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. Author manuscript; available in PMC: 2009 Sep 14.
Published in final edited form as: Peptides. 2006 Dec 20;28(4):893–899. doi: 10.1016/j.peptides.2006.12.009

ENHANCED ACTIVATION OF PRO-INFLAMMATORY CYTOKINES IN MICE LACKING NATRIURETIC PEPTIDE RECEPTOR-A

Elangovan Vellaichamy 1, Kiran Kaur 1, Kailash N Pandey 1
PMCID: PMC2743377  NIHMSID: NIHMS20455  PMID: 17267074

Abstract

Natriuretic peptide receptor-A (NPRA) is the principal receptor for the cardiac hormones ANP and BNP. Mice lacking NPRA develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for hypertrophic growth in the absence of NPRA signaling are not yet known. In the present study, we determined whether deficiency of NPRA/cGMP signaling alters the cardiac pro-inflammatory cytokines gene expression in Npr1 (coding for NPRA) gene-knockout (Npr1−/−) mice exhibiting cardiac hypertrophy and fibrosis as compared with control wild-type (Npr1+/+ ) mice. A significant up-regulation of cytokine genes such as TNF-α (5- fold), IL-6 (3-fold) and TGF-β1 (4-fold) were observed in mutant mice hearts lacking NPRA as compared with the age-matched wild-type mice. In parallel, NF-κB binding activity was almost 5-fold greater in the nuclear extract of Npr1−/− mutant mice hearts as compared with wild-type Npr1+/+ mice hearts. Guanylyl cyclase (GC) activity and cGMP levels were drastically reduced by 10-fold and 6-fold, respectively, in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates inflammatory cytokines, probably via NF-κB mediated signaling pathway, and is associated with hypertrophic growth of null mutant mice hearts.

Keywords: Natriuretic peptide receptor-A, GC activity, cGMP signaling, and cytokines

1. Introduction

Recent studies suggest that pro-inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-β), and interleukin-6 (IL-6) are activated within the myocardium during the experimental load-induced stress (2, 23). Additionally, transforming growth factor-β1 (TGF-β1), TNF-α, and IL-6 have been shown to stimulate myocyte growth in vitro culture conditions (7, 27). The aforementioned cytokines play a dual role, activating apoptosis in myocytes (13), while also functioning in a cytoprotective manner (15). Furthermore, inappropriate activation of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) have been recognized as an important mediator in the development of endothelial dysfunction, cardiac hypertrophy, and heart failure in experimental animal models and in humans (9, 17, 28, 30). Atrial natriuretic peptide (ANP) has been shown to inhibit the TNF-α induced adhesion molecule expression in endothelial cells (32). ANP has also been shown to inhibit TNF-α production in interferon-gamma-activated macrophages (29).

Accumulating evidences have suggested that the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) signaling pathway is important not only in maintaining blood pressure homeostasis but is also locally involved in antagonizing cardiac growth stimulated by hypertrophic stimuli (3, 4, 6, 24, 25). In particular, mice carrying targeted-disruption of Npr1 gene (encoding for NPRA) exhibit hypertension, marked cardiac hypertrophy, and congestive heart failure with sudden death after six months of age (12, 21). Conversely, over-expression of NPRA in cardiomyocytes inhibits the hypertrophic effects of isoproterenol and aortic constriction on mouse hearts (33), and rescues the cardiac hypertrophic phenotype (12). The ANP/NPRA system has been implicated as a negative regulator of inflammation and hypertorophic growth (12, 21, 32). However, in vivo studies have not been carried out to examine the role of NPRA signaling in regulating the expression of inflammatory cytokines. To our knowledge, this is the first report demonstrating that permanent ablation of NPRA signaling activates the expression of pro-inflammatory cytokines, which plays a critical role in the development of cardiac hypertrophy and congestive heart failure.

2. Materials and Methods

2.1 Materials

Trizol reagent was obtained from Life Technologies/Invitrogen (Carlsbad, CA). cGMP assay kit was obtained from Assay Designs, Inc (Ann Arbor, MI). Custom made cytokine multi-probe set containing lymphotoxin-β (LT-β), IL-6, TNF-α, transforming growth factor-β1 (TGF-β1), interferon -γ (IFN-γ), macrophage inhibitory factor (MIF), ribosomal RNA (L-32) and glyceraldehyde-3-phospahate dehydrogenase (GAPDH) were from BD Biosciences (San Diego, CA). Antibodies were obtained from Santa Cruz Biotechnology (San Diego, CA). T4 polynucleotide kinase, protein A-agarose, NAP-5 column, and [γ-32P] ATP (3000 Ci/mmol) and [α-32P] UTP (3000 Ci/mmol) were purchased from Amersham Biosciences (Piscataway, NJ).

2.2 Generation of Npr1 gene-knockout mice

Npr1 gene-knockout mice were generated by homologous recombination in embryonic stem cells as previously described (21). Animals were bred and maintained at the animal facility of Tulane University Health Sciences Center and handled under protocols approved by the Institutional Animal Care and Use Committee. The mice were housed under 12 h light/dark cycles at 25° C and fed regular chow (Purina Laboratory) and tap water ad libitum. Npr1 genotypes used in the present studies were littermate progenies of C57/Bl6 genetic background and have been designated as Npr1 gene-disrupted mutant allele (Npr1−/−) and wild-type allele (Npr1+/+). Experiments were performed using male adult (16 weeks) Npr1+/+ (wild-type) and Npr1−/− homozygous null mutant mice.

2.2 Northern blot analyses of hypertrophy marker genes

Total RNA was isolated from the left ventricular heart tissues from Npr1+/+ and Npr1−/− mice, using TRIzol reagent according to the manufacturer’s protocol (Invitrogen/Life Technologies). To remove genomic DNA contamination, RNA samples were treated with RNase free DNase I (1 unit/μg RNA) at 37° C for 30 min. The RNA integrity was confirmed by visualization of distinct 28 S and 18 S bands after electrophoresis on 1.5 % agarose gel. Total RNA (10 μg) was fractionated on a 1 % formaldehyde-agarose gel and transferred to Hybond nylon membrane (Amersham Biosciences) by capillary action in 10 x standard saline citrate (SSC). Blots were pre-hybridized in a hybridization solution containing 7 % SDS, 0.5 M NaHPO4 (pH 7.2) and 250 μg/ml salmon sperm DNA, for 5 h at 65° C, and hybridized with [γ-32P] ATP-labeled oligonucleotide probes for 16 h at 65° C. Blots were washed three times in 2 × SSC/0.2 % SDS at room temperature for 30 min and then in 0.5 × SSC/0.2 % SDS at 65° C for 30 min before exposure to X-ray film. The sequence of oligonucleotide probes were as follows: β-myosin heavy chain (β-MHC): 5′-GAG GGC TTC ACG GGC ACC CTT AGA GCT GGG AGC ACA AGA TCT ACT CCT CAT TCA TTC AGG CC-3′, Sarco/endoplasmic reticulum Ca2+-ATPase-2a (SERCA-2a): 5′-TCA GTC ATG CAG AGG CTG GTA GAT GTG TTG CTA ACA ACG CAC ATG CAC GCA CCC GAA CA-3′ and GADPH: 5′-GAG TGG CAG TGA TGG CAT GGA CTG TAG TCA TGT AGC CCT TCC ACG ATG CGA AAG AGT TGT -3′ The intensity of the bands were quantified using image density analysis software (Alpha Innotech, San Leandro, CA). The expression results of β-MHC, and SERCA-2a were normalized with GADPH.

2.3 RNase protection assay of cytokines gene expression

RNase protection assay (RPA) was carried out using multi-probe templates consisting of LT-β, TNF-α, IL-6, TGF-β 1, IFN-γ, MIF and the control genes L32, and GADPH. The multiple template set was labeled with [α-32P]-UTP using a T7 RNA polymerase as described by the manufacturer’s protocol (BD PharMingen, San Diego, CA). Labeled probes (3 × 105 cpm) were allowed to hybridize with 20 μg of total RNA for 16 h at 56° C. The hybridized mRNA probes were treated with RNase A, and extracted with phenol-chloroform. Protected hybrids were resolved on a 5% denaturing polyacrylamide gel and exposed to autoradiographic film overnight at −80° C. Densitometry was performed using Alpha Innotech Imaging System.

2.4 Preparation of cytosolic and nuclear extracts

Nuclear and cytosolic proteins were extracted by the method of Dignam et al., (5) from the left ventricular tissues of Npr1+/+ and Npr1−/−mice, immediately after the animals were sacrificed. Tissues were homogenized in an ice-cold 10 mM Tris-HCl buffer (pH 8.0) containing; 0.32 M sucrose, 3 mM CaCl2, 2 mM MgOAc, 0.1 mM EDTA, 0.5% Nonidet P-40 (NP- 40), 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 4.0 μg/ml each of leupeptin, aprotinin, and pepstatin. The homogenate was centrifuged at 800 × g, and the supernatant was separated and saved as a cytosolic fraction. The nuclear fraction was re-suspended in a low- salt buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 25 % glycerol, 0.5 mM DTT, and 0.5 mM PMSF), incubated on ice for 5 min, and then mixed with equal volume of high-salt buffer containing; 20 mM HEPES, 1.5 mM MgCl2, 800 mM KCl, 0.2 mM EDTA, 25% glycerol, 1% NP-40, 0.5 mM DTT, 0.5 mM PMSF, and 4.0 μg/ml each of leupeptin, aprotinin and pepstatin. The mixture was incubated on ice for 30 min and centrifuged at 14,000 × g for 15 min. The supernatant was separated and stored at −80° C until used.

2.5 Western blot analysis cytokines

Left ventricular tissue samples were homogenized in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1 mM EDTA, 1 % (v/v) NP-40, 0.5 % (w/v) deoxycholate, 50 mM NaF, 50 mM sodium vanadate, 0.5 mM PMSF, and 2 μg/ml each of aprotinin, pepstatin, and leupeptin. Tissue homogenate (20 μg proteins) was mixed with sample loading buffer and separated under reducing condition using 10 % SDS-PAGE. The separated proteins were electrotransferred onto a PVDF membrane. The membrane was blocked with 1x Tris-buffered saline-Tween 20 (TBST; 25 mM Tris, 500 mM NaCl and 0.05% Tween 20, pH 7.5) containing 5% fat-free milk, and incubated overnight in TBST containing 3% fat-free milk at 4° C with specific primary antibodies to TNF-α, IL-6, TGF-β1, and α-tubulin. The membrane was then treated with corresponding secondary anti-rabbit or anti-mouse HRP-conjugated antibodies (1:20,000 dilution). Protein bands were visualized using the ChemGlow Western blot detection reagent kit (Alpha Innotech).

2.6 Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) was performed as described previously (31). Double-stranded oligonucleotides containing the consensus-binding site for NF-κB were end-labeled using [γ-32p]ATP and T4 polynucleotide kinase. Binding reaction was initiated by incubating 5 μg of nuclear proteins in 5 μl of binding buffer (50 mM Tris-HCl, pH 8.0, 750 mM KCl, 2.5 mM EDTA, 0.5 % Triton-X 100, 62.5 % glycerol, and 1 mM DTT) containing 2 μg of Poly dI-dC and radiolabeled oligonucleotide (50,000 cpm) at 22° C for 20 min. Cold competitor assays were performed by adding 100-fold excess molar concentrations of unlabeled NF-κB. The DNA-protein complex was resolved from the free-labeled DNA using 4% native polyacrylamide gel electrophoresis and autoradiography.

2.7 Ventricular guanylyl cyclase (GC) activity and cGMP assay

Ventricular plasma membranes were prepared as described previously (10). The GC activity was assayed as described by Leitman et al (16) with slight modification (10). An aliquot of plasma membrane (25 μg) from the mutant and wild-type mice were added to a 100 μl reaction mixture containing, Tris-HCl buffer (50 mM, pH 7.6), MnCl2 (4 mM), GTP (0.2 mM), BSA (1 mg/ml), creatine phosphate (7.5 mM), IBMX (2 mM), and creatine phosphokinase (3 units). The membranes were incubated at 37° C for indicated time. The reaction was stopped with the addition of 900 μl of sodium acetate buffer (50 mM, pH 6.2) and subsequently by boiling the samples for 3 min. The amount of cGMP generated was determined by direct cGMP enzyme immunoassay kit (Assay Designs, Inc., Ann Arbor, MI). To determine the total cGMP content, the frozen ventricular tissue samples were homogenized in 10 volumes of 0.1 M HCl containing 1% Triton X-100. Homogenate was heated at 95° C for 5 min, centrifuged at 600 × g for 20 min at 22° C, supernatants collected, and stored at −80° C until used for cGMP assay. Ventricular tissue cGMP levels were analyzed using a direct cGMP enzyme immunoassay kit.

2.8 Statistical analysis

Statistical analysis was performed using GraphPad Software, Inc. (San Diego, CA). The results are presented as mean ± SEM. Differences between groups were determined by one-way analysis of variance (ANOVA) with student’s t test. The probability value of p<0.05 was considered significant.

3. Results

The results presented in Fig. 1 show that mutant mice Npr1−/− displayed an increased mRNA expression of various cytokines; including: TNF-α (5- fold), IL-6 (3-fold), and TGF-β1 (4-fold) as compared with wild-type Npr1+/+ mice (Fig. 1A, and B). LT-β and IFN-γ genes expression was found to be slightly increased in the mutant mice hearts, while MIF gene expression was not altered as compared with the wild-type mice. As depicted in Fig. 2A, there was a significant increase in the protein levels of TNF-α (4-fold), IL-6 (3-fold) and TGF-β1 (3-fold) was noted in the null mutant mice homogenate as compared with the age-matched wild-type type control mice.

Figure 1. Expression profiles of ventricular cytokine genes expression in adult Npr1+/+ and Npr1−/− mice hearts.

Figure 1

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A) Representative autoradiograph showing the cytokines gene expression in adult Npr1+/+ and Npr1−/− mice hearts. Labeled probe (3× 105 cpm) was allowed to hybridize with 20 μg of RNA at 56°C for 16 h. Protected hybrids were resolved on a 5% denaturing polyacrylamide gel and exposed to radiographic film overnight at −80° C. B) Relative expression of cytokine genes normalized to the expression of L32 in adult Npr1+/+ and Npr1−/− mice hearts. Values are expressed as means ±SEM; N=8 mice/group; *p<0.05, **p<0.01 and ***p<0.001; Npr1+/+ vs Npr1−/−.

Figure 2. Analyses of ventricular TNF- α, IL-6, and TGF- β protein levels in adult Npr1+/+ and Npr1−/− mice hearts.

Figure 2

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A) Representative Western blot of cytokines (TNF- α, IL-6 and TGF- β) in Npr1+/+ and Npr1−/− mice hearts. B) Densitometric analysis of protein bands normalized with α-tubulin. Values are expressed as means ±SEM; N=8 mice /group; ***p<0.001; Npr1+/+ vs Npr1−/−.

As shown in Table 1, ventricular GC activity and cGMP concentrations were drastically reduced by 10-and 5-fold, respectively, in the Npr1−/− mutant mice, as compared with Npr1+/+ wild-type mice. Data presented in Fig. 3A, show that the expression of hypertrophic marker gene such as β-MHC was increased by more than 4-fold in Npr1 null mutant mice hearts as compared with wild-type control mice hearts. In contrast, SERCA-2a mRNA expression was significantly reduced by almost 3-fold in mutant mice Npr1−/− mice as compared with Npr1+/+ wild-type controls (Fig. 3B). NF-κB binding activity was analyzed in the nuclear extract isolated from adult Npr1−/− mutant mice hearts using electrophoretic mobility shift assay (EMSA) (Fig. 4A, and B). EMSA analysis demonstrated an increased NF-κB binding activity (5-fold) in the mutant mice hearts as compared with wild-type mice, confirming the increased activation of NF-κB signaling.

Table 1.

Guanylyl cyclase activity and total cGMP content in the left ventricular tissue of Npr1+/+ wild-type and Npr1−/− homozygous null mutant mice

Genotype Guanylyl cyclase activity
(pmoles cGMP/mg protein/30 min)		 Intracellular cGMP
(pmoles/mg protein)
Npr1+/+ 68 ± 9 28 ± 4
Npr1−/− 6 ± 1.1*** 5 ± 0.8***
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Guanylyl cyclase (GC) activity analysis was performed as described under the Materials and methods section. cGMP levels in wild-type and mutant Npr1 mice hearts were analyzed using direct cGMP enzyme immunoassay kit. Values are expressed as means ±SEM; N=8 mice/group;

***

p<0.001; Npr1+/+ vs Npr1−/−.

Figure 3. Hypertrophy marker gene expression in adult Npr1+/+ and Npr1−/− mice hearts.

Figure 3

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A) Representative Northern blots showing the mRNA expression of β-MHC and SERCA-2a in Npr1−/− and Npr1+/+ mice. B) Densitometric analysis of mRNA transcripts normalized with GADPH mRNA expression. Values are expressed as means ±SEM; N=8 mice/group; ***p<0.001; Npr1+/+ vs Npr1−/−.

Figure 4. Nuclear NF-κB binding activity in adult Npr1+/+ and Npr1−/− mice hearts.

Figure 4

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A) EMSA analysis of NF-κB binding activity in the nuclear extracts of Npr1+/+ and Npr1−/− mice hearts. The DNA-protein complex was resolved from the free-labeled DNA by electrophoresis using 4% (w/v) native polyacrylamide gel electrophoresis and autoradiography. B) Densitometric analysis of NF-κB protein bands. Values are expressed as means ±SEM; N=8 mice/group; ***p<0.001; Npr1+/+ vs Npr1−/−.

4. Discussion

The results obtained from this study demonstrate that left ventricular pro-inflammatory cytokines gene expression was increased in mice null mutant lacking NPRA. Among the genes analyzed, TNF-α, IL-6, and TGF-β1 were found to be highly up-regulated by almost 3- to 5-fold in the mutant mice hearts. Myocardial cytokine genes such as TNF-α, IL-1β and IL-6 expression were found to be higher in patients with compensated heart failure condition, and it has been suggested that increased cytokine gene expression has adaptive role in the left ventricular remodeling process (22). Experimental induction of hemodynamic overload in the adult mammalian heart has been shown to provoke a transient increase in pro-inflammatory cytokines (2). Furthermore, cardiac-specific over expression of IL-6 and TNF-α in mice hearts has been shown to develop cardiac hypertrophy and ventricular dysfunction similar to that seen in human disease conditions, suggesting that cytokines are critically involved in the cardiac remodeling process (9, 30).

In the present study, Npr1 null mutant mice showed a drastic reduction in the ventricular GC activity (10-fold) as well as cGMP content (6-fold) as compared with wild-type mice. Anti-proliferative and anti-hypertrophic effects of the ANP/NPRA system have been reported to be mediated through the generation of cGMP (3, 12, 21, 25, 33). Furthermore, ANP/NPRA/cGMP signaling have been shown to oppose the hypertrophic agonist such as Ang II and ET-1 mediated biological effects (14, 29). Hypertrophic agonist such as angiotensin II and norepinephrine, have been shown to mediate hypertrophic responses in neonatal and adult myoctes by activating IL-6 and TGF-β1 genes (7, 27). Additionally, recent evidences suggest that ANP/NPRA system has anti-inflammatory activity, and reported to inhibit the bacterial toxin (LPS) and IFN- gamma-induced pro-inflammatory cytokines expression in macrophages by suppressing signal transduction pathway leading to NF-κB activation (29, 32). It has been suggested that ANP plays important role in regulating inflammatory signaling in endothelial cells, leading to reduced TNF-α–induced expression of adhesion molecules (11). The deficiency of ANP/NPRA/cGMP signaling may leads to the activation of inflammatory cytokines in hearts that may provokes the hypertrophic remodeling process in mice lacking NPRA.

SERCA-2a has been implicated as a marker for progressive hypertrophic growth and heart failure (8). It has been suggested that a decreased level of SERCA-2a is associated with impaired uptake of Ca2+ into sarcoplasmic reticulum and contributes to slowing of relaxation in the failing human hearts (20). Thus, an altered level of SERCA-2a seems to be associated with contractile dysfunction of the heart. In the present study, SERCA-2a gene expression was found to be significantly decreased in Npr1 null mutant mice hearts as compared with the wild-type counterparts, suggesting a defect in Ca2+ handling and decreased cardiac performance in the mutant mice hearts.

In the present study, the increased NF-κB binding activity was positively correlated with the observed increased expression of cytokine genes in the Npr1 null mutant mice hearts. Recent studies have suggested that NF-κB signaling seems to be involved in a number of cytokines gene expressions, and development of cardiac hypertrophy in vivo and in vitro conditions (18, 26). The exact intracellular mechanisms underlying the activation of cytokines in mice lacking NPRA are not well understood. However, it can be suggested that NF-κB-mediated signaling is involved in the synthesis of pro-inflammatory cytokines in mice lacking NPRA. ANP/NPRA/cGMP signaling attenuates the production of inflammatory mediators such as TNF-α by regulating the NF-κB pathway (29, 32). Furthermore, ANP and cGMP analogues have been reported to suppress NF-κB activation by inhibiting the phosphorylation of IκB-α and its subsequent degradation in cultured cells (11). ANP has also been reported to inhibits the activation of Akt and MAPK/ERK signaling pathways (1, 25). Interestingly, it has been shown that AkT and MAPK/ERK signaling directly phosphorylates p65 protein in a non-classical manner (without phosphorylation and degradation of IkB-α) and thereby increases NF-κB transcriptional activity (19), implicating the regulatory role of ANP/NPRA signaling in NF-κB transactivation. Thus, it is conceivable that deficiency of ANP/NPRA signaling leads to an unbalanced activation of NF-κB and in turn activates various cytokines and promotes hypertrophic process in mice lacking NPRA.

In summary, the present results demonstrate that disruption of NPRA/cGMP signaling results in an increased activation of cytokines in hypertrophied hearts of Npr1 homozygous null mutant mice. The data provide strong evidence that reduced NPRA/cGMP signaling in mice lacking NPRA increases the activation of pro-inflammatory mediators, which are implicated to play a critical role in promoting cardiac hypertrophy and congestive heart failure. The findings provide novel mechanisms to demonstrate the central role of NPRA/GC/cGMP signaling in the regulation of inflammatory cytokines and development of cardiac hypertrophy.

Acknowledgments

Authors wish to thank L. Charlene Binford and Gevoni Bolden for technical assistance and Kamala Pandey for her assistance during the preparation of this manuscript. We are indebted to Dr. Oliver Smithies for providing initial breeding pairs of Npr1 gene-targeted mice colonies. Our special thanks are due to Dr. Bharat B. Aggarwal, Department of Experimental Therapeutics and Cytokine Research Laboratory at MD Anderson Cancer Center and Dr. Susan L. Hamilton, Department of Molecular Physiology and Biophysics at Baylor College of Medicine for providing their facilities during our displacement period due to Hurricane Katrina. This research was supported by the National Institutes of Health Grant HL-62147 and Louisiana Board of Regents Health Excellence Fund.

Footnotes

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