Effect of nitric oxide deficiency on the pulmonary PTHrP system

Abstract

Nitric oxide (NO) deficiency is common in pulmonary diseases, but its effect on pulmonary remodelling is still controversial. As pulmonary parathyroid hormone-related protein (PTHrP) expression is a key regulator of pulmonary fibrosis and development, the effect of chronic NO deficiency on the pulmonary PTHrP system and its relationship with oxidative stress was addressed. NO bioavailability in adult rats was reduced by systemic administration of L-NAME via tap water. To clarify the role of NO synthase (NOS)-3-derived NO on pulmonary expression of PTHrP, NOS-3-deficient mice were used. Captopril and hydralazine were used to reduce the hypertensive effect of L-NAME treatment and to interfere with the pulmonary renin-angiotensin system (RAS). Quantitative RT-PCR and immunoblot techniques were used to characterize the expression of key proteins involved in pulmonary remodelling. L-NAME administration significantly reduced pulmonary NO concentration and caused oxidative stress as characterized by increased pulmonary nitrite concentration and increased expression of NOX2, p47phox and p67phox. Furthermore, L-NAME induced the pulmonary expression of PTHrP and of its corresponding receptor, PTH-1R. Expression of PTHrP and PTH-1R correlated with the expression of two well-established PTHrP downstream targets, ADRP and PPARγ, suggesting an activation of the pulmonary PTHrP system by NO deficiency. Captopril reduced the expression of PTHrP, profibrotic markers and ornithine decarboxylase, but neither that of PTH-1R nor that of ADRP and PPARγ. All transcriptional changes were confirmed in NOS-3-deficient mice. In conclusion, NOS-3-derived NO suppresses pulmonary PTHrP and PTH-1R expression, thereby modifying pulmonary remodelling.

Keywords: ADRP; PPARγ; elastin; lung fibrosis.

Figure 1

Effect of L‐NAME administration on pulmonary NO levels and nitrotyrosine content. Data are mean ± S.D. from n = 10 samples.

Figure 2

Effect of L‐NAME administration on the pulmonary mRNA expression of components of the NADPH oxidase complex. Data points represent the expression of the individual samples (n = 6). The bar indicates the median. Exact P‐values are given.

Figure 3

Effect of L‐NAME administration on the pulmonary mRNA expression of PTHrP, PTH‐1 receptor, and PTHrP‐downstream targets ADRP and PPARγ, and of fibrotic markers. Data points represent the expression of the individual samples (n = 6). The bar indicates the median. Exact P values are given.

Figure 4

Effect of L‐NAME administration on the pulmonary protein expression of PTHrP, PTH‐1 receptor (PTH‐R), surfactant protein C (SP‐C), and α‐smooth muscle actin. Representative immunoblot is shown (A, D); Data are mean ± S.E.M. from n = 4–6 samples.

Figure 5

Effect of captopril and hydralazine on L‐NAME‐induced changes in the pulmonary mRNA expression of PTHrP, PTH‐1 receptor, and PTHrP downstream targets ADRP and PPARγ and that of fibrotic markers. Data are mean ± S.D. from n = 6 samples. Exact P values are given *, P < 0.05 vs. control.

Figure 6

Effect of eNOS deficiency on pulmonary expression of PTHrP (A), PTH‐1 receptor (B), ADRP (C), PPARγ (D), elastin (E); collagen‐1 (F), TGF‐β1 (G), and ODC (H) in wild‐type mice (+/+), heterozygous mice (±) and eNOS knockout mice (−/−). *, P < 0.05 vs. eNOS +/+ Data are mean ± S.D. from n = 9–23 mice.

Figure 7

Conclusive summary of the data obtained in this study. NO constitutively represses the pulmonary expression of PTHrP in alveolar type II cells (1) and PTH‐1 receptors (2) in lipofibroblasts, thereby controlling their expression of ADRP and PPARγ, two proteins required for proper formation of surfactant. RAS seems to modify pulmonary fibrosis independent of a potential role of PTHrP for the transition of alveolar lipofibroblasts to myofibroblasts.