Functional Polymorphism of the Anpep Gene Increases Promoter Activity in the Dahl Salt-Resistant Rat
We have reported that aminopeptidase N/CD13, which metabolizes angiotensin III to angiotensin IV, exhibits greater renal tubular expression in the Dahl salt-resistant (SR/Jr) rat than its salt-sensitive (SS/Jr) counterpart. In this work, aminopeptidase N (Anpep) genes from SS/Jr and SR/Jr strains were compared. The coding regions contained only silent single nucleotide polymorphisms between strains. The 5′ flanking regions also contained multiple single nucleotide polymorphisms, which were analyzed by electrophoretic mobility-shift assay using renal epithelial cell (HK-2) nuclear extracts and oligonucleotides corresponding with single nucleotide polymorphism–containing regions. A unique single nucleotide polymorphism 4 nucleotides upstream of a putative CCAAT/enhancer binding protein motif (nucleotides −2256 to −2267) in the 5′ flanking region of the SR/Jr Anpep gene was associated with DNA-protein complex formation, whereas the corresponding sequences in SS rats were not. A chimeric reporter gene containing ≈4.4 Kb of Anpep 5′ flank from the Dahl SR/Jr rat exhibited 2.5- to 3-fold greater expression in HK-2 cells than the corresponding construct derived from the SS strain (P<0.05). Replacing the CCAAT/enhancer binding protein cis-acting element from the SS rat with that from the SR strain increased reporter gene expression by 2.5-fold (P<0.05) and abolished this difference. CCAAT/enhancer binding protein association was confirmed by chromatin immunoprecipitation and correlated with expression, suggesting selection for a functional CCAAT/enhancer binding protein polymorphism in the 5′ flank of Anpep in the Dahl SR/Jr rat. These results highlight a possible association of the Anpep gene with hypertension in Dahl rat and raise the prospect that increased Anpep may play a mechanistic role in adaptation to high salt.
Aminopeptidase N (Anpep; EC 18.104.22.168; also known as CD13, gp150, microsomal aminopeptidase, and aminopeptidase M), is a homodimeric, membrane-bound, zinc-dependent aminopeptidase that preferentially releases neutral amino acids from the amino terminus of oligopeptides and has specificity similar to that of cytosolic leucine aminopeptidase (reviewed in References 1,2). Anpep belongs to the M1 family of the MA family of peptidases, also known as gluzincins, and includes membrane-bound type II glycoproteins. It has been cloned from 6 different mammalian species.3,4 It is widely distributed in tissues and, in the kidney, concentrated in the brush border membrane of proximal tubule cells.5–7 Anpep has been implicated in the regulation of enkephalins and pain, angiogenesis, tumor metastasis and invasion, inflammation, secretion, and apoptosis (reviewed in Reference 2).
Among Anpep substrates, which include neuropeptides (Met and Leu enkephalins, neurokinin A, Met-lys-bradykinin, and edorphins), vasoactive peptides (kallidin, somatostatin, and angiotensins), and chemotactic peptides (monocyte chemotactic protein/MCP-1 and N-formyl-methionine leucine phenylalanine/f-MLP), is the heptapeptide Ang III, which is metabolized to the natriuretic hexapeptide angiotensin IV (Val-Tyr-IIe-His-Pro-Phe) by deletion of the NH2-terminal arginine (reviewed in References 7,8). Unlike Ang II or Ang III, angiotensin IV is a natriuretic peptide capable of inhibiting renal Na uptake via blockade of ouabain-sensitive Na-K ATPase activity, increasing cortical blood flow, and reducing blood pressure in the rat.9–11
The expression of the Anpep gene, which consists of 20 exons, is regulated by alternate tissue-specific promoters, that is, an epithelial-specific proximal promoter and distal myeloid-specific promoter 8 kb upstream.7,12 Both promoters drive the expression of transcripts encoding identical cognate proteins. Differences in 5′ untranslated sequences derived from alternate noncoding first exons account for the reported size differences between myeloid-specific (≈3.7 kb) and epithelial-specific (≈3.4 kb) transcript sizes.12 The immediate 5′ flank of the human Anpep gene contains a TATA-like element (ATATAA) 22 bases upstream of the transcriptional start site (+1) that is conserved with the orthologous porcine gene. The proximal epithelial-specific promoter is also characterized by conserved binding sites for several trans-acting factors, including hepatocyte nuclear factor 1 and Sp1, which are thought to play important roles in Anpep proximal promoter activity.13,14 An enhancer region controlling proximal promoter function has also been identified within ≈2.7 kb of the start site, which contains canonical binding sites for members of the winged helix Ets family, including CCAAT/enhancer binding protein (C/EBP).7
From the rat sequencing data, it can be deduced that the Anpep gene lies within quantitative trait loci (QTLs) for blood pressure identified in both Dahl salt-sensitive (SS/Jr)-Lewis and Dahl SS/Jr–salt-resistant (SR/Jr) crosses (15,16 available online at http:/hyper.ahajournals.org). We have reported that Anpep transcript abundance, protein abundance, and activity are greater in the kidneys of Dahl SR/Jr than in SS/Jr rats, raising the possibility that increased Anpep-mediated signaling, perhaps by angiotensin IV, reduces renal tubular Na uptake in adaptation to high salt.15 Consistent with this is the finding that the nonclipped kidney in the Goldblatt 2-kidney 1-clip model has a highly significant increase in membrane-bound Anpep activity.17,18 Based on these results, we have proposed that the Anpep gene may be linked to salt-sensitive hypertension in the Dahl rat. The present study was performed to further examine this possibility by comparing the Anpep gene between Dahl SS/Jr and SR/Jr rat strains.
Rat Strains and Reagents
Male Dahl SS (SS/Jr), Dahl SR (SR/Jr), and Lewis rats were purchased from Harlan Sprague–Dawley (Indianapolis). Animal studies were approved by the University of Illinois at Chicago animal care committee. Culture medium (DMEM F12) and T4 polynucleotide kinase were purchased from Gibco BRL. Luciferase reporter and β-galactosidase assay systems and plasmid pGL3 were from Promega. [γ-32P] ATP was from Amersham Biosciences. Automated DNA sequencing was done by Davis Sequencing. Oligonucleotides were purchased from Qiagen. Rabbit polyclonal C/EBPα and C/EBPβ antibodies were purchased from Santa Cruz Biotechnology.
Mycoplasma-free human renal epithelial (HK-2) cells were obtained from the American Type Culture Collection and were maintained in DMEM-F12 medium supplemented with 10% FBS in a humidified atmosphere of 5% CO2.
Analysis of Anpep Gene
Genomic DNA was isolated by phenol–chloroform extraction. The 4.4-kb 5′ flanking regions of the Anpep gene were PCR amplified using express suquence tag-based sequences and cloned into the TA vector (Invitrogen) for automated sequencing. Polymorphisms were confirmed by sequencing both strands of DNA derived from 5 or 6 rats from each strain. DNA sequences were compared using DNAsis software for Windows (Molecular Biology Insights).
Transfections and Luciferase Assays
Anpep promoter/enhancer constructs were generated by inserting 4.4-kb 5′ flanking regions of Dahl SS/Jr and SR/Jr rats as SacI/XhoI fragments into a pGL3-basic promoter-less vector digested with SacI/XhoI. pAnpep SS/SR construct was generated by replacing the −2232 to −2394 sequence of the SS/Jr rat with that of the SR/Jr rat by digesting the SR/Jr rat promoter sequence with MscI and NheI restriction enzymes. To generate a SR/Jr Anpep promoter with mutant C/EBPα binding site (SR/C/EBP mutant), the −2232 to −2394 region was amplified using a primer 5′CTGAGCGAAGTTTagGAAagTGA 3′ and then cloned into the MscI and NheI site of SR/Jr Anpep promoter plasmid. C/EBPα expression vector was described previously.19 C/EBPα dominant-negative mutant (C’30) was a HindIII and AscI deletion of wild-type C/EBPα provided by Dr Daniel G. Tenen (Harvard Medical School, Boston, MA).20
Transient transfection of HK-2 cells was performed using the Lipofectamine 2000 reagent according to the manufacturer’s instructions (Invitrogen). Approximately 48 hours after transfection, luciferase reporter activity was measured in fresh whole-cell lysates using a commercially available luciferase assay system (Promega) and TD20/20 luminometer. To control for variations in transfection efficiency, cells were cotransfected with a control β-galactosidase expression vector (pSV-βGal, Pharmacia), and luciferase activity was normalized for β-galactosidase activity, measured by the β-galactosidase assay kit (Promega), in the same samples.
Electrophoretic Mobility Shift Assays
Nuclear extracts from HK-2 cells were prepared as described previously.21 Probes for gel-shift analysis were generated by PCR using specific primers flanking each single nucleotide polymorphism (SNP; details in an online supplement available http:/hyper.ahajournals.org) and were end-labeled with [γ-32P] ATP using polynucleotide kinase (Promega). Oligonucleotides corresponding with the −2277 to −2255 sequence, containing a C/EBP-reported canonical binding site, −2256 to −2266, and the −2272 T SNP (5′ CTGAGTGAAGTTTCAGAACATGA 3′; wild type; WT) and a mutant form containing 4 mutated bp (5′ CTGAGTGAAGTTTagGAAagTGA 3′), were used as probes to examine fragment 9. Electrophoretic mobility-shift assay (EMSA) was performed by incubating (30 minutes, room temperature) nuclear extract (25 μg) from HK-2 cells in a total volume of 30 μL of EMSA buffer with 32P-labeled double-stranded oligonucleotides (8 fmol). For competition assays, a 100-fold excess of unlabeled oligonucleotide was added for 15 minutes before the addition of the labeled probe. Samples were run on a 6% nondenaturing polyacrylamide gel. DNA–protein complexes were visualized by autoradiography. Supershift assay was performed by adding 2 μg of C/EBPα antibody (Abcam) 10 minutes before adding the labeled probe.
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation assay was performed using a kit assay (Upstate). Chromatin fragments were isolated from nuclei by sonication in SDS lysis buffer (1% SDS, 10 mmol/L of EDTA, and 50 mmol/L of Tris; pH 8.1). Fragments were precleared with protein A agarose beads and salmon sperm DNA followed by overnight incubation with continuous shaking at 4°C either in the presence of control normal rabbit IgG or polyclonal C/EBPα antibody. The beads were washed once each with a low-salt immune complex, high-salt immune complex, and LiCl wash buffer followed by 2 washes with 10 mM Tris/1 mM EDTA (pH 8.0) buffer and incubated at 65°C overnight in a 50-μL buffer consisting of 20 mmol/L of Tris·HCl (pH 8.0), 100 mmol/L of NaCl, and 10 mg/mL of proteinase K. The resulting DNA fragments were subjected to real-time PCR using primers flanking the C/EBP putative cis-binding sequence (palindrome) and −2272 T/C SNP in the Anpep promoter region (forward: 5′ GATTGGAA AAGGAAGACA 3′ and reverse: 5′ GGAGCCATCAGAAGCC 3′) and control primers flanking the PDE4B promoter that do not contain any C/EBP or C/EBP-related transcription factor binding sites (forward: 5′ AGTGTTTATTAACCTAGGTCTTTCT 3′ and reverse: 5′ CCAATAAACCAGTGCATTCAAGATC 3′). Quantitative real-time PCR analysis for chromatin immunoprecipitation assays and quantitation of C/EBPα protein binding to the 5′ flanking region was calculated as described previously.22 ΔCT is derived by subtracting the threshold cycles (CT) of SS/Jr and SR/Jr rat chromatin immunoprecipitates amplified using control primers from corresponding samples (CT) using primers flanking the C/EBPα site and the 2272 T to C polymorphism. ΔCT of 1 is equivalent to a 2-fold change in sensitivity.
Comparisons were made by ANOVA. Data are expressed as mean±SD. Experimental values were significantly different at P<0.05.
Identification of Anpep Gene Polymorphisms
Silent SNPs in Coding Region of Anpep Gene Between SS/Jr and SR/Jr Rats
Anpep cDNA was sequenced in 7 SR/Jr and 8 SS/Jr rats. The sequences corresponded with that in Genbank (Accession No. 205108 M25073) with the exception of silent SNPs (see online Appendix). Two silent SNPs were identified at positions 2306 and 2292, that is, T/C and G/A, respectively.
Anpep Gene Polymorphisms Within the 5′ Promoter Region Between SS/Jr and SR/Jr Rats
The 4.4-kb 5′ flanking region of the Anpep gene was sequenced from the Dahl SR/Jr and SS/Jr strains. A total of 11 SNPs were identified between SR/Jr and SS/Jr strains (Figure 1).
CEBPα Associates With 5′ Flanking Region of Anpep Gene in SR/Jr but Not SS/Jr Rats
EMSA was performed using 11 PCR-generated fragments, each containing 1 of the 11 SNPs detected between SR/Jr and SS/Jr strains (Figure 1). Inclusion of these PCR-generated DNA fragments with nuclear extract from HK-2 cells revealed complex formation only in fragment 9 from SR/Jr but not from the SS/Jr strain (Figure 2, C2272 + protein and arrow), suggesting that the T −2272 C SNP increases binding affinity of nuclear protein for that region of the promoter. Fragment 9 from the Lewis rat was also sequenced, because it was used in Dahl SS/Jr crosses to identify the blood pressure QTL to which the ANPEP gene maps.4 It also contained the −2272 C SNP (no other changes).
A database search (TRANSFAC) of fragment 9 identified a putative canonical C/EBP DNA binding site: RTTGCGYAAY (−2256 to −2267 bp). The C/T SNP is 4 nucleotides upstream (−2272) of this C/EBP binding site. No other putative cis-acting DNA elements were identified in fragment 9.
C/EBPα association with fragment 9 was examined by chromatin immunoprecipitation assay. C/EBPα and C/EBPβ were detected by immunoblot analysis in rat kidneys (see the online supplement). Chromatin fragments were immunoprecipitated with C/EBPα antibody. Control primers without binding sites for any C/EBP confirmed chip assay specificity. C/EBPα binding to the endogenous Anpep promoter from the SR/Jr rat was ≈7-fold greater than that from the SS/Jr strain (Figure 3).
C/EBPα association to the canonical binding site (TTagGAA) in fragment 9 was examined by EMSA using HK-2 cell protein extracts (Figure 4). A DNA–protein complex was detected with a labeled WT but not mutant probe with CA/AG mutations (−2263/64 and −2258/59; Figure 4a, labeled WT lane 2 versus mutant probe lane 2). Competition with 100-fold excess of unlabeled WT oligonucleotide abolished the band, and incubation with C/EBPα antibody super shifted the complex (Figure 4a, labeled WT probe lanes 3 and 4, arrow). In contrast, 100-fold excess of mutant oligonucleotide did not abolish the binding of c/EBPα protein with WT probe, suggesting the specificity of c/EBPα binding (Figure 4a, right, lane 2).
In parallel experiments, luciferase promoter activity of the 4.4-kb 5′ flanking region of Anpep was reduced by ≈50% (P<0.05; Figure 4b) by introducing the same mutations, that is, C/A-2263/64 and A/G-2258/59, indicating coordinate decreases in C/EBPα–DNA association and promoter activity.
Anpep Promoter Activity Is Greater and Stimulated by c/EBPα in the SR/Jr Versus SS/Jr Rat Strain
Anpep promoter activity was measured using luciferase reporter constructs harboring the −4.4-kb 5′ Anpep region from SS/Jr and SR/Jr strains in HK-2 cells (Figure 5). Basal promoter activity was 2-fold greater in SR/Jr than SS/Jr strains (Figure 5, black bar versus striped bar). Cotransfection with C/EBPα increased activity in SR/Jr but not SS/Jr strains (Figure 5, + C/EBPα black versus striped bars). Cotransfection with dominant-negative C/EBPα reduced the basal promoter activity of the SR/Jr rat by 2-fold indicating the regulation of Anpep promoter activity of the SR/Jr rat by endogenous C/EBPα (Figure 5, striped bar versus + C/EBPα DN striped bar). The activity of an SS/SR hybrid Anpep promoter reporter plasmid (pAnpep-SS/SR) in which fragment 9 from the SR/Jr rat was inserted (−2232 to −2394) into the Anpep promoter from the SS/Jr rat was also greater than that of the SS/Jr alone (Figure 6, SS/Jr versus SS/SR). These results indicate that the −2272 T–C polymorphism confers increased promoter activity.
The present results identify a functional polymorphism of a C/EBPα cis-element in the promoter of Anpep gene in the Dahl SR/Jr rat. The C/EBP transcription factors are a highly conserved group within the basic leucine zipper family that control genes involved in the control of cellular growth and differentiation, immune and inflammatory responses, and neural function/memory.23 Six C/EBP members (α to δ) have been cloned. Our data show that both α and β forms of C/EBP are expressed in HK-2 cells, consistent with previous reports in the kidney.23
The essential C/EBP binding site has been reported to contain a dyad symmetrical repeat 5′-RTTGCGYAAY-3′, where R is A or G and Y is C or T,24 and its palindrome.24 The present results show that C/EBPα association with fragment 9 requires both TTCAGAA, a segment of the defining dyad repeat of C/EBP cis-elements, and a T SNP 4 bp upstream from this sequence. This is the first report, to our knowledge, of an SNP not within the dyad repeat regulating C/EBPα association. However, SNPs at a wide range of distances, ranging from a few to hundreds of base pairs,25,26 from the consensus binding sequences have been reported to be capable of influencing the DNA–protein association. The mechanisms of these regulations are not often clear, but may involve another protein, for example, enhancer or cofactor, binding to either DNA or C/EBPα.
Promoter activity for the 4.4-kb 5′ flanking region of the Anpep gene from SR/Jr rats is greater than that from SS/Jr strain in HK-2 cells, consistent with greater Anpep transcript and protein abundance in the kidneys.4 The difference in promoter activity was present even in the presence of a dominant-negative mutant of C/EBPα, suggesting other important cis-acting elements and/or a role of C/EBPβ. Eleven SNPs are identified in the 4.4-kb 5′ flanking region of the Anpep gene. EMSA was used to screen for effects of these SNPs on the DNA–protein interaction. C/EBPα was the only trans-acting factor identified in this manner as physically associating with the functionally significant SNP-containing region. This does not, however, exclude binding by other factors with different binding affinities or in a combinatorial context in vivo.
The results raise the possibility that the Anpep gene is a salt-sensitive hypertension susceptibility gene in the Dahl rat model. The 2272 C SNP is present in the SR/Jr but not SS/Jr rat and can be inferred to map to a previously mapped QTL identified in the Dahl SS/Jr × SR/Jr cross. The 2272 C SNP is also present in the Lewis rat, a less salt-sensitive strain. We have reported previously that Anpep maps to a QTL defined in a Dahl SS/Jr–Lewis cross,15 suggesting that this SNP is important in differentiating the Lewis from Dahl SS/Jr rat strains. Additional strategic congenic and transgenic rat experiments are necessary to confirm a link between adaptation to high salt and the Anpep gene.
The present results identify Anpep as a new candidate gene in the Dahl rat. Although many genes have been tested, the specific genes underlying salt-sensitive hypertension in the Dahl rat remain elusive. Whether Anpep is truly important will require complementary strategies. Transgenic, congenic, and consomic strains are likely to be especially useful in the further judgment of Anpep.
A critical component to evaluating Anpep as a candidate gene is demonstrating a functional or physiological mechanism that links Anpep to the regulation of renal salt handling. In this regard, Anpep is especially intriguing, because it has been linked to RAS by metabolizing angiotensin III to angiotensin IV, a “diuretic” peptide. This raises the spectra of another level of complexity in the regulation of RAS. Significantly, it may lead to the discovery of a physiological role for angiotensin IV, which has been reported to be, as opposed to angiotensin II, a diuretic peptide, which reduces Na+ uptake in tubule cells. Time will tell whether this Anpep “pans out” as a cause of salt-sensitive hypertension in the Dahl rat and/or plays a role in human forms of hypertension.
We appreciate the critical discussion and input of Dr Anne Kwitek (Medical College of Wisconsin, Milwaukee, WI).
Sources of Funding
These studies were supported by National Institutes of Health Awards 5R21DK065628-02 and DK54687-06 (to R.S.D. and R.H.C., respectively), American Heart Association Grant-in-Aid (R.S.D.), and the Phillip Morris Research Institute (R.S.D.).
- Received October 12, 2006.
- Revision received November 2, 2006.
- Accepted December 13, 2006.
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