US EPA

2264290 

Journal Article 

Abstract 

Nitric oxide stimulates activation of focal adhesion kinase and lung fibroblast migration 

Guzman, SL; Brown, R; Baron, RM; Schissel, S 

2010 

Yes 

American Journal of Respiratory and Critical Care Medicine
ISSN: 1073-449X
EISSN: 1535-4970 

181 

A3567 

English 

10.1164/ajrccm-conference.2010.181.1_MeetingAbstracts.A3567 

WOS:000208771002677 

is part of a larger document 3452678 Proceedings of the American Thoracic Society 2010 International Conference, May 14-19, 2010, New Orleans

RATIONALE: Fibroblast migration and proliferation are key events in the pathogenesis of idiopathic pulmonary fibrosis. Activation of focal adhesion kinase (FAK) by phosphorylation leads to increased cell migration and induces pro-proliferative cell signaling via Src kinase. Nitric oxide (NO) and its metabolites are elevated in pulmonary fibrosis and regulate FAK in endothelial cells. Here we investigate the effects of controlled NO expression and NO treatment on FAK signaling in lung fibroblasts and its effects on fibroblast migration.

METHODS: Cultured NIH 3T3 fibroblasts over-expressing inducible nitric oxide synthase (NOS2) or cells treated with the NO-donor DETA NONOate were analyzed for activation and phosphorylation of FAK and Src using immunoblotting. Fibroblast migration was assayed by measuring the closure of wounds induced in confluent MRC-5 human lung fibroblast cultures in the absence or presence of DETA NONOate.

RESULTS: NOS2 over-expression resulted in increased phosphorylation and activation of both FAK and Src by 24h and reached a maximum effect by 48h; total levels of FAK and Src were not altered by NOS2 over-expression. Similarly, fibroblasts treated with a low dose (10 micromolar) of the NO-donor NONOate had increased levels of phosphorylated FAK. In contrast fibroblast treatment with high doses (>100 micromolar) of NONOate had decreased levels of phosphorylated FAK. To test the functional consequences of our findings, we measured migration of lung fibroblasts in the presence of low- or high dose NONOate. Fibroblasts treated with 10 micromolar (low dose) NONOate migrated markedly faster by 24 hours with an 82.1% wound closure compared to 52% in control cells, and 45% in cells treated with 100 micromolar (high dose) NONOate.

CONCLUSIONS: NOS2-derived NO and low-dose exogenous NO induces FAK phosphorylation and activation, as well as downstream Src signaling in lung fibroblasts. Moreover, low-dose exogenous NO stimulates lung fibroblast migration. Manipulation of lung NO levels by exogenous treatment or controlled NOS2 expression may alter the pro-fibrotic actions of lung fibroblasts and be a target of future treatments for pulmonary fibrosis. 

American Thoracic Society 2010 International Conference 

New Orleans, LA 

May 14-19, 2010