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| IJCEM Copyright © 2008-All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711 |
Int J Clin Exp Med 2(1),87-94;2009
Original Article
Hypoxic upregulation of preproendothelin-1 gene expression is associated with
protein tyrosine kinase-PI3K signaling in cultured lung vascular endothelial cells
Jianliang Zhang, Vikram M. Narayan, Noah Juedes, Jawaharlal M. Patel
Department of Medicine, University of Florida College of Medicine; and Research Service, Malcom Randall Department of Veterans
Affairs Medical Center, Gainesville, FL 32608-1197, USA
Received February 25, 2009; accepted April 8, 2009; available online April 15, 2009
Abstract: Hypoxia-increased endothelin-1 (ET-1) expression contributes to vasoconstriction and vessel wall thickening, often seen in
the progression of pulmonary hypertension. We sought to investigate whether hypoxic modulation of preproET-1 transcription is
associated with protein tyrosine kinase and phosphatidylinositol-3-kinase (PI3K). ET-1 is predominantly produced in and secreted from
the vascular endothelium. Cultured human pulmonary artery endothelial cells (PAEC) in basic medium EBM-2 were exposed to hypoxia
(1% oxygen, 5% CO2, 37oC) or normoxia (room air containing 5% CO2) for 0-48 hr. RNA was extracted from the treated cells and
subjected to quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Hypoxia increases the relative levels of steady-state
preproET-1 mRNA. The results of actinomycin D chase studies suggest that hypoxia-increased levels of preproET-1 mRNA are unlikely
to be caused by increased RNA stability. A modified nuclear run-on method coupled with the sensitive qRT-PCR technique was used to
assess preproET-1 gene transcription. The synthesis rate of preproET-1 mRNA in the cells exposed to hypoxia is higher than that in
normoxic cells. The inhibitors of protein tyrosine kinases and PI3K, genistein and PI3Kγ inhibitor II, were used to elucidate the role of
protein tyrosine kinase and PI3K in hypoxic modulation of preproET-1 expression. Pre-incubation of human PAEC with genistein or
PI3Kγ inhibitor II abolishes hypoxia-increased levels of preproET-1 mRNA. Our observations support the notion that hypoxia increases
the level of preproET-1 mRNA through upregulation of RNA synthesis, which is associated with protein tyrosine kinase- and PI3K-
mediated signal transduction pathways. This implies that therapeutic interventions targeting protein tyrosine kinases and/or PI3K might
be used to treat hypoxic pulmonary hypertension. (IJCEM902007).
Key Words: Preproendothelin-1 gene (PreproET-1), protein tyrosine kinase (PI3K), lung vascular endothelial cells, hypoxia, signal
transduction
Full Text PDF
Address all correspondence to:
Jianliang Zhang, PhD
Pulmonary Division
Department of Medicine
University of Florida College of Medicine
1600 S. W. Archer Road, Gainesville
FL 32610-0225, USA
Tel: 353-376-1611, Ext. 6265, Fax: 352-374-6170
E-mail: Zhangjl@medicine.ufl.edu
Original Article
Hypoxic upregulation of preproendothelin-1 gene expression is associated with
protein tyrosine kinase-PI3K signaling in cultured lung vascular endothelial cells
Jianliang Zhang, Vikram M. Narayan, Noah Juedes, Jawaharlal M. Patel
Department of Medicine, University of Florida College of Medicine; and Research Service, Malcom Randall Department of Veterans
Affairs Medical Center, Gainesville, FL 32608-1197, USA
Received February 25, 2009; accepted April 8, 2009; available online April 15, 2009
Abstract: Hypoxia-increased endothelin-1 (ET-1) expression contributes to vasoconstriction and vessel wall thickening, often seen in
the progression of pulmonary hypertension. We sought to investigate whether hypoxic modulation of preproET-1 transcription is
associated with protein tyrosine kinase and phosphatidylinositol-3-kinase (PI3K). ET-1 is predominantly produced in and secreted from
the vascular endothelium. Cultured human pulmonary artery endothelial cells (PAEC) in basic medium EBM-2 were exposed to hypoxia
(1% oxygen, 5% CO2, 37oC) or normoxia (room air containing 5% CO2) for 0-48 hr. RNA was extracted from the treated cells and
subjected to quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Hypoxia increases the relative levels of steady-state
preproET-1 mRNA. The results of actinomycin D chase studies suggest that hypoxia-increased levels of preproET-1 mRNA are unlikely
to be caused by increased RNA stability. A modified nuclear run-on method coupled with the sensitive qRT-PCR technique was used to
assess preproET-1 gene transcription. The synthesis rate of preproET-1 mRNA in the cells exposed to hypoxia is higher than that in
normoxic cells. The inhibitors of protein tyrosine kinases and PI3K, genistein and PI3Kγ inhibitor II, were used to elucidate the role of
protein tyrosine kinase and PI3K in hypoxic modulation of preproET-1 expression. Pre-incubation of human PAEC with genistein or
PI3Kγ inhibitor II abolishes hypoxia-increased levels of preproET-1 mRNA. Our observations support the notion that hypoxia increases
the level of preproET-1 mRNA through upregulation of RNA synthesis, which is associated with protein tyrosine kinase- and PI3K-
mediated signal transduction pathways. This implies that therapeutic interventions targeting protein tyrosine kinases and/or PI3K might
be used to treat hypoxic pulmonary hypertension. (IJCEM902007).
Key Words: Preproendothelin-1 gene (PreproET-1), protein tyrosine kinase (PI3K), lung vascular endothelial cells, hypoxia, signal
transduction
Full Text PDF
Address all correspondence to:
Jianliang Zhang, PhD
Pulmonary Division
Department of Medicine
University of Florida College of Medicine
1600 S. W. Archer Road, Gainesville
FL 32610-0225, USA
Tel: 353-376-1611, Ext. 6265, Fax: 352-374-6170
E-mail: Zhangjl@medicine.ufl.edu
