Analysis of the mechanism of ultraviolet irradiation on epidermal keratinocytes,

紫外线照射对表皮角质细胞作用机制分析，

基本信息

批准号：
15591181
负责人：
MURAKAMI Shinji
金额：
$ 2.24万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591181/
关键词：
UVB EGF receptor transactivation shedding HB-EGF knockout mouse keratinocyte serum free culture EFG receptor

项目摘要

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

紫外线照射对皮肤有多种生物学效应，其中之一是表皮生长因子受体（EGFR）磷酸化并随后激活MAP激酶，从而导致表皮增生。然而，近年来，紫外线照射与EGFR磷酸化之间的相互作用机制仍不清楚。报道称，G 偶联蛋白受体对 EGFR 的反式激活是通过 HB-EGF 脱落介导的，即将 HB-EGF 从膜锚定形式转化为因此，我们探索了紫外线诱导的 EGFR 磷酸化是通过 HB-EGF 脱落介导的。首先，我们研究了紫外线照射对人角质形成细胞中 EGFR 磷酸化的影响。时间依赖的方式，最佳为 20 分钟时的 2.1 倍接下来，我们检查了紫外线照射是否会诱导 HB-EGF。紫外线照射后 10 分钟，细胞表面的生物素化膜锚定 HB-EGF 几乎完全消失。通过生物测定，可溶性 EGFR 配体在 5 时增加了 1.5 倍。紫外线照射后 1 分钟，抑制测定鉴定出 77% 的可溶性 EGFR 配体为 HB-EGF。 R8301 是 HB-EGF 脱落的特异性抑制剂，在 1 μM 时可完全抑制紫外线诱导的 HB-EGF 脱落。我们还证实了 HB-EGF 抑制剂，例如抗 HB-EGF 阻断抗体 R8301 和可溶性特异性抑制剂 CRM197。 HB-EGF，完全抑制紫外线诱导的 EGFR 磷酸化最后，我们使用 HB-EGF 来研究 HB-EGF 在紫外线诱导的表皮增生中的作用。 Cre/loxP 技术产生的角质形成细胞特异性 HB-EGF 敲除小鼠在 HB-EGF 敲除小鼠中，紫外线诱导的表皮增生被完全抑制。总之，我们清楚地证明了紫外线照射诱导的 EGFR 磷酸化和表皮增生是通过介导的。 HB-EGF 脱落，HB-EGF 从膜锚定形式转化为可溶形式