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. 2007 Apr;28(4):893-9.
doi: 10.1016/j.peptides.2006.12.009. Epub 2006 Dec 20.

Enhanced activation of pro-inflammatory cytokines in mice lacking natriuretic peptide receptor-A

Elangovan Vellaichamy  1 , Kiran Kaur, Kailash N Pandey
Affiliations

Enhanced activation of pro-inflammatory cytokines in mice lacking natriuretic peptide receptor-A

Elangovan Vellaichamy et al. Peptides. 2007 Apr.

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-alpha (five-fold), IL-6 (three-fold) and TGF-beta1 (four-fold) were observed in mutant mice hearts lacking NPRA as compared with the age-matched wild-type mice. In parallel, NF-kappaB binding activity was almost five-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- and 5-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-kappaB mediated signaling pathway, and is associated with hypertrophic growth of null mutant mice hearts.

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Figures

Figure 1
Figure 1. Expression profiles of ventricular cytokine genes expression in adult Npr1+/+ and Npr1−/− mice hearts
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
Figure 2. Analyses of ventricular TNF- α, IL-6, and TGF- β protein levels in adult Npr1+/+ and Npr1−/− mice hearts
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−/−.
Figure 3
Figure 3. Hypertrophy marker gene expression in adult Npr1+/+ and Npr1−/− mice hearts
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
Figure 4. Nuclear NF-κB binding activity in adult Npr1+/+ and Npr1−/− mice hearts
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−/−.
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