 inhibited renin mRNA expression more than treatment with either of these agents alone (Fig. 3A). Similar findings were observed in luciferase reporter assays with p5-6 transfection (Fig. 3B). This synergistic effect suggests that vitamin D likely counters the cAMP-PKA pathway in the regulation of renin gene expression; however, vitamin D is unlikely to directly target PKA.

1,25(OH)2D3 Inhibits Nuclear Protein Binding to CRE Site—In the 78-bp distal fragment that is required for vitamin D transrepression, a few cis-DNA elements have been identified that are crucial for the basal renin gene transcription, including the CRE and RARE (12, 13) (Fig. 4A). To explore the molecular mechanism underlying the vitamin D-induced transrepression, we used EMSA to investigate the effect of 1,25(OH)2D3 on the interaction between nuclear proteins and cis-DNA elements. For the EMSA, nuclear extracts isolated from ethanolor 1,25(OH)2D3-treated As4.1-hVDR cells were incubated with 32P-labeled probe A or probe B that covered the RARE or CRE, respectively (Fig. 4A). The results showed that 1,25(OH)2D3 had no effects on the protein-DNA interaction on the RAR/RXR binding sites (Fig. 4B), consistent with the above observation that mutation of the RARE had no effect on renin transrepression by vitamin D. In contrast, 1,25(OH)2D3 markedly reduced the formation of DNA-protein complex on the CRE site (Fig. 4C). The complex was competed off with an excess amount (50x and 100x) of unlabeled probe B, confirming the specificity of the DNA-protein interaction. Moreover, inclusion of antibody against CREB or CREM in the incubation reaction either reduced the formation of the complex (for anti-CREB antibody, lanes 4 and 6, Fig. 4D) or created a supershift band (for anti-CREM antibody, lanes 5 and 7, Fig. 4D) in both ethanol- and 1,25(OH)2D3-treated nuclear extracts, confirming the presence of CREB and CREM in the complex (13). These observations suggest that 1,25(OH)2D3 directly interferes with protein binding to the CRE, leading to a reduction of renin gene transcription.


Figure 4
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  		FIGURE 4.
1,25(OH)2D3 reduces the binding of nuclear proteins to the CRE. A, nucleotide sequence from –2725 to –2647 in the Ren-1c gene promoter. The CRE, RAR, and RXR binding sites are bolded and underlined, and the sequences of Probe A or Probe B that contains the RAR and RXR sites or the CRE, respectively, are overlined. B, EMSA using Probe A and nuclear extracts isolated from As4.1-hVDR cells treated with ethanol (E) or 1,25(OH)2D3 (2 x 10–8 M) (VD) for 24 h. FP, free probe. Arrows indicate specific bands. C, EMSA using Probe B and nuclear extracts isolated from ethanol- or 1,25(OH)2D3-treated As4.1-hVDR cells. The DNA-protein interaction was competed with 50-fold (50x) or 100-fold (100x) unlabeled Probe B. D, EMSA with Probe B and ethanol- or 1,25(OH)2D3-treated nuclear extracts, in the presence or absence of anti-CREB-1 or anti-CREM-1 antibody as indicated. Note anti-CREB-1 antibody reduces the DNA-protein interaction (compare lanes 2 and 3 with 4 and 6), whereas anti-CREM-1 antibody produces a supershift (lanes 5 and 7).

 


Figure 5
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  		FIGURE 5.
Liganded VDR blocks the formation of CRE-protein complex in renin gene promoter by directly binding to CREB. A, schematic illustration of the PCR primers used in the ChIP assays that flank the CRE in the Ren-1c gene promoter. The numbers indicate the positions of the most 5' and the most 3' nucleotides of the two primers, respectively. B, ChIP assays to detect the proteins bound to the CRE in the Ren-1c promoter. C, ChIP assays to detect VDR binding to the VDRE in the Cyp24a1 gene promoter. For ChIP assays, As4.1-hVDR cells were treated with ethanol (–) or 2 x 10–8 M 1,25(OH)2D3 (+) for 24 h. Sonicated chromatin was precipitated with antibody (Ab) against CREB, CREM, CBP, or VDR, or with normal IgG as indicated. Input represents 5% of the total amount of chromatin. All PCRs were carried out for 35 cycles. D, Western blot analysis of the protein level of VDR, CREB, CREM, p-CREB, and RXR in As4.1-hVDR cells treated with ethanol (E) or 1,25(OH)2D3 (VD). E, purification of bacterially expressed GST and GST-LBD-VDR fusion proteins. The purified proteins were stained with Coomassie Blue on SDS-PAGE gel. F, analysis of VDR-CRE interaction by GST pull-down assay. Cell lysates of HEK293 cells transfected with pCMV-CREB plasmid were incubated with GST or GST-LBD-VDR beads in the presence of ethanol or 10–7 M 1,25(OH)2D3 as indicated. The beads were then subject to SDS-PAGE and Western blot analysis with anti-CREB antibody. G-VDR, GST-LBD-VDR. G, As4.1-hVDR cells were co-transfected with p4-6 reporter and 50 µgof pcDNA3.1, CREB, or VP16-CREB expression plasmid. Transfected cells were then treated with ethanol (E) or 1,25(OH)2D3 (VD), and luciferase activity was determined after 24 h. *, p < 0.01 versus corresponding E; **, p < 0.01 versus pcDNA3.1 control.

 
Liganded VDR Blocks the Formation of CRE-Protein Complex by Directly Binding to CREB—We further utilized ChIP assays to explore the mechanism underlying the EMSA results. The PCR primers for the ChIP assays were designed to flank the CRE site in the distal fragment of the Ren-1c gene (Fig. 5A). As shown in Fig. 5, in the absence of 1,25(OH)2D3, the chromatin fragment that contained this CRE was precipitated by antibody against CREB, CREM, or CBP, but not by anti-VDR antibody or non-immune IgG (Fig. 5B), indicating that the CRE was bound by CREB, CREM and CBP, but not by VDR, in the basal state to drive renin transcription. These data confirmed that the cAMP-PKA pathway is indeed constitutively active in As4.1 cells. Remarkably, treatment of the As4.1-hVDR cells with 1,25(OH)2D3 completely blocked the binding of CREB, CREM, and CBP to the CRE site (Fig. 5B), indicating that 1,25(OH)2D3 suppressed renin gene transcription, at least in part, by direct inhibition of CRE-mediated transcriptional activity.

Because VDR was not associated with the CRE regardless of the presence or absence of 1,25(OH)2D3, VDR was unlikely involved in the formation of the protein-DNA complex on the CRE site. As a positive control for VDR, 1,25(OH)2D3 induced VDR binding to the VDRE in the Cyp24a1 gene promoter in As4.1 cells (Fig. 5C), consistent with our previous observation that 1,25(OH)2D3 induced vitamin D 24-hydroxylase expression in these cells (32). Moreover, 1,25(OH)2D3 treatment did not seem to markedly change the protein level of CREB, CREM, or RXR or the phosphorylation of CREB (p-CREB) in As4.1-hVDR cells (Fig. 5D).

VDR is known to physically interact with a variety of regulatory proteins such as Smad3, beta-catenin, and p65 NF-{kappa}B (33–36). To explore the possibility that VDR interacts with CREB to block CRE complex formation, we generated and purified GST-LBD-VDR fusion protein that contains the LBD of the VDR protein (Fig. 5E). GST and GST-LBD-VDR were then incubated with cell lysates transfected with CREB. As expected, GST did not pull down CREB regardless the presence or absence of 1,25(OH)2D3; interestingly, GST-LBD-VDR interacted with CREB and the interaction was markedly enhanced in the presence of 1,25(OH)2D3 (Fig. 5F). This finding suggests that liganded VDR blocks CREB binding to CRE by directly interacting with CREB.

To confirm that 1,25(OH)2D3 functionally blocks CREB binding to CRE, we compared the effect of 1,25(OH)2D3 on CREB and VP16-CREB activity. VP16-CREB consists of the transcriptional activation domain of the herpes simplex protein VP16 fused to the basic/leucine zipper dimerization and DNA-binding domain of CREB (37). As expected, co-transfection of As4.1-hVDR cells with CREB or VP16-CREB expression plasmid led to significant increase in p4-6 luciferase activity, which was suppressed by 68 and 57%, respectively, in the presence of 1,25(OH)2D3 (Fig. 5G). Thus the magnitude of CREB and VP16-CREB inhibition is essentially the same, suggesting that 1,25(OH)2D3 suppresses renin transcription by blocking CREB DNA binding.

VDR LBD Is Required for Renin Transrepression—Because CREB binds to the LBD of VDR, we asked whether the LBD is required for renin repression. First we stably transfected As4.1 cells with hVDR-(1–413) lacking the AF-2 domain (Fig. 6A), and selected two stable clones (70 and 84) that expressed hVDR-(1–413) at the same level as the wild-type hVDR (clone 57) for study (Fig. 6B). The hVDR-(1–413) protein retains the DNA- and ligand-binding capacity but lacks the capacity to interact with beta-catenin (38). In contrast to the clone (57) harboring the full-length hVDR, treatment of clones expressing hVDR(–AF-2) with 1,25(OH)2D3 did not reduce renin mRNA expression (Fig. 6C). Then we tested two LBD mutants at R391C and R274L (Fig. 6A) using p4-6-Luc luciferase reporter assays in As4.1 cells. As expected, 1,25(OH)2D3 had little effect on renin promoter activity in cells co-transfected with pcDNA3.1 empty vector; however, the promoter activity was markedly reduced by 1,25(OH)2D3 when the cells were co-transfected with wild-type hVDR. Interestingly, renin promoter activity was not significantly affected by 1,25(OH)2D3 in cells co-transfected with hVDRR391C or hVDRR274L (Fig. 6D). These results indicate that a functional LBD is required to mediate the transrepression by 1,25(OH)2D3.

1,25(OH)2D3 Suppression Is Attenuated by CREB, CBP, or p300 Overexpression—We further tested whether an excess amount of CREB can overcome the suppression of CREB binding to CRE by co-transfecting As4.1-hVDR cells with p4-6 reporter and CREB, CBP or p300. As shown in Fig. 7, 1,25(OH)2D3 suppressed the reporter activity by about 80% in the absence of co-transfection, and CREB, CBP, or p300 co-transfection overcame the repression dose-dependently. Interestingly, the suppression was completely reversed by low doses (100 ng) of CREB expression plasmid (Fig. 7A); in the cases of CBP and p300, high doses (500 ng) of plasmid were needed to reverse the repression (Fig. 7B). This observation is consistent with the fact that CREB directly binds to the CRE, whereas CBP and p300 rely on CREB for action. These results further support that liganded VDR suppresses renin gene transcription by blocking CREB DNA binding.


Figure 6
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  		FIGURE 6.
VDR LBD is required to mediate renin gene transrepression induced by 1,25(OH)2D3. A, schematic illustration of the hVDR mutations. The DNA-binding, ligand-binding, and AF-2 domains are indicated. Stars indicate point mutations. B, Western blot analyses of parental As4.1 cells (P) and As4.1 stable clones expressing wild-type hVDR (57) and hVDR-(1–413) that lacks the AF-2 domain (70 and 84) with anti-VDR antibody. C, renin mRNA expression in clones 57, 70, and 84 treated with ethanol (E) or 1,25(OH)2D3 (2 x 10–8 M) for 24 h (VD). D, Luciferase reporter assays to determine the activity of hVDR mutants. As4.1 cells were co-transfected with p4-6-Luc and pcDNA3.1, pcDNA-hVDR, pcDNA-hVDRR391C, or pcDNA-hVDRR274L as indicated, and the cells were then treated with ethanol (E) or 1,25(OH)2D3 (2 x 10–8 M) (VD). Luciferase activity was determined after 24 h. *, p < 0.01 versus corresponding E control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous studies have demonstrated that 1,25(OH)2D3 negatively regulates renin expression in JG cells (24). Through deletion analysis of the 5'-flanking sequence of the Ren-1c gene, we identified two short DNA fragments in the gene promoter that are sufficient to mediate the transcriptional repression. The proximal fragment is the minimal Ren-1c gene promoter, and the distal fragment is within a 242-bp enhancer previously identified to be important for basal renin expression (8). Most recently, this enhancer was shown to be critical for the control of renin expression in vivo, and mice lacking this enhancer have renin depletion in JG cells and develop low blood pressure (39). Therefore, 1,25(OH)2D3 must target important cis-elements within these fragments in the Ren-1c gene promoter to regulate renin. In the present study we focused on the distal enhancer and identified the CRE as a critical element that mediates the transrepression by vitamin D.

1,25(OH)2D3 regulates gene expression by interaction with the VDR. For positive regulation, liganded VDR heterodimerizes with RXR and binds to the vitamin D response element (VDRE) to stimulate gene transcription. A typical VDRE is composed of two hexanucleotide direct repeats separated by three nucleotides (25). On the other hand, 1,25(OH)2D3 also acts as a negative transcription regulator, but the mechanism of the negative regulation is far more complicated and diverse and only partially understood. For instance, in the case of vitamin D suppression of IL-2 and GM-CSF gene expression, liganded VDR-RXR heterodimer or VDR monomer inhibits the formation of NFAT-1/AP-1 transcriptional complex in the gene promoter (40, 41). In the promoter of PTH and PTHrP genes, VDR binds to a negative VDRE (nVDRE), with the involvement of Ku antigen, to suppress gene transcription (42). Vitamin D suppresses Cyp27B1 transcription via interaction with VDIR (43). The VDR-RXR heterodimer can recruit nuclear co-repressors, such as NCoR and Alien, to directly mediate transcriptional repression (44). Moreover, liganded VDR can also directly interact with key regulatory factors, such as Smad3, beta-catenin, and p65 NF-{kappa}B (33, 34, 36), to interfere gene expression.


Figure 7
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  		FIGURE 7.
Overexpression of CREB, CBP, or p300 rescues the suppression of renin gene transcription by 1,25(OH)2D3. As4.1-hVDR cells were co-transfected with p4-6 reporter plasmid and increasing amounts of expression plasmid of CREB (A), CBP, or p300 (B) as indicated. Transfected cells were cultured in the presence (+) or absence (–) of 1,25(OH)2D3 (2 x 10–8 M) for 24 h before the luciferase activity was measured.

 


Figure 8
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  		FIGURE 8.
Model of 1,25(OH)2D3-induced transrepression of renin gene expression. The cAMP-PKA pathway plays a key role in stimulation of renin gene expression. This pathway activates CREB by phosphorylation, leading to recruitment of CBP/p300. In the presence of 1,25(OH)2D3, liganded VDR interacts with CREB and blocks its binding to CRE, leading to reduction of renin gene transcription. P, phosphorylation; D, 1,25(OH)2D3; Pol II, RNA polymerase II.

 
There are no recognizable conventional VDREs within the two DNA fragments in the Ren-1c promoter. Although the distal fragment contains a few hexanucleotide half-sites (i.e. RAR and RXR binding sites) that may bind to VDR, we did not detect VDR binding to these sites by EMSA or ChIP assays. Moreover, neither mutation of the RARE (Fig. 2K) nor deletion of the entire RAR/RXR binding sites (e.g. construct p4-7 does not contain the RAR/RXR sites, Fig. 1B) had any effects on the transrepression induced by 1,25(OH)2D3. Therefore, the RAR/RXR sites in the distal fragment are unlikely involved in the VDR-mediated transrepression.

Our study provides several lines of evidence demonstrating that the CRE within the distal enhancer plays a key role in mediating the vitamin D transrepression. This CRE (5'-TGACATCA-3') is one nucleotide different from the canonical consensus CRE (5'-TGACGTCA-3'), is bound by CREB and CREM in As4.1 cells and is crucial for the basal renin expression (13). Although we could not detect stimulation of CRE activity in As4.1 cells because the cAMP-PKA pathway is constitutively active, using HEK293 and MCs we confirmed that this CRE is functional in response to forskolin stimulation. We showed that 1,25(OH)2D3 inhibits CRE-mediated luciferase activity in both the basal and forskolin-stimulated states, and mutation of this CRE not only abolishes forskolin stimulation but also eliminates 1,25(OH)2D3 suppression of the reporter activity. Interestingly, in As4.1 cells, mutation of the CRE dramatically diminishes but not completely eliminates vitamin D repression, whereas in MCs CRE mutation completely eliminates the transrepression. This observation suggests the existence of other cis-element(s) that is (are) also important for the vitamin D action in a JG cell-specific manner. Further studies are needed to identify the cis-DNA element(s).

Our data strongly suggest that 1,25(OH)2D3 suppresses CRE-mediated transcriptional activity by blocking CREB binding to the CRE (Fig. 8). Because a combined treatment with 1,25(OH)2D3 and PKA inhibitor H89 additively or synergistically inhibits renin expression, it is unlikely that 1,25(OH)2D3 directly targets PKA. Moreover, 1,25(OH)2D3 does not appear to affect the protein levels of CREB and CREM or the phosphorylation of CREB. ChIP assays demonstrate that CREB, CREM, and CBP are binding to the CRE in the basal state in As4.1 cells, which helps to drive a high level of renin expression. In the presence of 1,25(OH)2D3, liganded VDR binds to CREB, thus blocks CREB binding to the CRE and disrupts the formation of CRE-CREB-CBP/p300 complex, leading to reduction in renin gene expression (Fig. 8). This model is supported by the data obtained from EMSA, ChIP, and GST pull-down assays as well as by VP16-CREB transfection and CREB overexpression experiments. VDR interacts with CREB by the LBD. However, our data do not exclude other regulatory mechanisms. For example, 1,25(OH)2D3 may affect the CRE activity through regulation of other proteins.

The role of CREM is unclear. CREM can function as a transcription activator in response to cAMP. CREM phosphorylated at Ser-117 recruits CBP, and unphosphorylated CREM can recruit ACT in transactivation (18). Isoforms of CREM can act as suppressors of cAMP-induced transcription and modulate the final CRE response (45). Specific knockdown of CREB and CREM by RNA interference may help further delineate the role of CREB and CREM in renin regulation. The fact that liganded VDR suppresses renin transcription by interacting with CREB suggests that CREB is a predominant regulator in renin expression. Whether liganded VDR also interacts with CREM remains to be determined. It is noteworthy that a previous study failed to detect CREB-2 (i.e. CRE-BP1 (46)), ATF-1, ATF-2, or ATF-3 binding to this CRE by EMSA (13).

As the rate-limiting enzyme of the renin-angiotensin cascade, renin biosynthesis is tightly regulated. Among the most important physiological factors that influence renin production and release from JG cells are renal perfusion pressure, tubular sodium chloride load and sympathetic nerve activity (47). It is believed that intracellular cAMP is critically involved in the induction of renin expression stimulated by activation of sympathetic nerve activity (mediated by beta-adrenergic receptor) or by low sodium chloride concentration in renal distal tubules (mediated by the macula densa cells) (48). Therefore, the cAMP-dependent PKA signaling pathway is a major regulatory pathway involved in renin production. In the present study, we demonstrated that 1,25(OH)2D3 down-regulates renin gene transcription by suppressing, at least in part, CRE-mediated transcriptional activity in the renin gene promoter. As CRE is activated by cAMP-PKA signaling, our study identifies a major regulatory pathway as the target in vitamin D inhibition of renin synthesis.


   FOOTNOTES
 
* This work was supported in part by American Heart Association Grant-in-aid 0350503Z and National Institutes of Health Grants DK062072 and HL085793 (to Y. C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Medicine, The University of Chicago, MC 4080, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 773-702-2477; Fax: 773-702-5790; E-mail: cyan{at}medicine.bsd.uchicago.edu.

2 The abbreviations used are: RAS, renin-angiotensin system; VDR, vitamin D receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; cAMP, cyclic AMP; CRE, cAMP response element; PKA, protein kinase A; CREB, CRE-binding protein; CREM, CRE modulator; CBP, CREB-binding protein; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; JG, juxtaglomerular; 8-Br-cAMP, 8-bromo-cAMP; GST, glutathione S-transferase. Back



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