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Abstract
Background Transforming growth factor (TGF)-β is a key mediator of epithelial-mesenchymal transition (EMT), a pathogenetic mechanism in idiopathic pulmonary fibrosis (IPF). Nitric oxide (NO) may potentiate TGF-β/Smad-signalling and increased levels of NO and one isoform of its generating enzyme, inducible nitric oxide synthetase (iNOS), are observed in experimental models of IPF. Asymmetric dimethylarginine (ADMA) competitively inhibits iNOS, and is hydrolysed by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2. Our prior data suggests that regulation of NO production via inhibitory methylarginines may play a role in IPF. The role of NOS inhibition on the NO-ADMA-DDAH axis in TGF-β mediated EMT is unknown.
Methods Human type II alveolar epithelial cells (A549) were serum starved for 24 hrs before stimulation with 5 ng/ml TGF-β (control); co-treatment with TGF-β 5 ng/ml and 100 uM/ml 1400 W (highly selective iNOS inhibitor); and 1400 W treatment alone. A separate experiment was performed with 100 uM/ml exogenous ADMA (pan-NOS inhibitor). A profile indicating transformation from an epithelial to mesenchymal phenotype (E-cadherin, a-SMA), and expression and protein levels of DDAH isoforms and NOS enzymes was assessed by qRT-PCR and Western blotting.
Results TGF-β mediated EMT was confirmed by significant changes in protein levels in both 1400 W and ADMA experiments respectively: decreased E-cadherin (p = 0.0002, p = 0.0005) and increased a-SMA (p = 0.009, p = 0.003). Protein levels of DDAH2 (p = 0.01, p = 0.0024) and iNOS (p = 0.01, p = 0.0083) were increased. In the presence of either TGF-β and 1400 W or TGF-β and ADMA co-treatments; the mesenchymal pattern of changes in E-cadherin and α-SMA, and the elevation in DDAH2 and iNOS levels, were not attenuated. Treatment with either 1400 W or ADMA alone resulted in significantly elevated E-cadherin levels compared to TGF-β control or co-treatments (Figure 1). Pan-NOS inhibition with ADMA alone resulted in a several fold increase in E-cadherin levels compared to no treatment (p = 0.0005) (Figure 1b).
Fold change in band intensity of E-cadherin levels corrected for tubulin on Wastern blotting: 1a) Treatment with 140W, 1b) Treatment with ADWA (p values = 0.0002 and 0.0005 respectively on one-way ANOVA)
Conclusion TGF-β mediated transition towards a mesenchymal phenotype is not attenuated by NOS inhibition. However, our results suggest that regulation of NO production by ADMA promotes E-cadherin expression via an iNOS independent mechanism and may play a role in the maintenance of an epithelial phenotype in IPF.