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H. Yang, Z. Wang, J. E. Capó-Aponte, J. M. Wolosin, P. S. Reinach; DUSP6/MKP3 Overexpression in Corneal Epithelial Cells Inhibits EGF-induced Erk1/2, NKCC1 Phosphorylation and Cell Proliferation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):694.
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Mitogen activated protein kinase (MAPK) signaling depends on the magnitude and duration of kinase phosphorylation whereas signaling termination is elicited by dual-specificity phosphatases (DUSPs).DUSP6 selectively dephosphorylates cytosolic Erk1/2. In human corneal epithelial cells (HCEC), mitogenic responses to epidermal growth factor (EGF) are elicited through ERK pathway signaling and Na:K:2Cl cotransporter (NKCC1) activation. To determine if EGF-induced increases in HCEC proliferation are modulated by DUSP6, the time dependent effects of DUSP6 overexpression were evaluated on changes in the phosphorylation patterns of Erk1/2 and NKCC1.
A HCEC subline strongly overexpressing DUSP6 (DUSP6+) was created using the pLEX lentivector (Open Biosystems). Lentiviral particles were produced in 293 cells, concentrated and used to transduce SV40-HCEC with the aid of polybrene. Transduced cells were selected based on the strong puromycin resistance conferred by the pLEX transduction. Western blots were used to monitor Erk1/2 and NKCC1 phosphorylation status. [3H] thymidine incorporation evaluated cell proliferation.
In unmodified cells EGF (5 ng/ml) induces transient increases in Erk1/2 phosphorylation that peak at ~ 15 min and are followed by a gradual decline to near baseline levels 60 min later. Similar biphasic phosphorylation changes occurred with a slight delay in NKCC1. Cell proliferation increased 1.6-fold. Preincubation with 50 µM bumetanide blocked the EGF-linked NKCC1 phosphorylation without affecting the time course and magnitude of Erk1/2 phosphorylation, indicating the critical role of NKCC1 activation in cell cycling. In the DUSP6+ cells, EGF failed to induce transient changes in Erk1/2 or NKCC1 phosphorylation and simultaneously abolished the proliferative response to EGF. The EGF effects on the other two terminal MAPK, JNK and p38 in the DUSP6+ cells were essentially identical to those in the native cells.
DUSP6 controls the magnitude and duration of EGF-induced Erk1/2 phosphorylation. EGF-induced HCEC mitogenesis is dependent on Erk1/2 mediated phosphorylation of NKCC1.
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