help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1381
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/3/1424    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Itani, H. A.
Right arrow Articles by Sigmund, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Itani, H. A.
Right arrow Articles by Sigmund, C. D.
Endocrinology Vol. 148, No. 3 1424-1430
Copyright © 2007 by The Endocrine Society

Functional Characterization of Polymorphisms in the Kidney Enhancer of the Human Renin Gene

Hana A. Itani, Xuebo Liu, J. Howard Pratt and Curt D. Sigmund

Molecular and Cellular Biology Interdisciplinary Graduate Program (H.A.I., C.D.S.), Department of Internal Medicine (X.L., C.D.S.), Department of Molecular Physiology and Biophysics (C.D.S.), Center on Functional Genomics of Hypertension (C.D.S.), Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242; and Department of Internal Medicine (J.H.P.), Indiana University School of Medicine, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Curt D. Sigmund, Ph.D., Departments of Internal Medicine and Physiology and Biophysics, 3181B Medical Education and Biomedical Research Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242. E-mail: curt-sigmund{at}uiowa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The renin gene is regulated by an enhancer located 2.6 kb upstream of the transcription start site in the mouse and 11 kb upstream in humans. Despite extensive sequence conservation, the mouse renin enhancer is transcriptionally more active than the human renin enhancer. We report that the mechanism accounting for this is a result of sequence variation in the promoter proximal half-site of a retinoic-acid response element present in the enhancer. This sequence difference also prompted us to search for naturally occurring polymorphisms in the renin enhancer among normal and hypertensive human subjects. We sequenced the kidney enhancer from 90 samples derived from the Coriell Polymorphism Discovery Resource and 95 severely hypertensive Caucasian and African-American individuals. A single relatively frequent polymorphism (7, 2, and 7%, respectively in the Coriell, African-American, and Caucasian) was identified in the enhancer, one nucleotide downstream of the promoter distal half-site of the retinoic-acid response element. This variant was transcriptionally silent in transfection assays performed in renin-expressing As4.1 cells, a model of renal juxtaglomerular cells. A singleton polymorphism in the promoter was also identified in a single African-American individual. This polymorphism was located between binding sites for CBF1 and homeobox D10 but was also transcriptionally silent either in the presence or absence of the enhancer. Our study demonstrates the presence of silent polymorphisms in the renin promoter and enhancer, thus underscoring the critical importance of performing functional analyses before initiating expensive clinical studies seeking association between polymorphisms and complex diseases such as hypertension.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RENIN-ANGIOTENSIN SYSTEM (RAS) is a critical regulator of electrolyte homeostasis and arterial pressure in mammals and a prime candidate in the development and maintenance of hypertension. Renin, an aspartyl protease, catalyzes the rate-limiting step in the RAS by cleaving angiotensinogen into angiotensin (Ang)-I, which subsequently undergoes cleavage by angiotensin-converting enzyme to form Ang-II. Ang-II, the central effector of the RAS, increases blood pressure directly by vasoconstriction and indirectly by stimulating aldosterone release, thus promoting sodium and fluid retention. Although the RAS system has been studied for years, the mechanisms by which renin is regulated remains unclear. Renin (REN) is principally expressed in the juxtaglomerular cells of the kidney and is released in response to physiological cues such as sympathetic stimulation, renal artery hypotension, and decreased sodium delivery to the distal tubules.

Two critical regions identified in the 5'-flanking region of the REN gene that are required for its transcriptional regulation are the proximal promoter and an enhancer element located at –2866 to –2625 bp. The proximal promoter was originally reported to consist of binding sites for cAMP response element-binding protein (CREB) and pituitary-specific transcription factor 1 (Pit-1) (1, 2, 3). Liver X receptor-{alpha} has also been reported to be a cAMP-responsive factor that, through the cAMP response element (CRE), is required for maximal renin expression (4, 5). More recently, transcription factors important in fetal development including homeobox D10 (HoxD10), pre-ß-cell leukemia transcription factor 1b, CBF1, and Ets-1 were found to map to conserved regions in the renin proximal promoter and are required for high-level renin transcription (6, 7). The importance of renin expression developmentally may be underscored by the observation that gene-targeted deletion of renin leads to severe renal abnormalities and lethality (8).

The mouse renin enhancer (mE) was identified by deletion mutagenesis and transient transfection analysis in mouse kidney renin-expressing As4.1 cells and is now recognized to be a highly conserved transcriptional regulatory element required for high-level expression in As4.1 cells (9, 10). The enhancer consists of several tightly linked regulatory elements that can bind either stimulatory or inhibitory transcription factors. Stimulatory transcription factors include retinoic acid (RA) receptor-{alpha}/retinoid X receptor, E-box proteins upstream stimulatory factor-1 and -2, and CREB/CRE modulator, whereas the inhibitory factors include nuclear factor Y and nuclear receptor 2f6 (9, 11, 12, 13, 14, 15). The enhancer has also been implicated as the target of inflammatory cytokines such as IL-1ß and TNF{alpha}, which attenuate renin expression and act through mechanisms, including opposition of CREB activity at the CRE by nuclear factor-{kappa}B, and by indirect effects of signal transducer and activator of transcription-3 and -5 through mechanisms that remain unclear (16, 17, 18, 19).

Previous studies have shown that a homologous sequence to mE has been identified approximately 11 kb upstream of the human REN promoter (10, 20). Despite extensive sequence homology, the mE exhibits a markedly higher transcriptional stimulatory activity than the human renin enhancer (hE). However, both appear to be required to maintain baseline expression of renin in vivo (21, 22). The purpose of this study was 2-fold. First, we sought to identify the mechanism accounting for the disparity in transcriptional activity stimulated by the mE and hE. Second, we sought to determine whether natural sequence variation (polymorphism) exists in the hE that may differentially bind transcription factors and alter renin expression in humans. Our data show that a single nucleotide change in one of the transcription factor binding sites in hE can account for the difference in activity between hE and mE and that a common polymorphism found in both the normal and hypertensive population mapping directly between two transcription factor binding sites in hE is functionally silent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture, transient transfection, and luciferase assay
As4.1 cells (CRL2193; American Type Culture Collection, Rockville, MD) were maintained in DMEM (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were plated on 10-cm dishes 24 h before transfection in DMEM containing 10% FBS. Before transfection, the medium was removed and replaced with fresh medium containing 1% FBS. Cells were then transfected with a master mix containing luciferase reporter vector using Fugen-6 (Roche, Indianapolis, IN) and split by trypsin and replated into six-well plates approximately 5 h later. Thapsigargin (Thap) (100 nmol/liter), IL-1ß (1 ng/µl), and all-trans RA (atRA) (1 µmol/liter) were added to the medium 24 h after transfection (19, 23). The FBS used for the retinoic-treated cells was charcoal treated. Forty to 48 h after transfection, the cells were harvested and lysed, and luciferase activity was determined using the dual-luciferase reporter (DLR) kit (Promega, Madison, WI). RSV-LUC was used as a positive control and pRL-SV40 (Promega) used as an internal control. Luciferase activity was normalized to total cellular protein in lysates and then calculated as a percentage of RSV promoter activity. Luciferase activity assays in each independent experiment were performed in duplicate, and the average of the two readings became one data point (n = 1).

Plasmids
The luciferase (LUC) reporter vectors m2.6, mE2.6, hE2.6, hEB(G->A)2.6, h896, hE896, and mE896 were described previously (10, 11, 12). We PCR amplified the mE and hE with ApaI sites on both primers and subcloned into linearized plasmids m2.6 and h896. Site-directed mutagenesis was performed by use of the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The sequence of all mutants was confirmed by DNA sequencing and restriction digestion analysis. The mutant constructs hES(G->A), SDM1, SDM2, and h896(C->A) were generated with the following oligonucleotides (mutated bases are italic and deleted bases indicated by dashes): 5'-CAGATGGTGACCTAGCCATACTGGCCTCTCAGATCCTTGG-3', 5'-CAGATGGTGACCTGGCTGTA—CTGACCTCTGAGTGGCTGG-3', 5'-CGCAGATGGTGACCTGGCCATA—CTGACCTCTGAGTGGC-3', and 5'-GAAACCTGGGTACCCTTCACCCACCTAGATCTGTCCCGCAGTG-3', respectively.

RNA isolation and RNase protection assay
As4.1 cells were treated with Thap (100 nmol/liter), IL-1ß (1 ng/µl), atRA (1 µmol/liter), or vehicle (in 1% FBS). Total RNA was extracted using the QIAGEN (Valencia, CA) RNeasy kit. T3 RNA polymerase was used to prepare antisense RNA transcripts as RNase protection probes. The protected probe for mouse REN mRNA was 326 nucleotides, and the protected probe for cyclophilin was 105 nucleotides. RNase protection was performed using the RPAIII (Ambion Inc, Austin, TX). The protected fragments were resolved on 5% polyacrylamide denaturing gel (containing 8 M urea), and incorporated radioactivity was quantified with phosphorimager (GE Healthcare, Sunnyvale, CA).

Restriction fragment length polymorphism (RFLP)
Genomic DNA samples were identified as heterozygous by means of a PCR, followed by a RFLP. The PCR product amplifying 300 bp encompassing the hE using 5'-CTGATGTGGACACTGGGAGAAGAC-3' and 5'-CAAGTAGGACGTGGCTGTGGATAG-3' with the G->A polymorphism resulted in a loss of the SfiI restriction site, whereas homozygous samples were digested by both Bgl1 and Sfi1 at 37 C (New England Biolabs, Beverly, MA). The digested fragments were separated on a 1.5% agarose gel.

DNA, PCR, and sample sequencing
Ninety genomic DNA samples were obtained from the Coriell DNA Polymorphism Discovery Resource (http://ccr.coriell.org/nigms/products/pdr.html). The samples were derived from normal subjects of European, African, Mexican, Asian, and Native American ancestry. DNA samples from 95 severely hypertensive black and 95 severely hypertensive white individuals were obtained in a completely blinded fashion that prevented identification of the original patient by either the provider or recipient of the DNA. Thus, this research was classified as nonhuman subjects research by the University of Iowa Institutional Review Board. PCR were performed to amplify 300 bp of the human enhancer and 400 bp of the human promoter using the primers 5'-CTGATGTGGACACTGGGAGAAGAC-3' and 5'-CAAGTAGGACGTGGCTGTGGATAG-3' and 5'-ACGCTTGTCCCAGTTTTGAT-3' and 5'-AGCGAGGCATCCTTCTCC-3', respectively, in a volume of 50 µl. Final reaction conditions were 20 ng DNA, 0.2 µM each primer, 1x PCR buffer, 2 mM MgSO4, 0.2 mM dNTP, and 1 U Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA) using a Bio-Rad (Hercules, CA) thermal cycler. PCR was initiated with a hot start at 94 C for 2 min, and standard cycling conditions were carried out at 94 C for 30 sec, 55 C for 30 sec, and 68 C for 30 sec (30 cycles) with a final extension at 68 C for 5 min. PCR products for sequencing were purified with the QIAquick PCR purification kit (QIAGEN). Sequencing was performed at the University of Iowa Genome Center using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA). The sequences were analyzed using the Phred algorithm with the base cutoff set at Q > 60 (error rate <1 in 1 x 106). All sequences found to have a single-nucleotide polymorphism (SNP) were validated by resequencing in both directions from independent PCR products. The enhancer and promoter SNPs were deposited in the NCBI SNP database.

Statistical analysis
Our data are presented as means ± SE. Group comparisons of the data were analyzed by two-way ANOVA using the Bonferroni correction for multiple testing using SigmaStat (SPSS Scientific, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The renin enhancer is required for maximal activity of the renin promoter and was initially reported as a 241-bp sequence located 2.6 kb and approximately 11 kb upstream of the mouse and human REN genes, respectively (9, 10). Extensive deletion and mutagenesis studies revealed that most of the activity of the enhancer can be attributed to a 55-bp sequence consisting of several closely linked binding sites for transcription factors (Fig. 1AGo). Mutagenesis studies revealed that elimination of the CRE (element-d), E-box (element-e), and what has been termed the RA response element (RARE) because it binds RA receptor-{alpha}/retinoid X receptor (elements b+c), markedly lowers transcriptional activity (11, 13). On the contrary, deletion of the binding site for nuclear factor Y (element-a), or increasing the spacing between element-a and element-b increases transcriptional activity (12). These data suggest the enhancer may act as a complex regulatory element that can either stimulate or inhibit renin gene expression. In vivo, the enhancer is required to maintain the baseline level of renin expression but is dispensable for tissue- and cell-specific expression (21, 22).


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 1. Schematic representation of the renin enhancer. A, Sequence alignment of the region of the mouse and human renin enhancers (mREN, hREN) required for maximal activity. The regulatory elements Ea–Ee in the enhancer are shown with their corresponding transcription factors binding sites underlined. Both Ec and Eb with the spacer sequence between them form the RARE. Dashes indicate missing bases; vertical lines show identical nucleotides, and asterisks indicates a natural polymorphism. B, Schematic map of the mouse and human renin gene constructs used in this study. The location of the mE and hE is indicated by the cross-hatched and checkerboard rectangles, respectively. C, SDM1 was generated by a two-nucleotide deletion in the spacer region between Eb and Ec within the 2.6-kb 5'-flanking sequence of the mouse Ren-1c gene cloned upstream of luciferase. SDM2 was next generated by a two-nucleotide mutation using SDM1 as a template. Asterisks indicate the deleted and mutated bases. D, hEB(G->A) contains a G->A mutation in the TGACCT motif. E, hES(G->A) was generated by site-directed mutagenesis of the first base in the RARE spacer in hE.

 
An alignment of mouse and human renin enhancer sequences revealed them to be identical in the CRE, E-box, and one half-site of the RARE (element-c), whereas a SNP was evident in the other half-site of the RARE (element-b) (Fig. 1AGo). The core sequence of the RARE is conserved in the enhancers upstream of the renin genes of 11 different strains of inbred mice. This led us to hypothesize that this change may have functional significance. There were also species-specific differences noted in both length and sequence of the spacer separating the two RARE half-sites. To examine the mechanism for the difference in enhancer activity, we performed transient transfections in renin-expressing As4.1 cells. Constructs were generated that consisted of the mE or hE fused to either 2.6 kb of the mouse REN promoter or 896 bp of the human REN promoter (Fig. 1BGo). We then examined transcriptional activity of the constructs at baseline or after treatment with atRA, which stimulates renin promoter activity, and IL-1ß and Thap, which both decrease renin promoter activity. We first verified the effects of these treatments on endogenous renin gene expression in As4.1 cells (Fig. 2Go). IL-1ß and Thap decreased renin expression, whereas renin expression was modestly induced in response to atRA. Quantification of three independent samples revealed an 83% decrease in renin mRNA by Thap, a 32% decrease by IL-1ß, and a 53% increase in renin mRNA by atRA.


Figure 2
View larger version (87K):
[in this window]
[in a new window]

  		FIG. 2. Endogenous renin mRNA in As4.1 cells. RNase protection assay of total RNA isolated from As4.1 cells incubated with 100 nmol/liter Thap, 1 ng/µl IL-1ß, and 1 µmol/liter atRA for 24 h. NS indicates nonstimulated As4.1 cells. The treatment was performed in triplicate. The mouse REN protected fragment runs at 326 nucleotides and cyclophilin at 105 nucleotides.

 
Compared with a construct lacking the enhancer (m2.6), mE stimulated promoter activity 18 ± 2.3-fold compared with 3.6 ± 0.4-fold for hE (P < 0.001, Fig. 3Go). The mE was similarly more powerful than hE under atRA-stimulated (49 ± 9- vs. 6 ± 1-fold, P = 0.002 conditions). To determine whether sequence variation in the RARE spacer could explain the difference in renin enhancer activity between mouse and human, mutagenesis was performed on mE2.6 to generate SDM1 and SDM2 (Fig. 1CGo). SDM1 is a deletion of two nucleotides in the spacer so that the size of the hE and mE spacers are the same, whereas SDM2 contains the same deletion as SDM1 but also mutates two additional nucleotides so that the spacer sequence matches that of hE. Compared with mE2.6, the spacer mutations did not decrease mE-induced promoter activity (13.6 ± 1.7-fold for SDM1 and 35.7 ± 1-fold for SDM2, Fig. 3Go). Both spacer mutants also roughly retained the same magnitude of induction by RA. These data suggest that differences in the RARE spacer cannot account for the difference in mE and hE activity.


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 3. Transcriptional effect of mE spacer mutation. A, Transcriptional activity (percent RSV-Luc) of m2.6, hE, mE, SDM1, and SDM2 is shown. Cells were treated with vehicle (white bars) or tRA (1 µmol/liter) (black bars). *, P < 0.05, RA vs. no RA; {dagger}, P < 0.05 vs. hE; {dagger}{dagger}, P < 0.05 vs. hE plus RA (n = 5 for all constructs).

 
Next, to assess whether the difference in enhancer activity can be attributed to sequence variation in the RARE half-site (element-b), site-directed mutagenesis was performed to alter element-b of hE to match that of mE (Fig. 1DGo). Compared with m2.6, which lacks the enhancer, hEB(G->A) and mE stimulated promoter activity 12.5 ± 1-fold and 8 ± 2-fold, respectively, compared with 1.9 ± 0.4-fold for the unaltered hE (P < 0.001, Fig. 4AGo). Similarly, hEB(G->A) retained the decrease in renin promoter activity by Thap and IL-1ß and the increase in promoter activity by RA. Similar results were obtained when the various wild-type and mutant enhancers were fused to the human renin 896-bp promoter (Fig. 4BGo). A two-way ANOVA of the entire data set revealed that hEB(G->A) acts much more like mE than hE. Interestingly, when fused to the mouse renin 2.6-kb promoter, hEB(G->A) exhibited significantly higher activity than mE. This may be due to the absence of the negative regulatory element in hE normally encompassed by element-a in mE (Fig. 1AGo), thus causing a superinduction above that of mE.


Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 4. Transcriptional activity of mouse and human REN enhancer. Luciferase reporter vectors were transfected into As4.1 cells using a mouse 2.6-kb promoter (A) and a human 896-bp promoter (B). Shown are the transcriptional activities (percent RSV-Luc) of pGL2Basic (pGL2B) and constructs lacking the enhancer 2.6 or 896, containing the wild-type enhancer (mE and hE), or containing a G->A mutation in Eb of the human REN enhancer [hEB(G->A)]. Cells were either treated with vehicle (white bars), 100 nmol/liter Thap (gray bars), 1 ng/µl IL-1ß (cross-hatched bars), or 1 µmol/liter atRA (black bars). *, P < 0.05 vs. hE; {dagger}, P < 0.05 vs. hE treated with RA (n = 5 for all constructs). Two-way ANOVA was performed to assess significance between the complete data sets of hEB(G->A) and mE vs. hE. P values are indicated.

 
To determine whether a natural variation exists in the sequence of the renin E in humans, we obtained 90 genomic DNA samples from normal individuals of variable ethnicity from the Coriell Polymorphism Discovery Resource. We also obtained genomic DNA from 95 white and 95 black severely hypertensive subjects (Table 1Go). We used high-throughput PCR sequencing to identify any naturally occurring SNPs in the hE (Table 2Go). Sequencing revealed that 84 of the 90 Coriell samples, 88 of the 95 white severely hypertensive samples, and 93 of the 95 black severely hypertensive samples were totally conserved and identical to the consensus hE (Table 2Go). Interestingly, six of the Coriell, seven of the white hypertensive, and two of the black hypertensive samples all had the same G->A SNP within the RARE spacer, one nucleotide downstream of element-c (Fig. 5AGo). Both sequencing of PCR products (Fig. 5BGo) and RFLP (Fig. 5CGo) revealed the G->A change to be heterozygous in all subjects.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Characteristics of the hypertensive populations

 

View this table:
[in this window]
[in a new window]

  		TABLE 2. Summary of genotyping analysis

 

Figure 5
View larger version (35K):
[in this window]
[in a new window]

  		FIG. 5. Identification of a SNP in the human renin enhancer. A, A single G->A variation in the spacer of the hE RARE was identified in six of 90 samples of the Coriell DNA and two black hypertensive and seven white hypertensive individuals; B, sequence diagram showing the sequence of wild-type and heterozygous sample indicated by the arrow at the double peak; C, RFLP assay distinguishing the SNP revealed them to be heterozygous in all samples. The PCR fragment amplified in wild-type samples was digested with both Bgl1 and Sfi1, but the G->A polymorphism in heterozygous samples caused a loss in the Sfi1 restriction site.

 
To examine the effect of the G->A SNP in the RARE spacer, we mutated the hE construct to hES(G->A) and fused it to both the m2.6-kb and h896-bp promoters (Fig. 1EGo). Compared with m2.6, hES(G->A) had similar promoter activity as hE at baseline (1.8 ± 0.16-fold vs. 1.9 ± 0.4-fold) and after RA stimulation (Fig. 6AGo). Activity of mE was greater than either hE or hES(G->A) at baseline or in response to RA stimulation. Similar results were obtained when hE and hES(G->A) were fused to the human renin promoter (Fig. 6BGo). These results suggest that a relatively frequent polymorphism exists in the hE but that it is functionally silent.


Figure 6
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 6. Transcriptional activity of the hE spacer SNP. Transcriptional activities (percent RSV-Luc) of pGL2Basic (pGL2B) and constructs lacking the enhancer (2.6 or 896) or containing the wild-type enhancers (hE and mE) and the spacer mutation [hES(G->A)] placed upstream of the m2.6 promoter (A) or h896 promoter (B) are shown. Cells were treated with vehicle (white bars), 100 nmol/liter Thap (gray bars), 1 ng/µl IL-1ß (cross-hatched bars), or 1 µmol/liter atRA (black bars). *, P < 0.05 vs. hE; {dagger}, P < 0.05 vs. hE treated with RA (n = 5 for all constructs). Two-way ANOVA was performed to assess significance between the complete data sets comparing hES(G->A), mE vs. hE. P values are indicated.

 
In addition to the enhancer, a region of the human renin promoter encompassing –324 to +71 was also sequenced, and a single polymorphism located at –130 was identified from a single black hypertensive individual (Fig. 7Go, A and B). To examine the transcriptional effect of the singleton polymorphism, site-directed mutagenesis was performed on both 896C and hE896C constructs to create 896A and hE896A. There was no difference in promoter or hE-induced activity irrespective of the nucleotide at position –130 under baseline or RA-stimulated conditions (Fig. 7CGo). Moreover, Thap and IL-1ß had little effect on promoter activity of any of the constructs tested. As above, this polymorphism appears silent.


Figure 7
View larger version (32K):
[in this window]
[in a new window]

  		FIG. 7. Transcriptional activity of the human renin promoter SNP. A, C->A variation in the human promoter sequence located between the CBF1 and TATA box identified from a single black hypertensive individual; B, sequence diagram showing the sequence of wild-type and heterozygous sample indicated by the arrow at the double peak; C, transcriptional activities (percent RSV-Luc) of pGL2Basic (pGL2B), 896C, 896A, hE896C, and hE896A are shown. Cells were treated with vehicle (white bars), 100 nmol/liter Thap (gray bars), 1ng/µl IL-1ß (cross-hatched bars), or 1 µmol/liter atRA (black bars); n = 5 for all constructs. Two-way ANOVA was performed to assess significance between the complete data sets comparing all four constructs. P values are indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hypertension is a multifactorial disorder where genetic and environmental factors contribute to its incidence. Given that the RAS is a critical regulator of blood pressure and a prime candidate in the development of hypertension, that renin mediates the rate-limiting step in the system, and that a potent evolutionarily conserved enhancer regulates transcription of the renin gene, we hypothesized that natural genetic variation in the renin enhancer might predispose to altered renin transcription and potentially hypertension. Therefore, as a prelude to a clinical association study, we examined the molecular mechanism differentiating the strength of the mouse and human renin enhancers and assessed the functional consequence on transcriptional activity of a naturally occurring polymorphism in the human renin enhancer. Showing a functional effect of a promoter polymorphism is a critical first step if one is to fully interpret an association between the polymorphism and a pathological condition.

The mouse renin gene is regulated by a classical enhancer that acts in a position- and orientation-independent manner (9). A sequence with about 85% identity to the 55-bp minimal mE sequence was identified approximately 11 kb upstream of the human promoter (10). Conserved noncoding sequences in the 5'-flanking region of genes are typically indicative of important transcriptional regulatory sequences. Indeed, the CRE, E-box, and upstream half-site of the RARE are 100% identical in the mouse and human renin enhancer, and mutagenesis and transfection studies clearly indicate their requirement for full enhancer activity (11, 13). Differences in the RARE spacer and downstream half-site therefore became obvious candidates to explain the 10-fold difference in mouse vs. human enhancer activity. Whereas the spacer was not causative of this difference, a reversion mutation changing human element-b to match mouse element-b increased enhancer activity up to a level similar to mE. This implies that the variant in element-b of the RARE may account for the difference in enhancer strength observed in vitro. Interestingly, this variant in the human enhancer is perfectly conserved in the 280 human DNA samples sequenced and retains retinoid-induced transcription of the renin promoter in As4.1 cells. What then is the physiological significance of the difference in mouse and human renin enhancer activity? One possibility is that this variant provides a molecular explanation for why the level of circulating renin in mice is much higher than in humans. Of course, we cannot rule out other potentially important factors including the distance from the enhancer to the promoter, which is much greater in the human renin (11 kb) than mouse renin (<3 kb) gene, and the experimental system (As4.1 cells), which is derived from the mouse (24). Although there is one human cell line (Calu-6) that expresses renin endogenously, it is not derived from the kidney and the regulation of renin mRNA is largely post-transcriptional (25, 26).

The identification of a naturally occurring renin enhancer variant among species suggested the possibility that naturally occurring variants might be present in the human renin promoter and enhancer of people of differing ethnicities and in hypertensives. One has to be very cautious when interpreting the presence of polymorphisms in the promoter of a gene especially when the transcriptional regulation of that gene is poorly characterized or when the functional transcription factor binding sites are unknown. Indeed, numerous SNPs in promoter regions of candidate genes for many complex diseases have been uncovered. In our study, we examined only those sequences where functional data implicate the presence and physiological relevance of a transcription factor binding site.

The initial 90 DNA samples were obtained from the Coriell DNA Polymorphism Discovery Resource and include unrelated individuals of European-American, African-American, Mexican-American, Native American, and Asian-American descent, representing approximately 27, 27, 13, 7, and 27% of the samples, respectively. The other 190 were anonymous DNA samples obtained from severely hypertensive black and white individuals. The only polymorphism identified was a single frequent SNP in the first base of the RARE spacer. Its presence in the spacer just a single nucleotide downstream from the fully conserved upstream half-site of the RARE suggested the possibility it may modulate enhancer activity. However, both this polymorphism and the singleton polymorphism identified to lie between the binding sites for CBF1 and HOXD10.PBX in the human renin promoter were both silent in transcriptional assays in As4.1 cells.

In a previous study, polymorphisms in the rat renin promoter were identified when comparing sequences from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats (27). However there was no consistent variants observed in hypertensive strains that were not present in normotensive strains, and no differences in transcription factor binding was reported. Similarly, variants in the renin promoter could not explain differences in renin expression observed when comparing angiotensin-responsive aldosterone-producing adenomas with angiotensin-unresponsive adenomas (28). In addition to the kidney enhancer located 2.6 kb upstream of mouse renin and 11 kb upstream of human renin, there was a report of a second enhancer highly active in choriodecidual cells (29). This enhancer (termed the chorionic enhancer) was reported to lie between –5777 and –5552 upstream of the human renin promoter. A recent study identified two variants in healthy French individuals downstream of the chorionic enhancer at coordinates –5870 and –5312 (30). Although downstream of the original reported location of the enhancer, DNA sequences including the 225-bp enhancer and extending to include these variants had higher transcriptional activity than the 225-bp enhancer alone. Moreover, one of the variants, –5312T, had 45% greater activity than did –5312C. Although we cannot rule out the importance of this enhancer, it is not conserved upstream of the Ren-1c or Ren-2 mouse renin gene and therefore is not an evolutionarily conserved noncoding sequence.

In conclusion, our results cannot be used to justify a study to assess an association between a polymorphism in the human renin kidney enhancer with hypertension. Indeed, we strongly feel it is critical to first evaluate the functional significance of a polymorphism in the regulatory region of a gene before embarking on a clinical study.


   Acknowledgments
 
We graciously thank Natasha Widmer for assistance with the initial identification of the spacer SNP in the Coriell samples, Dr. Henry Keen for bioinformatic support, and Dr. Val Sheffield and Gretel Beck for high-throughput sequencing. We gratefully acknowledge the generous research support of the Roy J. Carver Trust.


   Footnotes
 
This work was supported by grants from the National Institutes of Health (HL48058, HL61446, and HL55006 to C.D.S. and HL35795 to J.H.P.).

Disclosures: H.A.I., X.L., J.H.P., and C.D.S. have nothing to declare.

First Published Online December 7, 2006

Abbreviations: Ang, Angiotensin; atRA, all-trans retinoic acid; CRE, cAMP response element; CREB, CRE-binding protein; FBS, fetal bovine serum; hE, human renin enhancer; HoxD10, homeobox D10; mE, mouse renin enhancer; RA, retinoic acid; RARE, RA response element; RAS, renin-angiotensin system; RFLP, restriction fragment length polymorphism; SNP, single-nucleotide polymorphism; Thap, thapsigargin.

Received October 11, 2006.

Accepted for publication November 29, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Germain S, Konoshita T, Philippe J, Corvol P, Pinet F 1996 Transcriptional induction of the human renin gene by cyclic AMP requires CREB and a factor binding to pituitary-specific trans-acting factor (Pit-1) motif. Biochem J 316:107–113
  2. Ying L, Morris BJ, Sigmund CD 1997 Transactivation of the human renin promoter by the cyclic AMP/protein kinase A pathway is mediated by both CREB-dependent and CREB-independent mechanisms in Calu-6 cells. J Biol Chem 272:2412–2420[Abstract/Free Full Text]
  3. Sun J, Oddoux C, Lazarus A, Gilbert MT, Catanzaro DF 1993 Promoter activity of human renin 5'-flanking DNA sequences is activated by the pituitary-specific factor Pit-1. J Biol Chem 268:1505–1508[Abstract/Free Full Text]
  4. Tamura K, Chen YE, Horiuchi M, Chen Q, Daviet L, Yang Z, Lopez-Ilasaca M, Mu H, Pratt RE, Dzau VJ 2000 LXR{alpha} functions as a cAMP-responsive transcriptional regulator of gene expression. Proc Natl Acad Sci USA 97:8513–8518[Abstract/Free Full Text]
  5. Tamura K, Chen YE, Tanaka Y, Sakai M, Tsurumi Y, Koide Y, Kihara M, Pratt RE, Horiuchi M, Umemura S, Dzau VJ 2004 Nuclear receptor LXR{alpha} is involved in cAMP-mediated human renin gene expression. Mol Cell Endocrinol 224:11–20[CrossRef][Medline]
  6. Pan L, Xie Y, Black TA, Jones CA, Pruitt SC, Gross KW 2001 An Abd-B class HOX.PBX recognition sequence is required for expression from the mouse Ren-1c gene. J Biol Chem 276:32489–32494[Abstract/Free Full Text]
  7. Pan L, Glenn ST, Jones CA, Gross KW 2005 Activation of the rat renin promoter by HOXD10.PBX1b.PREP1, Ets-1, and the intracellular domain of notch. J Biol Chem 280:20860–20866[Abstract/Free Full Text]
  8. Takahashi N, Lopez ML, Cowhig Jr JE, Taylor MA, Hatada T, Riggs E, Lee G, Gomez RA, Kim HS, Smithies O 2005 Ren1c homozygous null mice are hypotensive and polyuric, but heterozygotes are indistinguishable from wild-type. J Am Soc Nephrol 16:125–132[Abstract/Free Full Text]
  9. Petrovic N, Black TA, Fabian JR, Kane CM, Jones CA, Loudon JA, Abonia JP, Sigmund CD, Gross KW 1996 Role of proximal promoter elements in regulation of renin gene transcription. J Biol Chem 271:22499–22505[Abstract/Free Full Text]
  10. Shi Q, Black TA, Gross KW, Sigmund CD 1999 Species-specific differences in positive and negative regulatory elements in the renin gene enhancer. Circ Res 85:479–488[Abstract/Free Full Text]
  11. Shi Q, Gross KW, Sigmund CD 2001 Retinoic acid-mediated activation of the mouse renin enhancer. J Biol Chem 276:3597–3603[Abstract/Free Full Text]
  12. Shi Q, Gross KW, Sigmund CD 2001 NF-Y antagonizes renin enhancer function by blocking stimulatory transcription factors. Hypertension 38:332–336[Abstract/Free Full Text]
  13. Pan L, Black TA, Shi Q, Jones CA, Petrovic N, Loudon J, Kane C, Sigmund CD, Gross KW 2001 Critical roles of a cyclic AMP responsive element and an E-box in regulation of mouse renin gene expression. J Biol Chem 276:45530–45538[Abstract/Free Full Text]
  14. Liu X, Huang X, Sigmund CD 2003 Identification of a nuclear orphan receptor (Ear2) as a negative regulator of renin gene transcription. Circ Res 92:1033–1040[Abstract/Free Full Text]
  15. Pan L, Glenn ST, Jones CA, Gronostajski RM, Gross KW 2003 Regulation of renin enhancer activity by nuclear factor I and Sp1/Sp3. Biochim Biophys Acta 1625:280–290[Medline]
  16. Baumann H, Wang Y, Richards CD, Jones CA, Black TA, Gross KW 2000 Endotoxin-induced renal inflammatory response. Oncostatin M as a major mediator of suppressed renin expression. J Biol Chem 275:22014–22019[Abstract/Free Full Text]
  17. Pan L, Wang Y, Jones CA, Glenn ST, Baumann H, Gross KW 2005 Enhancer-dependent inhibition of mouse renin transcription by inflammatory cytokines. Am J Physiol Renal Physiol 288:F117–F124
  18. Todorov VT, Volkl S, Muller M, Bohla A, Klar J, Kunz-Schughart LA, Hehlgans T, Kurtz A 2004 Tumor necrosis factor-{alpha} activates NF{kappa}B to inhibit renin transcription by targeting cAMP-responsive element. J Biol Chem 279:1458–1467[Abstract/Free Full Text]
  19. Liu X, Shi Q, Sigmund CD 2006 Interleukin-1ß attenuates renin gene expression via a mitogen-activated protein kinase kinase-extracellular signal-regulated kinase and signal transducer and activator of transcription 3-dependent mechanism in As4.1 cells. Endocrinology 147:6011–6018[Abstract/Free Full Text]
  20. Yan Y, Jones CA, Sigmund CD, Gross KW, Catanzaro DF 1997 Conserved enhancer elements in human and mouse renin genes have different transcriptional effects in As4.1 cells. Circ Res 81:558–566[Abstract/Free Full Text]
  21. Adams DJ, Head GA, Markus MA, Lovicu FJ, van der WL, Kontgen F, Arends MJ, Thiru S, Mayorov DN, Morris BJ 2006 Renin enhancer is critical for control of renin gene expression and cardiovascular function. J Biol Chem 281:31753–31761[Abstract/Free Full Text]
  22. Zhou X, Davis DR, Sigmund CD 2006 The human renin kidney enhancer is required to maintain baseline renin expression but is dispensable for tissue-specific, cell-specific and regulated expression. J Biol Chem 281:35296–35304[Abstract/Free Full Text]
  23. Klar J, Sigl M, Obermayer B, Schweda F, Kramer BK, Kurtz A 2005 Calcium inhibits renin gene expression by transcriptional and posttranscriptional mechanisms. Hypertension 46:1340–1346[Abstract/Free Full Text]
  24. Sigmund CD, Okuyama K, Ingelfinger J, Jones CA, Mullins JJ, Kim U, Kane-Haas C, Wu C, Kenney L, Rustum Y, Dzau V, Gross KW 1990 Isolation and characterization of renin expressing cell lines from transgenic mice containing a renin promoter viral oncogene fusion construct. J Biol Chem 265:19916–19922[Abstract/Free Full Text]
  25. Lang JA, Yang G, Kern JA, Sigmund CD 1995 Endogenous human renin expression and promoter activity in a pulmonary carcinoma cell line (Calu-6). Hypertension 25:704–710[Abstract/Free Full Text]
  26. Sinn PL, Sigmund CD 1999 Human renin mRNA stability is increased in response to cAMP in Calu-6 cells. Hypertension 33:900–905[Abstract/Free Full Text]
  27. Leckie BJ 2001 Polymorphisms of the renin gene promoter in spontaneously hypertensive and Wistar-Kyoto rats. Clin Exp Pharmacol Physiol 28:60–63[CrossRef][Medline]
  28. Ballantine DM, Klemm SA, Tunny TJ, Stowasser M, Gordon RD 1996 PCR-SSCP analysis of the promoter region of the renin gene in patients with aldosterone-producing adenomas. Clin Exp Pharmacol Physiol 23:584–586[Medline]
  29. Germain S, Bonnet F, Philippe J, Fuchs S, Corvol P, Pinet F 1998 A novel distal enhancer confers chorionic expression on the human renin gene. J Biol Chem 273:25292–25300[Abstract/Free Full Text]
  30. Fuchs S, Philippe J, Germain S, Mathieu F, Jeunemaitre X, Corvol P, Pinet F 2002 Functionality of two new polymorphisms in the human renin gene enhancer region. J Hypertens 20:2391–2398[CrossRef][Medline]



This article has been cited by other articles:


Home page
 HypertensionHome page
N. Moore, P. Dicker, J. K. O'Brien, M. Stojanovic, R. M. Conroy, A. Treumann, E. T. O'Brien, D. Fitzgerald, D. Shields, and A. V. Stanton
Renin Gene Polymorphisms and Haplotypes, Blood Pressure, and Responses to Renin-Angiotensin System Inhibition
Hypertension, August 1, 2007; 50(2): 340 - 347.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/3/1424    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Itani, H. A.
Right arrow Articles by Sigmund, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Itani, H. A.
Right arrow Articles by Sigmund, C. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals