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P38 and erk1/2coordinate corneal epithelial wound healing by activating migration and proliferation respectively induction of map kinase phosphatase by hgf may regulate erk1/2


, : P38 and erk1/2coordinate corneal epithelial wound healing by activating migration and proliferation respectively induction of map kinase phosphatase by hgf may regulate erk1/2. ARVO Annual Meeting Abstract Search & Program Planner : Abstract No 3816

Purpose: HGF stimulation of corneal epithelial cells induces activation of p38 and ERK1/2. Inhibiting one of these kinases activates the other, suggesting cross-talk activation (ARVO 2002, No. 1634). We studied both kinases in two wound-healing processes, migration and proliferation, and possible downstream control of ERK1/2 by HGF-stimulated MKP-1 induction. Methods: Migration of rabbit corneal epithelial (RCE) cells was measured in the presence of HGF and/or ERK1/2 and p38 inhibitors. Cells were removed from one side of a reference line and stimulated with HGF (20 ng/ml) for 24 hr. The specific MEK inhibitor PD98059 and p38 inhibitor SB203580 were added 1 hr before HGF. Numbers of migrating cells were randomly photographed (8-10 areas/dish), counted, and averaged. Cell proliferation was measured by CyQUANT kit in octiplate using 96-well plates. MKP-1 protein synthesis was evaluated by Western blot with specific antibodies and ECL chemiluminescence detection. Results: With HGF, 480 + 22 RCE/area migrated across the reference line, nearly a 4-fold increase versus no HGF. SB203580 prevented migration and SB203580 plus HGF reduced migration by 80-90% versus HGF alone, to 50 + 3 cells/area. In contrast, PD98059 did not affect migration. Pretreatment with PD98059 plus HGF increased migration (520 + 26 cells/area). Proliferation studies showed that HGF significantly stimulated proliferation at 48 and 72 hr versus no HGF. Pretreatment with PD98059 (1 hr) followed by HGF significantly (p<0.05) inhibited proliferation by almost 14% and 27% at 48 and 72 hr, respectively, versus HGF alone. But pretreatment of cells with SB203580 (1 hr) followed by HGF had no noticeable effect on cell proliferation versus HGF alone. HGF induced MKP-1 protein synthesis after 60-120 minutes. To determine whether HGF-mediated MKP-1 stimulation involves ERK1/2, we pre-incubated cells with PD98059 then co-treated with HGF. This inhibited MKP-1 expression. Conclusions: Inhibition of p38 but not ERK1/2reduces corneal epithelial cell migration, but cell proliferation is stimulated by ERK1/2 and is unaffected by p38. Induction of MKP-1 by HGF may provide a proliferation-control mechanism by ERK1/2 in corneal epithelial cells, as inhibition of ERK1/2 down-regulates MKP-1. The results suggest that multiple MAP-kinase signaling interactions modulate corneal epithelial wound healing.

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