Hypoxia-induced proliferation of human pulmonary microvascular endothelial cells depends on epidermal growth factor receptor tyrosine kinase activation.
We hypothesized that hypoxia would activate epidermal growth factor receptor (EGFR) tyrosine kinase, leading to increased arginase expression and resulting in proliferation of human pulmonary microvascular endothelial cell (hPMVEC). To test this hypothesis, hPMVEC were incubated in normoxia (20% O(2), 5% CO(2)) or hypoxia (1% O(2), 5% CO(2)). Immunoblotting for EGFR and proliferating cell nuclear antigen was done, and protein levels of both total EGFR and proliferating cell nuclear antigen were greater in hypoxic hPMVEC than in normoxic hPMVEC. Furthermore, hypoxic hPMVEC had greater levels of EGFR activity than did normoxic hPMVEC. Hypoxic hPMVEC had a twofold greater level of proliferation compared with normoxic controls, and this increase in proliferation was prevented by the addition of AG-1478 (a pharmacological inhibitor of EGFR). Immunoblotting for arginase I and arginase II demonstrated a threefold induction in arginase II protein levels in hypoxia, with little change in arginase I protein levels. The hypoxic induction of arginase II protein was prevented by treatment with AG-1478. Proliferation assays were performed in the presence of arginase inhibitors, and hypoxia-induced proliferation was also prevented by arginase inhibition. Finally, treatment with an EGFR small interfering RNA prevented hypoxia-induced proliferation and urea production. These findings demonstrate that hypoxia activates EGFR tyrosine kinase, leading to arginase expression and thereby promoting proliferation in hPMVEC.
Pulmonary Hypertension Group, Center for Perinatal Research, Research Institute at Nationwide Children's Hospital, and Department of Pediatrics, Ohio State University, Columbus, Ohio, USA.
Inimary T
IT
Toby
Louis G
LG
Chicoine
Hongmei
H
Cui
Bernadette
B
Chen
Leif D
LD
Nelin
D001119:Arginase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000098:blood supply*; D055806:Microvessels / Q000166:cytology*; D008954:Models, Biological; D011799:Quinazolines; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D020032:Tyrphostins / Q000494:pharmacology; D014508:Urea / Q000378:metabolism
D011799:Quinazolines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020032:Tyrphostins; C101044:RTKI cpd; D004815:Epidermal Growth Factor; D014508:Urea; D066246:ErbB Receptors; D001119:Arginase