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natriuretic peptide receptor-A gene
peptides 27 (2006) 1762–1769
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Transcriptional regulation of guanylyl cyclase/natriuretic peptide receptor-A gene Prerna Kumar, Kiran K. Arise, Kailash N. Pandey * Department of Physiology, SL-39, Tulane University Health Sciences Center and School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112, USA
article info
abstract
Article history:
Activation of natriuretic peptide receptor-A (NPRA) produces the second messenger cGMP,
Received 4 January 2006
which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. In
Accepted 5 January 2006
the present study, we have examined the role of trans-acting factor Ets-1 in transcriptional
Published on line 3 March 2006
regulation of Npr1 gene (coding for NPRA). Using deletional analysis of the Npr1 promoter, we have defined a 400 base pair (bp) region as the core promoter, which contains consensus
Keywords:
binding sites for transcription factors including: Ets-1, Lyf-1, and GATA-1/2. Overexpression
Natriuretic peptides
of Ets-1 in mouse mesangial cells (MMCs) enhanced Npr1 gene transcription by 12-fold.
Npr1 promoter
However, overexpression of GATA-1 or Lyf-1 repressed Npr1 basal promoter activity by 50%
Gene transcription
and 80%, respectively. The constructs having a mutant Ets-1 binding site or lacking this site
Ets-1
failed to respond to Ets-1 activation of Npr1 gene transcription. Collectively, the present
Mesangial cells
results demonstrate that Ets-1 greatly stimulates Npr1 gene promoter activity, implicating its critical role in the regulation and function of NPRA at the molecular level. # 2006 Elsevier Inc. All rights reserved.
1.
Introduction
Natriuretic peptides belong to a family of three homologous endogenous hormones: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) [10,25]. ANP is a circulating hormone which plays a pivotal role in regulation of sodium excretion, fluid volume, steroidogenesis, and cell proliferation; important factors in the control of blood pressure and cardiovascular homeostasis [25,32,2]. The action of ANP is mediated by binding to natriuretic peptide receptor-A (NPRA), which contains guanylyl cyclase (GC) catalytic activity and produces the intracellular second messenger cGMP [10,32]. The activity and expression of NPRA are regulated by a number of factors including: hormones such as glucocorticoids [24] and angiotensin II (Ang II) [16,19], growth factors [14,1], pathophysiological conditions [4,39], and
changes in extracellular ion composition [23,6]. Down-regulation of NPRA has been demonstrated both in vitro [20,5] and in vivo [37]. Additionally, increased cGMP down-regulates receptor density [20]. A cGMP response element has been characterized in the Npr1 gene (coding for NPRA) promoter [22]. Repression of Npr1 gene transcription by the vasoactive peptide hormone Ang II has been shown to be mediated through a region from 916 to 496 in the Npr1 promoter [16]. Previous studies have shown that Sp1 plays a dominant role in basal expression of Npr1 gene [27], and functional and physical interaction of NF-Y with Sp1 is essential for optimal transcription of its promoter in vascular smooth muscle cells [28]. However, studies to elucidate the molecular regulation of Npr1 gene transcription are limited, and the molecular machinery that regulates NPRA expression is not well understood.
* Corresponding author. Tel.: +1 504 988 1628; fax: +1 504 988 2675. E-mail address: [email protected] (K.N. Pandey). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.01.004
peptides 27 (2006) 1762–1769
The 50 -flanking regulatory region of the murine Npr1 gene contains three potential Sp1 binding sites, one inverted CCAAT box, but lacks the TATA box [15]. This region contains several putative cis-acting motifs of known transcription factors, suggesting that these multiple regulatory elements may exert a cooperative influence in order to closely regulate Npr1 gene expression. The 500 base pair (bp) region upstream of the transcription start site (TSS) contains the basal promoter elements to allow the expression of this gene [16]. In order to investigate the transcriptional regulatory mechanisms of the Npr1 gene promoter activity, we have characterized cis-acting sequences, which either activate or repress Npr1 gene transcription and have defined the minimal promoter region of Npr1 gene necessary for efficient transcription in mouse mesangial cells (MMCs). This region contains several putative cis-acting motifs including; Ets-1, p300, three Sp1 sites, Lyf-1, GATA 1/2, and AML-1. Among these cis-acting motifs we demonstrate that Ets-1 effectively upregulates the Npr1 promoter activity and enhances its gene transcription.
2.
Materials and methods
2.1.
Materials
The pGL3-basic vector, pRL-TK, pGL3-control plasmids, and dual luciferase assay system were purchased from Promega (Madison, WI). Plasmid isolation kit was obtained from Qiagen Inc., (Valencia, CA). Sequence-specific oligonucleotides were received from Midland Certified Reagent Company Inc. (Midland, TX). The cell culture media, fetal calf serum, ITS (insulin, transferrin, and sodium selenite), and Lipofectamine-2000 were purchased from Invitrogen (Carlsbad, CA). T4 polynucleotide kinase, protein A-agarose, and [g-32P]-ATP (3000 Ci/ mmol) were purchased from Amersham Biosciences (Piscataway, NJ). The Ets-1 expression vector (pEVRF0-Ets-1) and its empty vector (pEVRF0) were kindly provided by Dr. Paul Brindle [42]. The expression vectors pEF for wild-type GATA-1 and GATA-2 were generously provided by Dr. Adam N. Goldfarb [11]. Expression vector for Lyf-1 (IK-6 in pCDNA3) was a kind gift from Dr. Stephen Smale. All other chemicals were reagent grade and were obtained from Sigma Chemical Co. (St. Louis, MO) and Bio-Rad (Hercules, CA).
2.2.
Plasmid construction
The promoter-luciferase reporter constructs were generated by cloning the PCR-amplified DNA fragments of various lengths of mouse Npr1 gene promoter upstream of the promoter-less firefly luciferase gene in the pGL3-basic vector. The positions marked in the promoter constructs are relative to transcription start site (TSS). The cloning of constructs 1982/+55 (pNPRAluc1) and 496/+55 (pNPRA-luc5) has been previously described [16]. The constructs having 356 or 199 nucleotide position at their 50 end were amplified using forward PCR primers 356 (50 TAC GGA ACG CGT GAG GGG GGG CAG CTT CCT CAC 30 ), and 199 (50 TAC GGA ACG CGT CCA GCC ATA GTC AGG GCT GGG 30 ). The constructs having +55 or 46 nucleotide positions at their 30 ends were PCR-amplified using reverse primers +55 (50 TAC GGA AGA TCT CAG CGA GCG CAG CGA CGG AGC 30 ) and 46 (50 TAC
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GGA AGA TCT AGG CGG GCG GGG GCG GGG TGG 30 ). Underlined nucleotides indicate the MluI and BglII restriction sites. All forward primers contained the MluI restriction site, and the reverse primers contained BglII restriction site at their 50 ends. The plasmid having 496/+55 region was used as a template for amplification of all constructs. These amplified products were then subcloned into MluI/BglII-restricted pGL3-basic vector, and DNA used for transfections was purified by Qiagen columns. All the plasmid constructs were sequenced across both the junctions to confirm the nucleotide sequence and the predicted orientation.
2.3.
In vitro site-directed mutagenesis
Mutations in the Ets-1 binding motifs in the 356/+55 construct, were introduced utilizing Quick Change Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. Complementary sense and antisense strands containing the specific mutations for both the sites were designed separately. The sequence of the forward primer for Ets-1 site A is 50 GCC CCC GCC CGC CTC ATT CAC GGC CGG AGA CTT GG 30 and for Ets-1 site B is 50 CCG GAG ACT TGG GCC AGC ATT CCG CCC CTT CTG GC 30 . Underlined nucleotides indicate the mutated sequence. The Ets-1A mutant construct was used as a template for creating double mutant at Ets-1 site B. All the mutations were confirmed by sequencing the constructs.
2.4.
Cell transfection and luciferase assay
Mouse mesangial cells were isolated and cultured as previously described [33]. MMCs were grown in Dulbecco modified Eagle’s medium supplemented with 10% fetal calf serum. The cultures were maintained at 37 8C in an atmosphere of 5% CO2/ 95% O2. All the experiments were performed utilizing cells between 4 and 16 passages. The MMCs were seeded in a 24well plate at a density of 5 104 cells in each well producing 70–80% confluence after 24 h. The cells were transfected using Lipofectamine-2000 reagent with 1 mg of promoter-reporter construct and 300 ng of pRL-TK carrying the renilla luciferase gene downstream of thymidine kinase promoter, which was used as an internal transfection control. Cells were harvested after 48 h by using passive lysis buffer. The cell lysate was centrifuged at 10,000 rpm for 3 min at 4 8C, and the supernatant was used to measure firefly luciferase and renilla luciferase activities. The luciferase activities were measured by mixing 10 ml cell extract with 50 ml of luciferase firefly assay reagent. The fluorescence intensity was integrated for 10 s using a TD 20/20 luminometer (Turner Designs). Subsequently, the resultant mixture was used for renilla luciferase activity. The results were normalized for the transfection efficiency relative to light units per renilla luciferase activity.
2.5.
Whole cell lysate preparation and immunoblot assay
MMCs were harvested by trypsinization and electroporated with 5 mg of either empty vector DNA (pEVRF0) or Ets-1 expression vector at 220 V with a capacitance setting of 960 mF using a gene pulser (Bio-Rad, Hercules, CA). After transfection, cells were seeded into 100 mm2 petri dishes. Forty-eight hours
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after transfection, cells were washed with PBS and lysed in buffer containing: 25 mM HEPES, pH 7.5, 0.05% 2-mercaptoethanol, 1% Triton X-100, 1 mM sodium vanadate, 10 mm NaF, 0.2 mm PMSF, and 10 mg/ml each of aprotinin and leupeptin. Cell extract was passed through a 1 cc syringe with a 21-guage needle and centrifuged at 14,000 rpm for 10 min. The clear cell lysate was collected and stored at 80 8C until used. The protein concentrations of the lysate were estimated using a Bradford protein detection kit (Bio-Rad). Whole cell lysate (40 mg) from each sample was mixed with sample loading buffer and resolved by 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were electrotransferred onto a PVDF membrane. The membrane was blocked with 1 Tris–buffered saline-Tween 20 (TBST) containing 5% fat-free milk for 2 h at room temperature, and incubated overnight at 4 8C in TBST containing 3% fat-free milk with primary antibodies (1:1000 dilution). The membrane was then treated with corresponding secondary anti-rabbit or anti-mouse HRP-conjugated antibodies (1:10,000 dilutions). Protein bands were visualized by enhanced chemiluminescence (ECL) plus detection system.
2.6.
Nuclear extract preparation
Nuclear extracts were prepared from MMCs as described by Dignam [8]. The cells were harvested and centrifuged at 250 g for 10 min. Pellet was washed with PBS and centrifuged as described above. The resulting pellet was resuspended in five volumes of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM PMSF) and incubated on ice for 10 min and centrifuged as above. The pellet was again resuspended in three volumes of buffer A, to which Nonidet P-40 (0.05%, v/v) was added and the suspension was homogenized with 20 strokes of a tight-fitting Dounce homogenizer to release the nuclei. The suspension was then centrifuged at 250 g for 10 min to pellet the nuclei. The pellet was resuspended in 100–200 ml of buffer C (5 mM HEPES, pH 7.9, 26% glycerol v/v, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM PMSF). To the suspension, NaCl was added to a final concentration of 300 mM. After mixing, the suspension was incubated on ice for 30 min, and then centrifuged at 24,000 g for 20 min. The protein concentration of the nuclear extract was estimated using a Bradford protein detection kit (Bio-Rad). All centrifugation steps were carried out at 4 8C. Aliquots of the supernatant were stored at 70 8C until used.
2.7.
UV cross-linking assay
To determine the molecular mass of the nuclear protein bound to the Ets-1 binding sites, a UV cross-linking assay was performed. Sense and antisense primers spanning the binding sites for Ets-1 were synthesized. The forward primers for Ets-1 site A (50 CCG CCC GCC TCC GGA ACG GCC GGA 30 ) and for Ets-1 site B (50 TGG GCC AGC CGG ACG CCC CTT CTG 30 ) were endlabeled using [g-32P]-ATP and T4 polynucleotide kinase, and annealed with antisense strand. Binding reactions were carried out in a total volume of 15 ml containing 1 binding buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 50 mM NaCl, 0.5 mM EDTA, 4% glycerol v/v, and 0.5 mM DTT), 8 mg poly (dIdC) as a non specific competitor, and 20 mg MMC nuclear
extract. The reaction mixture was incubated on ice for 5 min, after which [g-32P]-ATP labeled probe (150,000 cpm) was added, and the mixture was further incubated on ice for 30 min. The reaction mixture was then subjected to UV radiation of 254 nm using a UV Stratalinker (Stratagene, La Jolla, CA) in auto-crosslinking mode for five exposures (each exposure is of 120,000 mJ for 40 s). The reaction mixture was then mixed with SDS-loading buffer, boiled, and resolved using 10% SDS–PAGE and autoradiography.
2.8. Electrophoretic mobility shift assay (EMSA) and supershift assay Protein–DNA complexes were detected using 50 biotin endlabeled double-stranded DNA probes prepared by annealing complementary oligonucloetides. Sequence of the probes is same as given in UV cross-linking assay. The binding reaction was performed using LightShift kit (Pierce). Briefly, MMC nuclear extracts (1.5 mg of protein) and binding buffer were incubated on ice for 5 min in a volume of 20 ml, then biotinlabeled probe of Ets-1A or Ets-1B (40 or 20 fmol, respectively) was added, respectively, and reaction was allowed to incubate for an additional 25 min at room temperature. In super-shift experiments, the extracts were preincubated with anti-Ets-1 polyclonal antibody for 60 min on ice. Protein–DNA complexes were separated on nondenaturing polyacrylamide gels and observed by the LightShift Chemiluminescent EMSA kit following the manufacturer’s procedure.
2.9.
Statistical analysis
The results are presented as mean S.E. The statistical significance was evaluated by Student t-test and 1-way ANOVA, followed by Dunnett’s multiple comparison test using the computer software PRISM (GraphPad Software Inc., San Diego, CA). The probability values of P < 0.05 were considered significant.
3.
Results
The results presented in Fig. 1 demonstrate that a deletion of 150 bp in Npr1 promoter construct 496/+55 (356/+55) towards its 50 end significantly increased basal promoter activity ( p < 0.01) in MMCs as compared with the construct 496/+55. The construct 356/+55 showed a maximum of a 75fold increase in luciferase activity, while the full length promoter (1982/+55) exhibited only a 28-fold induction in luciferase activity as compared with the pGL3-basic vector. In the construct 356/46, a deletion of 90 bp from the 30 end significantly decreased the transcriptional activity as compared with the construct 356/+55 ( p < 0.001), indicating that the region 46 to +55 in the core promoter is essential for Npr1 transcriptional activity (Fig. 1). We further localized the transcription factor binding sites in the region 356 to +55 using the TRANSFAC 3.2 database, which revealed the presence of many putative transcription factor binding sites including: Ets-1, Lyf-1, and GATA-1/2 and coactivator p300 (Fig. 2). The region 356 to +55 (from TSS), which contains binding sites for transcription factors Ets-1, GATA-1/2, and Lyf-1, are
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Fig. 1 – Luciferase activity of murine Npr1 core promoter deletion constructs in mouse mesangial cells. Left hand side of the figure shows the diagrammatic representation of 50 Npr1 promoter-reporter deletion constructs. The numbers shown next to the construct indicate the 50 and 30 ends of the constructs, respectively. Right side represents the promoter activity of the deletion constructs cotransfected with pRLTK vector into MMCs. pGL3-Basic was taken as control. Firefly luciferase activity was normalized to Renilla luciferase activity. Luc represents the luciferase gene. Values represent the mean W S.E. of four independent experiments in triplicates. *P < 0.05, ** P < 0.01, ***P < 0.001.
shown in Fig. 3A. To evaluate the relative contributions of these binding sites to the core promoter activity, we overexpressed these transcription factors in a dose dependent manner and cotransfected them with the basal promoter construct 356/ +55 in MMCs (Fig. 3B). Overexpression of Ets-1 vector DNA (100 and 250 ng) in MMCs elicited almost 10-fold and 12-fold increase in the transcriptional activity of Npr1 gene, respectively (Fig. 3B). Conversely, overexpression of GATA-1 and Lyf-1 vector DNA at similar concentrations repressed the Npr1 basal promoter
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activity by almost 50% and 80%, respectively. To conclusively demonstrate the role of Ets-1 in the activation of Npr1 gene promoter activity, we utilized site-specific Ets-1 point mutations and deletion constructs. A diagrammatic representation of constructs having wild-type, point mutations, and deletion of Ets-1 binding sites are shown in Fig. 4A. The construct having mutant Ets-1 sites or the construct lacking Ets-1 binding sites (+356/46) failed to respond to Ets-1 activation of Npr1 gene transcription, whereas, wild-type Ets-1 construct stimulated full activation (12-fold) of Npr1 gene transcription (Fig. 4B). Furthermore, each site was mutated separately in constructs 356/+55 Ets-1Am and 356/+55 Ets-1Bm to see their effect. Basal expression was reduced to 40–50% in cells transfected with construct having either site mutated as compared with the construct 356/+55 WT taken as 100% (Fig. 4C). On the other hand additive effect in loss of transcriptional activity (90%) was seen when both the sites were mutated together (Fig. 4C). The Western blot analysis showed that the expression of Ets-1 in transfected cells exhibited the production of Ets-1 protein, corresponding to the molecular mass of 54 kDa (Fig. 4D). In order to confirm whether endogenous Ets-1 protein present in the nuclear extract of MMCs binds to its consensus sequence, a UV cross-linking assay was performed. [g-32P]ATP-labeled oligonucleotides containing Ets-1 binding motifs A and B were incubated with MMCs nuclear extract, UV cross-linked, and resolved by SDS–PAGE and autoradiography. A DNA–protein complex of Ets-1 (54 kDa) was found to bind to both of the Ets-1 consensus binding sites in Npr1 promoter (Fig. 5, lanes b and d) as compared with the free probes (Fig. 5, lanes a and c). Antibody supershift assays were also performed to confirm the specificity of the protein binding to Ets-1 motifs in the Npr1 promoter. When incubated with MMC nuclear extract wild type Ets-1A and Ets-1B oligonucleotides supported the formation of specific nucleoprotein complexes (Fig. 6A and B, lanes 2 and 6). Whereas, Ets-1 antibody disrupted the DNA protein complex hence abolishing the specific band (Fig. 6A and B, lanes 4 and 8).
Fig. 2 – Nucleotide sequence of the 50 -flanking region of murine Npr1 gene spanning from S356 to +55. The transcription start site (TSS) is indicated by a vertical arrow. Nucleotide positions are numbered with respect to the TSS+1. Consensus sequence for known transcription factor binding site and cis-acting elements are indicated with an arrow and in bold face. GenBank accession no. AJ307712.
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Fig. 3 – Effect of overexpression of Ets-1, Lyf-1, and GATA1/2 on Npr1 basal promoter activity in MMCs. (A) Schematic representation of the putative cis-acting elements present in the region S356 to +55 of Npr1 gene promoter. (B) MMCs were cotransfected with 1 mg of S356/+55 Npr1 promoter construct along with increasing amount (50, 100, and 250 ng) of indicated expression vectors. Luciferase activity is represented as fold induction with respect to corresponding empty vector used as control. Firefly luciferase activity was normalized to Renilla luciferase activity. Values represent the mean W S.E. of three independent experiments in triplicates. *P < 0.05, **P < 0.01, ***P < 0.001.
4.
Discussion
The findings of this study show that Ets-1 plays an important role in mediating the activity of Npr1 gene promoter. Ets-1 binding sites are present in the core promoter of Npr1 gene spanning from 356 to +55 along with several other putative cis-acting elements for transcription factors including: Lyf-1, MZF-1, GATA 1/2, and SP1. Our results demonstrate that Ets-1 mediates the transcriptional activation of murine Npr1 gene promoter through its binding sites present in the region 46 to +55. Ets proteins have been shown in the regulation of numerous genes involved in diverse cellular processes, such as proliferation, differentiation, development, transformation, and apoptosis [38,43,26]. The Ets family of transcription factors specifically recognizes DNA sequences that contain GGAA/T as a core element [31,9]. Interestingly, NPRA/cGMP signaling has been implicated in various anti-mitogenic and cell proliferation activities, due to, perhaps in part, the regulation exhibited by the Ets-1 activity demonstrated in the present studies. Additionally, ANP has been reported to be involved in NPRA-mediated apoptosis of cardiac myocytes, endothelial, and vascular smooth muscle cells [12]. Among its diverse functions, Ets proteins play a significant role in regulating genes involved in both cell cycle progression and programmed cell death, including: cdc2, jun B, c-fos, c-myc, Rb, p53 and bcl-2 [3]. In addition, the function of Ets-1 in the
regulation of apoptosis has recently been demonstrated in lymphoid and colon cancer cells [21]. Thus, it is evident that Ets-1 upregulates Npr1 gene, and this process may play an important role in Npr1-mediated apoptosis. Cell- and tissue-specific gene expression is established through the combinatorial action of restricted and more widely expressed transcription factors. The architecture of a regulatory element directs the assembly of these factors into higher-order structures that ultimately determine the transcriptional readout. Our results showed that overexpression of Ets-1 in MMCs greatly enhanced the Npr1 promoter activity, whereas, the overexpression of GATA-1 and Lyf-1 significantly repressed Npr1 transcriptional activity. At present it is not known that the transcription factors GATA-1 and Lyf-1 are involved in any transcriptional repression. GATA-1 is a tissuespecific transcription factor, which is expressed in erythrocytes, megakaryocytes, eosinophils, and mast cells [30,35]. All erythroid genes are positively regulated by GATA-1 but the full extent of its transcriptional actions is currently unknown [41]. On the other hand Lyf-1 is reported to be transcriptional activator of lymphoid-specific genes [29] and Lyf-1 protein is known to be involved in the regulation of gene expression during early stages of B- and T- cell development [18]. Their expression and role in mesangial cells are currently unknown and it might be a possibility that these factors play a role in modulation and tissue-specific regulation of Npr1 gene.
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Fig. 5 – Endogenously expressed Ets-1 binds to its consensus site in the Npr1 promoter. Twenty micrograms of MMC nuclear extract (NE) were incubated with [g-32P]ATP-labeled probes and UV cross-linked. The product was electrophoresed on 10% SDS-PAGE and visualized by autoradiography. Mr and position of UV cross-linked product of Ets-1 site A and site B in the absence (lanes a and c) and in the presence (lanes b and d) of MMC nuclear extract are indicated by an arrow. Bars on the left margin show the Mr of marker proteins. The autoradiogram is the representative of four independent experiments.
Fig. 4 – Effect of Ets-1 binding sites on the functional activation of Npr1 promoter. (A) Diagrammatic representation of the Npr1 core promoter construct harboring wild type (WT), mutant type (MT) and deleted Ets-1 binding sites. (B) One microgram of the constructs S356/+55 Ets-1 WT, S356/+55 Ets-1 MT, and S356/S46 was cotransfected with 50 or 250 ng of Ets-1 expression vector or the empty control vector pEVRF0. Luciferase activity is represented as fold induction with respect to corresponding empty vector used as control. (C) One microgram of the constructs S356/+55 WT, S356/+55 Ets1Am, S356/+55 Ets-1Bm and S356/+55 Ets-1 MT was transiently transfected in MMCs and luciferase activity was measured after 48 h. Value of construct S356/+55 WT is taken as 100%. (D) Western blot analysis demonstrates overexpression of Ets-1 protein in MMCs when transfected with pEVRF0-Ets-1. Cells were transfected with 10 mg of pEVRF0 or pEVRF0-Ets-1, and whole cell lysate was prepared after 48 h and developed with Ets-1 polyclonal antibody. Lower panel shows b-actin used as a loading control. This experiment was repeated three times independently. Values given in luciferase activity (B and C) represent the mean W S.E. of three independent experiments in triplicates. *P < 0.01, **P < 0.001.
Several agents that are known to upregulate Ets-1 transcription, include RA, TNF-alpha, VEGF, and TPA [7,17,40]. Ets-1 is upregulated at exposure to agonists such as serum in vitro and is expressed in injured vasculature [36]. MAPK-mediated phosphorylation positively regulates the transcriptional activation functions of Ets-1 by recruiting CBP/p300 [13]. Not much is known about Ets-1 expression or regulation in mesangial cells. A temporal increase of mesangial cell Ets-1 expression has been reported which correlates with mesangial cell activation in mesangioproliferative glomerulonephritis suggesting involvement of PDGF-B [34]. There might be a possibility that during glomerulonephritis increased Ets-1 expression upregulates Npr1 gene as a protective mechanism. Npr1 gene has been shown to negatively regulate mitogen-activated protein kinase and proliferation of mesangial cells [33]. In conclusion, our results demonstrate that the precise control of Npr1 gene transcriptional activity is achieved through a synergy of activators and repressors in which Ets1 plays an integral role as a transcriptional activator. Comparatively, Lyf-1 and GATA-1 act as repressors, inhibiting and regulating the transcriptional activity of Npr1 gene promoter. The present findings suggest that Ets-1 plays a critical role in enhancing Npr1 gene transcription and may have an important influence in hypertension and cardiovascular homeostasis at the molecular level.
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Fig. 6 – Ets-1 antibody supershift assay. Ets-1 antibody supershift assay in nuclear extract from MMCs A and B, MMC nuclear extract was incubated with labeled Ets-1 site A and Ets-1 site B probe, respectively, in presence of 2 mg of anti Ets-1 antibody. Arrow shows Ets-1 nuclear protein complex binding with Ets-1A (lane 2) and Ets-1B sites (lane 6), respectively. Star shows disruption of the respective binding in the presence of Ets-1 antibody (lanes 4 and 8).
Acknowledgements The authors wish to thank Huong T. Nguyen and Gevoni Bolden for their valuable technical assistance during the course of this work and Mrs. Kamala Pandey for her assistance in typing the manuscript. We sincerely thank Dr. Paul Brindle, Dr. Adam N. Goldfarb, and Dr. Stephen Smale for the kind gifts of expression vectors. Our special thanks are due to Dr. Susan L. Hamilton, Department of Molecular Physiology and Biophysics at Baylor College of Medicine and to Dr. Bharat B. Aggarwal, Department of Experimental Therapeutics and Cytokine Research Laboratory at MD Anderson Cancer Center for providing their facilities during our displacement period due to Hurricane Katrina. This research was supported by the National Institutes of Health Grants HL57531 and HL62147.
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alternatively spliced mRNAs derived from the Ikaros gene. Mol Cell Biol 1994;14:7111–23. Haneda M, Kikkawa R, Maeda S, Togawa M, Koya D, Horide N, et al. Dual mechanism of angiotensin II inhibits ANPinduced mesangial cGMP accumulation. Kidney Int 1991;40:88–94. Hirata Y, Hirose S, Takata S, Takagi Y, Matsubara H. Downregulation of atrial natriuretic peptide receptor and cyclic GMP response in cultured rat vascular smooth muscle cells. Eur J Pharmacol 1987;35:439–42. Huang CC, Papas TS, Bhat NK. A variant form of ETS1 induces apoptosis in human colon cancer cells. Oncogene 1997;15:851–6. Hum D, Besnard S, Sanchez R, Devost D, Gossard F, Hamet P, et al. Characterization of a cGMP-response element in the guanylyl cyclase/natriuretic peptide receptor A gene promoter. Hypertension 2004;43:1270–8. Katafuchi T, Mizuno T, Hagiwara H, Itakura M, Ito T, Hirose S. Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells. J Biol Chem 1992;267:7624–9. Lanier-Smith KL, Currie MG. Glucocorticoid regulation of atrial natriuretic peptide receptors on cultured endothelial cells. Endocrinology 1991;129:2311–7. Levin ER, Gardner DG, Samson WK. Natriuretic peptides. N Engl J Med 1998;339:321–8. Li R, Pei H, Watson DK. Regulation of Ets function by protein–protein interactions. Oncogene 2000;19:6514–23. Liang F, Schaufele F, Gardner DG. Sp1 dependence of natriuretic peptide receptor A gene transcription in rat aortic smooth muscle cells. Endocrinology 1999;140:1695– 701. Liang F, Schaufele F, Gardner DG. Functional interaction of NF-Y and Sp1 is required for type A natriuretic peptide receptor gene transcription. J Biol Chem 2001;276:1516–22. Lo K, Landau NR, Smale ST. LyF-1, a transcriptional regulator that interacts with a novel class of promoters for lymphocyte-specific genes. Mol Cell Biol 1991;11:5229–43. Martin DI, Zon LI, Mutter G, Orkin SH. Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages. Nature 1990;344:444–7. Nye JA, Petersen JM, Gunther CV, Jonsen MD, Graves BJ. Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif. Genes Dev 1992;6:975–90.
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[32] Pandey KN. Vascular action: natriuretic peptide receptor. In: Sowers JR, editor. Contemporary endocrinology: endocrinology of the vasculature. Totowa, NJ: Humana; 1996. p. 255–67. [33] Pandey KN, Nguyen HT, Li M, Boyle JW. Natriuretic peptide receptor-A negatively regulates mitogen-activated protein kinase and proliferation of mesangial cells: role of cGMPdependent protein kinase. Biochem Biophys Res Commun 2000;271:374–9. [34] Raffetseder U, Wernert N, Ostendorf T, van Roeyen C, Rauen T, Behrens P, et al. Mesangial cell expression of proto-oncogene Ets-1 during progression of mesangioproliferative glomerulonephritis. Kidney Int 2004;66:622–32. [35] Romeo PH, Prandini MH, Joulin V, Mignotte V, Prenant M, Vainchenker W, et al. Megakaryocytic and erythrocytic lineages share specific transcription factors. Nature 1990;344:447–9. [36] Santiago FS, Khachigian LM. Ets-1 stimulates plateletderived growth factor A-chain gene transcription and vascular smooth muscle cell growth via cooperative interactions with Sp1. Circ Res 2004;95:479–87. [37] Schiffrin EL, Tremblay J, Hamet P, St-Louis J. Vascular binding sites for atrial natriuretic peptide in rats: downregulation in volume expanded states is mediated by ANP. Clin Res 1986;34:486A. [38] Sementchenko VI, Watson DK. Ets target genes: past, present and future. Oncogene 2000;19:6533–48. [39] Tsutamoto T, Kanamori T, Morigami N, Sugimoto Y, Yamaoka O, Kinoshita M. Possibility of down-regulation of atrial natriuretic peptide receptor coupled to guanylate cyclase in peripheral vascular beds of patients with chronic severe heart failure. Circulation 1993;87:70Y–5Y. [40] Wang DY, Yang VC, Chen JK. Oxidized LDL inhibits vascular endothelial cell morphogenesis in culture. In Vitro Cell Dev Biol Anim 1997;33:248–55. [41] Welch JJ, Watts JA, Vakoc CR, Yao Y, Wang H, Hardison RC, et al. Global regulation of erythroid gene expression by transcription factor GATA-1. Blood 2004;104:3136–47. [42] Yang C, Shapiro LH, Rivera M, Kumar A, Brindle PK. A role for CREB binding protein and p300 transcriptional coactivators in Ets-1 transactivation functions. Mol Cell Biol 1998;18:2218–29. [43] Yordy JS, Muise-Helmericks RC. Signal transduction and the Ets family of transcription factors. Oncogene 2000;19:6503–13.
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Transcriptional regulation of guanylyl cyclase/natriuretic peptide receptor-A gene Prerna Kumar, Kiran K. Arise, Kailash N. Pandey * Department of Physiology, SL-39, Tulane University Health Sciences Center and School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112, USA
article info
abstract
Article history:
Activation of natriuretic peptide receptor-A (NPRA) produces the second messenger cGMP,
Received 4 January 2006
which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. In
Accepted 5 January 2006
the present study, we have examined the role of trans-acting factor Ets-1 in transcriptional
Published on line 3 March 2006
regulation of Npr1 gene (coding for NPRA). Using deletional analysis of the Npr1 promoter, we have defined a 400 base pair (bp) region as the core promoter, which contains consensus
Keywords:
binding sites for transcription factors including: Ets-1, Lyf-1, and GATA-1/2. Overexpression
Natriuretic peptides
of Ets-1 in mouse mesangial cells (MMCs) enhanced Npr1 gene transcription by 12-fold.
Npr1 promoter
However, overexpression of GATA-1 or Lyf-1 repressed Npr1 basal promoter activity by 50%
Gene transcription
and 80%, respectively. The constructs having a mutant Ets-1 binding site or lacking this site
Ets-1
failed to respond to Ets-1 activation of Npr1 gene transcription. Collectively, the present
Mesangial cells
results demonstrate that Ets-1 greatly stimulates Npr1 gene promoter activity, implicating its critical role in the regulation and function of NPRA at the molecular level. # 2006 Elsevier Inc. All rights reserved.
1.
Introduction
Natriuretic peptides belong to a family of three homologous endogenous hormones: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) [10,25]. ANP is a circulating hormone which plays a pivotal role in regulation of sodium excretion, fluid volume, steroidogenesis, and cell proliferation; important factors in the control of blood pressure and cardiovascular homeostasis [25,32,2]. The action of ANP is mediated by binding to natriuretic peptide receptor-A (NPRA), which contains guanylyl cyclase (GC) catalytic activity and produces the intracellular second messenger cGMP [10,32]. The activity and expression of NPRA are regulated by a number of factors including: hormones such as glucocorticoids [24] and angiotensin II (Ang II) [16,19], growth factors [14,1], pathophysiological conditions [4,39], and
changes in extracellular ion composition [23,6]. Down-regulation of NPRA has been demonstrated both in vitro [20,5] and in vivo [37]. Additionally, increased cGMP down-regulates receptor density [20]. A cGMP response element has been characterized in the Npr1 gene (coding for NPRA) promoter [22]. Repression of Npr1 gene transcription by the vasoactive peptide hormone Ang II has been shown to be mediated through a region from 916 to 496 in the Npr1 promoter [16]. Previous studies have shown that Sp1 plays a dominant role in basal expression of Npr1 gene [27], and functional and physical interaction of NF-Y with Sp1 is essential for optimal transcription of its promoter in vascular smooth muscle cells [28]. However, studies to elucidate the molecular regulation of Npr1 gene transcription are limited, and the molecular machinery that regulates NPRA expression is not well understood.
* Corresponding author. Tel.: +1 504 988 1628; fax: +1 504 988 2675. E-mail address: [email protected] (K.N. Pandey). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.01.004
peptides 27 (2006) 1762–1769
The 50 -flanking regulatory region of the murine Npr1 gene contains three potential Sp1 binding sites, one inverted CCAAT box, but lacks the TATA box [15]. This region contains several putative cis-acting motifs of known transcription factors, suggesting that these multiple regulatory elements may exert a cooperative influence in order to closely regulate Npr1 gene expression. The 500 base pair (bp) region upstream of the transcription start site (TSS) contains the basal promoter elements to allow the expression of this gene [16]. In order to investigate the transcriptional regulatory mechanisms of the Npr1 gene promoter activity, we have characterized cis-acting sequences, which either activate or repress Npr1 gene transcription and have defined the minimal promoter region of Npr1 gene necessary for efficient transcription in mouse mesangial cells (MMCs). This region contains several putative cis-acting motifs including; Ets-1, p300, three Sp1 sites, Lyf-1, GATA 1/2, and AML-1. Among these cis-acting motifs we demonstrate that Ets-1 effectively upregulates the Npr1 promoter activity and enhances its gene transcription.
2.
Materials and methods
2.1.
Materials
The pGL3-basic vector, pRL-TK, pGL3-control plasmids, and dual luciferase assay system were purchased from Promega (Madison, WI). Plasmid isolation kit was obtained from Qiagen Inc., (Valencia, CA). Sequence-specific oligonucleotides were received from Midland Certified Reagent Company Inc. (Midland, TX). The cell culture media, fetal calf serum, ITS (insulin, transferrin, and sodium selenite), and Lipofectamine-2000 were purchased from Invitrogen (Carlsbad, CA). T4 polynucleotide kinase, protein A-agarose, and [g-32P]-ATP (3000 Ci/ mmol) were purchased from Amersham Biosciences (Piscataway, NJ). The Ets-1 expression vector (pEVRF0-Ets-1) and its empty vector (pEVRF0) were kindly provided by Dr. Paul Brindle [42]. The expression vectors pEF for wild-type GATA-1 and GATA-2 were generously provided by Dr. Adam N. Goldfarb [11]. Expression vector for Lyf-1 (IK-6 in pCDNA3) was a kind gift from Dr. Stephen Smale. All other chemicals were reagent grade and were obtained from Sigma Chemical Co. (St. Louis, MO) and Bio-Rad (Hercules, CA).
2.2.
Plasmid construction
The promoter-luciferase reporter constructs were generated by cloning the PCR-amplified DNA fragments of various lengths of mouse Npr1 gene promoter upstream of the promoter-less firefly luciferase gene in the pGL3-basic vector. The positions marked in the promoter constructs are relative to transcription start site (TSS). The cloning of constructs 1982/+55 (pNPRAluc1) and 496/+55 (pNPRA-luc5) has been previously described [16]. The constructs having 356 or 199 nucleotide position at their 50 end were amplified using forward PCR primers 356 (50 TAC GGA ACG CGT GAG GGG GGG CAG CTT CCT CAC 30 ), and 199 (50 TAC GGA ACG CGT CCA GCC ATA GTC AGG GCT GGG 30 ). The constructs having +55 or 46 nucleotide positions at their 30 ends were PCR-amplified using reverse primers +55 (50 TAC GGA AGA TCT CAG CGA GCG CAG CGA CGG AGC 30 ) and 46 (50 TAC
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GGA AGA TCT AGG CGG GCG GGG GCG GGG TGG 30 ). Underlined nucleotides indicate the MluI and BglII restriction sites. All forward primers contained the MluI restriction site, and the reverse primers contained BglII restriction site at their 50 ends. The plasmid having 496/+55 region was used as a template for amplification of all constructs. These amplified products were then subcloned into MluI/BglII-restricted pGL3-basic vector, and DNA used for transfections was purified by Qiagen columns. All the plasmid constructs were sequenced across both the junctions to confirm the nucleotide sequence and the predicted orientation.
2.3.
In vitro site-directed mutagenesis
Mutations in the Ets-1 binding motifs in the 356/+55 construct, were introduced utilizing Quick Change Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. Complementary sense and antisense strands containing the specific mutations for both the sites were designed separately. The sequence of the forward primer for Ets-1 site A is 50 GCC CCC GCC CGC CTC ATT CAC GGC CGG AGA CTT GG 30 and for Ets-1 site B is 50 CCG GAG ACT TGG GCC AGC ATT CCG CCC CTT CTG GC 30 . Underlined nucleotides indicate the mutated sequence. The Ets-1A mutant construct was used as a template for creating double mutant at Ets-1 site B. All the mutations were confirmed by sequencing the constructs.
2.4.
Cell transfection and luciferase assay
Mouse mesangial cells were isolated and cultured as previously described [33]. MMCs were grown in Dulbecco modified Eagle’s medium supplemented with 10% fetal calf serum. The cultures were maintained at 37 8C in an atmosphere of 5% CO2/ 95% O2. All the experiments were performed utilizing cells between 4 and 16 passages. The MMCs were seeded in a 24well plate at a density of 5 104 cells in each well producing 70–80% confluence after 24 h. The cells were transfected using Lipofectamine-2000 reagent with 1 mg of promoter-reporter construct and 300 ng of pRL-TK carrying the renilla luciferase gene downstream of thymidine kinase promoter, which was used as an internal transfection control. Cells were harvested after 48 h by using passive lysis buffer. The cell lysate was centrifuged at 10,000 rpm for 3 min at 4 8C, and the supernatant was used to measure firefly luciferase and renilla luciferase activities. The luciferase activities were measured by mixing 10 ml cell extract with 50 ml of luciferase firefly assay reagent. The fluorescence intensity was integrated for 10 s using a TD 20/20 luminometer (Turner Designs). Subsequently, the resultant mixture was used for renilla luciferase activity. The results were normalized for the transfection efficiency relative to light units per renilla luciferase activity.
2.5.
Whole cell lysate preparation and immunoblot assay
MMCs were harvested by trypsinization and electroporated with 5 mg of either empty vector DNA (pEVRF0) or Ets-1 expression vector at 220 V with a capacitance setting of 960 mF using a gene pulser (Bio-Rad, Hercules, CA). After transfection, cells were seeded into 100 mm2 petri dishes. Forty-eight hours
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after transfection, cells were washed with PBS and lysed in buffer containing: 25 mM HEPES, pH 7.5, 0.05% 2-mercaptoethanol, 1% Triton X-100, 1 mM sodium vanadate, 10 mm NaF, 0.2 mm PMSF, and 10 mg/ml each of aprotinin and leupeptin. Cell extract was passed through a 1 cc syringe with a 21-guage needle and centrifuged at 14,000 rpm for 10 min. The clear cell lysate was collected and stored at 80 8C until used. The protein concentrations of the lysate were estimated using a Bradford protein detection kit (Bio-Rad). Whole cell lysate (40 mg) from each sample was mixed with sample loading buffer and resolved by 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were electrotransferred onto a PVDF membrane. The membrane was blocked with 1 Tris–buffered saline-Tween 20 (TBST) containing 5% fat-free milk for 2 h at room temperature, and incubated overnight at 4 8C in TBST containing 3% fat-free milk with primary antibodies (1:1000 dilution). The membrane was then treated with corresponding secondary anti-rabbit or anti-mouse HRP-conjugated antibodies (1:10,000 dilutions). Protein bands were visualized by enhanced chemiluminescence (ECL) plus detection system.
2.6.
Nuclear extract preparation
Nuclear extracts were prepared from MMCs as described by Dignam [8]. The cells were harvested and centrifuged at 250 g for 10 min. Pellet was washed with PBS and centrifuged as described above. The resulting pellet was resuspended in five volumes of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM PMSF) and incubated on ice for 10 min and centrifuged as above. The pellet was again resuspended in three volumes of buffer A, to which Nonidet P-40 (0.05%, v/v) was added and the suspension was homogenized with 20 strokes of a tight-fitting Dounce homogenizer to release the nuclei. The suspension was then centrifuged at 250 g for 10 min to pellet the nuclei. The pellet was resuspended in 100–200 ml of buffer C (5 mM HEPES, pH 7.9, 26% glycerol v/v, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM PMSF). To the suspension, NaCl was added to a final concentration of 300 mM. After mixing, the suspension was incubated on ice for 30 min, and then centrifuged at 24,000 g for 20 min. The protein concentration of the nuclear extract was estimated using a Bradford protein detection kit (Bio-Rad). All centrifugation steps were carried out at 4 8C. Aliquots of the supernatant were stored at 70 8C until used.
2.7.
UV cross-linking assay
To determine the molecular mass of the nuclear protein bound to the Ets-1 binding sites, a UV cross-linking assay was performed. Sense and antisense primers spanning the binding sites for Ets-1 were synthesized. The forward primers for Ets-1 site A (50 CCG CCC GCC TCC GGA ACG GCC GGA 30 ) and for Ets-1 site B (50 TGG GCC AGC CGG ACG CCC CTT CTG 30 ) were endlabeled using [g-32P]-ATP and T4 polynucleotide kinase, and annealed with antisense strand. Binding reactions were carried out in a total volume of 15 ml containing 1 binding buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 50 mM NaCl, 0.5 mM EDTA, 4% glycerol v/v, and 0.5 mM DTT), 8 mg poly (dIdC) as a non specific competitor, and 20 mg MMC nuclear
extract. The reaction mixture was incubated on ice for 5 min, after which [g-32P]-ATP labeled probe (150,000 cpm) was added, and the mixture was further incubated on ice for 30 min. The reaction mixture was then subjected to UV radiation of 254 nm using a UV Stratalinker (Stratagene, La Jolla, CA) in auto-crosslinking mode for five exposures (each exposure is of 120,000 mJ for 40 s). The reaction mixture was then mixed with SDS-loading buffer, boiled, and resolved using 10% SDS–PAGE and autoradiography.
2.8. Electrophoretic mobility shift assay (EMSA) and supershift assay Protein–DNA complexes were detected using 50 biotin endlabeled double-stranded DNA probes prepared by annealing complementary oligonucloetides. Sequence of the probes is same as given in UV cross-linking assay. The binding reaction was performed using LightShift kit (Pierce). Briefly, MMC nuclear extracts (1.5 mg of protein) and binding buffer were incubated on ice for 5 min in a volume of 20 ml, then biotinlabeled probe of Ets-1A or Ets-1B (40 or 20 fmol, respectively) was added, respectively, and reaction was allowed to incubate for an additional 25 min at room temperature. In super-shift experiments, the extracts were preincubated with anti-Ets-1 polyclonal antibody for 60 min on ice. Protein–DNA complexes were separated on nondenaturing polyacrylamide gels and observed by the LightShift Chemiluminescent EMSA kit following the manufacturer’s procedure.
2.9.
Statistical analysis
The results are presented as mean S.E. The statistical significance was evaluated by Student t-test and 1-way ANOVA, followed by Dunnett’s multiple comparison test using the computer software PRISM (GraphPad Software Inc., San Diego, CA). The probability values of P < 0.05 were considered significant.
3.
Results
The results presented in Fig. 1 demonstrate that a deletion of 150 bp in Npr1 promoter construct 496/+55 (356/+55) towards its 50 end significantly increased basal promoter activity ( p < 0.01) in MMCs as compared with the construct 496/+55. The construct 356/+55 showed a maximum of a 75fold increase in luciferase activity, while the full length promoter (1982/+55) exhibited only a 28-fold induction in luciferase activity as compared with the pGL3-basic vector. In the construct 356/46, a deletion of 90 bp from the 30 end significantly decreased the transcriptional activity as compared with the construct 356/+55 ( p < 0.001), indicating that the region 46 to +55 in the core promoter is essential for Npr1 transcriptional activity (Fig. 1). We further localized the transcription factor binding sites in the region 356 to +55 using the TRANSFAC 3.2 database, which revealed the presence of many putative transcription factor binding sites including: Ets-1, Lyf-1, and GATA-1/2 and coactivator p300 (Fig. 2). The region 356 to +55 (from TSS), which contains binding sites for transcription factors Ets-1, GATA-1/2, and Lyf-1, are
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Fig. 1 – Luciferase activity of murine Npr1 core promoter deletion constructs in mouse mesangial cells. Left hand side of the figure shows the diagrammatic representation of 50 Npr1 promoter-reporter deletion constructs. The numbers shown next to the construct indicate the 50 and 30 ends of the constructs, respectively. Right side represents the promoter activity of the deletion constructs cotransfected with pRLTK vector into MMCs. pGL3-Basic was taken as control. Firefly luciferase activity was normalized to Renilla luciferase activity. Luc represents the luciferase gene. Values represent the mean W S.E. of four independent experiments in triplicates. *P < 0.05, ** P < 0.01, ***P < 0.001.
shown in Fig. 3A. To evaluate the relative contributions of these binding sites to the core promoter activity, we overexpressed these transcription factors in a dose dependent manner and cotransfected them with the basal promoter construct 356/ +55 in MMCs (Fig. 3B). Overexpression of Ets-1 vector DNA (100 and 250 ng) in MMCs elicited almost 10-fold and 12-fold increase in the transcriptional activity of Npr1 gene, respectively (Fig. 3B). Conversely, overexpression of GATA-1 and Lyf-1 vector DNA at similar concentrations repressed the Npr1 basal promoter
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activity by almost 50% and 80%, respectively. To conclusively demonstrate the role of Ets-1 in the activation of Npr1 gene promoter activity, we utilized site-specific Ets-1 point mutations and deletion constructs. A diagrammatic representation of constructs having wild-type, point mutations, and deletion of Ets-1 binding sites are shown in Fig. 4A. The construct having mutant Ets-1 sites or the construct lacking Ets-1 binding sites (+356/46) failed to respond to Ets-1 activation of Npr1 gene transcription, whereas, wild-type Ets-1 construct stimulated full activation (12-fold) of Npr1 gene transcription (Fig. 4B). Furthermore, each site was mutated separately in constructs 356/+55 Ets-1Am and 356/+55 Ets-1Bm to see their effect. Basal expression was reduced to 40–50% in cells transfected with construct having either site mutated as compared with the construct 356/+55 WT taken as 100% (Fig. 4C). On the other hand additive effect in loss of transcriptional activity (90%) was seen when both the sites were mutated together (Fig. 4C). The Western blot analysis showed that the expression of Ets-1 in transfected cells exhibited the production of Ets-1 protein, corresponding to the molecular mass of 54 kDa (Fig. 4D). In order to confirm whether endogenous Ets-1 protein present in the nuclear extract of MMCs binds to its consensus sequence, a UV cross-linking assay was performed. [g-32P]ATP-labeled oligonucleotides containing Ets-1 binding motifs A and B were incubated with MMCs nuclear extract, UV cross-linked, and resolved by SDS–PAGE and autoradiography. A DNA–protein complex of Ets-1 (54 kDa) was found to bind to both of the Ets-1 consensus binding sites in Npr1 promoter (Fig. 5, lanes b and d) as compared with the free probes (Fig. 5, lanes a and c). Antibody supershift assays were also performed to confirm the specificity of the protein binding to Ets-1 motifs in the Npr1 promoter. When incubated with MMC nuclear extract wild type Ets-1A and Ets-1B oligonucleotides supported the formation of specific nucleoprotein complexes (Fig. 6A and B, lanes 2 and 6). Whereas, Ets-1 antibody disrupted the DNA protein complex hence abolishing the specific band (Fig. 6A and B, lanes 4 and 8).
Fig. 2 – Nucleotide sequence of the 50 -flanking region of murine Npr1 gene spanning from S356 to +55. The transcription start site (TSS) is indicated by a vertical arrow. Nucleotide positions are numbered with respect to the TSS+1. Consensus sequence for known transcription factor binding site and cis-acting elements are indicated with an arrow and in bold face. GenBank accession no. AJ307712.
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Fig. 3 – Effect of overexpression of Ets-1, Lyf-1, and GATA1/2 on Npr1 basal promoter activity in MMCs. (A) Schematic representation of the putative cis-acting elements present in the region S356 to +55 of Npr1 gene promoter. (B) MMCs were cotransfected with 1 mg of S356/+55 Npr1 promoter construct along with increasing amount (50, 100, and 250 ng) of indicated expression vectors. Luciferase activity is represented as fold induction with respect to corresponding empty vector used as control. Firefly luciferase activity was normalized to Renilla luciferase activity. Values represent the mean W S.E. of three independent experiments in triplicates. *P < 0.05, **P < 0.01, ***P < 0.001.
4.
Discussion
The findings of this study show that Ets-1 plays an important role in mediating the activity of Npr1 gene promoter. Ets-1 binding sites are present in the core promoter of Npr1 gene spanning from 356 to +55 along with several other putative cis-acting elements for transcription factors including: Lyf-1, MZF-1, GATA 1/2, and SP1. Our results demonstrate that Ets-1 mediates the transcriptional activation of murine Npr1 gene promoter through its binding sites present in the region 46 to +55. Ets proteins have been shown in the regulation of numerous genes involved in diverse cellular processes, such as proliferation, differentiation, development, transformation, and apoptosis [38,43,26]. The Ets family of transcription factors specifically recognizes DNA sequences that contain GGAA/T as a core element [31,9]. Interestingly, NPRA/cGMP signaling has been implicated in various anti-mitogenic and cell proliferation activities, due to, perhaps in part, the regulation exhibited by the Ets-1 activity demonstrated in the present studies. Additionally, ANP has been reported to be involved in NPRA-mediated apoptosis of cardiac myocytes, endothelial, and vascular smooth muscle cells [12]. Among its diverse functions, Ets proteins play a significant role in regulating genes involved in both cell cycle progression and programmed cell death, including: cdc2, jun B, c-fos, c-myc, Rb, p53 and bcl-2 [3]. In addition, the function of Ets-1 in the
regulation of apoptosis has recently been demonstrated in lymphoid and colon cancer cells [21]. Thus, it is evident that Ets-1 upregulates Npr1 gene, and this process may play an important role in Npr1-mediated apoptosis. Cell- and tissue-specific gene expression is established through the combinatorial action of restricted and more widely expressed transcription factors. The architecture of a regulatory element directs the assembly of these factors into higher-order structures that ultimately determine the transcriptional readout. Our results showed that overexpression of Ets-1 in MMCs greatly enhanced the Npr1 promoter activity, whereas, the overexpression of GATA-1 and Lyf-1 significantly repressed Npr1 transcriptional activity. At present it is not known that the transcription factors GATA-1 and Lyf-1 are involved in any transcriptional repression. GATA-1 is a tissuespecific transcription factor, which is expressed in erythrocytes, megakaryocytes, eosinophils, and mast cells [30,35]. All erythroid genes are positively regulated by GATA-1 but the full extent of its transcriptional actions is currently unknown [41]. On the other hand Lyf-1 is reported to be transcriptional activator of lymphoid-specific genes [29] and Lyf-1 protein is known to be involved in the regulation of gene expression during early stages of B- and T- cell development [18]. Their expression and role in mesangial cells are currently unknown and it might be a possibility that these factors play a role in modulation and tissue-specific regulation of Npr1 gene.
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Fig. 5 – Endogenously expressed Ets-1 binds to its consensus site in the Npr1 promoter. Twenty micrograms of MMC nuclear extract (NE) were incubated with [g-32P]ATP-labeled probes and UV cross-linked. The product was electrophoresed on 10% SDS-PAGE and visualized by autoradiography. Mr and position of UV cross-linked product of Ets-1 site A and site B in the absence (lanes a and c) and in the presence (lanes b and d) of MMC nuclear extract are indicated by an arrow. Bars on the left margin show the Mr of marker proteins. The autoradiogram is the representative of four independent experiments.
Fig. 4 – Effect of Ets-1 binding sites on the functional activation of Npr1 promoter. (A) Diagrammatic representation of the Npr1 core promoter construct harboring wild type (WT), mutant type (MT) and deleted Ets-1 binding sites. (B) One microgram of the constructs S356/+55 Ets-1 WT, S356/+55 Ets-1 MT, and S356/S46 was cotransfected with 50 or 250 ng of Ets-1 expression vector or the empty control vector pEVRF0. Luciferase activity is represented as fold induction with respect to corresponding empty vector used as control. (C) One microgram of the constructs S356/+55 WT, S356/+55 Ets1Am, S356/+55 Ets-1Bm and S356/+55 Ets-1 MT was transiently transfected in MMCs and luciferase activity was measured after 48 h. Value of construct S356/+55 WT is taken as 100%. (D) Western blot analysis demonstrates overexpression of Ets-1 protein in MMCs when transfected with pEVRF0-Ets-1. Cells were transfected with 10 mg of pEVRF0 or pEVRF0-Ets-1, and whole cell lysate was prepared after 48 h and developed with Ets-1 polyclonal antibody. Lower panel shows b-actin used as a loading control. This experiment was repeated three times independently. Values given in luciferase activity (B and C) represent the mean W S.E. of three independent experiments in triplicates. *P < 0.01, **P < 0.001.
Several agents that are known to upregulate Ets-1 transcription, include RA, TNF-alpha, VEGF, and TPA [7,17,40]. Ets-1 is upregulated at exposure to agonists such as serum in vitro and is expressed in injured vasculature [36]. MAPK-mediated phosphorylation positively regulates the transcriptional activation functions of Ets-1 by recruiting CBP/p300 [13]. Not much is known about Ets-1 expression or regulation in mesangial cells. A temporal increase of mesangial cell Ets-1 expression has been reported which correlates with mesangial cell activation in mesangioproliferative glomerulonephritis suggesting involvement of PDGF-B [34]. There might be a possibility that during glomerulonephritis increased Ets-1 expression upregulates Npr1 gene as a protective mechanism. Npr1 gene has been shown to negatively regulate mitogen-activated protein kinase and proliferation of mesangial cells [33]. In conclusion, our results demonstrate that the precise control of Npr1 gene transcriptional activity is achieved through a synergy of activators and repressors in which Ets1 plays an integral role as a transcriptional activator. Comparatively, Lyf-1 and GATA-1 act as repressors, inhibiting and regulating the transcriptional activity of Npr1 gene promoter. The present findings suggest that Ets-1 plays a critical role in enhancing Npr1 gene transcription and may have an important influence in hypertension and cardiovascular homeostasis at the molecular level.
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Fig. 6 – Ets-1 antibody supershift assay. Ets-1 antibody supershift assay in nuclear extract from MMCs A and B, MMC nuclear extract was incubated with labeled Ets-1 site A and Ets-1 site B probe, respectively, in presence of 2 mg of anti Ets-1 antibody. Arrow shows Ets-1 nuclear protein complex binding with Ets-1A (lane 2) and Ets-1B sites (lane 6), respectively. Star shows disruption of the respective binding in the presence of Ets-1 antibody (lanes 4 and 8).
Acknowledgements The authors wish to thank Huong T. Nguyen and Gevoni Bolden for their valuable technical assistance during the course of this work and Mrs. Kamala Pandey for her assistance in typing the manuscript. We sincerely thank Dr. Paul Brindle, Dr. Adam N. Goldfarb, and Dr. Stephen Smale for the kind gifts of expression vectors. Our special thanks are due to Dr. Susan L. Hamilton, Department of Molecular Physiology and Biophysics at Baylor College of Medicine and to Dr. Bharat B. Aggarwal, Department of Experimental Therapeutics and Cytokine Research Laboratory at MD Anderson Cancer Center for providing their facilities during our displacement period due to Hurricane Katrina. This research was supported by the National Institutes of Health Grants HL57531 and HL62147.
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