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Z. Wang, J. M. Wolosin, H. Yang, J. E. Capó-Aponte, P. S. Reinach; Changes in JNK-1 Phosphorylation Status Modulate IL-6 and IL-8 Responses Induced by TRPV1 in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5899.
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Capsaicin (CAP) is a transient receptor potential vanilloid 1 (TRPV1) agonist, which induces increases in IL-6 and IL-8 release in human corneal epithelial cells (HCEC). It has been shown that induction involves activation of all three of the MAPK cascades, leading to phosphorylation of ERK, p38 and JNK. Dual specificity phosphatase, DUSP1, modulates the cascades by controlling the phosphorylation status of these terminal kinases. Now we sought to establish the relative role of the JNK-DUSP1 interaction in the TRPV1 linked pathways.
DUSP1i and JNK1i SV40-HCEC sublines expressing shRNAs for inhibition of DUSP1 and JNK1 gene expression, respectively, were generated using pGIPz lentivectors (Open Biosystems). Viral particles were produced in 293T cells and transduced into HCEC target cells with the aid of polybrene. Transduced cells were selected based on strong puromycin (15 µg/ml) resistance. Western blots were used to confirm the effect of shRNA expression. Interleukins expression was measured by ELISA.
The JNK1i line showed >90 % reduction of expression of this kinase and complete inhibition of CAP-elicited IL-6 and 8 release. In DUSP1i, a) rises in DUSP1 protein expression in response to EGF or CAP were largely curtailed. b) post-peak decreases in the dramatic phosphorylation of Erk1/2, p38 α/β and JNK1/2 MAPK in response to EGF or CAP were abolished; and c) CAP-induced increases of IL-6 and 8 were 8-fold higher than those elicited in the native cells. The TRPV1 antagonist, capsazepine (10 µM), fully suppressed the effects of CAP in both the nontransfected and transfected cell lines.
CAP-induced increases in IL-6 and IL-8 occur primarily through phosphorylated JNK1. Since at the MAPK level CAP and EGF elicited seemingly identical phosphorylation changes, activation of TRPV1 must involve a second signal transduction event not elicited by EGF.
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