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1.  Regulation of Bcl-xL Expression in Lung Vascular Smooth Muscle 
Pulmonary hypertension is characterized by thickened pulmonary arterial walls due to increased number of pulmonary artery smooth muscle cells (PASMC). Apoptosis of PASMC may play an important role in regulating the PASMC number and may be useful for reducing pulmonary vascular thickening. The present study examined the regulation of an anti-apoptotic protein Bcl-xL. Bcl-xL expression was found to be increased in the pulmonary artery of chronic hypoxia–treated rats with pulmonary vascular remodeling. Adenovirus-mediated gene transfer of Bcl-xL indeed showed that this protein has anti-apoptotic activities in PASMC. Treatment of remodeled pulmonary artery with sodium nitroprusside (SNP) reduced Bcl-xL expression by targeting the bcl-xL promoter. The bcl-xL promoter contains two GATA elements, and SNP decreases the GATA-4 DNA-binding activity. Overexpression of GATA-4 attenuated the SNP-mediated suppression of Bcl-xL expression, providing direct evidence for the role of GATA-4 in Bcl-xL gene transcription. We established that SNP targets the 250 proximal region of the gata4 promoter and suppresses its gene transcription. Thus, inducers of pulmonary hypertension enhance anti-apoptotic Bcl-xL gene transcription, which can be suppressed by targeting gata4 gene transcription.
doi:10.1165/rcmb.2006-0359OC
PMCID: PMC1868666  PMID: 17272823
apoptosis; genes; pulmonary hypertension; smooth muscle
2.  Retinoic Acid Inhibits Airway Smooth Muscle Cell Migration 
Airway remodeling in chronic asthma is characterized by increased smooth muscle mass that is associated with the reduction of the bronchial lumen as well as airway hyperresponsiveness. The development of agents that inhibit smooth muscle growth is therefore of interest for therapy to prevent asthma-associated airway remodeling. All-trans retinoic acid (ATRA) suppresses growth of vascular smooth muscle cells (SMCs) from the systemic and pulmonary circulation. The present study investigated the effects of ATRA on human bronchial (airway) SMCs. Human bronchial SMCs were found to express mRNAs for retinoic acid receptor (RAR)-α, -β, -γ, and retinoid X receptor (RXR)-α, -β, but not RXR-γ. Although ATRA was not effective in inhibiting proliferation or in inducing apoptosis in airway SMCs, we found that ATRA (0.2–2 μM) inhibited the SMC migration in response to platelet-derived growth factor (PDGF), as determined in a modified Boyden chamber assay. Both RAR and RXR agonists also blocked PDGF-induced airway SMC migration. ATRA also inhibited PDGF-induced actin reorganization associated with migration. PDGF-induced actin reorganization and migration were blocked by inhibitors of phosphatidylinositol 3 kinase (PI3K) and Akt. However, migration was blocked by inhibitors of the MEK/ERK pathway, with no effect on cytoskeletal reorganization. ATRA suppressed PDGF-induced Akt activation without influencing ERK activation. RAR was found to form protein–protein interactions with the p85 PI3K subunit. These results suggest that retinoic acid inhibits airway SMC migration through the modulation of the PI3K/Akt pathway.
doi:10.1165/rcmb.2005-0306OC
PMCID: PMC1831536  PMID: 16456186
airway; migration; retinoic acid; signal transduction; smooth muscle
3.  Regulation of Bcl-xL expression in lung vascular smooth muscle 
Pulmonary hypertension is characterized by thickened pulmonary arterial walls due to increased number of pulmonary artery smooth muscle cells (PASMC). Apoptosis of PASMC may play important roles in regulating the PASMC number and may be useful for reducing pulmonary vascular thickening. The present study examined the regulation of an anti-apoptotic protein Bcl-xL. Bcl-xL expression was found to be increased in the pulmonary artery of chronic hypoxia treated rats with pulmonary vascular remodeling. Adenovirus-mediated gene transfer of Bcl-xL indeed showed that this protein has anti-apoptotic activities in PASMC. Treatment of remodeled pulmonary artery with sodium nitroprusside (SNP) reduced Bcl-xL expression by targeting the bcl-xL promoter. The bcl-xL promoter contains two GATA elements, and SNP decreases the GATA-4 DNA binding activity. Overexpression of GATA-4 attenuated the SNP-mediated suppression of Bcl-xL expression, providing direct evidence for the role of GATA-4 in Bcl-xL gene transcription. We identified that SNP targets the 250 proximal region of the gata4 promoter and suppresses its gene transcription. Thus, inducers of pulmonary hypertension enhance anti-apoptotic Bcl-xL gene transcription, which can be suppressed by targeting the gata4 gene transcription.
doi:10.1165/rcmb.2006-0359OC
PMCID: PMC1868666  PMID: 17272823
Apoptosis; Genes; Pulmonary hypertension; Smooth muscle
4.  Retinoic Acid Inhibits Airway Smooth Muscle Cell Migration 
Airway remodeling in chronic asthma is characterized by increased smooth muscle mass that is associated with the reduction of the bronchial lumen as well as airway hyperresponsiveness. The development of agents that inhibit smooth muscle growth is therefore of interest for therapy to prevent asthma-associated airway remodeling. All-trans retinoic acid (ATRA) suppresses growth of vascular smooth muscle cells (SMCs) from the systemic and pulmonary circulation. The present study investigated the effects of ATRA on human bronchial (airway) SMCs. Human bronchial SMCs were found to express mRNAs for retinoic acid receptor (RAR)-α, -β, -γ, and retinoid X receptor (RXR)-α, -β, but not RXR-γ. Although ATRA was not effective in inhibiting proliferation or in inducing apoptosis in airway SMCs, we found that ATRA (0.2–2 μM) inhibited the SMC migration in response to platelet-derived growth factor (PDGF), as determined in a modified Boyden chamber assay. Both RAR and RXR agonists also blocked PDGF-induced airway SMC migration. ATRA also inhibited PDGF-induced actin reorganization associated with migration. PDGF-induced actin reorganization and migration were blocked by inhibitors of phosphatidylinositol 3 kinase (PI3K) and Akt. However, migration was blocked by inhibitors of the MEK/ ERK pathway, with no effect on cytoskeletal reorganization. ATRA suppressed PDGF-induced Akt activation without influencing ERK activation. RAR was found to form protein–protein interactions with the p85 PI3K subunit. These results suggest that retinoic acid inhibits airway SMC migration through the modulation of the PI3K/Akt pathway.
doi:10.1165/rcmb.2005-0306OC
PMCID: PMC1831536  PMID: 16456186
airway; migration; retinoic acid; signal transduction; smooth muscle
5.  Transforming Growth Factor-β1 Effects on Endothelial Monolayer Permeability Involve Focal Adhesion Kinase/Src 
Transforming growth factor (TGF)-β1 activity has been shown to increase vascular endothelial barrier permeability, which is believed to precede several pathologic conditions, including pulmonary edema and vessel inflammation. In endothelial monolayers, TGF-β1 increases permeability, and a number of studies have demonstrated the alteration of cell–cell contacts by TGF-β1. We hypothesized that focal adhesion complexes also likely contribute to alterations in endothelial permeability. We examined early signal transduction events associated with rapid changes in monolayer permeability and the focal adhesion complex of bovine pulmonary artery endothelial cells. Western blotting revealed rapid tyrosine phosphorylation of focal adhesion kinase (FAK) and Src kinase in response to TGF-β1; inhibition of both of these kinases using pp2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), ameliorates TGF-β1–induced monolayer permeability. Activation of FAK/Src requires activation of the epidermal growth factor receptor downstream of the TGF-β receptors, and is blocked by the epidermal growth factor receptor inhibitor AG1478. Immunohistochemistry showed that actin and the focal adhesion proteins paxillin, vinculin, and hydrogen peroxide–inducible clone-5 (Hic-5) are rearranged in response to TGF-β1; these proteins are released from focal adhesion complexes. Rearrangement of paxillin and vinculin by TGF-β1 is not blocked by the FAK/Src inhibitor, pp2, or by SB431542 inhibition of the TGF-β type I receptor, anaplastic lymphoma kinase 5; however, pp1 (4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which inhibits both type I and type II TGF-β receptors, does block paxillin and vinculin rearrangement. Hic-5 protein rearrangement requires FAK/Src activity. Together, these results suggest that TGF-β1–induced monolayer permeability involves focal adhesion and cytoskeletal rearrangement through both FAK/Src-dependent and -independent pathways.
doi:10.1165/rcmb.2006-0439OC
PMCID: PMC2176121  PMID: 17585111
focal adhesion complex; hydrogen peroxide–inducible clone 5; nuclear translocation; paxillin; vinculin

Results 1-5 (5)