Melatonin Suppresses Hypoxia-Induced Migration Of Human Umbilical Vein Epithelial Cells

Part I Melatonin Suppresses Hypoxia-Induced Migration of and Expression of HIF-1α in Human Umbilical Vein Epithelial Cells Objective: To investigate the role of melatonin on hypoxia-induced migration of and HIF-1α expression in human umbilical vein epithelial cells(HUVECs). Methods: 1. Wound closure assay was used to test migration rate of HUVECs culcured in normoxia or hypoxia in the presence or absence of melatonin. 2. Western blot analysis was carried out to determine levels of HIF-1α protein in HUVECs in normoxic or hypoxic condition in the presence or absence of melatonin. 3. Wound closure assay was performed to test the migration rate of HUVECs transfected with HIF-1α si RNA and then cultured in hypoxic condition.Results:1. The migration rate of HUVECs was significantly increased in hypoxic condition compared to that of HUVECs in normoxic condition. Melatonin inhibited cell migratory ability in a dose-dependent manner. 2. Hypoxia resulted in significant and rapid increase in HIF-1α protein levels in HUVECs; this increase, however, was suppressed by melatonin treatment dose-dependently. 3. The hypoxia-induced increase in migration rate was attenuated by knockdown of HIF-1α in HUVECs. Conclusion:Melatonin inhibits hypoxia-induced cell migration in a dose-dependent manner. Hypoxia results in significant increase in HIF-1α expression, which can be suppressed by melatonin. We thus conclude that melatonin inhibits cell migration in hypoxic condition, possibly by inhibiting HIF-1α expression in HUVECs.Part II The Role of ERK and Rac1 activation in Hypoxia-Induced Cell Migration and the Effect of Melatonin on These Cellular Events Objective: To determine whether Rac1 is involved in hypoxia-induced cell migration and in melatonin-mediated suppression of HIF-1α expression in HUVECs. Methods: 1. To evaluate HIF-1a protein levels, VEGF m RNA levels, and cell migration in HUVECs expressing ectopic active(Rac1-V12) or dominant negative RAC-1(Rac1-T17N). 2. To assess endogenous RAC-1 GTPase activity and cell migration in hypoxic condition in the presence or absence of melatonin and to know the effect of dominant negative RAC1 on cell migration. 3. To determine changes of RAC1 cellular localization induced by hypoxia and the effect of melatonin on this change. 4. To elucidate ERK phosphorylation induced by hypoxia and the effect of ERK activtion on RAC1 GTPase activity and cell migration in the presence or absence of melatonin. Results: 1. Expression of exogenous active, but not dominant negative, RAC1 was sufficient to induce the expression of HIF-1a and its downstream target VEGF and to trigger cell migration; melatonin had no effect on RAC1-mediated cell migration. 2. Melatonin inhibited hypoxia-induced cell migration, likely through suppression of Rac1 activation. 3. Hypoxic treatment resulted in translocation of Rac1 from the cytosol to the cytoskeleton; however, this change in cell localization was reversed by melatonin treatment. 4. ERK phosphorylation and activation lied upstream of Rac1 activation; melatonin inhibited hypoxia-induced Rac1 activation and cell migration, possibly by blocking ERK phosphorylation. Conclusion: Inhibition of hypoxia-induced expression of HIF-1α, VEGF, and cell migration by melatonin require Rac1 GTPase activity. ERK activation is necessary for hypoxia-induced Rac1 activation and cell migration. Based on our results, we conclude that melatonin can inhibit these cellular events and cell migration in hypoxic conditionmost likely by blocking ERK phosphorylation.