COOPERATIVE ACTIVATION OF Npr1 GENE TRANSCRIPTION AND EXPRESSION BY INTERACTION OF ETS-1 AND P300

Abstract

The objective of the present study was to gain insight into the cooperative role of Ets-1 and p300 in transcriptional regulation and expression of Npr1 gene (coding for guanylyl cyclase-A/natriuretic peptide receptor-A; GC-A/NPRA). Overexpression of Ets-1 and p300 in mouse mesangial cells (MMCs) increased Npr1 promoter activity by 12-fold, NPRA mRNA levels by 5-fold, and ANP-dependent intracellular accumulation of cGMP by 26-fold. Knockdown of Ets-1 and p300 expression by small interfering RNA (siRNA) inhibited Npr1 gene transcription by 90%. Sequential chromatin immunoprecipitation assay demonstrated a direct physical association between p300 and Ets-1 upon binding to Npr1 promoter, suggesting that a physical interaction between Ets-1 and p300 is important to enhance Npr1 gene transcription. Mutant p300 lacking histone acetyltransferase (HAT) activity did not show functional effect with Ets-1, suggesting that HAT activity of p300 is required for the cooperative interaction in modulating Npr1 gene transcription. Overexpression of wild-type adenovirus E1A significantly decreased the Npr1 promoter activity by 40%, whereas mutant E1A, which is incapable of binding to p300, did not show any effect. The results indicate that Npr1 gene transcription is critically controlled by histone acetyltransferase p300 and Ets-1. The present findings should yield important insights into the molecular signaling governing Npr1 gene transcription, an important regulator in the control of hypertension and cardiovascular events.

Introduction

Atrial and brain natriuretic peptides (ANP, BNP) principally mediate natriuresis/diuresis, vasorelaxation, and antimitogenic responses, largely directed to the reduction of blood pressure and blood volume ^1–3. The actions of ANP and BNP are mediated by binding to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA), which produces the intracellular second messenger cGMP ^4–6. It has been demonstrated that Npr1 gene (coding for GC-A/NPRA) lowers arterial blood pressure and increases GC activity in a gene-dose-dependent manner in Npr1 gene-targeted mice ^7–9. Previous studies have also shown that the disruption of Npr1 gene leads to renal insufficiency and cardiac hypertrophy and fibrosis in Npr1 null mutant mice ^{8, 10–13}. Earlier studies have provided evidence for a significant association for Npr1 gene variants with hypertensive family history and with increased left ventricular mass index as well as left ventricular septal wall thickness in human essential hypertension ^14–16. Extracellular osmolality and vitamin D have been shown to stimulate Npr1 gene promoter activity in inner medullary collecting duct cells ¹⁷. On the other hand, angiotensin II (Ang II) has been found to repress the Npr1 gene transcription in mouse mesangial cells ^{18, 19}. However, the mechanistic regulation of Npr1 gene transcription and expression in target cells is not well understood.

Ets-1 protein, which is expressed in a variety of cell types including endothelial cells, mesangial cells, and vascular smooth muscle cells, has been implicated in the transcriptional regulation of several genes involved in angiogenesis and remodeling of the extracellular matrix proteins ^{20, 21}. Similarly, transcriptional coactivator p300 and CREB-binding protein (CBP) play a central role in coordinating multiple signal-development events with the transcription apparatus, allowing appropriate level of gene activity to occur in response to diverse physiological stimuli ²². Many Ets family proteins including Elk-1, Ets-1, Ets-2, and ER81 interact with co-activator CBP/p300 to stimulate gene transcription ^{23, 24}. The glomerular mesangial cells provide an attractive model system to delineate the synergistic regulation of Npr1 gene transcription because these cells modulate kidney function, contain functional GC-A/NPRA, and express both Ets-1 and p300 proteins ^{20, 25}. The present study demonstrates that Ets-1 and p300 play an essential role in modulating the transcription and expression of Npr1 gene, which plays a critical role in the control of kidney function and regulation of blood pressure and cardiovascular homeostasis.

Methods

Plasmids and promoter constructs

The promoter-luciferase reporter constructs were generated by cloning PCR-amplified DNA fragments of murine Npr1 promoter upstream of the promoterless firefly luciferase gene in the pGL3-basic vector (Promega) as described earlier ²⁶. The cloning of the construct −356/+29 base pairs (bp) was performed using −356 forward (5′-tacggaacgcgtgagggggggcagcttcctcac-3′) and +29 reverse (5′-tacggaagatctgtgtgcgctcgcgcttgcccc-3′) primers. The vectors pEVRF0-Ets-1 and pEVRF0 were received from Paul Brindle (St. Jude Children’s Research Hospital, Memphis, TN), E1A 12Swt and E1A 12SΔ2–36 were obtained from Elizabeth Moran (UNDNJ, Newark, NJ), and pcDNA3.1-p300 WT and pcDNA3.1-p300-(HAT-) were received from Warner Greene (UCSF, San Francisco, CA) as kind gifts.

Cell Transfection and Luciferase Assay

Mouse mesangial cells (MMCs) were isolated and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FBS) and insulin-transferrin-sodium selenite (ITS) as previously described ²⁷. Rat thoracic aortic smooth muscle cells (RTASMCs) were cultured in DMEM supplemented with 10% FBS as earlier described ²⁸. The cells (between 4^th to 15^th passages) were transfected using Lipofectamine-2000 reagent (Invitrogen) and luciferase activity in cell lysate was measured as previously described ²⁶. The results were normalized for the transfection efficiency as relative to light units per Renilla luciferase activity. Transfection efficiency was assessed using in situ beta-galactosidase staining kit (Stratagene) and was achieved 80% and 85% in MMCs and RTASMCs, respectively.

Real-time RT-PCR Assay

Total RNA was extracted from transfected cells using RNeasy mini-kit (Qiagen) and the first-strand cDNA was synthesized from 1 μg of total RNA using RT² First Strand kit (SABiosciences). Real-time RT-PCRs were performed using the Mx3000P Real-Time PCR System and data was analyzed with MxPro software (Stratagene). PCR amplification (in triplicates) was carried out in a 25 μl reaction volume using RT² real-time™ SYBR Green/ROX PCR Master Mix. The reaction conditions were: 95°C for 10 min; followed by 45 cycles at 95°C for 15 s and 60°C for 1 min; followed by 1 cycle at 95°C for 1 min, 55°C for 30 s and 95°C for 30 s for the dissociation curve. Beta-actin was amplified from all samples on each plate as housekeeping gene to normalize expression levels of targets between different samples, and to monitor assay reproducibility. The reaction mixture without template cDNA was used as negative control. Threshold cycle number (C_T) was determined with MxPro QPCR Software and transformed using the ΔC_T comparative method. The Npr1 gene expression values were normalized to expression values of beta-actin within each sample, and relative expression of Npr1 gene was determined by the comparative C_T analysis (ΔΔC_T).

Immunoblot Assay

Forty-eight hours after transfection, cells were lysed and immunoblot assay was performed as earlier described ²⁶. In brief, cell lysate (80 μg proteins) was electrophoresed for 2 h and then transferred to nylon membrane. The Membrane was incubated with primary antibodies (anti-Ets-1, anti-p300, or anti-NPRA) and treated with corresponding secondary anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibodies (Santa Cruz). Protein bands were visualized by ChemiGlow West (Alpha Innotech).

Sequential Chromatin Immunoprecipitation Assay

Chromatin Immunoprecipitation (ChIP) was performed using the ChIP-IT Express kit (Active Motif). Briefly, cells (1.5×10⁷ ) were cross-linked in 1% formaldehyde for 10 min at 22°C and the reaction was quenched with 0.1 M glycine. Cells were scraped, resuspended in 1 ml lysis buffer on ice and homogenized with dounce homogenizer. The homogenate was centrifuged at 5,000 rpm for 10 min at 4°C to pellet the nuclei. The pellet was resuspended in 1 ml digestion buffer and 50 μl of enzymatic shearing cocktail and incubated at 37°C for 10 min. The reaction was stopped by adding 20μl of 0.5 M EDTA followed by chilling on ice for 10 min. Sheared DNA was centrifuged at 13,000 rpm at 4°C for 10 min and supernatant was collected. Immunoprecipitation was performed using protein G magnetic beads and 5 μg antibody of either Ets-1, p300, or control IgG at 4°C with rotation for overnight. Beads were pelleted and washed sequentially once with ChIP buffer 1 and twice with ChIP buffer 2. The bound-protein was eluted from the beads by incubation with 10 mM dithiothrietol at 37°C for 30 min, and again immunoprecipitated with second antibody overnight at 4°C. After washing the magnetic beads, bound-protein was eluted by gentle rotation for 15 min in elution buffer AM2 at 22°C. After elution of protein/DNA complex, crosslinking was reversed at 65°C overnight to release DNA. Immunoprecipitated DNA was sequentially treated with RNase A and proteinase K and then purified. The Npr1 promoter containing Ets-1 and p300 binding sites was PCR-amplified using purified DNA as a template and the forward (5′-ctctcttgtcgccgaatctg-3′) and the reverse (5′-gtggagagcgagagaacgaga -3′) primers.

Immunoprecipitation of Acetylated Ets-1

Cells were transfected with p300 expression plasmid and cell lysate was prepared after 48 h of transfection. Cell lysate (100 μg proteins) was incubated with 2 μg of polyclonal Ets-1 antibody and immunoprecipitated as described above. For detection of acetylated Ets-1, membranes were incubated with anti-Ac-lys (AKL5C1) antibody (Santa Cruz) and treated with anti-mouse horseradish peroxidase-conjugated secondary antibody.

Transfection of Small Inhibitory RNA

Cells were cultured to 70%–80% confluence and transfected with Ets-1 and p300 small inhibitory RNA (siRNA; a pool of 3 target-specific 20–25 nucleotide sequence siRNA) using Lipofectamine-2000 reagent. A nontargeting 20–25 nucleotide sequence siRNA was used as a negative control. Four-hours after transfection, fresh medium was added to the plates and after 48 h, cells were lysed to measure firefly and Renilla luciferase activities.

cGMP Assay

Forty-eight hours after transfection, cells were treated with ANP at 37°C for 20 min in the presence of 0.2 mM 3-isobutyl-1-methylxanthine as previously described ²⁹. Cells were washed three times with phosphate-buffered saline (PBS) and scraped into 0.5 N HCl, subjected to 5 cycles of freeze and thaw, and then centrifuged at 10,000 rpm for 15 min. The supernatant thus collected was used for cGMP assay using the direct cyclic-GMP assay kit (Assay Designs).

Statistical analysis

The statistical significance was evaluated by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison tests using PRISM software (GraphPad Software, San Diego, CA). The results are expressed as mean ± SE and a p value of < 0.05 was considered significant.

Results

The Npr1 promoter construct (−356/+55 bp) containing Ets-1 and p300 binding sites exhibited 60- to 70- fold increase in luciferase activity in RTASMCs and MMCs, respectively, as compared with the pGL3-basic vector (Fig. 1A). Mutation of Ets-1 binding sites in the construct −356/+55 and deletion of +29 to +55 bp region containing p300 binding site from this construct significantly decreased the Npr1 promoter activity in both MMCs and RTASMCs. The deletion of both Ets-1 and p300 binding sites in the construct −356/−46, reduced the promoter activity by almost 80% and 90% in RTASMCs and MMCs, respectively (Fig. 1A). An interspecies comparison of Ets-1 and p300 consensus sites present in Npr1 promoter showed that these sites are highly conserved in mouse, rat, and human (Fig. 1B). Overexpression of Ets-1 and p300 plasmids with construct −356/+55 enhanced Npr1 promoter activity by almost 12-fold (Fig. 2A). Construct having mutations in both Ets-1 binding sites showed only 4-fold stimulation in luciferase activity with overexpression of Ets-1 and p300 as compared with the untransfected controls. The deletion of p300 sites also showed only 7-fold stimulation in luciferase activity with overexpression of Ets-1 and p300 as compared with the wild-type construct (Fig. 2A). The construct −356/−46 (lacking Ets-1 and p300 binding motifs) failed to respond to Ets-1 and p300 protein expression further confirming that Ets-1 and p300 utilize their consensus sites to activate Npr1 gene transcription. A significant expression of Ets-1 and p300 proteins (2-fold) in MMCs was confirmed by Western blot analysis using anti-Ets-1 and anti-p300 antibodies (Fig. 2B). Cotransfection of p300 and Ets-1 siRNA along with the Npr1 promoter reduced the luciferase activity by 50% and 75%, respectively, as compared with untransfected cells, however, transfection of control siRNA showed no change in luciferase activity (Fig. 2C). Transfection of Ets-1 and p300 siRNA together repressed luciferase activity by more than 90%. Western blot analysis showed that Ets-1 and p300 protein expression was markedly reduced in siRNA-transfected cells as compared with control siRNA-transfected cells (Fig. 2D).

Figure 1. Luciferase activity of 3′ deletion constructs of Npr1 promoter.

(A) Left panel shows the schematic representation of deletion construct of Npr1 promoter. Right panel shows transcriptional activity of these constructs in MMCs and RTASMCs. (B) A direct interspecies comparison of Ets-1 and p300 sites in mouse, rat, and human Npr1 gene promoter. Sequence in bold shows the consensus binding sites of Ets and p300. Bars represent the mean ± SE of four independent experiments in triplicates. ** p < 0.01; †† p < 0.01; *** p < 0.001; ††† p < 0.001.

Figure 2. Effect of overexpression and knockdown of Ets-1 and p300 on Npr1 promoter activity.

(A) Luciferase activity of 3′ deletion constructs (−356 to +55 bp) of Npr1 promoter cotransfected with Ets-1, p300, or empty vector (−Ets-1/-p300) in MMCs. (B) Western blot analysis of Ets-1 and p300 in transfected MMCs. (C) Luciferase activity of Npr1 promoter cotransfected with Ets-1, p300, and control (ctrl) siRNA. (D) Western blot analysis of knockdown effect of Ets-1, p300, and control siRNA in MMCs. Beta-actin was used as loading control. Data represent the mean ± SE of four independent experiments. UT, untransfected; WB, Western blot. *, p< 0.05; **, p < 0.01; ***, p < 0.001.

To examine whether Ets-1 and p300 coexist within the same protein complex resident in the Npr1 promoter, sequential ChIP assay was performed. The initial ChIP assay was carried out with Ets-1 antibody and the sequential ChIP showed the occupancy of p300 in the same protein-DNA complex (Fig. 3A). Conversely, initial ChIP with p300 antibody followed by second IP with Ets-1 antibody further confirmed the coexistence of Ets-1 and p300 in the protein-DNA complex binding to the Npr1 promoter (Fig 3B). To investigate the effect of overexpression of Ets-1 and p300 on the endogenous Npr1 gene expression, we analyzed the mRNA levels of NPRA by real time RT-PCR assay. Experiments were performed in MMCs and RTASMCs to determine the effect of Ets-1 and p300 in a cell-type specific manner. There was almost 5-fold induction in NPRA mRNA levels in Ets-1 and p300 transfected MMCs as compared with untransfected controls (Fig. 4A). The Western blot analysis exhibited an enhanced level of NPRA protein expression in Ets1-/p300 transfected MMCs as compared with untransfected control cells (Fig. 4B). The treatment of Ets-1 transfected RTASMCs and MMCs with 100 nM ANP showed an increase in intracellular accumulation of cGMP by 7-fold and 12-fold, respectively, compared with untransfected control cells (Fig. 4, C and D). Coexpression of Ets-1 and p300 and treatment with 100 nM ANP significantly increased cGMP levels by almost 19-fold in RTASMCs and 26-fold in MMCs as compared with untransfected controls.

Figure 3. Sequential ChIP showing the interaction of Ets-1 and p300 in Npr1 promoter.

Sequential ChIP assay was performed after an initial immunoprecipitation (IP) with anti-Ets-1 (A) and ant-p300 antibody (B). A protein-DNA complex was recovered after a second IP with Ets-1 and p300 antibody. Representative results of 3 experiments are shown. M, DNA ladder; Ab, antibody.

Figure 4. Effect of Ets-1/p300 on NPRA mRNA expression and intracellular accumulation of cGMP.

(A) Npr1 mRNA in transfected MMCs was determined by real time RT-PCR. Values indicate the fold-induction of Npr1 mRNA levels relative to untransfected cells with beta-actin as an internal control. (B) Western blot analysis of NPRA protein levels in transfected MMCs. (C) and (D) The intracellular accumulation cGMP in transfected MMCs and RTASMCs. Bars represent the mean ± SE of four independent experiments in triplicate. UT, untransfected. *, p < 0.05, **, p < 0.01; *** p < 0.001.

We examined the cooperative interactive role of Ets-1 and p300 in the regulation of Npr1 gene transcription and expression in MMCs and RTASMCs, which were transfected with either Ets-1 alone, wild-type, or mutant p300 construct lacking HAT activity. Cotransfection of Ets-1 and mutant p300-(-HAT) construct did not show any significant increase in promoter activity as compared with Ets-1 transfected MMCs (Fig. 5A). We confirmed the overexpression of Ets-1 and p300-HAT mutant in MMCs by Western blot analysis (Fig. 5B). Immunoprecipitation assay showed that endogenous Ets-1 exhibited measurable levels of lysine acetylation in untransfected MMCs (Fig. 5C). However, there was a significant increase in the level of Ets-1 acetylation in cells transfected with p300 expression plasmid. Bottom panel in Fig. 5C shows the direct Western blot analysis of total Ets-1 on a different gel. We further confirmed the role of p300 and its HAT activity in cooperation with Ets-1 on Npr1 gene transcription. Cotransfection of MMCs with construct −356/+55 and wild-type E1A significantly reduced luciferase activity, whereas mutant construct showed no inhibition in the luciferase activity as compared with the controls (Fig. 6). There was a significant reduction by approximately 40% in Npr1 promoter activity in cells transfected with Ets-1/p300 and wild-type E1A as compared with Ets-1/p300. On the other hand, cotransfection of Ets-1/p300 and mutant E1A showed no change in luciferase activity as compared with Ets-1/p300-transfected cells (Fig. 6).

Figure 5. Effect of p300 HAT activity on Npr1 transcription in MMCs.

A) Cells were transiently transfected with Npr1 promoter construct 356/+55 bp and Ets-1 expression plasmid (250 ng) with wild-type p300 or mutant p300 (250 ng) plasmid and luciferase activity was measured. (B) Western blot analysis of Ets-1 and p300-HAT in transfected MMCs. (C) After immunoprecipitation, acetylated Ets-1 and total Ets-1 were determined. The complex formation was not detected in lysates immunoprecipitated with control mouse IgG. Lower panel shows total Ets-1 detected by direct Western blot of the lysate. Representative blots of 3 experiments are shown. Bars represent the mean ± SE of four independent experiments in triplicate. UT, untransfected; IP, immunoprecipitation; WB, Western blot; Ac, acetylated. *, p < 0.05.

Figure 6. Effect of adenovirus E1A on Npr1 gene transcription.

Luciferase activity of Npr1 promoter construct −356/+55 with wild-type or mutant adenovirus E1A expression plasmid was measured. Wild-type or mutant adenovirus E1A plasmids (100 ng) were transfected alone or with Ets-1 (250 ng) and p300 (250 ng) expression vector with Npr1 promoter construct in MMCs. Bars represent the mean ± SE of four independent experiments. Ad, adenovirus. *, p < 0.05, **, p < 0.01.

Discussion

The results of the present study demonstrate that Npr1 promoter activity is regulated by cooperative interaction of Ets-1 and p300, which activate Npr1 gene transcription through the consensus sites present in region −46 to +55 bp of the Npr1 promoter. Sequential ChIP assay further demonstrated that Ets-1/p300 activation of Npr1 gene transcription involves a direct physical association between these two factors. Overexpression of Ets-1 and p300 greatly stimulated NPRA mRNA and protein levels, and increased intracellular accumulation of second messenger cGMP in both MMCs and RTASMCs. Ets-1 seems to be essential for normal development of mammalian kidneys and for maintenance of glomerular integrity ^{30, 31}. It is implicated that Ets-1 is critical in hematopoiesis and angiogenesis during the earliest stages of embryogenesis, and in later stages it is important in organ formation and tissue remodeling in kidneys, liver, and vasculature ^{32, 33}. In the nephritic kidney Ets-1 has been shown to decrease extracellular matrix deposition and type I collagen expression ²⁰. Similarly, endogenous p300 seems to be critical in the integration of signal transduction pathways in mesangial cells, and interference with p300 alters apoptotic signals ²⁵. Several lines of evidence suggest that p300 is critical for the development of the embryonic heart and plays a central role in quantitative control of physiologically adaptive cardiac hypertrophy that is dependent on its HAT activity ^{34, 35}. The present results indicate that Npr1 gene is under tight control of Ets-1 and p300 transcription factors. The overexpression of Ets-1 gene in human colon cancer cell lines has been shown to reduce the rate of anchorage-independent growth in a dose-dependent manner ³⁶. On the other hand, Ets-1 expression has been suggested to regulate endothelial cell proliferation during angiogenesis and is essential for normal coronary and myocardial development ^37–39. Based on those previous findings and the present results, it is possible that Ets-1 plays a dual role during pathological conditions and upregulates Npr1 signaling as a protective regulatory mechanism in renal and cardiovascular disease states.

Histone acetyltransferases, including p300, are able to acetylate several transcription factors including p53 and GATA 4 to enhance their transcriptional activity, and regulate gene expression patterns by affecting chromatin structure ⁴⁰. Our present results demonstrate that HAT activity of p300 is required for the cooperative interaction of Ets-1 and p300 is regulating Npr1 gene. Detection of acetylated Ets-1 in immunoprecipitated complex from p300-transfected cells showed that p300 hyperacetylates endogenously expressed Ets-1 protein. It has been shown that under in vivo conditions acetylated Ets-1 preferentially associates with p300 complexes ⁴¹. Our data demonstrate that adenovirus E1A disrupted the Ets-1/p300-dependent transcription, whereas mutant E1A lacking p300-binding domain showed no significant effect on Npr1 gene transcription. These results suggest that E1A inhibition is exclusively dependent on its binding to p300. Apart from its HAT activity, p300/CBP provides a link between specific transcription factors and general transcriptional machinery; thus, E1A may inhibit p300/CBP, and thereby Npr1 promoter either by inhibiting intrinsic HAT activity of p300/CBP or by diminishing the interaction between p300 and Ets-1. The present results are compatible with the findings that E1A interferes in the association of p300/CBP and inhibits acetylation of transcription factors p53 and GATA-4 ^{42, 43}.

Our previous studies have shown that Ang II represses whereas Ets-1 stimulates Npr1 promoter activity ^{18, 19, 44}. It has also been suggested that Ang II increases the expression of Ets-1 in mesangial cells as well as in vascular smooth muscle cells, thereby Ets-1 provokes gene activation in response to vascular inflammation ^{45, 46}. Furthermore, Ets-1 induces the expression of caspase-1, which plays a prominent role in the apoptotic induction of inflammatory response cells ^{47, 48}. Since Ets-1 is stimulated by pro-inflammatory mediators and it also enhances Npr1 gene transcription, it is implicated that Ets-1 may have a dual function involving p300 in cardiovascular disease states. On one hand, it plays a role in inflammatory responses downstream of Ang II signaling pathway, and on the other hand in cooperative interaction with p300, it stimulates Npr1 gene transcription and expression, which seem to inhibit pro-inflammatory responses in hypertension and cardiovascular disease states.

Perspectives

The present findings provide direct evidence that Ets-1 and p300 are essential for Npr1 gene transcription and expression. The functional and physical interaction between Ets-1 and p300 is necessary for their action in regulating the optimum level of Npr1 gene transcription. Further understanding of the regulation of DNA binding ability and turnover of these transcription factors in regulation of NPRA expression would provide the framework to develop strategies to regulate the expression levels of NPRA. The results of this present study will greatly enhance our understanding of the pathways involved in the transcriptional regulation of Npr1 gene, an important locus in the control of hypertension and cardiovascular events.