We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVECs) depends on
epidermal growth factor receptor (EGFR) activation. To determine downstream signaling leading to proliferation, we tested the hypothesis that
hypoxia-induced proliferation in hPMVECs would require EGFR-mediated activation of
extracellular signal-regulated kinase (ERK) leading to
arginase II induction. To test this hypothesis, hPMVECs were incubated in either normoxia (21% O2, 5% CO2) or
hypoxia (1% O2, 5% CO2) and Western blotting was performed for EGFR,
arginase II, phosphorylated-ERK (pERK), and total ERK (ERK).
Hypoxia led to greater EGFR, pERK, and
arginase II
protein levels than did normoxia in hPMVECs. To examine the role of EGFR in these
hypoxia-induced changes, hPMVECs were transfected with
siRNA against EGFR or a scrambled
siRNA and placed in
hypoxia. Inhibition of EGFR using
siRNA attenuated
hypoxia-induced pERK and
arginase II expression as well as the
hypoxia-induced increase in viable cell numbers. hPMVECs were then treated with vehicle, an EGFR inhibitor (
AG1478), or an ERK pathway inhibitor (
U0126) and placed in
hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the
hypoxia-induced increase in pERK level. Both
AG1478 and
U0126 also significantly attenuated the
hypoxia-induced increase in viable hPMVECs numbers. hPMVECs were transfected with an adenoviral vector containing
arginase II (AdArg2) and overexpression of
arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVECs. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic induction of
arginase II and cellular proliferation.