Natriuretic peptide receptor-3 underpins the disparate regulation of endothelial and vascular smooth muscle cell proliferation by C-type natriuretic peptide

Abstract

Background and purpose: C-type natriuretic peptide (CNP) is an endothelium-derived vasorelaxant, exerting anti-atherogenic actions in the vasculature and salvaging the myocardium from ischaemic injury. The cytoprotective effects of CNP are mediated in part via the G(i) -coupled natriuretic peptide receptor (NPR)3. As GPCRs are well-known to control cell proliferation, we investigated if NPR3 activation underlies effects of CNP on endothelial and vascular smooth muscle cell mitogenesis.

Experimental approach: Proliferation of human umbilical vein endothelial cells (HUVEC), rat aortic smooth muscle cells (RAoSMC) and endothelial and vascular smooth muscle cells from NPR3 knockout (KO) mice was investigated in vitro.

Key results: CNP (1 pM-1 µM) facilitated HUVEC proliferation and inhibited RAoSMC growth concentration-dependently. The pro- and anti-mitogenic effects of CNP were blocked by the NPR3 antagonist M372049 (10 µM) and the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 (30 µM) and were absent in cells from NPR3 KO mice. Activation of ERK 1/2 by CNP was inhibited by Pertussis toxin (100 ng·mL⁻¹) and M372049 (10 µM). In HUVEC, ERK 1/2 activation enhanced expression of the cell cycle promoter, cyclin D1, whereas in RAoSMC, ERK 1/2 activation increased expression of the cell cycle inhibitors p21(waf1/cip1) and p27(kip1) .

Conclusions and implications: A facet of the vasoprotective profile of CNP is mediated via NPR3-dependent ERK 1/2 phosphorylation, resulting in augmented endothelial cell proliferation and inhibition of vascular smooth muscle growth. This pathway may offer an innovative approach to reversing the endothelial damage and vascular smooth muscle hyperplasia that characterize many vascular disorders.

Figure 1

The mitogenic effects of CNP are mediated via NPR3. BrdU incorporation in (A) RAoSMC and (B) HUVEC treated with increasing concentrations of CNP (1 pM–1 µM; 24 h), (C) RAoSMC treated with CNP (1 µM; 24 h) in the absence or presence of M372049 (10 µM) and (D) HUVEC treated with CNP (100 pM; 24 h) in the absence or presence of M372049 (10 µM). Data shown are means ± SEM expressed as a percentage of basal growth. *P < 0.05 versus basal, #P < 0.05 versus CNP alone. n≥ 12 from ≥4 separate experiments.

Figure 2

The mitogenic effects of CNP are absent in NPR3 KO cells. BrdU incorporation in WT and NPR3 KO mouse AoSMC and PMEC under basal conditions (A and B) or treated with CNP (1 µM AoSMC; 100 pM PMEC; 24 h; C and D). Data shown are means ± SEM expressed as a percentage of basal growth. *P < 0.05 versus basal or WT. n≥ 12 from ≥4 separate experiments.

Figure 3

NPR expression and cGMP formation in RAoSMC and HUVEC. RT-PCR analysis of NPR1 (labelled A), NPR2 (B) and NPR3 (C) mRNA expression in (A) RAoSMC and (B) HUVEC (gels representative of three separate experiments). Formation of cGMP in response to CNP in RAoSMC (1 µM; C) and HUVEC (100 pM; D) in the absence and presence of M372049 (10 µM). *P < 0.05 versus basal. n = 9 observations from three separate experiments.

Figure 4

ERK 1/2 phosphorylation underlies the effects of CNP on cell growth. BrdU incorporation (A and B) and levels of total and phospho-ERK 1/2 (C and D) in RAoSMC (A and C) and HUVEC (B and D) treated with CNP (RAoSMC 1 µM; HUVEC 100 pM; 24 h) in the absence and presence of PD98059 (30 µM). Total ERK 1/2 and phospho-ERK 1/2 were analysed by Western blot, and bands were quantified by densitometry. Data shown are means ± SEM expressed as a percentage of basal growth or ERK 1/2 phosphorylation. *P < 0.05 versus basal, #P < 0.05 versus CNP alone. n≥ 8 from ≥6 separate experiments.

Figure 5

The NPR3 antagonist M372049 and the G_i-protein inhibitor Pertussis toxin attenuate CNP-induced ERK 1/2 phosphorylation. Levels of total ERK 1/2 and phospho-ERK 1/2 in (A and C) RAoSMC and (B and D) HUVEC treated with CNP (RAoSMC 1 µM, 10 min; HUVEC 100 pM, 30 min) in the absence and presence of M372049 (10 µM) or Pertussis toxin (PTx; 100 ng·mL⁻¹). Total ERK 1/2 and phospho-ERK 1/2 were analysed by Western blot, and bands were quantified by densitometry. Data shown are means ± SEM expressed as a percentage of basal ERK 1/2 phosphorylation. *P < 0.05 versus basal; #P < 0.05 versus CNP alone. n = 3–4.

Figure 6

CNP disparately regulates cell cycle protein expression in RAoSMC and HUVEC. Expression of (A) p21^waf1/cip1 and (B) p27^kip1 in RAoSMC treated with CNP (1 µM; 24 h) and (C) cyclin D1 in HUVEC treated with CNP (100 pM; 24 h). p21^waf1/cip1, p27^kip1 and cyclin D1 were analysed by Western blot, and bands were quantified by densitometry. Data shown are means ± SEM expressed as a percentage of basal cell cycle protein expression. *P < 0.05 versus basal. n = 3–4.

Figure 7

The ERK 1/2 inhibitor PD98059 attenuates the effects of CNP on altered cell cycle protein expression in RAoSMC and HUVEC. Expression of (A) p21^waf1/cip1 and (B) p27^kip1 in RAoSMC treated with CNP (1 µM; 24 h) in the absence and presence of PD98059 (30 µM) and (C) cyclin D1 in HUVEC treated with CNP (100 pM; 6 h) in the absence and presence of PD98059 (30 µM). p21^waf1/cip1, p27^kip1 and cyclin D1 were analysed by Western blot, and bands were quantified by densitometry. Data shown are means ± SEM expressed as a percentage of basal cell cycle protein expression. *P < 0.05 versus basal, #P < 0.05 versus CNP alone. n = 3–5.