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(Hypertension. 2003;41:730.)
© 2003 American Heart Association, Inc.

Scientific Contributions

Angiotensin II–Mediated Negative Regulation of Npr1 Promoter Activity and Gene Transcription

From the Department of Physiology and Hypertension and Renal Center of Excellence, Tulane University Health Sciences Center and School of Medicine, New Orleans, La.

Correspondence to Dr Kailash N. Pandey, Department of Physiology SL-39, Tulane University Health Sciences Center, 1430 Tulane Ave, New Orleans, LA 70112. E-mail kpandey{at}tulane.edu

	Abstract

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Atrial natriuretic peptide receptor A (NPRA) plays important role(s) in the control of extracellular fluid volume and blood pressure homeostasis. We have determined and analyzed the functional promoter region of Npr1 gene (coding for NPRA) and studied the effect of angiotensin (Ang) II on its promoter activity and expression in cultured mouse mesangial cells. The promoter analysis of Npr1 gene revealed the presence of positive regulatory cis-elements in the regions -1982 to -1841 bp and -916 to -496 bp and of the repressor elements in the regions -1841 to -916 bp and 56 to 382 bp relative to transcription start site. The Ang II pretreatment of cultured mouse mesangial cells transiently transfected with the promoter construct pNPRA-luc1 significantly inhibited the promoter activity in a time- and dose-dependent manner, with a maximum inhibition at 24 hours. The Ang II–dependent repression of Npr1 promoter activity was partially blocked by both angiotensin type 1 and type 2 antagonists candesartan and PD 123,319, respectively. The mRNA level of NPRA was also downregulated by Ang II treatment as determined by semiquantitative reverse transcriptase–polymerase chain reaction assay. The deletion analysis showed that the promoter region 916 bp upstream of transcription start site contains the cis-elements involved in Ang II–mediated repression of transcription of Npr1 gene. The present study thus reveals the presence of functional cis-regulatory elements in the promoter region of the murine Npr1 gene and its transcriptional downregulation by vasoactive peptide Ang II.

Key Words: natriuretic peptide receptor • angiotensin II • gene transcription • promoter analysis • gene expression • mesangial cells

	Introduction

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Atrial natriuretic peptide (ANP) is a member of the natriuretic peptide family, which is comprised of ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), each derived from a separate gene.¹ ANP elicits a number of vascular, renal, and endocrine effects, resulting in the maintenance of blood pressure and extracellular fluid volume by binding to the specific cell surface receptors.^2–5 Three subtypes of natriuretic peptide receptors have been characterized, purified, and cloned, ie, natriuretic peptide receptors A, B, and C (also designated as NPRA, NPRB, and NPRC, respectively).^4,5 ANP specifically binds NPRA and NPRC, of which NPRA contains guanylyl cyclase catalytic activity and produces intracellular second-messenger cGMP in response to hormone binding, and NPRC lacks the guanylyl cyclase activity. NPRA is considered the biological receptor of ANP and BNP because most of the physiological effects of these peptide hormones are triggered by generation of cGMP or its cell-permeable analogs.⁶ The activity and expression of NPRA, assessed primarily through ANP-dependent guanylyl cyclase activity and cGMP accumulation, are regulated by a number of factors, including ANP itself, ^7–9 other hormones such as glucocorticoids¹⁰ and angiotensin (Ang) II,^11–13 growth factors,^14,15 pathophysiological conditions,^16,17 and changes in extracellular ion composition.^18,19

The peptide hormone Ang II is an important component of renin-angiotensin system and exerts its biological effects such as blood pressure control, vasoconstriction, and cell proliferation in many tissues, including in the kidney, adrenal glands, brain, and vasculature. The 2 vasoactive peptide hormones Ang II (vasoconstrictive) and ANP (vasodilatory) interact and mutually antagonize the physiological effects of each other at various levels. For example, ANP has been reported to inhibit Ang II–induced contraction of isolated glomeruli and cultured mesangial cells.²⁰ ANP also inhibits Ang II–stimulated activation of protein kinase C and mitogen-activated protein kinase in vascular smooth muscle and mesangial cells in a cGMP-dependent manner.^21,22 Furthermore, ANP has been shown to decrease Ang II–evoked secretion and steroidogenesis in cultured glomerulosa cells.²³ Conversely, Ang II has been shown to downregulate guanylyl cyclase activity of NPRA by activating protein kinase C¹² and/or by stimulating protein tyrosine phosphatase activity,¹³ thereby inhibiting the ANP-induced cGMP accumulation. Ang II also reduces the ANP-dependent cGMP levels by stimulating cGMP hydrolysis, apparently via a calcium-dependent cGMP phosphodiesterase.^11,12,24 In contrast, Ang II pretreatment has also been reported to increase cGMP production elicited by ANP.²⁵

The glomerular mesangial cells are an attractive model for investigating a potential interaction among ANP, Ang II, and their receptors, because these cells contain functional receptors for both of these hormones.^26,27 Although Ang II has been shown to downregulate NPRA at the protein level,^12,13 it has not been reported to exert an effect on the transcriptional regulation of Npr1 gene. Thus, the objectives of the present study were (1) to determine the functional promoter region of the mouse Npr1 gene, and (2) to investigate the effect of Ang II on the promoter activity and the expression of this gene in cultured mouse mesangial cells. To our knowledge, this is the first report to demonstrate that Ang II modulates the transcriptional regulation of Npr1 gene.

	Methods

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Materials
The pGL3-basic vector, pRL-TK, pGL3-control plasmids, and dual luciferase assay system were purchased from Promega. The TRIzol reagent, elongase enzyme, and restriction endonucleases were obtained from Life Technologies/Invitrogen. Plasmid isolation kit was obtained from Qiagen. Sequence-specific oligonucleotides were purchased from MWG-Biotech. The cell culture media and fetal calf serum were purchased from Life Technologies. Transfection reagent lipofectamine-2000 was purchased from Invitrogen, and LT-1 was obtained from Panvera. BSA was obtained from Sigma. Ang II was purchased from Peninsula Laboratories Inc. Candesartan was generously provided as a gift by Astra Zeneca (Molndal, Sweden). PD 123,319 was a gift from Parke-Davis (Plymouth, Mich). MuLV reverse transcriptase and RNasin were purchased from Perkin-Elmer. All other chemicals were molecular biology reagent grade.

Plasmid Construction
All the promoter-luciferase reporter constructs were made by cloning the DNA fragments of various lengths of mouse Npr1 gene promoter region, upstream of the promoter-less firefly luciferase gene in the vector pGL3-basic. All the positions in the following promoter constructs are relative to transcription start site (TSS). The pNPRA-luc4 (-916 to 55 bp), pNPRA-luc5 (-496 to 55 bp), and pNPRA-luc7 (-1 to 359 bp) were generated by polymerase chain reaction (PCR) by using the p^eGFP-5'NPRA²⁸ as template and DNA polymerase (elongase). The PCR primers used in this study were as follows: F4 (-916 to -893 bp), 5'-ATC GGA ACG CGT ACT GGC ACT TGA CAC AGC TGG TCC-3'; F5 (-496 to -472 bp), 5'-TAC GGA ACG CGT CTG GCT CGC CTC TAC TTG ATT GCC-3'; F8 (-1 to 24 bp), 5'-ACT GGA ACG CGT CAT ACT CCT GGG GCA AGC GCG AGC G-3'; R1 (359 to 337 bp), 5'-TAC GGA AGA TCT CAG CGA GCG CAG CGA CGG AGC-3'; and R2 (55 to 33 bp), 5'-TAC GGA AGA TCT GCG GGT GCG CCA GCG AGG AAA GG-3'. All the 3 forward primers (F4, F5, and F8) contained MluI restriction site, whereas the reverse primers contained BglII restriction site at the 5' ends (shown as underlined). These restriction sites were used for cloning of the amplified fragments into MluI-BglII–restricted pGL3-basic vector. Four of the deletion constructs were generated by using the unique restriction sites present in the promoter region for the 5'-linkage: SacI for pNPRA-luc1 (-1982 to 55 bp), PstI for pNPRA-luc2 (-1841 to 55 bp) and pNPRA-luc6 (-1841 to 382 bp), and NdeI for pNPRA-luc3 (-1346 to 55 bp). The 3'-linkage in pNPRA-luc1, -luc2, and -luc3 used an internal BstEII restriction site at -508-bp position. The 0.43-kb fragment of pNPRA-luc4 generated by either SacI-BstEII or KpnI-BstEII double digestions was replaced with the fragments -1982 to -508, -1841 to -508, and -1346 to -508 bp for the generation of pNPRA-luc1, -luc2, and -luc3, respectively. For the construction of pNPRA-luc6, -1841- to 382-bp fragment generated by PstI-MluI restriction, was subcloned into pGL3-basic vector. All the plasmid constructs were sequenced across both the junctions to confirm the nucleotide sequence and the predicted orientation.

Cell Culture
Mouse mesangial cells (MMCs) were isolated and cultured as previously described.^22,29 MMCs were grown in Dulbecco modified Eagle’s medium supplemented with 10% fetal calf serum and ITS (insulin, transferrin, and sodium selenite). The cultures were maintained at 37°C in a 5% CO₂/95% O₂ humidified atmosphere. For all the experiments, cells were used between 4 to 15 passages.

Transient Transfection and Luciferase Assay
The cells were seeded in 12-well plates at a density producing 70% to 80% confluence on the next day. After 18 to 24 hours, the cells were transfected by using lipofectamine-2000 reagent according to manufacturer’s instructions, with 3 µg of test plasmid and 1 µg of pRL-TK carrying the renilla luciferase gene downstream of thymidine kinase promoter, which was used as internal transfection control. The medium was changed after 24 hours, and the cells were harvested after 48 hours by using passive lysis buffer (Promega). The supernatant obtained by centrifugation for 1 minute at 4°C was used to measure firefly luciferase and renilla luciferase activities. The luciferase activities were measured by TD 20/20 luminometer (Turner Designs) with 20 µL cell extract using dual luciferase reporter assay system. In the transfection experiments, a pGL3-control vector containing both the SV40 promoter and enhancers was used as a positive control, and the empty pGL3-basic vector was used as a negative control. The experiments were performed in triplicates 4 to 6 times independently. The results were normalized for the transfection efficiency as relative light units per renilla luciferase activity.

Hormonal Treatment
To study the effect of Ang II, cells were seeded in 24-well plates at 80% to 90% confluence and were transfected with 1 µg test plasmid and 0.3 µg pRL-TK using either lipofectamine-2000 or LT-1 reagent according to manufacturer’s instructions. After 24 hours, the cells were washed twice with serum-free medium containing 0.1% BSA (assay medium) and were treated with Ang II or other reagents in fresh assay medium. The cells were harvested at the indicated time intervals, and dual luciferase assay was performed as described above. All the experiments were performed in triplicates 4 to 5 times independently. The results were normalized for the transfection efficiency as relative light units per renilla luciferase activity.

Reverse Transcriptase–PCR Assay
The confluent MMCs were treated with or without 10^-8mol/L Ang II in assay medium for 16 hours. The cells were harvested, and total RNA was extracted by TRIzol reagent. One microgram of total RNA was reverse transcribed by using 50 U of MuLV reverse transcriptase in a reaction mixture containing 2.5 mmol/L dNTPs, 20 U Rnasin, and 100 ng of oligo (dT)₁₆ at 42°C. An aliquot of cDNA was amplified by using 2.5 U of Taq DNA polymerase and Npr1 gene–specific primers. The upstream primer (5'-ATC TAT TTC AGT GAT ATC GTG GGC-3'; 2964 to 2987 bp) and downstream primer (5'-CAT CGA ACT CTT CCA GCA CAG-3'; 3420 to 3400 bp) were designed from published mouse cDNA sequence.³⁰ The amplification (45 seconds at 94°C, 45 seconds at 54°C, and 2 minutes at 72°C, followed by an extension cycle of 10 minutes at 72°C) was performed for 35 cycles as the amount of PCR product increased linearly up to 40 cycles (data not shown). Control experiments were performed with RNA samples but without reverse transcriptase. PCR product of 456 bp was electrophoresed through 1.5% agarose gel and stained with ethidium bromide. The gel was digitized, and signal intensities of the corresponding bands were quantified by use of Alpha Imager 3.24 software. The specific primers for ß-actin were included in the PCR as an internal control.

Statistical Analysis
The results are expressed as mean±SE. The statistical significance was evaluated by the 1-way ANOVA with Student t test, and differences were considered significant if the probability value was <0.05.

	Results

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Functional Analysis of the Npr1 Gene Promoter
To determine the nucleotide sequence essential for the transcription of Npr1 gene, a series of Npr1 promoter-luciferase chimeric plasmids were constructed by cloning various lengths of the 5'-flanking promoter region of Npr1 gene upstream of the promoter-less firefly luciferase gene in the vector pGL3-basic (Figure 1A). These chimeric constructs were transiently transfected into MMCs. As shown in Figure 1B, all of the promoter constructs showed significant promoter activity compared with the promoter-less vector, pGL3-basic, and the maximum activity was observed in pNPRA-luc4 deletion construct. The smallest construct (pNPRA-luc5) containing only -496 bp was also active and sufficient for Npr1 expression in MMCs. To understand the functions of 5'-flanking region downstream of TSS in promoting transcription of Npr1 gene, 2 promoter-luciferase reporter plasmids containing the region between TSS and the start codon (pNPRA-luc6 and pNPRA-luc7) were constructed. The addition of the region (56 to 382 bp) to the upstream promoter region caused a significant decrease in the transcription activity, as evident by comparing the activities of pNPRA-luc2 and pNPRA-luc6 (Figure 1B). This suggests the presence of cis-acting repressor elements in 56- to 382-bp region. The pNPRA-luc7 construct containing only the 5'-flanking untranslated region (1 to 359 bp) also showed some basal promoter activity (3-fold compared with that of vector), suggesting the presence of some proximal promoter-like activity in this region.

View larger version (18K): [in this window] [in a new window]   Figure 1. Deletion analysis of Npr1 gene promoter. A, Diagrammatic representation of different lengths of the 5'-flanking region of mouse Npr1 gene, inserted upstream of firefly luciferase gene. The numbers shown next to the schematics indicate the nucleotide positions for the 5' and 3' ends of the constructs, respectively. The arrows indicate the TSS designated as 1. B, The chimeric constructs were transiently transfected into MMCs. Normalized luciferase activity is shown as a percentage of the activity of pNPRA-luc1. The results are expressed as mean±SE from 6 independent experiments. *P<0.01 vs pNPRA-luc5; P<0.01 vs pNPRA-luc4; and P<0.01, P<0.05 vs pNPRA-luc2.

The comparison of the relative luciferase activities of various deletion constructs showed some significant differences among them, revealing the presence of some cis-acting positive and negative regulatory elements in the promoter region of Npr1 gene. The promoter activities of pNPRA-luc1 and pNPRA-luc4 were significantly higher than those of pNPRA-luc2 and pNPRA-luc5, respectively, suggesting the presence of cis-acting enhancer elements in the regions -1982 to -1841 and -916 to -496 bp relative to TSS (Figure 1B). On the other hand, the activity of pNPRA-luc2 was significantly lower than that of pNPRA-luc4, revealing negative regulatory sequence(s) to be contained in the region -1841 to -916 bp relative to TSS. The positive pGL3-control plasmid, containing both the strong SV40 promoter and enhancer elements, had very high activity (144-fold) compared with that of the largest Npr1 promoter construct, pNPRA-luc1 (data not shown), suggesting that the Npr1 gene is expressed normally at moderately low levels in MMCs.

Effect of Ang II on the Promoter Activity of Npr1 Gene
To determine the effect of Ang II on the promoter activity of Npr1 gene, MMCs transiently transfected with pNPRA-luc1 (the largest promoter construct) were treated with Ang II at different doses (10^-10 to 10^-6 mol/L) for 24 hours. Figure 2 shows that Ang II caused a dose-dependent inhibition of the Npr1 promoter activity, and the maximal inhibition occurred at 10^-8 mol/L Ang II concentration, whereas the effect of Ang II was attenuated at higher concentration (10^-7 mol/L) of Ang II. The Ang II–dependent inhibition of Npr1 promoter activity was also studied at different time intervals. The inhibitory effect of Ang II increased from 8 to 24 hours, with a maximum inhibition at 24 hours (Figure 3A). The effect of Ang II was attenuated greatly after 24 hours, and the Npr1 promoter activity returned to almost normal levels after 48 hours (Figure 3B). To determine the specificity of the inhibitory effect of Ang II and to investigate the receptors involved in this process, selective receptor antagonists were utilized. The cells were transiently transfected with pNPRA-luc1 and treated simultaneously with Ang II (10^-8 mol/L) and the angiotensin type 1 (AT₁) receptor antagonist candesartan (10^-6 mol/L) or the angiotensin type 2 (AT₂) receptor antagonist PD 123,319 (10^-6 mol/L) for 24 hours. Both the AT₁ and AT₂ receptor antagonists partially reversed the inhibitory action of Ang II on the promoter activity of Npr1 gene (Figure 4), but both of these inhibitors were unable to abolish the inhibition completely. This suggests a possible involvement of both AT₁ and AT₂ receptor subtypes in the inhibitory action of Ang II on the promoter activity of Npr1 gene.

View larger version (85K): [in this window] [in a new window]   Figure 2. Effect of Ang II treatment on Npr1 gene promoter activity in MMCs. Cultured MMCs were cotransfected with 1 µg of pNPRA-luc1 and 0.3 µg of pRL-TK. After 24 hours, cells were treated with Ang II at the indicated doses for 24 hours. Normalized luciferase activity is shown as a percentage of the activity of untreated control. The results are expressed as mean±SE from 3 independent experiments. *P<0.05, **P<0.01 vs control group.

View larger version (23K): [in this window] [in a new window]   Figure 3. Time course of the Ang II–mediated repression of Npr1 gene promoter activity in MMCs. A, MMCs transiently transfected with 1 µg of pNPRA-luc1 and 0.3 µg of pRL-TK, were treated with or without 10 nmol/L Ang II for indicated time periods, and normalized luciferase activity is shown as a percentage of the activity of untreated control group. B, MMCs transiently transfected as above were treated with Ang II at indicated concentrations for different time periods. All values are expressed as a percentage of luciferase activity in control (untreated) group. The results are expressed as mean±SE from 3 to 5 independent experiments. *P<0.05, **P<0.01 vs control group.

View larger version (83K): [in this window] [in a new window]   Figure 4. Effect of Ang II antagonists on the inhibitory action of Ang II on pNPRA-luc1 promoter activity. Transiently transfected cells were treated with 10 nmol/L Ang II, 1 µmol/L candesartan, and 1 µmol/L PD 123,319 either alone or in combination for 24 hours as indicated. All values are expressed as a percentage of normalized luciferase activity in untreated control group. The results are mean±SE from 4 independent experiments. *P<0.05 vs control.

Effect of Ang II Treatment on the Expression of Npr1 Gene
To investigate the effect of Ang II treatment on the expression of Npr1 gene in mesangial cells, MMCs were treated with or without Ang II (10^-8 mol/L) for 16 hours, and the mRNA level of NPRA was analyzed by semiquantitative reverse transcription-PCR assay using the expression of ß-actin as an internal control. As seen in Figure 5, the amount of Npr1-specific PCR product was significantly decreased in Ang II–treated samples compared with control samples. However, the ß-actin specific transcript levels were comparable between the 2 samples. This further confirmed that Ang II mediates the downregulation of Npr1 gene transcription.

View larger version (30K): [in this window] [in a new window]   Figure 5. Ang II decreases NPRA mRNA level in MMCs. A, Representative example of reverse transcription–PCR experiment evaluating the transcript level of NPRA and ß-actin in MMCs after treatment with 10 nmol/L Ang II for 16 hours. B, Results are expressed as percentage of untreated control of the ratio of optical densities of NPRA RT-PCR product versus ß-actin product. Vertical bars represent the mean±SE from 3 independent experiments. *P<0.05 vs control.

Localization of the Ang II Responsive Promoter Region
To localize the cis-acting element(s) responsible for conferring the inhibitory action of Ang II, MMCs were transiently transfected with different promoter deletion variants and treated with Ang II (10^-8 mol/L) for 24 hours. As seen in Figure 6, the inhibition of the relative luciferase activity was observed in constructs containing the promoter region upstream of -916 bp relative to TSS (pNPRA-luc1 and pNPRA-luc3), whereas there was no inhibition in the smaller promoter constructs (pNPRA-luc4 and pNPRA-luc5). Interestingly, the construct pNPRA-luc3 exhibited the Ang II–dependent repression in Npr1 promoter activity, whereas pNPRA-luc4 did not show Ang II–mediated effect, suggesting the presence of Ang II responsive element(s) in the region -1346 to -916 bp, upstream of the TSS of Npr1 gene promoter.

View larger version (45K): [in this window] [in a new window]   Figure 6. Deletion analysis reveals a region important for Ang II–mediated repression of Npr1 gene promoter activity. MMCs were transiently transfected with 1 µg of different deletion variants of Npr1 gene promoter-luc reporter constructs and 0.3 µg of pRL-TK. After 24 hours, cells were treated with or without 10 nmol/L Ang II for 24 hours. The results are mean±SE from 3 independent experiments. *P <0.05 vs untreated control.

	Discussion

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The current studies were designed to analyze the functional promoter region of murine Npr1 gene and to investigate the effect of Ang II on its expression and promoter activity. The determination of the activities of Npr1 promoter-reporter construct and its deletion variants showed that all the constructs are active, and the -496-bp region upstream of TSS contains all the basal promoter elements to allow the expression of this gene. The promoter analysis also revealed the presence of some negative and positive regulatory cis-elements in the promoter region of Npr1 gene. The 5'-untranslated region downstream of the TSS caused a significant decrease in the promoter activity when it was included with the upstream -1846-bp promoter region, suggesting the presence of cis-acting repressor elements in this region (56 to 382 bp). This promoter region contains the putative cis-element for the repressor EF1 as previously suggested,²⁸ which might be involved in this significant repression of the Npr1 promoter activity. Another strong repressor element is present in the region -1841 to -916 bp, as revealed by the comparison of promoter activities of pNPRA-luc2 and pNPRA-luc4. The promoter activity of pNPRA-luc4 was significantly higher compared with that of pNPRA-luc5, suggesting some activator element(s) to be present in the region -916 to -496 bp relative to TSS. This region contains some putative cis-elements for the enhancers AP1, AP4, GATA1/2, and C/EBP- and -ß, but the functional involvement of these elements in activating the promoter activity of Npr1 gene needs further experimental confirmation. Interestingly, the activity of pNPRA-luc1 was significantly higher than that of pNPRA-luc2. Because the region from -1982 to -1841 bp upstream of TSS contains a tandem repeat element of (CA)₁₈, it is probable that this tandem repeat is involved in regulating the promoter activity in a positive manner.

The mesangial cells are one of the main targets of Ang II and ANP in the renal cortex; thus, we studied the effect of Ang II on the Npr1 promoter activity in transiently transfected MMCs. Ang II treatment of MMCs caused a significant inhibition of Npr1 promoter activity in a dose-dependent manner (0.1 to 10 nmol/L). However, at higher concentration (100 nmol/L), the inhibitory effect of Ang II was attenuated to some extent. Ang II mediates its biological effects such as vasoconstriction, vascular remodeling, and cell proliferation primarily through AT₁ receptor.³¹ However, AT₂ receptor subtype has been shown to be involved in specific tissues and pathologic conditions, and regulates blood pressure control, diuresis/natriuresis, cell growth inhibition, and renal inflammatory cell infiltration.³² Also, the mechanisms of signal transduction after stimulation of AT₁ and AT₂ receptor subtypes are different. The stimulation of AT₁ evokes several intracellular signals like calcium mobilization and activation of protein kinases, including protein kinase C and mitogen-activated protein cascade, whereas AT₂ activates one or several tyrosine phosphatases and mitogen-activated protein kinase phosphatase, resulting in the inhibition of specific kinases and apoptosis.^31,32 However, some of the Ang II responses could be mediated by both the receptor subtypes, including NO release³³ and collagen synthesis.³⁴ In cultured rat mesangial cells and vascular smooth muscle cells, both AT₁ and AT₂ receptors mediate Ang II–induced nuclear factor–B activity.^35,36 Also, Ang II induces leukocyte–endothelial cell interactions via both AT₁- and AT₂ receptor-mediated P-selectin upregulation.³⁷ Similarly, in case of Ang II–dependent inhibition of ANP-stimulated particulate guanylyl cyclase activity and cGMP accumulation, the role of both AT₁ and AT₂ receptor subtypes has been implicated.^11,13,24 In cultured vascular smooth muscle cells, which predominantly contain only AT₁ receptors, Ang II has been shown to decrease cGMP accumulation through NPRA by activation of calcium-dependent cGMP phosphodiesterase.¹¹ In rat adrenal glomerulosa cells, AT₂ receptor negatively regulates basal and ANP-stimulated guanylyl cyclase activity by stimulating protein tyrosine phosphatase activity,¹³ whereas in neuron cultures, AT₂ receptors decrease the cellular concentration of cGMP by a mechanism involving calcium and activation of a phosphodiesterase.²⁴

Because the mesangial cells have been shown to harbor both AT₁ and AT₂ receptor subtypes, ³⁶ we investigated the inhibitory effect of Ang II on the expression of Npr1 in the presence of nonpeptide receptor blockers specific to AT₁ and AT₂ receptor subtypes. Both of the AT₁ and AT₂ receptor antagonists, candesartan and PD 123,319, respectively, partially blocked the inhibitory effect of Ang II on the promoter activity of Npr1 gene; thus, it appears that both receptors are involved in mediating the observed effect of Ang II. Because both the receptors have different signaling mechanisms and cellular targets, it is probable that they mediate the inhibitory effect of Ang II through different pathways. The effect of Ang II on the expression of Npr1 was further confirmed by reverse transcription–PCR assay, which showed a significant decrease in the NPRA mRNA levels by Ang II treatment, whereas there was no apparent change in constitutively expressed ß-actin mRNA levels. Because the promoter activity of Npr1 gene is repressed by Ang II treatment, the reduction in the transcript level might be owing to an inhibition of Npr1 gene transcription rather than an increase in the degradation of transcripts.

To localize the promoter region that contains the cis-elements involved in the Ang II–mediated repression of Npr1 gene expression, the effect of Ang II was examined on different deletion mutants of the Npr1 promoter region. The effect of Ang II was not observed with the deletion mutants pNPRA-luc4 and -luc5, whereas pNPRA-luc1 and -luc3 showed Ang II–mediated repression in promoter activity. Thus, the promoter region responsive to the Ang II in Npr1 gene is about 916 bp upstream of TSS. This region contains a putative cAMP-response element, TGCCGTCA, (at -932 bp position) recognized by the transcription factor CREB, which is one of the few reported cis-elements through which Ang II has been shown to regulate the gene expression. Although CREB is mostly described as a positive transcription factor, several reports have shown that it can also inhibit the transcription activity of diverse promoters such as those of c-fos,³⁸ 5-amino-leavulinate synthase,³⁹ and somatostatin⁴⁰ genes. Lemaigre et al⁴¹ showed that CREB, after dimerization, inhibits diverse transcriptional activators. Previous studies have also shown that Ang II inhibits adenylyl cyclase activity and intracellular accumulation of cAMP.⁴² Thus, this putative cis-element for CREB-binding might be involved in the Ang II–mediated repression of Npr1 gene expression but needs further experimental confirmation. Another possible candidate is transforming growth factor-ß (TGF-ß) because Ang II has been shown to induce the expression of TGF-ß in cultured mesangial cells and vascular smooth muscle cells.^43,44 On the other hand, TGF-ß has been shown to downregulate the NPRA expression in thymic stromal cell line and cultured aortic vascular smooth muscle cells.^14,15 Therefore, it can also be a potential mechanism of the Ang II–mediated repression of Npr1 gene expression in mesangial cells.

Perspectives
In summary, the present study reveals the presence and locations of negative and positive regulatory cis-elements in the promoter region of Npr1 gene. Also, this is the first report to demonstrate that Ang II augments the transcriptional repression of Npr1 gene, as evident by a decrease in both its mRNA level and the promoter activity. This type of specific interaction between 2 physiological peptidic hormones is an intriguing model of heterologous receptor regulation having important physiological and pathophysiological implications. Ang II–mediated downregulation of Npr1 gene expression should contribute not only to hypertension but also to the onset and progression of the vascular damage associated with disease states in which plasma levels of Ang II are elevated. The findings of this study will help in further understanding the roles of Ang II in the ANP/NPRA pathway mediating the physiology and pathophysiology of hypertension and cardiovascular homeostasis. These results also provide an example of novel Ang II response via both AT₁ and AT₂ receptors in the mesangial cells.

	Acknowledgments

 
We thank Dr L. Gabriel Navar for helpful discussion and Huong T. Nguyen for expert technical assistance during the course of this work. This study was supported by grants from the National Institutes of Health (HL 62147) and Louisiana Board of Regents Health Excellence Funds.

Received September 19, 2002; first decision October 25, 2002; accepted December 3, 2002.

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