| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
UV irradiation has various biological effects on skin. One is EGF receptor(EGFR) phosphorylation and the subsequent activation of MAP kinase, leading to epidermal hyperplasia. However, the cross-talk mechanism linking UV irradiation and EGFR phosphorylation remains unclear. Recently, it was reported that EGFR transactivation by G-coupled-protein receptors is mediated via HB-EGF shedding, the conversion of HB-EGF from a membrane anchored form to a soluble form. So we hypothesized that UV-induced EGFR phosphorylation is mediated via HB-EGF shedding. First we examined the effect of UV irradiation on EGFR phosphorylation in human keratinocytes. UV irradiation (30 mJ/cm^2) induced EGFR phosphorylation in a time dependent manner, optimally 2.1 fold at 20 min. Next we examined whether UV irradiation induces HB-EGF shedding from the keratinocyte cell surface. Biotinylated membrane-anchored HB-EGF on the cell surface disappeared almost completely 10 min after UV irradiation. The soluble form of EGFR ligands in culture medium was measured by bioassay. Soluble EGFR ligands increased optimally 1.5 fold at 5 min after UV irradiation. An inhibition assay identified 77% of the soluble EGFR ligands as HB-EGF. Furthermore, R8301, a specific inhibitor of HB-EGF shedding, inhibited UV induced HB-EGF shedding completely at 1 μM. We also confirmed the HB-EGF inhibitors such as R8301, anti-HB-EGF blocking antibody and CRM197, a specific inhibitor of soluble HB-EGF, completely inhibited UV-induced EGFR phosphorylation. Finally, we examined the involvement of HB-EGF in UV-induced epidermal hyperplasia using keratinocyte-specific HB-EGF knockout mice generated by Cre/loxP technology. In HB-EGF knockout mice, UV-induced epidermal hyperplasia was completely inhibited. In conclusion, we clearly demonstrated that UV irradiation-induced EGFR phosphorylation and epidermal hyperplasia are mediated via HB-EGF shedding, the conversion of HB-EGF from a membrane-anchored form to a soluble form
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