1,25 Dihydroxyvitamin D Amplifies Type A Natriuretic Peptide Receptor Expression and Activity in Target Cells
Songcang Chen,
Xi-Ping Ni,
Michael H. Humphreys and
David G. Gardner
Diabetes Center and Department of Medicine, University of California at San Francisco, San Francisco, California
Address correspondence to: Dr. David G. Gardner, 1109 HSW, Diabetes Center, University of California at San Francisco, 3rd and Parnassus Avenue, San Francisco, CA 94143-0540. Phone: 415-476-2729; Fax: 415-564-5813; E-mail: gardner{at}itsa.ucsf.edu
1,25 dihydroxyvitamin D (VD) has been shown to exert a numberof beneficial effects on cardiovascular function, includingreduction in BP and inhibition of cardiac hypertrophy. In aneffort to identify a possible mechanistic link between VD andthese salutary effects, the role of VD in controlling the activityand expression of the type A natriuretic peptide receptor (NPR-A),a receptor that signals reductions in BP and suppression ofcellular growth in the myocardium and vascular wall, was investigated.VD, as well as the nonhypercalcemic analogue RO-25-6760, increasedNPR-A–dependent cyclic guanosine monophosphate productionand NPR-A gene expression in cultured rat aortic smooth musclecells. The increase in NPR-A expression was associated withan increase in NPR-A gene promoter activity that was criticallydependent on the presence of a functional VD receptor responseelement located approximately 495 bp upstream from the transcriptionstart site of the gene. This element was associated with theVD receptor/retinoid X receptor complex in vitro. Mutation ofthis element resulted in complete elimination of the VD-dependentinduction of the NPR-A gene promoter but did not affect osmoticstimulation of the promoter. Treatment of rats with RO-25-6760for 7 d increased the atrial natriuretic peptide–dependentexcretion of sodium and cyclic guanosine monophosphate withoutaffecting mean arterial BP or plasma calcium levels. This wasassociated with a twofold increase in NPR-A mRNA levels in theinner medulla. Amplification of NPR-A activity represents aplausible mechanism to account for at least some of the beneficialeffects that VD exerts on cardiovascular function.
The natriuretic peptides constitute a family of closely relatedvasoactive hormones that play an important role in the regulationof cardiovascular, renal, and endocrine homeostasis (1). Atrialnatriuretic peptide (ANP) and brain natriuretic peptide (BNP)are produced predominantly in the heart, whereas C-type natriureticpeptide (CNP) is produced in endothelial cells of the vasculature(2), reproductive tissues (3), growth plate of long bones (4),and the central nervous system (5). The natriuretic peptidesexert their biologic effects through association with specificmembrane-bound receptors. Natriuretic peptide receptor typesA and B (NPR-A and NPR-B, respectively) each have a large extracellularligand-binding domain connected to the intracellular signalingdomain through a single membrane-spanning segment. The intracellulardomain harbors a noncatalytic, kinase-like region linked toa particulate guanylyl cyclase at the carboxy terminus of themolecule (1). NPR-A serves as the cognate receptor for bothANP and BNP, whereas NPR-B binds selectively to CNP. Most ofthe important physiologic activities of these peptides seemto be mediated through activation of these guanylyl cyclase–linkedreceptors. A third receptor subtype, NPR-C, is thought to functionlargely, albeit not entirely (6), as a clearance receptor. Blockadeof NPR-C has been shown to increase levels of endogenous natriureticpeptides in circulating plasma (7).
Vitamin D is a seco-steroid that bears a number of importantsimilarities to hormonal ligands that act through nuclear mechanismsto control gene expression. Its most polar metabolite, 1,25dihydroxyvitamin D (VD), binds specifically and with high affinityto a member of the nuclear receptor gene family termed the vitaminD receptor (VDR). The liganded VDR, when complexed with itsheterodimeric partner, the retinoid X receptor (RXR), associateswith specific regulatory elements on DNA called vitamin D responseelements (VDRE). One such VDRE, DR3, is a direct repeat of thesequence AGGTCA separated by a three-nucleotide spacer (AGGTCANNNAGGTCA).DR3 displays selectivity in preferentially binding to the VDR-RXRheterodimer versus other nuclear receptor dimeric complexes(8).
A number of studies have identified VD as a potentially importantregulator of BP and cardiovascular homeostasis. The mechanismunderlying the inverse relationship between VD levels and BPis not completely understood; however, the recent studies ofLi et al. (9) suggest that the renin-angiotensin system maybe involved. Using genetically engineered VDR knockout mice,they showed that these animals were hypertensive relative totheir VDR +/+ littermates and that this increase in BP was accompaniedby apparent activation of the plasma renin-angiotensin system(RAS).
The data are equally supportive for an important regulatoryrole for VD in the heart. Rats made vitamin D–deficientdisplay elevations not only in BP but also in ventricular hypertrophy(10). The former seems to be related, at least in part, to theattendant hypocalcemia because calcium supplementation correctsthe hypertension; however, the cardiac hypertrophy persists.Subsequent studies from the same group indicated that a majorcomponent of the hypertrophy seen in vitamin D–deficientanimals reflects expansion of the interstitial compartment inthe heart (predominantly cardiac fibroblasts) with increasedproduction of extracellular matrix proteins (11). VD has beenshown to reduce endothelin-stimulated ANP and BNP gene expression(markers of myocyte hypertrophy) and transcription in culturedneonatal rat atrial (12) and ventricular (13) myocytes and tosuppress the hypertrophic response to endothelin, a well knownhypertrophic agonist in the cultured ventricular myocyte model(13). It is interesting that this suppressive activity seemsto require structural features of the VDR that are more typicallyassociated with the activation function of this receptor (14,15).
Like the liganded VDR, activated NPR-A has been shown to beboth antihypertrophic and antihypertensive (16,17) in a numberof different model systems. In the present study, we exploredthe possibility that VD might exert beneficial effects on thecardiovascular system through NPR-A. Our findings support thishypothesis in demonstrating that VD increases both the activityof the NPR-A protein and the expression of the NPR-A gene incultured rat aortic smooth muscle (RASM) cells.
Materials
[-32P]dCTP and the cyclic guanosine monophosphate (cGMP) RIAkit were purchased from Perkin-Elmer Life Sciences (Boston,MA). ANP was obtained from Phoenix Pharmaceuticals, Inc. (MountainView, CA). RNeasy minikit was from Qiagen Inc. (Santa Clara,CA). Primer-it RMT kit, hybridization solution, and NucTrappush columns were purchased from Stratagene (La Jolla, CA).RO-25-6760 was provided by M. Uskokovic (Roche Pharmaceuticals).Other reagents were obtained through standard commercial suppliers.
Culture of Vascular Smooth Muscle Cells
Neonatal RASM cells, originally isolated by P. Lynch at theUniversity of Southern California, were obtained at passage20 from H. Ives at University of California San Francisco. Cellswere cultured at 37°C in a 5% CO2-humidified incubator inDMEM supplemented with 10% FBS, 100 units/ml penicillin, 100µg/ml streptomycin, and 2% (vol/vol) broth, tryptose phosphate(18).
Measurement of ANP-Stimulated cGMP Levels
RASM cells were grown to approximately 80% confluence, thenchanged to serum-substitute media (19) for 24 h. At that point,cells were treated with 10–10 to 10–7 M VD for 48h. For measurement of ANP-stimulated cGMP accumulation, cellswere washed three times with prewarmed PBS and incubated with0.5 ml of DMEM that contained 0.5 mM isobutylmethylxanthineand 10 mM HEPES (pH 7.4) for 10 min at 37°C. 10–7M ANP was added to the medium, and the incubation was continuedfor another 10 min. The reaction was stopped by the removalof medium and addition of 0.3 ml of 12% TCA. The extractionwas continued for 30 min at 4°C. The contents of the platewere collected and centrifuged to pellet particulate material.The supernatant fraction was extracted four times with 0.5 mlof water-saturated ether. cGMP levels were determined by RIAafter acetylation of the sample and standard using a commercialantibody and [125I]cGMP as tracer.
RNA Isolation and Northern Blot Analysis
RASM cells were plated in 10-cm dishes, cultured, and treatedwith VD or the nonhypercalcemic vitamin D analogue RO-25-6760(20,21) at different concentrations, as indicated in the individualfigure legends. Total RNA was extracted from cells using theRNeasy minikit according to instructions provided by the manufacturer.Total RNA was denatured and separated on a gel that contained2.2% formaldehyde, transferred to nitrocellulose filters, andhybridized to radiolabeled cDNA as described previously (22).A 1.2-kb EcoRI fragment of the rat NPR-A cDNA was isolated fromvector sequence, radiolabeled using the Primer-it RMT kit (Stratagene),and separated from free nucleotide using NucTrap push columns(Stratagene). The membranes were prehybridized for 30 min at68°C and hybridized with 32P-labeled NPR-A cDNA (107 cpm/2ml of hybridization solution) for 1 h at 68°C in hybridizationsolution provided by Stratagene. All membranes were subsequentlystripped and rehybridized with a radiolabeled 1150-bp BamHI/EcoRIfragment of 18S rDNA to permit normalization among samples fordifferences in RNA loading and/or transfer to the filter. Hybridizationsignal was detected by autoradiography and quantified usingthe NIH Image program.
Plasmid Constructions
–1575 rat NPR-A LUC was generated by cloning 1575 bp ofNPR-A 5' flanking sequence into BamH1/NarI sites of pFOXLUCvector as described previously (23). Six of the deletion mutantswere generated through the use of convenient restriction sites.In each case, the 3' linkage used the NarI site of the pFOXLUCvector. The 5' linkage used unique restriction sites (NdeI for–1290LUC, BclI for –716LUC, HindIII for –387LUC,SstII for –273LUC, and DraII for –77LUC) for placementin the polylinker of the parent vector. The shortest construct,–9LUC was generated using an upstream sense oligonucleotide(5'-GGGGAT CCTTCTGGCACACTCCTG-3') that incorporated a BamHIsite at its 5' terminus and a downstream antisense oligonucleotide(5'-TAGAGGATAGAATGGCGCCGG-3') derived from 3' linker sequenceof pFOXLUC containing the NarI site. A PCR product of appropriatesize was restricted with BamHI/NarI and cloned into compatiblesites of pFOXLUC. All of the deletion constructs were sequencedto confirm the predicted structure.
Transfection and Luciferase Assay
RASM cells were transfected by electroporation at 250 V and960 µF using 1 µg of –1575 NPR-A LUC with0.2 µg of cytomegalovirus--galactosidase. After transfection,cells were plated on six-well plates in growth medium for 24h. Medium was then changed to DMEM/serum substitute for theensuing 24 h, at which point various concentrations of VD, or10–8 M VD at different time intervals, were added. Luciferaseactivity was measured using the luciferase assay system (Promega).-Galactosidase activity was assayed using the Galactolight Pluschemiluminescence assay (Tropix, Bedford, MA). Luciferase levelswere normalized for -galactosidase activity in the individualcultures.
Preparation of Nuclear Extracts
RASM cells were transfected with 5 µg of hVDR and hRXRor sham transfected using Lipofectin Reagent (InVitrogen) for24 h. Cells were changed to serum substitute media and treatedwith vehicle or 10–8 M VD for 48 h. Nuclear extracts wereprepared as described previously (24).
Electrophoretic Mobility Shift Assay 35S-labeled hVDR and unlabeled hVDR and hRXR were translatedin vitro using a cell-free translation kit (TNT T7 Quick MasterMix kit; Promega) with [35S]methionine and unlabeled methionine,respectively. Double-stranded DR3 (5'-AGCTTCAGGTCAAGGAGGTCAGAG-3';consensus VDRE half sites indicated in bold letters) and wild-typeor mutant oligonucleotides derived from NPR-A promoter sequencespanning the region between –504 and –472 were generatedfor use in the electrophoretic mobility shift assay (EMSA).Two different protocols were used for EMSA.
Protocol 1
Two microliters each of unlabeled recombinant VDR and hRXR or3 µl of RASM cell nuclear extract (see above) were incubatedin binding reaction buffer (10 mM HEPES [pH 7.9], 50 mM KCl,0.2 mM EDTA, 2.5 mM DTT, 10% glycerol, and 0.05% NP-40) thatcontained 0.5 µg of poly(dI-dC) and 32P-end-labeled, double-strandedoligonucleotide, and 10 nM VD at room temperature for 30 min.For competition experiments, a 10- or 100-fold molar excessof unlabeled, double-stranded oligonucleotide was added to thebinding reaction. All samples were resolved on 5% nondenaturingpolyacrylamide gels. Gels were dried and exposed to x-ray film.
Protocol 2
Two microliters of 35S-labeled VDR (synthesized in vitro) wasincubated, in the presence of 2 µl of unlabeled hRXR and0.5 µg poly(dI-dC), with 20 ng of unlabeled, double-strandedDR3 or NPR-A oligonucleotides and 10 nM VD. Reactions were carriedout in binding buffer (10 mM NaHPO4 [pH 7.6], 0.25 mM EDTA,0.5 mM MgCl2, and 5% glycerol) for 20 min at 23°C. DNA–proteincomplexes were resolved by electrophoresis on 5% nondenaturingpolyacrylamide gels run in TEA buffer (67 mM Tris-HCl [pH 7.5],10 mM EDTA, and 33 mM sodium acetate) at 240 V for 3 h at 4°C.The gel was washed three times with 30% methanol and 10% glacialacetic acid, amplified for 30 min (Amplifier, Amersham PharmaciaBiotech), dried, and subjected to autoradiography.
Site-Directed Mutagenesis
Mutations (M1, M2) were created in –1575 NPR-A-Luc bysite-directed mutagenesis using a commercial kit (Stratagene).Briefly, mixtures that contained 50 ng of –1575 NPR-A-Luc,two mutagenic primers, dNTP, and Pfu DNA polymerase were addedto the PCR buffer.
PCR was carried out for 18 cycles using 30 s of denaturationat 95°C, 1 min of annealing at 55°C, and 2 min/kb extensionat 68°C. After PCR, 1 µl of Dpn I was added to thereaction to cut parental DNA template, and 5 µl of thisdigest was used for transformation. Several candidate cloneswere identified and characterized by DNA sequencing.
Analysis of Renal ANP Activity In Vivo
Under anesthesia (intraperitoneal injection of Brevital sodium50 mg/kg), a midscapular incision was made and Alzet osmoticminipump was implanted subcutaneously into adult male Sprague-Dawleyrats. In the treatment group, the pumps were loaded with RO-25-6760(0.8 µg/kg per d dissolved in 98% propylene glycol + 2%ethanol). In the control group, pumps were filled out with vehiclealone. The wound was closed with sutures, and the rats werereturned to their cages after recovery from anesthesia. Overthe ensuing 7 d, they were observed for any signs of calciumtoxicity: Tremors, muscle spasms, seizures, anorexia, or weightloss. One week after pump implantation, each rat received anintraperitoneal injection of Inactin (100 mg/kg) to induce anesthesia.Animals were placed on a heated operating table. A tracheotomywas performed, and a segment of PE205 tubing was inserted intothe trachea to facilitate clearance of secretions from the upperrespiratory tract. The left jugular vein was exposed, and acatheter (PE50) was placed. A solution of normal saline thatcontained 1 mg/ml BSA was infused continuously at 25 µl/minthrough this catheter. The carotid artery was identified througha small paratracheal incision, and a tapered polyethylene catheter(PE50) was inserted for measurement of arterial pressure witha P23id Statham BP transducer attached to a direct writing recorder.A small catheter (PE10) was inserted into the right ureter nearthe hilum through a right flank incision for collection of urinefrom the right kidney. A catheter (PE50 tubing) was suturedinto the dome of the urinary bladder via a suprapubic incisionfor the timed collection of urine from the left kidney. A curved30-G needle attached to PE10 tubing was inserted into the leftrenal artery near its takeoff from the aorta through a leftflank incision under the rib cage. Prompt reflux of arterialblood into the catheter was used to indicate successful needleplacement. After completion of this preparative surgery, therats received an infusion of normal saline that contained 5%BSA (total volume equal to 0.7% of total body weight) over 15min to replace surgical fluid losses. BP was measured over thesubsequent 30 min, and an average value of the electrical meanwas taken to record mean arterial pressure. At that point, ANP(250 fmol/L per min) was infused into the left renal arteryfor 20 min. Urine was collected from both kidneys in the 20-minperiod before and during the 20-min period of intrarenal ANPinfusion. At the end of study, the rats were killed, blood wascollected and processed for measurement of plasma calcium, andthe renal inner medulla from both kidneys was isolated for measurementof NPR-A mRNA. Urine volume was determined, and urinary sodiumwas measured by flame photometry (Model 943; InstrumentationLaboratories, Lexington, MA). Urine samples were then diluted50-fold before measurement of cGMP (2-µl sample) usingthe method described above. All procedures were approved bythe IAUCC at UCSF.
Real-Time PCR Analysis
Total RNA was prepared from frozen inner medullas of controland RO-25-6760–treated rats using the RNaeasy kit. Twomicrograms of total RNA was used to reverse transcribe cDNAusing the Clontech reverse transcription kit. Rat NPR-A andglyceraldehyde phosphate dehydrogenase (GAPDH) (25) primer pairsand probes were synthesized by Applied Biosystems. Real-timePCR was performed using Taqman Master mix (A&B Applied Biosystems)with an ABI Prism 7700 (A&B Applied Biosystems). Negativecontrols without input cDNA were used to assess signal specificity.NPR-A transcript levels were quantified and normalized for GAPDHtranscript levels in each sample.
Statistical Analyses
Data were analyzed by ANOVA using Bonferroni test to assesssignificance.
To determine whether VD has a role in the regulation of NPR-Aexpression/activity, we examined the functional response ofthe receptor to ligand (ANP-dependent cGMP generation) in thepresence or absence of VD. Forty-eight hours of VD treatmenthad no effect on basal cGMP levels in RASM cells (Chen et al.,data not shown). ANP treatment of control cells led to an approximatelyfivefold increment in cGMP levels, relative to basal, over a10-min incubation period. VD pretreatment nearly doubled thisresponse with a net stimulation of cGMP levels of approximately10-fold after ANP treatment (Figure 1A). A similar amplificationof the response to ANP was seen after pretreatment with thenonhypercalcemic VD analogue RO-25-6760. This increment in NPR-Aactivity resulted, at least in part, from an increase in NPR-Agene expression. As shown in Figure 1B, VD pretreatment ledto an approximately five- to sixfold increase in steady-stateNPR-A mRNA levels. Of note, the nonhypercalcemic analogue RO-25-6760led to a similar increment (approximately fivefold) in NPR-AmRNA levels in cultured RASM cells (Figure 1C).
Figure 1. Vitamin D (VD) effects on natriuretic peptide receptor A (NPR-A) activity and expression. (A) Rat aortic smooth muscle (RASM) cells were cultured in serum substitute medium for 24 h and then treated with VD (10–10 to 10–7 M) or RO-25-6760 (10–8 M) for 48 h. Cells were washed extensively and incubated with 10–7 M atrial natriuretic peptide (ANP) in the presence of isobutylmethylxanthine (0.5 M) for 10 min. ANP-stimulated cyclic guanosine monophosphate (cGMP) was determined as described in the Materials and Methods section. Pooled data from three independent experiments are shown. **P < 0.01 versus control in the presence of ANP. Control cGMP levels were 198 ± 21 pmol/mg soluble protein. (B) RASM cells were treated with various concentrations of VD for 48 h. Total RNA was isolated, and Northern blot analysis was performed as described in the materials and Methods section. Representative autoradiographs are shown. The bar graph displays findings (NPR-A mRNA/18S rRNA) from three separate experiments. **P < 0.01 versus control. (C) RASM cells were exposed to various concentrations of RO-25-6760 for 48 h. Total RNA was prepared, and Northern hybridization was carried out. NPR-A mRNA levels were normalized to 18S rRNA. VD and RO-25-6760 concentrations in all three panels presented as log [M]. Pooled data from three independent experiments are shown. **P < 0.01 versus control.
The increment in gene expression, in turn, seems to reflectan increase in NPR-A gene transcription. As shown in Figure 2,VD, as well as RO-25-6760, treatment of cultured RASM cellsled to a significant increase in the activity of a transfectedNPR-A promoter-luciferase reporter construct that was not seenwith the transfected background vector (pGL3). This stimulationwas ligand dependent (Figure 2A), VD receptor dependent (Figure 2B),and time-dependent (Figure 2B) and amplifiable with co-transfectedRXR, the heterodimeric partner of VDR (Figure 2C).
Figure 2. VD upregulates NPR-A promoter activity in RASM cells. (A) RASM cells were transfected with –1595 NPR-A LUC or pFOX LUC, and cytomegalovirus--galactosidase (CMV--gal) and cultured for 24 h. Medium was then changed to DMEM/serum substitute for the ensuing 24 h, at which point VD or RO-25-6760, at the concentrations indicated (all concentrations presented as log [M]), was added for the next 48 h. (B) –1595 NPR-A LUC and CMV--gal were co-transfected with increasing concentrations of VD receptor (VDR) expression vector into RASM cells. After transfection, cells were treated with 10–8 M VD for indicated time intervals. (C) A total of 0.5 µg of VDR and retinoid X receptor (RXR) was co-transfected with –1595 NPR-A LUC/CMV--gal into RASM cells. The transfected cells were incubated with vehicle or 10–8 M VD for 48 h. In all cases, lysates were generated and luciferase and -gal activities were measured as described in the Materials and Methods section. Experiments were repeated three to six times. **P < 0.01, *P < 0.05 versus corresponding control.
A series of 5'-deletion mutants of the NPR-A promoter, extendingfrom –1575 to –77 relative to the transcriptionstart site, were generated and linked to the luciferase reporter.As shown in Figure 3, mutants that harbored as little as 706bp of the 5' flanking sequence retained the ability to respondto VD; however, the –387 NPR-A-luciferase mutant displayedno induction with VD treatment. This suggests that the VD-sensitiveregulatory element is positioned between –706 and –387in the NPR-A gene promoter.
Figure 3. Deletion analysis of VD induction of NPR-A promoter activity. Different deletions of NPR-A-LUC were made as described in the Materials and Methods section and transfected into RASM cells. Twenty-four hours later, cells were incubated in presence or absence of 10–8 M VD for 48 h before generating lysates for luciferase and -gal measurements. The experiments were repeated three to four times. **P < 0.01 versus corresponding control.
Examination of the DNA sequence spanning this region for thepresence of conventional VD regulatory elements revealed fivepotential candidates, each displaying moderate to high homologyto the DR3 motif (two AGGTCA motifs arrayed as direct repeatsand separated by three spacer nucleotides [8]; Figure 4). Double-strandedoligonucleotides spanning each of these regions were incubatedwith radiolabeled VDR, unlabeled RXR, and the VD ligand (10–8M) and fractionated on nondenaturing polyacrylamide gels toassess potential DNA–protein interactions (i.e., conventionalEMSA assay). As shown in Figure 5A, NPR-A-1, which displayedthe highest degree of homology to the consensus VDRE (homologousat 9 of 12 positions), failed to interact with the VDR-RXR complexin vitro. Similarly, NPR-A-2 and NPR-A-3 failed to associatewith this receptor complex (Figure 5B); however, NPR-A-4 displayeda level of VDR-RXR binding that was readily discernible, albeitat a fraction (approximately 50%) of the intensity seen withthe consensus DR3 motif. Binding to radiolabeled NPR-A-4 wasalmost completely inhibited by a 100-fold molar excess of unlabeledoligonucleotide (Figure 5C). Introduction of mutations intothe downstream candidate site (NPR-A-4 M1) did not impair theability of this oligonucleotide to inhibit VDR-RXR binding toNPR-A-4 (Figure 6A) (i.e., mutated sequence does not harborthe VDRE). Mutation of the upstream site (NPR-A-4 M2), however,resulted in near complete abrogation of the competition forNPR-A-4 binding to the heterodimeric receptor complex. The latterfinding suggests that the liganded VDR-RXR complex binds tothe upstream sequence that is targeted by the NPR-A-4 M2 mutation.This was confirmed in direct binding studies as shown in Figure6B. Whereas wild-type NPR-A-4 and the M1 mutant associated withradiolabeled VDR in this assay, the M2 mutant was devoid ofbinding activity.
Figure 4. Identification of candidate VD response elements (VDRE) in NPR-A promoter. Consensus VDRE half sites in DR3 motif are identified at the top. Candidate VDRE sequences in NPR-A promoter are identified in bold type. Termini identify positions of the DNA fragments relative to the transcription start site; orientation is indicated by arrows positioned above. Mutated bases in VDRE of NPR-A4 (M1 and M2) are indicated in lowercase letters.
Figure 5. Specific VDR/RXR binding activity resides in NPR-A-4 sequence. (A and B) 35S-VDR and unlabeled RXR were incubated with unlabeled oligonucleotides encoding a consensus DR3 motif or one of four putative VDRE in the NPR-A promoter. All incubations were carried out in the presence of VD (10–8 M). DNA-protein complexes were fractionated by electrophoretic mobility shift assays (EMSA) as described in the Materials and Methods section. (C) Unlabeled VDR and RXR, along with VD, were incubated with 32P-labeled NPR-A-4 oligonucleotide in the absence or presence of increasing concentrations of unlabeled oligonucleotide (10- or 100-fold molar excess). The reaction complexes were resolved on 5% nondenaturing polyacrylamide gels. The experiments were repeated two to three times. Representative autoradiographs are shown.
Figure 6. Selective mutation of the VDRE in NPR-A-4. (A) Unlabeled VDR and RXR, along with VD (10–8 M), were incubated with 32P-labeled NPR-A-4 oligonucleotide in the absence or presence of increasing concentrations (10- or 100-fold excess) of one of two mutant NPR-A oligonucleotides (see Figure 4 for identification). A 100-fold molar excess NPR-A1 oligonucleotide was included as negative control. (B) 35S-VDR, unlabeled RXR, and VD were incubated with the consensus DR3 element, wild-type NPR-A-1, wild-type NPR-A-4, or one of the two mutants of NPR-A-4 identified in Figure 4. EMSA was performed as described in the Materials and Methods section. Experiments were repeated three times; representative results are shown.
Binding of VDR to NPR-A-4 did not require ligand; binding ofrecombinant VDR/RXR to NPR-A-4, if anything, was reduced slightlyin the presence of 1,25 dihydroxyvitamin D (Figure 7A). It isinteresting that this differed from receptor expressed in atransfected cell. As shown in Figure 7B, extracts of cells thatwere transfected with hVDR plus hRXR showed a modest increasein binding to NPR-A-4 versus untransfected cells; however, theinclusion of ligand resulted in a dramatic increase in bindingof the extract to the oligonucleotide, suggesting that otherproteins in the extract may serve to stabilize the DNA-VDR/RXRcomplex.
Figure 7. VD promotes binding of VDR to VDRE in intact cells but not in cell-free extracts. (A) 35S-VDR and unlabeled RXR were incubated with unlabeled NPR-A-4 or consensus VDRE oligonucleotide in the presence or absence of 10–8 M VD and separated by EMSA. (B) Six micrograms of nuclear extract from sham- versus VDR/RXR-transfected RASM cells in the presence or absence of VD were incubated with 32P-labeled NPR-A-4 oligonucleotide and separated by EMSA. Each experiment was repeated three times. Representative autoradiographs are shown.
Extending this analysis to the functional assay, mutation ofthe M1 site in the context of –1575 NPR-A luciferase failedto influence the VD-dependent stimulation of this promoter,relative to the wild-type sequence, whereas introduction ofthe M2 mutation into the same promoter context resulted in completeloss of the VD-dependent stimulation (Figure 8A). Noteworthy,the M2 mutation did not prevent induction of the NPR-A promoterby osmotic stimuli such as NaCl or sucrose (22,26) (Figure 8B),indicating that the targeted sequence selectively transducesthe response to VD.
Figure 8. Mutation of the VDRE in the NPR-A gene promoter interferes with VD-induced NPR-A gene promoter activity. (A) Wild-type –1595 NPR-A-LUC or one of two individual mutants of the NPR-A-4 sequence (M1 and M2) was co-transfected with CMV--gal into RASM cells. (B) –1595 NPR-A-LUC and M2 mutant along with CMV--gal were co-transfected into RASM cells. After 24 h, the cells were treated with 10–8 M VD for 48 h. Alternatively, 48 h after transfection, cells were treated with 150 mM sucrose or 75 mM NaCl for 24 h. Luciferase and -gal activity was measured. Experiment was repeated three times. **P < 0.01 versus corresponding control.
To assess the physiologic relevance of the in vitro findings,we attempted to extend these analyses into an in vivo model.We chose a model that we have used previously to examine theeffects of ANP infused directly into the renal artery on urinaryvolume and sodium excretion (27). This model has the advantageof identifying ANP-induced changes in urinary sodium and waterhandling independent of changes in peripheral hemodynamics.As shown in Table 1, pretreatment of animals with the nonhypercalcemicanalogue RO-25-6760 for 7 d had no effect on basal mean arterialpressure relative to control. Serum calcium levels were measuredin both the control and the analogue-infused animals. Calciumlevels in the RO-25-6760–treated animals remained in thenormal range and were not statistically different from the controls(Table 1). Infusion of ANP (250 fmol/kg per min) into the leftrenal artery had no effect on systemic arterial pressure (Table 1).It did, however, promote an increase in urinary sodium excretionthat was not seen in the contralateral, noninfused kidney (Figure 9A).Pretreatment with RO-25-6760 for 7 d resulted in a significantincrease in ANP-stimulated urinary sodium excretion in the infusedkidney, and this was accompanied by a significant increase inANP-dependent urinary cGMP levels when compared with the controlgroup (Figure 9B). In both instances, the magnitude of the increases(in UNa and UcGMP) was larger in the RO-25-6760–treatedversus vehicle-treated rats. A modest elevation in UNaV andUcGMP was seen in urine that was collected from the contralateralkidney, presumably reflecting the effects of peptide that haseither found its way into the systemic circulation or refluxedinto the aorta from the infusion site. Collectively, these findingssupport our in vitro data in demonstrating that the VD analogueamplifies the activity of the natriuretic peptides in targetcells.
Figure 9. Effect of RO-25-6760 on ANP-stimulated UNaV, urinary cGMP, and NPR-A mRNA levels in vivo. (A) Intrarenal infusion of ANP (250 fmol/kg per min) for 20 min. Urine was collected from infused and contralateral kidney of control and RO-25-6760–treated rats before and after ANP infusion. Urine volume and sodium were measured. (B) Urinary cGMP levels were measured in aliquots of urine as described in the Materials and Methods section. Control cGMP levels were 533 ± 53 pmol/ml urine. (C) Real-time PCR analysis of NPR-A mRNA levels in rat inner medulla. Paired inner medullas from control and RO-25-6760–treated rats were isolated and snap-frozen in liquid nitrogen. Total RNA was prepared and reverse transcribed into cDNA. Real-time PCR was carried out as described in the Materials and Methods section. Pooled data (n = 8) are shown. **P < 0.01, *P < 0.05 versus ANP-infused control; +P < 0.01 versus corresponding control.
To determine whether the increase in natriuretic peptide activityresulted from increased NPR-A expression, as documented in thein vitro system above, we measured NPR-A mRNA transcripts ininner medulla of control versus RO-25-6760–treated ratsusing real-time PCR. As shown in Figure 9C, treatment with RO-25-6760for 7 d resulted in an approximately twofold increment in NPR-AmRNA levels in the inner medulla. Levels of the GAPDH transcriptwere essentially unchanged. This suggests that the incrementin activity of the liganded NPR-A noted above results from VD-dependentstimulation of NPR-A gene expression.
The studies described above provide definitive evidence fortranscriptional regulation of a cardiovascular gene by VD andidentify an important mechanism through which this hormone mayexert salutary effects in the cardiovascular system. Previousstudies have suggested regulation of NPR-C expression by glucocorticoids(28); however, transcriptional regulation was not documentedin those studies, and a specific glucocorticoid responsive elementwas not identified. The current study demonstrates VD1-dependentstimulation of NPR-A activity as well as NPR-A gene expression.The latter derives from an increase in NPR-A promoter activitythat is driven by a solitary VDRE located approximately 495bp upstream from the transcription start site.
Vitamin D activity is classically described in terms of itscalcitropic properties in the intestine, kidney, and bone (29);however, more recently increased attention has been devotedto its ability to regulate physiologic events through pathwaysindependent of those that it uses to control serum calcium andphosphorous concentrations. VD has immune-suppressant and anti-inflammatoryproperties (30), it reduces cell proliferation in a varietyof in vitro models through mechanisms that are only partiallyunderstood (31,32), and it seems to play an important role inthe growth and development of the epidermis and hair follicle(33,34). Data acquired through a number of different experimentaland clinical studies indicate that VD also plays an importantrole in cardiovascular homeostasis. 25-Hydroxyvitamin D levelsare inversely related to BP levels in selected forms of rodent(35) and human (36) hypertension, and administration of VD restoresBP to normal or near-normal levels (37,38). Of note, the recentstudy of Teng et al. (39) demonstrated improved survival ofpatients who had ESRD and were on hemodialysis when they weretreated with the nonhypercalcemic analogue of VD, paricalcitol,versus VD itself, suggesting the existence of important salutaryeffects of the seco-steroid that are independent of its calcitropicproperties. Because the majority of patients with ESRD die ofcardiovascular causes, the inference would be that the benefitaccrued secondary to the beneficial cardiovascular effects ofthese drugs.
True vitamin D–deficiency in the rat is accompanied byacquired hypertension and associated ventricular hypertrophy(10), although in this model, the BP elevation abates with correctionof the attendant hypocalcemia. The recent studies of Li et al.(9) suggest that hypertension results from defective VD actionin general, rather than solely from hypocalcemia. In VDR null(–/–) mice, systolic BP was elevated approximately20 mmHg versus controls, and this was associated with activationof the RAS. Because these animals were maintained on a high-calciumdiet to prevent the development of hypocalcemia, it seems likelythat the BP elevation is at least partially reflective of thedefect in VDR signaling. Li et al. also presented data suggestinga direct effect of the liganded VDR on the renin gene promoter,although a discrete functional VDRE was not identified.
NPR-A plays a major role in controlling BP and extracellularfluid volume (1). In fact, the studies of DuBois et al. (40)suggest that most, if not all, of the renal effects of ANP aremediated through this receptor. That it mediates vasodilation,increased urinary excretion of sodium and water, and suppressionof myocardial hypertrophy and fibrosis (17) makes it an attractivecandidate as a target of and mediator for VD action. Althoughthis does not exclude a parallel role for VD in suppressionof the RAS, it is noteworthy that the liganded NPR-A is alsocapable of suppressing the RAS at multiple points in the signalingcascade (41), including inhibition of renin gene expression.Therefore, it is conceivable that at least a portion of thereduction in renin expression could result from enhanced NPR-Aexpression and activity rather than through a direct ligandedVDR-dependent suppression of the renin gene promoter. In fact,Li et al. (42) reported that NPR-A gene expression is reducedin VDR –/– mice. Additional studies will be requiredto assess whether the direct versus indirect model is operativehere.
Finally, our previous demonstration that VD suppresses expressionand secretion of ANP (12,13,43) seems, at first glance, to bephysiologically inconsistent with the data presented here, becausea reduction in ligand levels would be predicted to offset theincrease in receptor activity. However, it is important to pointout that the VD-dependent inhibition of ANP expression is largelyconfined to hypertrophy-dependent expression (e.g., that inducedby endothelin treatment) and reflects a global antagonism ofthe hypertrophic process rather than isolated inhibition ofthe ANP gene. Activation of the ANP gene in hypertrophy representsan attempt, oftentimes futile (44), to reverse the hemodynamicstimuli that elicit the increase in myocardial mass and throughprimary (16,17) as well as secondary (1) effects control furtherprogression of the hypertrophic process. On the basis of itsability to increase NPR-A activity, VD would be predicted tosupport ANP effects in this setting. Thus, the VD effects maybe viewed as consistently aligned toward suppression of growthand hypertrophy in the cardiovascular system.
In summary, we have shown that the vasodilatory, antimitogenicreceptor NPR-A is under the transcriptional regulatory controlof 1,25 dihydroxyvitamin D. This effect may be responsible fora major component of the salutary effects that vitamin D exertsin the cardiovascular system.
Acknowledgments
These studies were supported by RO1 HL45637 and DK58812 fromthe National Institutes of Health.
We are grateful to M. Uskokovic of Roche Pharmaceuticals forproviding RO-25-6760.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Levin ER, Gardner DG, Samson WK: Natriuretic peptides.
N Engl J Med 339
: 321
–328, 1998[Free Full Text]
Suga S, Itoh H, Komatsu Y, Ogawa Y, Hama N, Yoshimasa T, Nakao K: Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells—Evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells.
Endocrinology 133
: 3038
–3041, 1993[Abstract]
Stepan H, Leitner E, Walter K, Bader M, Schultheiss H, Faber R, Walther T: Gestational regulation of the gene expression of C-type natriuretic peptide in mouse reproductive and embryonic tissue.
Regul Pept 102
: 9
–13, 2001[CrossRef][Medline]
Yasoda A, Ogawa Y, Suda M, Tamura N, Mori K, Sakuma Y, Chusho H, Shiota K, Tanaka K, Nakao K: Natriuretic peptide regulation of endochondral ossification. Evidence for possible roles of the C-type natriuretic peptide/guanylyl cyclase-B pathway.
J Biol Chem 273
: 11695
–11700, 1998[Abstract/Free Full Text]
Levin ER: Natriuretic peptide C-receptor: More than a clearance receptor.
Am J Physiol 264
: E483
–E489, 1993
Maack T, Suzuki M, Almeida FA, Nussenzveig D, Scarborough RM, McEnroe GA, Lewicki JA: Physiological role of silent receptors of atrial natriuretic factor.
Science 238
: 675
–678, 1987[Abstract/Free Full Text]
Umesono K, Murakami KK, Thompson CC, Evans RM: Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors.
Cell 65
: 1255
–1266, 1991[CrossRef][Medline]
Li YC, Kong J, Wei M, Chen ZF Liu SQ, Cao LP: 1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system.
J Clin Invest 110
: 229
–238, 2002[CrossRef][Medline]
Weishaar RE, Simpson RU: Vitamin D3 and cardiovascular function in rats.
J Clin Invest 79
: 1706
–1712, 1987
Weishaar RE, Kim SN, Saunders DE, Simpson RU: Involvement of vitamin D3 with cardiovascular function. III. Effects on physical and morphological properties.
Am J Physiol 258
: E134
–E142, 1990
Wu J, Garami M, Cao L, Li Q, Gardner DG: 1,25(OH)2D3 suppresses expression and secretion of atrial natriuretic peptide from cardiac myocytes.
Am J Physiol 268
: E1108
–E1113, 1995
Wu J, Garami M, Cheng T, Gardner DG: 1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes.
J Clin Invest 97
: 1577
–1588, 1996[Medline]
Chen S, Costa CH, Nakamura K, Ribeiro RC, Gardner DG: Vitamin D-dependent suppression of human atrial natriuretic peptide gene promoter activity requires heterodimer assembly.
J Biol Chem 274
: 11260
–11266, 1999[Abstract/Free Full Text]
Chen S, Cui J, Nakamura K, Ribeiro RC, West BL, Gardner DG: Coactivator-vitamin D receptor interactions mediate inhibition of the atrial natriuretic peptide promoter.
J Biol Chem 275
: 15039
–15048, 2000[Abstract/Free Full Text]
Holtwick R, van Eickels M, Skryabin BV, Baba HA, Bubikat A, Begrow F, Schneider MD, Garbers DL, Kuhn M: Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A.
J Clin Invest 111
: 1399
–1407, 2003[CrossRef][Medline]
Knowles JW, Esposito G, Mao L, Hagaman JR, Fox JE, Smithies O, Rockman HA, Maeda N: Pressure-independent enhancement of cardiac hypertrophy in natriuretic peptide receptor A-deficient mice.
J Clin Invest 107
: 975
–984, 2001[Medline]
Wilson E, Mai Q, Sudhir K, Weiss RH, Ives HE: Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF.
J Cell Biol 123
: 741
–747,
199
Bauer RF, Arthur LO, Fine DL: Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium.
In Vitro 12
: 558
–563, 1976[Medline]
Uskokovic MR, Norman AW, Manchand PS, Studzinski GP, Campbell MJ, Koeffler HP, Takeuchi A, Siu-Caldera ML, Rao DS, Reddy GS: Highly active analogs of 1,25-dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways.
Steroids 66
: 463
–471, 2001[CrossRef][Medline]
Pakkala S, de Vos S, Elstner E, Rude RK, Uskokovic M, Binderup L, Koeffler HP: Vitamin D3 analogs: Effect on leukemic clonal growth and differentiation, and on serum calcium levels.
Leuk Res 19
: 65
–72, 1995[CrossRef][Medline]
Chen S, Gardner DG: Osmoregulation of natriuretic peptide receptor signaling in inner medullary collecting duct. A requirement for p38 MAPK.
J Biol Chem 277
: 6037
–6043, 2002[Abstract/Free Full Text]
Cao L, Wu J, Gardner DG: Atrial natriuretic peptide suppresses the transcription of its guanylyl cyclase-linked receptor.
J Biol Chem 270
: 24891
–24897, 1995[Abstract/Free Full Text]
Winer J, Jung CK, Shackel I, Williams PM: Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro.
Anal Biochem 270
: 41
–49, 1999[CrossRef][Medline]
Chen S, Cao L, Intengan HD, Humphreys M, Gardner DG: Osmoregulation of endothelial nitric-oxide synthase gene expression in inner medullary collecting duct cells. Role in activation of the type A natriuretic peptide receptor.
J Biol Chem 277
: 32498
–32504, 2002[Abstract/Free Full Text]
Chen XW, Ying WZ, Valentin JP, Ling KT, Lin SY, Wiedemann E, Humphreys MH: Mechanism of the natriuretic action of gamma-melanocyte-stimulating hormone.
Am J Physiol 272
: R1946
–R1953, 1997
Herman JP, Dolgas CM, Marcinek R, Langub MC Jr: Expression and glucocorticoid regulation of natriuretic peptide clearance receptor (NPR-C) mRNA in rat brain and choroid plexus.
J Chem Neuroanat 11
: 257
–265, 1996[CrossRef][Medline]
Holick MF: Evolution and function of vitamin D. Recent results.
Cancer Res 164
: 3
–28, 2003
Hayes CE, Nashold FE, Spach KM, Pedersen LB: The immunological functions of the vitamin D endocrine system.
Cell Mol Biol (Noisy-le-grand) 49
: 277
–300, 2003
Godyn JJ, Xu H, Zhang F, Kolla S, Studzinski GP: A dual block to cell cycle progression in HL60 cells exposed to analogues of vitamin D3.
Cell Prolif 27
: 37
–46, 1994[Medline]
Kira M, Kobayashi T, Yoshikawa K: Vitamin D and the skin.
J Dermatol 30
: 429
–437, 2003[Medline]
Vegesna V, O’Kelly J, Uskokovic M, Said J, Lemp N, Saitoh T, Ikezoe T, Binderup L, Koeffler HP: Vitamin D3 analogs stimulate hair growth in nude mice.
Endocrinology 143
: 4389
–4396, 2002[Abstract/Free Full Text]
Thierry-Palmer M, Carlyle KS, Williams MD, Tewolde T, Caines-McKenzie S, Bayorh MA, Emmett NL, Harris-Hooker SA, Sanford GL, Williams EF: Plasma 25-hydroxyvitamin D concentrations are inversely associated with blood pressure of Dahl salt-sensitive rats.
J Steroid Biochem Mol Biol 66
: 255
–261, 1998[CrossRef][Medline]
Lind L, Hanni A, Lithell H, Hvarfner A, Sorensen OH, Ljunghall S: Vitamin D is related to blood pressure and other cardiovascular risk factors in middle-aged men.
Am J Hypertens 8
: 894
–901, 1995[CrossRef][Medline]
Lind L, Wengle B, Wide L, Ljunghall S: Reduction of blood pressure during long-term treatment with active vitamin D (alphacalcidol) is dependent on plasma renin activity and calcium status. A double-blind, placebo-controlled study.
Am J Hypertens 2
: 20
–25, 1989[Medline]
Pfeifer M, Begerow B, Minne HW, Nachtigall D, Hansen C: Effects of a short-term vitamin D(3) and calcium supplementation on blood pressure and parathyroid hormone levels in elderly women.
J Clin Endocrinol Metab 86
: 1633
–1637, 2001[Abstract/Free Full Text]
Teng M, Wolf M, Lowrie E, Ofsthun N, Lazarus JM, Thadhani R: Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy.
N Engl J Med 349
: 446
–456, 2003[Abstract/Free Full Text]
Dubois SK, Kishimoto I, Lillis TO, Garbers DL: A genetic model defines the importance of the atrial natriuretic peptide receptor (guanylyl cyclase-A) in the regulation of kidney function.
Proc Natl Acad Sci U S A 97
: 4369
–4373, 2000[Abstract/Free Full Text]
Franco-Saenz R, Atarashi K, Takagi M, Mulrow PJ: Effect of atrial natriuretic factor on renin and aldosterone.
J Cardiovasc Pharmacol 13[Suppl 6]
: S31
–S35, 1989
Li X, Zheng W, Li YC: Altered gene expression profile in the kidney of vitamin D receptor knockout mice.
J Cell Biochem 89
: 709
–719, 2003[CrossRef][Medline]
Li Q, Gardner DG: Negative regulation of the human atrial natriuretic peptide gene by 1,25-dihydroxyvitamin D3.
J Biol Chem 269
: 4934
–4939, 1994[Abstract/Free Full Text]
Charloux A, Piquard F, Doutreleau S, Brandenberger G, Geny B: Mechanisms of renal hyporesponsiveness to ANP in heart failure.
Eur J Clin Invest 33
: 769
–778, 2003[CrossRef][Medline]
Received for publication September 27, 2004.
Accepted for publication October 20, 2004.
Related Article
This Month’s Highlights
J. Am. Soc. Nephrol. 2005 16: 279-283.
[Full Text][PDF]
This article has been cited by other articles:
D. G. Gardner, S. Chen, D. J. Glenn, and C. L. Grigsby Molecular Biology of the Natriuretic Peptide System: Implications for Physiology and Hypertension
Hypertension,
March 1, 2007;
49(3):
419 - 426.
[Full Text][PDF]
C. P. Schmitt, T. Odenwald, and E. Ritz Calcium, Calcium Regulatory Hormones, and Calcimimetics: Impact on Cardiovascular Mortality
J. Am. Soc. Nephrol.,
April 1, 2006;
17(4_suppl_2):
S78 - S80.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
-32P]dCTP and the cyclic guanosine monophosphate (cGMP) RIA kit were purchased from Perkin-Elmer Life Sciences (Boston, MA). ANP was obtained from Phoenix Pharmaceuticals, Inc. (Mountain View, CA). RNeasy minikit was from Qiagen Inc. (Santa Clara, CA). Primer-it RMT kit, hybridization solution, and NucTrap push columns were purchased from Stratagene (La Jolla, CA). RO-25-6760 was provided by M. Uskokovic (Roche Pharmaceuticals). Other reagents were obtained through standard commercial suppliers. 
-galactosidase. After transfection, cells were plated on six-well plates in growth medium for 24 h. Medium was then changed to DMEM/serum substitute for the ensuing 24 h, at which point various concentrations of VD, or 10–8 M VD at different time intervals, were added. Luciferase activity was measured using the luciferase assay system (Promega). 
