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Nili Parekh, Zheng Wang, Hua Yang, Peter S. Reinach; Triptolide Stimulates Migration in Human Corneal Epithelial Cells via P38 MAPK and JNK. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2050.
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Triptolide, a derivative of the Chinese herbal medicinal vine tripterygium wilfordii hook F has immunomodulatory and anti-inflammatory effects on corneal fibroblasts. Since triptolide is under evaluation for use in treatment of corneal ulcers, we determined if triptolide stimulates human corneal epithelial cells (HCEC) migration through receptor-linked signaling pathways mediating corneal epithelial renewal.
SV-40 immortalized HCEC, MKP-1(DUSP-1) knockdown and JNK-1 knockdown cell sub lines were used .Cell Viability was measured using Cell Titer-Glo® Luminescent Viability Assay. Cell migration rates were determined with a scratch wound assay. Cell proliferation was inhibited with 2.5 mM hydroxyurea. HCEC were incubated with either triptolide (1nM) or EGF (10ng/ml) in the presence and absence of p38 and JNK1/2 MAPK pathway inhibitors-10µM SB203580 and 10µM SP600125, respectively. Images were taken with an inverted stage microscope over period of 24 hours and analyzed using Sigma Scan Software. Wound Closure rates were reported as mean percentage of remaining wound area open ± SEM.Western Blots determined kinetics of phosphorylation of the terminal kinases in the p38 and JNK pathways.
Triptolide stimulated wound closure through increases in migration without any effect on cell viability up to 10nM with maximal responses at 1 nM. At 24 hours, the remaining wound area was11 ± 2 % for Triptolide (1nM) and 14 ±2 % for EGF (10ng/ml) whereas 34 ± 1 % area of the initial wound remained open in the untreated control (n=6).Increases in migration were suppressed below that of untreated control when p38 or JNK MAPK activation was inhibited. In the MKP-1 knockdown cells triptolide failed to augment this response any further relative to control. In JNK-1 knockdown cells, migration is comparable to SV40 wild type untreated control. In JNK-1 knockdown cells, triptolide mediated increases are diminished completely in the presence of p38 inhibition.
Triptolide at concentrations up to 10 nM promotes cell migration in HCEC without compromising cell survival. Such promotion is mediated by loss of MKP-1 negative feedback control of p38 and JNK activation. Therefore, triptolide stimulates cell migration through inhibition of MKP-1 (DUSP1) stabilization induced by kinase mediated phosphorylation and is associated with prolongation of p38 MAPK and JNK activation resulting from declines in negative feedback control by MKP-1 of this response.
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