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Endocrinology, doi:10.1210/en.2008-0127
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Endocrinology Vol. 149, No. 9 4647-4657
Copyright © 2008 by The Endocrine Society

The Pal3 Promoter Sequence Is Critical for the Regulation of Human Renin Gene Transcription by Peroxisome Proliferator-Activated Receptor-{gamma}

Vladimir T. Todorov, Michael Desch, Thomas Schubert and Armin Kurtz

Institute of Physiology, University of Regensburg, D-93040 Regensburg, Germany

Address all correspondence and requests for reprints to: Dr. Vladimir T. Todorov, Institute of Physiology, University of Regensburg, D-93040 Regensburg, Germany. E-mail: vladimir.todorov{at}vkl.uni-regensburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently reported that human renin gene transcription is stimulated by the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-{gamma} in the renin-producing cell line Calu-6. The effect of PPAR{gamma} was mapped to two sequences in the renin promoter: a direct repeat hormone response element (HRE), which is related to the classical PPAR response element (PPRE) and a nonconsensus palindromic element with a 3-bp spacer (Pal3). We now find that PPAR{gamma} binds to the renin HRE. Neither the human renin HRE nor the consensus PPRE was sufficient to attain the maximal stimulation of renin promoter activity by the PPAR{gamma} agonist rosiglitazone. In contrast, the human renin Pal3 element mediates both the full PPAR{gamma}-dependent activation of transcription and the PPAR{gamma}-driven basal renin gene transcription. The human renin Pal3 sequence was found to selectively bind PPAR{gamma} and the retinoid X receptor-{alpha} from Calu-6 nuclear extracts. This is in contrast to the consensus PPRE, which can bind other nuclear proteins. PPAR{gamma} knockdown paradoxically did not attenuate the stimulation of the endogenous renin gene expression by rosiglitazone. Similarly, a deficiency of PPAR{gamma} did not attenuate the activation of the minimal human renin promoter, which contains the endogenous Pal3 motif. However, when the human renin Pal3 site was replaced by the consensus PPRE sequence, PPAR{gamma} knockdown abrogated the effect of rosiglitazone on renin promoter activity. Thus, the human renin Pal3 site appears to be critical for the PPAR{gamma}-dependent regulation of gene expression by mediating maximal transcription activation, particularly at the low cellular level of PPAR{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EXPRESSION OF the renin gene is controlled by a complex network of transcriptional and posttranscriptional mechanisms (1, 2, 3). The critical regulatory sequences involved in the regulation of renin transcription are basically grouped in two evolutionarily conserved 5'-flanking regions known as the (kidney) enhancer and the minimal promoter (3). The enhancer represents a 242-bp motif whose position varies in different species. The minimal promoter is an approximately 200-bp-long region located immediately upstream of the transcriptional starting site that displays high interspecies homology (3, 4, 5).

Many of the members of the nuclear receptor transcription factor superfamily are known to be involved in the control of renin transcription (6, 7, 8, 9, 10, 11). Among the nuclear receptor ligands, the mechanism of action of vitamin A (retinoic acid) was the first to be thoroughly characterized (7). Retinoic acid receptor (RAR)-{alpha} and retinoid X receptor (RXR)-{alpha} transmit the vitamin A stimulatory signal to the renin gene by binding to an enhancer direct repeat of the hexameric motif 5'-AGGTCA-3'. This motif is identical with the core sequence of the consensus DNA-binding site for the nuclear receptor superfamily and is therefore generally termed the hormone response element (HRE).

In a recent study, we found that the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-{gamma} is a transcription factor involved in the control of basal renin transcription and induces the renin gene upon agonist treatment (12). This effect might be of particular importance because the known endogenous PPAR{gamma} ligands, which are unsaturated fatty acids, play key roles in lipid homeostasis (13). Therefore, PPAR{gamma} is one of the molecular links between the disturbances of lipid metabolism (such as obesity) and the accompanying renin-related cardiovascular disorders (14, 15, 16, 17, 18). In addition, pharmacological PPAR{gamma} agonists (such as the glitazones) are known to have complex actions on the cardiovascular system, which might be partially mediated by the effect of PPAR{gamma} on renin gene expression (19, 20). Interestingly, we found that, unlike all other known PPAR{gamma}-regulated genes, the human renin gene is regulated by PPAR{gamma} through a palindrome with a 3-bp spacer (Pal3) located in the minimal promoter (12). The Pal3 motif was bound by PPAR{gamma}/PPAR{gamma} homodimers and by PPAR{gamma}/RXR{alpha} heterodimers (21). In contrast, the consensus PPAR response element (PPRE) consists of a direct repeat (DR) of the core hexamer with a single-base spacer (DR1) and is bound by PPAR{gamma} only as a heterodimer with the RXRs (21, 22). Thus, the canonical PPRE is closely related to the human renin enhancer HRE, with the exception that the latter is a DR8. Notably, the enhancer HRE appeared to be involved in the PPAR{gamma}-dependent activation of the human renin promoter (12). It remained unclear, however, whether PPAR{gamma} could bind to the renin enhancer HRE. Because the HRE can augment basal renin promoter activity, the presence of the HRE in a renin promoter construct with a mutated Pal3 sequence could have underscored some residual Pal3-dependent PPAR{gamma} effect (12). A more important issue to clarify was why the PPAR{gamma}-dependent control of the human renin gene operates through a Pal3 sequence but not a PPRE, which is in contrast to the typical PPAR{gamma}-driven genes. To answer these questions, we combined RNA interference with DNA-protein binding studies along with transient transfections of different human renin promoter fragments containing the endogenous Pal3 element or a substitution of Pal3 for PPRE. The regulation of promoter activity was studied in parallel with the regulation of the endogenous renin gene expression by PPAR{gamma} ligands in the human cell line Calu-6.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and chemicals
Calu-6 cells (ATCC-HTB-56) were cultured in Eagle’s MEM supplemented with 10% fetal bovine serum, sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% nonessential amino acids at 37 C in a humidified atmosphere containing 5% CO2. Rosiglitazone and pioglitazone were from Cayman Chemical (Ann Arbor, MI). The glitazones were dissolved in dimethyl sulfoxide and were applied for 20 h at 200 nM (rosiglitazone) or 1 µM (pioglitazone) unless otherwise indicated. Control cells were treated only with the vehicle.

Chromatin immunoprecipitation (ChIP)
DNA-protein complexes isolated from rosiglitazone-treated Calu-6 cells were analyzed in ChIP assays using the ChIP-IT kit from Active Motif as already described (12). In short, DNA-protein cross-linked complexes were precleared with Protein G agarose beads (input samples), or were further precipitated with preimmune antigoat antiserum, or anti-PPAR{gamma} antibody (Active Motif, Rixensart, Belgium). After reversal of the cross-linking and digestion of RNA and proteins, DNA was eluted and PCR amplified for 32–35 cycles. The primers used for the amplification of a 200-bp-long human renin enhancer fragment containing the HRE site were: sense, 5'-cagacttcctccacccctt-3', antisense, 5'-cttgagatagttctggagg-3'.

RNA isolation, reverse transcription, quantitative LightCycler PCR, standard PCR
Total RNA was isolated from Calu-6 with the RNeasy spin columns (QIAGEN, Hilden, Germany). A standard protocol for reverse transcription was used.

Real-time PCR (Light Cycler System 2.0; Roche, Mannheim, Germany) was used to quantitatively amplify renin and citoplasmatic β-actin cDNA fragments as already described (12). The expression of renin was normalized to β-actin.

Nuclear receptor cDNAs were amplified by standard PCR protocol of 35 cycles, each consisting of denaturation for 30 sec at 95 C, annealing for 30 sec at 60 C, and elongation for 60 sec at 72 C. The sequences of the primers are shown in Table 1Go (6, 12, 23, 24, 25, 26, 27).


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  		TABLE 1. Primers used to amplify NR cDNAs

 
RNA interference
Ready-to-use double-stranded small interfering (si) RNAs were synthesized by Dharmacon (Lafayette, CO) to target human PPAR{gamma}, or human RXR{alpha} (ON-TARGETplus siRNA SMARTpool L-003436–00 and L-003443, respectively). Fifty nanomoles of double-stranded nontargeting siControl siRNA (Dharmacon; used as control), RXR{alpha}-specific siRNA (siRXR{alpha}), or PPAR{gamma}-specific siRNA (siPPAR{gamma}) was transfected into Calu-6 cells using Dharmafect 2 (Dharmacon) according to the manufacturer’s protocol. The cells were harvested 72 h after transfection.

Plasmids
The plasmid constructs used in this study are shown in Table 2Go. The modified pGL3 vector (Promega, Madison, WI) encoding firefly luciferase under the control of the minimal human renin promoter (bases –199 to +23 relative to the transcription starting site, hRenMin construct) was originally described elsewhere (28). The human renin Pal3 sequence, 5'-GGGTACcctTCACCC-3', is located at –148 to –134 (the repeat is in uppercase and the spacer is in lowercase). All the following constructs were obtained from hRenMin through site-directed mutagenesis (QuikChange kit; Stratagene, La Jolla, CA). Construct hRenMinPPRE was derived from hRenMin by replacing the Pal3 element by the canonical PPRE motif, 5'-AGGTCAaAGGTCA-3'. The consensus Pal3 sequence, 5'-AGGTCAccgTGACCC-3', replaces the human renin Pal3 motif in construct hRenMinPal3consensus. The mouse (5'-GCTTATcctATACCT-3') or rat Pal3 (5'-GCTTATcccTCACCC-3') sequence replaces the human renin Pal3 motif in constructs hRenMin-mPal3 and hRenMin-rPal, respectively. Constructs GGGTCAcctTCACCC, GGGTACccgTCACCC and GGGTACcctTGACCC represent the minimal renin promoter fragment in which single bases of the endogenous Pal3 element (underlined) are changed to mimic the consensus Pal3 sequence.


Figure 10
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  		FIG. 10. Plasmids used for the experiments

The cis-regulatory elements in the proximal renin promoter (bases –199 to +23) are illustrated with different geometric figures. Filled figure indicates a mutation. The lines within the Pal3 element represent the single bases changed to mimic the consensus Pal3 sequence. The same bases are in lowercase in the titles of the corresponding constructs.

 
The rest of the plasmids are already described (12). In short, the constructs Pal3mut and HRE-like-mut contain mutations in the human renin Pal3 or the HRE-like site in the context of hRenMin. The constructs HRE-hRenMin and HREmut-hRenMin contain the human renin enhancer HRE or the mutated human renin enhancer HRE, respectively, inserted in front of the minimal human renin promoter hRenMin. The construct HRE-Pal3mut contains a mutation in the Pal3 sequence in the context of the HRE-hRenMin fragment.

Transient transfection and luciferase assay
Firefly luciferase reporter vector (0.2 µg) was transfected into Calu-6 cells together with 0.01 µg of plasmid encoding Renilla luciferase (pRL-0 vector; Promega) according to the protocol already described (12, 29, 30, 31). Twenty-four hours after transfection, medium was replaced with a fresh medium containing the vehicle (control) or the active substances. Cells were harvested 32 h after transfection. Relative luciferase activity (RLA) was calculated as the ratio of firefly luciferase to Renilla luciferase.

EMSA
The extraction of nuclear proteins from Calu-6 cells and the following shift/supershift assay are already described in detail (12, 32). The sequence of the human renin Pal3 probe was 5'-acctGGGTACCCTTCACCCacc t-3' and corresponded to the human renin promoter sequence from –152 to –130 containing the core endogenous Pal3 sequence (–148 to –134, in uppercase). The core Pal3 sequence was replaced by 5'-AGGTCACCGTGACCC-3' or 5'-AGGTCAAAGGTCA-3' in probes consensus Pal3 or consensus PPRE (DR1), respectively. The PPAR{gamma} antibody was from Active Motif.

Western blotting
Calu-6 total cellular protein was isolated in lysis buffer [10 mM Tris, 1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 5 mM Pefablock], and protein concentration was determined (MicroProtein determination kit; Sigma, Taufkirchen, Germany). The samples were boiled in Laemmli buffer and loaded on 10% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes (Bio-Rad, Munich, Germany) in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS, 20% methanol). The membranes were blocked and then incubated with 1:500-diluted rabbit polyclonal anti-PPAR{gamma} (Active Motif) or anti-RXR{alpha} antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The bound antibody was visualized with horseradish peroxidase-conjugated antirabbit IgG (DiaNova, Hamburg, Germany) followed by enhanced chemiluminescence detection (Santa Cruz) and exposure to Biomax MS film (Kodak, Rochester, NY).

Statistics
Experiments were carried out in duplicate with at least three samples per condition. Levels of significance were estimated by ANOVA before the Student’s unpaired t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PPAR{gamma} binds to the human renin HRE sequence
Although the human renin enhancer HRE was found to be targeted by PPAR{gamma} agonists to increase renin promoter activity, it remained unknown whether this motif is capable of binding PPAR{gamma} (12). The binding of PPAR{gamma} to the native renin enhancer was studied in ChIP experiments (Fig. 1Go). The PPAR{gamma}-specific antibody enriched the precipitated fraction of a human renin HRE-containing DNA fragment, compared with the nonspecific antibody (Fig. 1Go, compare lane 3 with lane 2).


Figure 1
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  		FIG. 1. PPAR{gamma} binds to human renin enhancer HRE. A 200-bp human renin enhancer DNA fragment containing the HRE was PCR amplified after cross-linking and precipitation with anti-PPAR{gamma} (lane 3) or nonspecific antibody (antigoat antibody, lane 2). The input sample (positive control) was diluted 1:10 (lane 4).

 
Human renin HRE or consensus PPRE are not necessary for the full stimulation of renin promoter activity by PPAR{gamma} agonists
We next studied whether the human renin HRE is necessary for the up-regulation of renin transcription by the PPAR{gamma} agonist rosiglitazone. Consistent with our previous findings (12), mutation of the HRE in a renin promoter construct containing the Pal3 element (HREmut-hRenMin) did prevent the increase of promoter activity induced by rosiglitazone (Fig. 2Go). Nonetheless, mutation of the Pal3 element in a construct containing the HRE (HRE-Pal3mut) decreased the stimulatory effect of rosiglitazone on renin promoter activity by about 3-fold (Fig. 2Go). These results imply that, although the HRE is targeted by PPAR{gamma} to up-regulate renin transcription, the Pal3 sequence is necessary to attain the maximal PPAR{gamma}-dependent increase in transcription. Additionally, the replacement of the human renin Pal3 sequence by a consensus PPRE (construct hRenMinPPRE) blunted the effect of rosiglitazone on renin promoter activity (Fig. 2Go). These data demonstrate that the human renin HRE and the consensus PPRE are functionally equipotent and are both insufficient to mediate the maximal stimulation of renin promoter activity by PPAR{gamma}.


Figure 2
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  		FIG. 2. Activation of human renin promoter constructs by the PPAR{gamma} agonist rosiglitazone. The effect of rosiglitazone is presented as percent increase of the RLA of the corresponding construct relative to the control, which was treated with vehicle only: [(RLARosiglitazone/RLAControl) – 1] x100. The data shown are means ± SEM.

 
Knockdown of PPAR{gamma} paradoxically does not impair the stimulation of renin gene expression by PPAR{gamma} agonists
We further used the RNA interference technique to study the role of PPAR{gamma} and the renin Pal3 motif in the stimulation of renin transcription. Surprisingly, the knockdown of PPAR{gamma} did not diminish the rate of stimulation of the endogenous renin gene expression by the PPAR{gamma} ligand rosiglitazone (Fig. 3Go). Similar results were obtained with other PPAR{gamma} agonists such as pioglitazone (data not shown).


Figure 3
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  		FIG. 3. Effect of PPAR{gamma} knockdown on the stimulation of renin expression by rosiglitazone. Calu-6 cells were transfected with nontargeting siRNA as control (siControl) or with PPAR{gamma} sequence-specific siRNA (siPPAR{gamma}). A, Efficacy of the knockdown PPAR{gamma} immunoblot, B, Effect of PPAR{gamma} deficiency on renin mRNA abundance. Renin and β-actin mRNAs were quantified by real-time PCR. The data shown are means ± SEM. *, P < 0.05.

 
The human renin Pal3 site has different protein-binding and functional properties, compared with both the consensus PPRE and Pal3 sequences
We hypothesized that the paradoxical effect of PPAR{gamma} knockdown on the PPAR{gamma}-stimulated renin gene expression could be due to the human renin promoter Pal3 sequence. Therefore, we first compared the transcription factor-binding pattern of the human renin Pal3 site and the consensus PPRE. Indeed, the complex bound to the PPRE probe showed a stronger shift than the complex bound to the human renin Pal3 probe as detected by EMSA (Fig. 4AGo, left and middle panels). PPAR{gamma} antibody produced two supershifted bands with the human renin Pal3 probe and a single supershifted band with the PPRE probe (Fig. 4AGo, left and middle panels). We have previously shown that the upper supershifted band revealed with the PPAR{gamma} antibody and the human renin Pal3 probe consists of PPAR{gamma}/RXR{alpha} heterodimers (12). The supershifted band with the PPRE probe should also consist of PPAR{gamma}/RXR{alpha} heterodimers because PPRE is the canonical binding site for these heterodimers and because this supershifted band migrated similarly to the upper supershifted band with the human renin Pal3 probe. We assumed that the lower supershifted band with the human renin Pal3 probe should be composed of PPAR{gamma}/PPAR{gamma} homodimers because the Pal3 motif was known to bind PPAR{gamma}/PPAR{gamma} homodimers (21). Unexpectedly, the PPAR{gamma} antibody increased the degree of the shift with the PPRE probe (Fig. 4AGo, middle panel). Although the preimmune serum did not stabilize the protein complex bound to the human renin Pal3 probe (12), it was still possible that the PPAR{gamma} antibody stabilized the protein complex bound to the PPRE probe in a nonspecific manner. To rule out this possibility, we studied protein binding to human renin Pal3 and PPRE when PPAR{gamma} or RXR{alpha} was knocked down by RNA interference. The deficiency of either PPAR{gamma} or RXR{alpha} decreased the protein binding to the human renin Pal3 probe in agreement with our earlier findings (Fig. 4BGo, left panel). In contrast, the protein binding to the PPRE motif was augmented by the deficiency of PPAR{gamma} or RXR{alpha}, suggesting that additional nuclear receptors bound to PPRE (Fig. 4BGo, middle panel).


Figure 4
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  		FIG. 4. Binding of nuclear PPAR{gamma} to human renin Pal3, consensus PPRE (DR1), and consensus Pal3 motifs. A, Nuclear proteins from Calu-6 cells were probed with 33P-labeled human renin Pal3 (left panel), consensus PPRE (DR1, middle panel), or consensus Pal3 (right panel). Anti-PPAR{gamma} antibody (Active Motif) was added where indicated. AB, Antibody; H, supershifted protein complex containing PPAR{gamma}/RXR{alpha} heterodimers; D, supershifted protein complex designated to contain PPAR{gamma}/PPAR{gamma} homodimers; NS, nonspecific; FP, free probe. Dotted arrows indicate the shifted protein complexes. Arrows indicate the supershifted protein complexes. B, Nuclear proteins from Calu-6 cells treated with nontargeting siRNA (siControl), PPAR{gamma}-specific siRNA (siPPAR{gamma}), or RXR{alpha}-specific siRNA (siRXR{alpha}) were probed with 33P-labeled human renin Pal3 (left panel), consensus PPRE (DR1, middle panel), or consensus Pal3 (right panel). NS, Nonspecific; FP, free probe. Dotted arrows indicate the shifted protein complexes. C, Expression array of nuclear receptors in Calu-6 cells. The expression of nuclear receptor mRNAs was determined by RT-PCR. VDR, Vitamin D receptor; TR, thyroid hormone receptor; COUP-TF, chicken ovalbumin upstream promoter transcription factor; GR, glucocorticoid receptor; ER, estrogen receptor; AR, androgen receptor; PR, progesterone receptor; St, DNA-length standard.

 
The human renin Pal3 motif differs by several bases from the core consensus Pal3 sequence as originally described (12, 21). Therefore, we were interested in testing whether these differences are important for DNA-protein interactions. To our surprise, the consensus Pal3 resembled PPRE rather than the human renin Pal3 (Fig. 4Go, A and B). Accordingly, the PPAR{gamma} antibody produced a single supershifted band and increased the intensity of the shift with the consensus Pal3 probe (Fig. 4AGo, right panel). Knockdown of PPAR{gamma} or RXR{alpha} did not considerably change protein binding to the consensus Pal3 (Fig. 4BGo, right panel). Moreover, the shift with the consensus Pal3 probe appeared to consist of two closely migrating bands, suggesting that there is binding of multiple proteins.

These data strongly suggested that the human renin Pal3 sequence is preferentially bound by PPAR{gamma} and RXR{alpha}. On the other hand, the consensus PPRE and Pal3 sequences appear to interact with other nuclear receptors that compete with PPAR{gamma} and RXR{alpha}. Accordingly, most of the typical members of the three major nuclear receptor classes that are capable of binding PPRE (DR1), Pal, or both are expressed in the Calu-6 cells (Fig. 4CGo).

The functional significance of different protein-binding patterns on the human renin Pal3, the consensus Pal3, and the consensus PPRE motifs for PPAR{gamma}-regulated transcription from the minimal renin promoter is shown in Fig. 5AGo. Two PPAR{gamma} agonists, rosiglitazone and pioglitazone, activated the hRenMin promoter more than the hRenMinPPRE (containing PPRE instead of the human renin Pal3) and the hRenMinPal3consensus (containing consensus Pal3 instead of the human renin Pal3). Constructs hRenMinPPRE and hRenMinPal3consensus were equally activated by the PPAR{gamma} ligands. These results are in accordance with the DNA-binding data and demonstrate that the cis-acting element that selectively binds PPAR{gamma} as homo- or heterodimer (i.e. human renin Pal3) is more strongly induced by PPAR{gamma} agonists. Because it was quite unexpected to discover that the human renin Pal3 sequence possesses substantially different features from the originally described consensus Pal3 sequence, we tried to identify the key bases responsible for this functional discrepancy (Fig. 5BGo). The consensus Pal3 site was defined as 5'-A/GGGTCAcngTGACCT/C-3', where n could be any base and the spacer is represented in lowercase (21). The sequence of the human renin Pal3 site is 5'-GGGTACcctTCACCC-3', where the bases underlined are those that differ from the consensus sequence and the spacer is again represented in lowercase (12). The hRenMin construct completely lost its PPAR{gamma} inducibility when the last two bases of the 5'-repeat were flipped or when the last base of the spacer was changed to guanosine to mimic the consensus Pal3 sequence. The cytosine to guanosine exchange in position 2 of the 3'-repeat was redundant. None of the single nucleotide changes could partially attenuate the PPAR{gamma}-dependent activation.


Figure 5
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  		FIG. 5. Functional characterization of the human renin Pal3 motif. A, Effect of the PPAR{gamma} agonists rosiglitazone and pioglitazone on the human renin Pal3-, consensus PPRE-, and consensus Pal3-driven promoter activity. The hRenMin construct (which contains the endogenous renin Pal3 sequence), hRenMinPPRE construct (in which Pal3 is replaced by consensus PPRE), or hRenMinPal3consensus construct (in which Pal3 is replaced by consensus Pal3) were transfected in Calu-6 cells, and the effect of rosiglitazone or pioglitazone was tested. B, Identification of the critical base differences between the human renin Pal3 and consensus Pal3. Single bases of the human renin Pal3 element were changed to mimic the consensus Pal3 in the context of the hRenMin construct, and the effect of rosiglitazone was tested. The changed bases are in bold. The hRenMin construct was termed by its endogenous Pal3 sequence (GGGTACCCTTCACCC). C, Mouse and rat Pal3 sequences are not targeted by PPAR{gamma}. The hRenMin construct (which contains the endogenous renin Pal3 sequence), hRenMin-mPal3 construct (in which the human Pal3 is replaced by the mouse renin Pal3 sequence), or hRenMin-Pal3 construct (in which the human Pal3 is replaced by the rat renin Pal3 sequence) was transfected in Calu-6 cells, and the effect of rosiglitazone was tested. The data shown are means ± SEM. *, P < 0.05.

 
These findings provided further evidence that the human renin Pal3 motif is functionally unique and essentially different from the consensus Pal3 site. Importantly, both mouse and rat renin Pal3 sequences differ from the human renin Pal3 (among others also) in the last two bases of the 5'-repeat and/or in the last base of the spacer (33), suggesting that they would not be targeted by PPAR{gamma}. To prove this hypothesis, we replaced the human renin Pal3 sequence by the mouse or rat renin Pal3 sequence (constructs hRenMin-mPal3 and hRenMin-rPal3, respectively) in the minimal human renin promoter hRenMin and tested the effect of the PPAR{gamma} agonist rosiglitazone on their activity. In contrast to hRenMin, the activity of hRenMin-mPal3 and hRenMin-rPal3 could not be induced by rosiglitazone (Fig. 5CGo). Thus, mouse and rat renin genes appear to be regulated by PPAR{gamma} independently of their proximal promoter Pal3 sites. We already reported that PPAR{gamma} agonists could stimulate mouse renin gene expression (12). This effect seemed to be mediated by the mouse renin enhancer HRE (data not shown).

The human renin Pal3 sequence plays a key role in the PPAR{gamma}-dependent stimulation of renin promoter activity
The results presented in Figs. 4Go and 5Go demonstrate that the human renin Pal3 site, compared with the consensus PPRE or Pal3 motifs, is unique in preferentially binding PPAR{gamma} homo- and heterodimers, thus allowing stronger PPAR{gamma}-dependent stimulation of gene expression. This feature explains the paradoxical lack of inhibition of PPAR{gamma}-induced renin gene expression when PPAR{gamma} is knocked down (Fig. 3Go). As already seen for the expression of the endogenous renin gene, the knockdown of PPAR{gamma} diminished the basal activity of the hRenMin construct (which contains the endogenous human renin Pal3) but not the rate of stimulation by rosiglitazone (Fig. 6AGo). In contrast, PPAR{gamma} knockdown increased the basal activity of the hRenMinPPRE construct (in which the human renin Pal3 is replaced by a consensus PPRE sequence) and abolished the stimulatory effect of the PPAR{gamma} agonist (Fig. 6BGo). These findings demonstrated that the human renin Pal3 sequence preserves the maximal rate of PPAR{gamma}-dependent induction of the renin promoter when the availability of PPAR{gamma} is minimal. These data agree with our previous results, showing that the human renin Pal3 site is selectively bound by PPAR{gamma} (Fig. 4BGo) and that PPAR{gamma} knockdown unexpectedly increased the intensity of the supershifted band that appeared to contain the PPAR{gamma}/PPAR{gamma} homodimers (12). In addition to PPAR{gamma}, human renin Pal3 is also selectively bound by RXR{alpha} (Fig. 4BGo). The knockdown of RXR{alpha} also increases the binding of the protein complex that should contain PPAR{gamma}/PPAR{gamma} homodimers to the human renin Pal3 site (12). Therefore, it could be predicted that RXR{alpha} knockdown would have similar effects on hRenMin and the hRenMinPPRE, compared with the knockdown of PPAR{gamma}. In fact, RXR{alpha} knockdown increased the PPAR{gamma}-dependent activation of hRenMin, whereas the activation of hRenMinPPRE was abrogated (Fig. 7Go). Interestingly, the basal activity of both constructs was induced by RXR{alpha} deficiency (Fig. 7Go). The overall data presented in Figs. 3–7GoGoGoGoGo strongly suggest that the human renin Pal3 motif permits PPAR{gamma} to effectively regulate renin gene expression.


Figure 6
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  		FIG. 6. Effect of PPAR{gamma} knockdown on the activation of hRenMin and hRenMinPPRE renin promoter constructs by rosiglitazone. Calu-6 cells were transfected with nontargeting siRNA as control (siControl) or with PPAR{gamma}-sequence-specific siRNA (siPPAR{gamma}) and with the hRenMin construct (which contains the endogenous renin Pal3 sequence, A) or the hRenMinPPRE construct (in which Pal3 is replaced by consensus PPRE, B). For the efficacy of the knockdown, see Fig. 3AGo. The data shown are means ± SEM. *, P < 0.05.

 

Figure 7
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  		FIG. 7. Effect of RXR{alpha} knockdown on the activation of hRenMin and hRenMinPPRE renin promoter constructs by rosiglitazone. A, Efficacy of the knockdown RXR{alpha} immunoblot. Calu-6 cells were transfected with nontargeting siRNA as control (siControl) or with RXR{alpha} sequence-specific siRNA (siRXR{alpha}) and with the hRenMin construct (which contains the endogenous renin Pal3 sequence, B) or with the hRenMinPPRE construct (in which Pal3 is replaced by consensus PPRE, C). The data shown are means ± SEM. *, P < 0.05.

 
The human renin Pal3 element is sufficient for the regulation of basal renin transcription by PPAR{gamma}
After we found that the human renin Pal3 sequence is necessary for the maximal PPAR{gamma}-dependent induction of renin promoter activity, we became interested in studying its role in PPAR{gamma}-dependent control of basal renin transcription. Knockdown of PPAR{gamma} by sequence-specific siRNA (siPPAR{gamma}) resulted in a decrease of steady-state renin mRNA, indicating that PPAR{gamma} is important for maintaining the basal level of renin expression (Fig. 3BGo). The human renin Pal3 motif could mediate the effect of PPAR{gamma} on basal renin transcription because its mutation in the minimal renin promoter resulted in decreased promoter activity (Fig. 8Go, siControl, constructs hRenMin and Pal3mut). Importantly, mutation of the human renin Pal3 site did not change basal renin promoter activity when PPAR{gamma} was knocked down (Fig. 8Go, siPPAR{gamma}, constructs hRenMin and Pal3mut). Mutation of the minimal renin promoter HRE-like site, which is not targeted by PPAR{gamma} but is also critical for basal activity (12), resulted in diminished construct activity, even when PPAR{gamma} was knocked down (Fig. 8Go, constructs hRenMin and HRE-like-mut). These findings indicate that the human renin Pal3 sequence is specifically involved in the PPAR{gamma}-dependent regulation of basal transcription from the renin promoter.


Figure 8
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  		FIG. 8. The Pal3 sequence mediates the effect of PPAR{gamma} on the basal renin promoter activity. Calu-6 cells were transfected with nontargeting siRNA as control (siControl) or with PPAR{gamma} sequence-specific siRNA (siPPAR{gamma}) and with the hRenMin construct (which contains the endogenous renin Pal3 sequence), the Pal3mut construct (which contains a mutated Pal3 sequence), or the HRE-like-mut construct (which contains a mutated HRE-like sequence). For the efficacy of the knockdown see Fig. 3AGo. The data shown are means ± SEM. ns, Not significant. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used the human renin-producing cell line Calu-6 to characterize the role of the renin promoter Pal3 sequence during PPAR{gamma}-dependent regulation of renin transcription. However, Calu-6 cells differ from native juxtaglomerular (JG) cells of the kidney, the main source of plasma renin in vivo, in that they are not of renal origin. The regulation of renin gene expression in Calu-6 cells is to some extent different, compared with other renin-producing cell lines (e.g. As4.1). Nevertheless, a board of experimental evidence supports the relevance of Calu-6 cells as a representative model for studying the cell-specific regulation of renin expression. The strong responsiveness of the renin gene to cAMP is a typical feature of JG and Calu-6 cells. In both cell types, the mechanism of cAMP-dependent induction involves transcriptional and posttranscriptional steps (1, 2, 34, 35, 36, 37, 38). Furthermore, we found that PPAR{gamma} agonists increase renin mRNA in JG and Calu-6 cells (12). Calu-6 cells express mineralocorticoid receptor (MR) similarly to native JG cells and As4.1 cells (39). Calu-6 cells have the additional advantage of being a clonal cell line in contrast to primary JG cell cultures, which contain many different types of renal cells (40).

The human renin enhancer HRE was found to bind PPAR{gamma}. This is in accordance with our earlier data showing that the PPAR{gamma}-dependent activation of the renin promoter could be partially mapped to the HRE sequence (12). This finding was important to confirm the role of the HRE motif in the regulation of renin transcription by PPAR{gamma}. We next found that the renin HRE mediates a similar rate of stimulation of renin promoter activity as the consensus PPRE. The canonical PPRE represents a direct repeat of the 5'-A/GGGTCA-3' motif with a single base as a spacer (DR1), and PPAR{gamma} binds to it as a heterodimer with RXRs (21, 22, 41). There are considerable variations in the repeat sequence of the naturally occurring PPREs. Because the known PPREs are generally characterized by spacers of up to 5 bp (42), the human renin HRE, which is a DR8, is different from these. Widely spaced DRs (up to 200 bp), however, are known to act as promiscuous response elements for a variety of nonsteroidal nuclear receptors (43).

Although the renin HRE and the consensus PPRE were similarly potent in transmitting the PPAR{gamma}-dependent up-regulation of the renin promoter activity, they were not sufficient for a maximal activation by PPAR{gamma} agonists. Instead, the maximal activation is mediated by the minimal promoter Pal3 sequence. The Pal3 motif represents an inverted repeat (IR, or palindrome) of the classical HRE hexamer with a 3-bp spacer. It can be bound by PPAR{gamma}/RXR{alpha} heterodimers and also PPAR{gamma}/PPAR{gamma} homodimers (12, 21). Whereas we could definitely identify the complex containing PPAR{gamma}/RXR{alpha} heterodimers (12), the evidence for the binding of PPAR{gamma}/PPAR{gamma} homodimers to the human renin Pal3 motif was rather indirect. A PPAR{gamma} antibody caused the appearance of two supershifted bands when added to nuclear extracts of Calu-6 cells probed with the human renin Pal3 in EMSA (12) (Fig. 4AGo). Because the intensity of the upper supershifted band diminished when either PPAR{gamma} or RXR{alpha} was knocked down, one could assume that this band contains PPAR{gamma}/RXR{alpha} heterodimers (12). We originally suggested that the lower supershifted band contains PPAR{gamma} as a homodimer based on the fact that Pal3 was shown to bind PPAR{gamma}/PPAR{gamma} homodimers in addition to the PPAR{gamma}/RXR{alpha} heterodimers (21). We next found that this supershifted band becomes stronger if RXR{alpha} was knocked down (12), which suggests enhanced binding of PPAR{gamma}/PPAR{gamma} homodimers. This finding was expected because the binding of PPAR{gamma}/PPAR{gamma} homodimers to Pal3 is facilitated when the molar ratio PPAR{gamma} to RXR{alpha} increases (21). That the PPAR{gamma} antibody recognizes PPAR{gamma} specifically is demonstrated by the fact that Calu-6 nuclear extracts probed with a consensus PPRE produced a single supershifted band. This band migrated similarly to the upper human renin Pal3 supershifted band that was shown to contain PPAR{gamma}/RXR{alpha} heterodimers (Fig. 4AGo). Importantly, the consensus PPRE is known to bind PPAR{gamma} only as a heterodimer but not as a homodimer.

The human renin Pal3 differs from the originally described consensus Pal3 by two flipped bases at the end of the 5'-repeat (CA->AC), by the third base of the spacer (G->T), and by the second base of the 3'-repeat (G->C). Surprisingly, our results demonstrated that only the human renin Pal3 sequence but not the consensus Pal3 sequence could bind PPAR{gamma} as part of two different protein complexes that contain PPAR{gamma} in either the homo- or heterodimer form. The consensus Pal3 sequence displayed similar binding properties to the consensus PPRE when tested with nuclear extracts from Calu-6 cells by EMSA. Both consensus Pal3 and PPRE appeared to bind PPAR{gamma} only as a heterodimer. Furthermore, in contrast to the human renin Pal3, these sequences appeared to bind additional nuclear receptor transcription factors (Fig. 4BGo). Moreover, the Calu-6 cells were found to express many representatives of all of the three main classes of nuclear receptors (Fig. 4CGo). Among the nuclear receptors tested only PPAR{delta}, RAR{alpha}, and the estrogen receptors (ERs) seemed not to be expressed in Calu-6 cells. IRs similar to the Pal3 sequence are generally recognized by the steroid hormone nuclear receptors (class III), whereas class I and II nuclear receptors bind to both DRs and IRs (42).

The protein-binding properties of the consensus Pal3 sequence characterized by nuclear extracts from Calu-6 cells are unexpectedly inconsistent with the original findings of Okuno and colleagues (21). The most probable reason for this discrepancy is in the different approaches used to prepare the protein extracts for the DNA-binding studies. Okuno et al. used purified FLAG-tagged PPAR{gamma} and RXR{alpha} overexpressed as fusion proteins in insect cells (21), whereas we applied native nuclear proteins from the human cell line Calu-6. Our results suggest that PPAR{gamma} interacts with the human renin Pal3 without competing with other nuclear receptors except RXR{alpha}. Therefore PPAR{gamma} may bind as homodimer to the human renin Pal3 sequence, but not to consensus Pal3 or PPRE.

We provide data showing that the human renin Pal3 site is functionally unrelated to the consensus Pal3, and mapped the critical bases to the 5'-repeat and the spacer. Based on these findings, we predicted that mouse and rat renin Pal3 sites would not function as PPAR{gamma} response elements, and confirmed experimentally that they are not targeted by the PPAR{gamma} agonist rosiglitazone.

The human renin Pal3 was found to be critical for the PPAR{gamma}-dependent regulation of the basal and the stimulated activities of the renin gene. This is in accordance with the unique characteristic of the human renin Pal3 sequence to selectively bind PPAR{gamma} as a heterodimer with RXR{alpha}, and most probably also as a homodimer. This feature of the human renin Pal3 motif would have two important consequences. First, it would result in "protection" of the motif from other nuclear receptors and the effects of their ligands. Accordingly, the activity of the minimal human renin promoter hRenMin was not affected by vitamin D or testosterone (data not shown), although their corresponding receptors are expressed in Calu-6 cells. Second, the human renin Pal3 would be expected to preserve its decisive role in the control of the PPAR{gamma}-induced gene expression, even at minimal levels of PPAR{gamma}. Consistently, stimulation of endogenous renin gene expression by PPAR{gamma} agonists was preserved when PPAR{gamma} was knocked down by RNA interference. Moreover, a similar effect resulting from PPAR{gamma} deficiency on renin promoter activity was dependent on the presence of the human renin Pal3, but not of the consensus PPRE sequence. To further support our findings, we studied the effect of RXR{alpha} knockdown, which was predicted to facilitate the interaction of PPAR{gamma} with the Pal3 sequence (12, 21). Similarly to PPAR{gamma} knockdown, a shortage of RXR{alpha} did not diminish, but actually increased the stimulation of the renin promoter activity by PPAR{gamma} agonists in the presence of the human renin Pal3 but not in the presence of the consensus PPRE.

Based on our current and earlier data from functional and DNA-binding studies, a model for the role of the human renin Pal3 sequence as described below could be proposed (Fig. 9Go). The cellular amount of RXR{alpha} is believed to be limiting because it forms heterodimers with many nuclear receptors (NRs) other than PPAR{gamma} (21, 42, 44, 45). These nuclear receptors, such as the vitamin D receptor, the thyroid hormone receptors, or the RARs, compete with PPAR{gamma} for the binding of RXR{alpha}. Thus, the bulk of RXR{alpha} is expected to bind to PPREs and other DRs as a heterodimer. However, the human renin Pal3 site could be selectively bound by PPAR{gamma}/PPAR{gamma} homodimers and PPAR{gamma}/RXR{alpha} heterodimers. At baseline conditions, the formation of PPAR{gamma}/PPAR{gamma} homodimers would be competed by RXR{alpha}, and therefore, the human renin Pal3 site would be predominantly bound by PPAR{gamma}/RXR{alpha} heterodimers (Fig. 9AGo) (12). The shortage of PPAR{gamma} would result in decreased formation of PPAR{gamma}/RXR{alpha} heterodimers and would enhance binding of NR/RXR{alpha} and NR/NR dimers to PPRE/DR1 motifs (Fig. 9BGo). Thus, the fraction of RXR{alpha} available to interfere with the formation of PPAR{gamma}/PPAR{gamma} homodimers bound to the human renin Pal3 would bind to PPRE/DR1 sequences instead. Importantly, other NRs could not interact with the human renin Pal3 to compete with the binding of PPAR{gamma} (Fig. 4BGo). The result would be a fractional increase of PPAR{gamma}/PPAR{gamma} homodimers bound to the human renin Pal3 (12). Such a model appears counterintuitive because it suggests increased DNA binding of PPAR{gamma}/PPAR{gamma} homodimers at a low cellular level of PPAR{gamma}. This idea seems to contradict the finding that PPAR{gamma}/PPAR{gamma} homodimers form preferentially if PPAR{gamma} is in excess over RXR{alpha} in a cell-free system (21). In contrast to the in vitro system, however, RXR{alpha} is competed by several NRs within the cell, and PPAR{gamma} is only one of them (42, 45). Therefore, the shortage of PPAR{gamma} would enhance the heterodimerization of RXR{alpha} with other NRs, which, in turn, would facilitate the formation of PPAR{gamma}/PPAR{gamma} homodimers at the human renin Pal3. The deficiency of RXR{alpha} (Fig. 9CGo) increases the intracellular PPAR{gamma} to RXR{alpha} molar ratio. This facilitates the binding of PPAR{gamma}/PPAR{gamma} homodimers and reduces the binding of PPAR{gamma}/RXR{alpha} heterodimers to the human renin Pal3 (12).


Figure 9
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  		FIG. 9. Suggested model for the binding of PPAR{gamma} and RXR{alpha} to the human renin Pal3 sequence. The NR binding to PPRE (DR1) is shown on the right as a comparison. The NRs are illustrated with different geometric figures. A, Under baseline conditions, RXR{alpha} heterodimerizes with PPAR{gamma} and other NRs (block arrows, NRs, nuclear receptors other than PPAR{gamma} or RXR{alpha}). PPAR{gamma} binds to the human renin Pal3 site predominantly in the form of PPAR{gamma}/RXR{alpha} heterodimers (bold arrow) and less frequently in the form of PPAR{gamma}/PPAR{gamma} homodimers (thin arrow). PPRE binds PPAR{gamma}/RXR{alpha} heterodimers, NR/RXR{alpha} heterodimers, and NR/NR dimers. B, Under PPAR{gamma} deficiency, RXR{alpha} heterodimerizes predominantly with NRs (bold block arrow) and less frequently with PPAR{gamma} (dotted block arrow). This indirectly facilitates the binding of PPAR{gamma} to the human renin Pal3 sequence in the form of PPAR{gamma}/PPAR{gamma} homodimers (bold arrow), whereas the binding of PPAR{gamma}/RXR{alpha} heterodimers is diminished (thin arrow). The binding of PPAR{gamma}/RXR{alpha} heterodimers to PPRE is also diminished, whereas the binding of NR/RXR{alpha} heterodimers and NR/NR dimers to PPRE is increased. C, Under RXR{alpha} deficiency, the formation of the RXR{alpha}-containing heterodimers is generally decreased (dotted block arrows). Therefore, PPAR{gamma} binds to the human renin Pal3 site predominantly in the form of PPAR{gamma}/PPAR{gamma} homodimers (bold arrow) and less frequently in the form PPAR{gamma}/RXR{alpha} heterodimers (thin arrow). The binding of PPAR{gamma}/RXR{alpha} and NR/RXR{alpha} heterodimers to PPRE is decreased, whereas the binding of NR/NR dimers to PPRE is drastically increased.

 
The shortage of either PPAR{gamma} or RXR{alpha} decreased the total protein binding to the renin Pal3 because PPAR{gamma}/RXR{alpha} heterodimers are essential part of the human renin Pal3 binding activity and because the human renin Pal3 sequence preferentially binds PPAR{gamma} and RXR{alpha} but not other NRs (Fig. 4Go). However, the effects of PPAR{gamma} and RXR{alpha} deficiency on hRenMin basal activity were discrepant. Knockdown of PPAR{gamma} diminished renin promoter activity, whereas knockdown of RXR{alpha} mildly increased it (Figs. 6Go and 7Go). The deficiency of PPAR{gamma}, however, led to a greater decrease in the binding of PPAR{gamma}/RXR{alpha} heterodimers to the human renin Pal3 motif, compared with the deficiency of RXR{alpha}, and the binding of the PPAR{gamma}/PPAR{gamma} homodimers seemed to be similarly potentiated (12). These findings suggest that the relative increase of the PPAR{gamma}/PPAR{gamma} homodimers could functionally (over)compensate for the reduction of the PPAR{gamma}/RXR{alpha} heterodimers bound to the human renin Pal3 when RXR{alpha}, but not PPAR{gamma}, was knocked down. The basal activity of the PPRE-driven renin promoter construct rose when PPAR{gamma} or RXR{alpha} was knocked down (Figs. 6Go and 7Go). This correlates with the increased binding of other nuclear receptors to PPRE (Fig. 4BGo). Notably, PPRE is practically identical with the retinoic acid response element and is closely related to the consensus DR binding sequences of the nonsteroidal nuclear receptors of classes I and II (42, 46).

The key finding of our study is that the human renin Pal3 sequence has unique protein-binding and functional features. It selectively binds PPAR{gamma}, thus amplifying the PPAR{gamma}-dependent stimulation of gene expression, especially at low cellular levels of PPAR{gamma}. These data are of particular importance in at least three different ways. First, the murine Pal3 motifs differ from the human renin Pal3 site by bases that are critical in determining its distinctive function and were consequently found not to be targeted by PPAR{gamma}. Thus, the mouse and rat may not be completely relevant as models for studying the impact of PPAR{gamma} on renin gene expression in humans. Second, the PPAR{gamma}-dependent regulation of gene expression appeared to be a complex function of the interaction between the distinct PPAR{gamma} binding motifs and the cell-specific expression pattern of the NRs. Therefore, different experimental settings for studying the mechanisms of action of PPAR{gamma} (e.g. purified transcription factors vs. total nuclear extracts, dominant-negative mutation without loss of DNA-binding vs. gene knockout, different cell types) may produce contradictory results. Finally, it could be predicted that in addition to human renin, other PPAR{gamma}-driven genes would be similarly regulated in tissues in which PPAR{gamma} is not strongly expressed.


   Acknowledgments
 
The authors thank Dr. Ralf Mrowka for the generous gift of the hRenMin construct.


   Footnotes
 
This work was supported by Grant SFB699/B1 from the Deutsche Forschungsgemeinschaft.

Disclosure Statement: All authors have nothing to declare.

First Published Online May 15, 2008

Abbreviations: ChIP, Chromatin immunoprecipitation; DR, direct repeat; ER, estrogen receptor; HRE, hormone response element; IR, inverted repeat; JG, juxtaglomerular; MR, mineralocorticoid receptor; NR, nuclear receptor; Pal3, palindrome with 3-bp spacer; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; RAR, retinoic acid receptor; RLA, relative luciferase activity; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; si, small interfering.

Received January 28, 2008.

Accepted for publication May 6, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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