Submitted on January 23, 2008
Revised on April 13, 2009
Accepted on April 26, 2009
ROLE OF cGMP-DEPENDENT PROTEIN KINASE (PKG) IN REGULATION OF PULMONARY VASCULAR SMOOTH MUSCLE CELL ADHESION AND MIGRATION: EFFECT OF HYPOXIA
Sewite Negash1*, Sudha Rani Narasimhan2, Weilin Zhou2, Jie Liu3, Felice Wei4, Jia Tian3, and J Usha Raj5
1 Harbor-UCLA Medical Center
2 Harbor-UCLA Medical Center, Los Angeles Biomedical Research Institute at Harbor-UCLA
3 Division of Neonatology, Harbor-UCLA Medical Center, Geffen School of Medicine at University of California, and Los Angeles Biomedical Research Institute, Los Angeles, California
4 UCLA
5 University of Illinois at Chicago
* To whom correspondence should be addressed. E-mail: SNegash{at}labiomed.org.
Exposure to prolonged hypoxia can result in pulmonary vascular remodeling and pulmonary hypertension. Hypoxia induces pulmonary vascular smooth muscle cell (PVSMC) proliferation and vascular remodeling by affecting cell adhesion, migration and secretion of extracellular matrix proteins. We have previously shown that acute hypoxia decreases cGMP-dependent protein kinase (PKG) activity in PVSMC and that PKG plays a role in maintaining the differentiated contractile phenotype in normoxia. In this study, we investigated the effect of hypoxia on PVSMC adhesion and migration and the role of PKG in these functions. Ovine fetal pulmonary artery SMC were incubated in normoxia (pO2 ~ 100 torr), hypoxia (pO2 ~ 30-40 torr), or treated with a PKG inhibitor DT-3 for 24 h in normoxia. To further study the role of PKG in the modulation of adhesion and migration, PVSMCs were transiently transfected with a full-length PKG1
(PKG-GFP) or a dominant negative construct (G1R-GFP). Cell adhesion to extracellular matrix proteins was determined, and integrin-mediated adhesion assessed using /
integrin-mediated cell adhesion array. Exposure to hypoxia (24h) and pharmacological inhibition of PKG1 by DT-3 significantly promoted adhesion mediated by 4, 1 and 51 integrins to fibronectin, laminin and tenacin and also resulted in increased cell migration. Likewise, inhibition of PKG by expression of a dominant negative PKG1 construct increased cell adhesion and migration, comparable to that induced by hypoxia. Dynamic actin reorganization associated with integrin-mediated cell adhesion is partly regulated by actin-binding protein cofilin, the (Ser3) phosphorylation of which inhibits its actin-severing activity. We found that increased PKG expression and activity is associated with decreased cofilin (Ser3) phosphorylation, implying a role for PKG in the modulation of cofilin activity and actin dynamics. Taken together, these findings identify cGMP/PKG1 signaling as central to the functional differences between PVSMC exposed to normoxia versus hypoxia.
