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

紫外线照射对皮肤具有各种生物学作用。一种是EGF受体（EGFR）磷酸化和随后的MAP激酶激活，导致表皮增生。然而，连接UV辐射和EGFR磷酸化的串扰机制尚不清楚。最近，据报道，G偶联蛋白受体通过HB-EGF脱落进行EGFR反式激活，这是HB-EGF的转化，HB-EGF从膜锚固形式转化为固体形式。因此，我们假设UV诱导的EGFR磷酸化是通过HB-EGF脱落介导的。首先，我们检查了紫外线照射对人角质形成细胞中EGFR磷酸化的影响。紫外线照射（30 mJ/cm^2）以时间依赖的方式诱导EGFR磷酸化，在20分钟时以最佳的2.1倍。接下来，我们检查了紫外线辐射是否诱导角质形成细胞表面脱落的HB-EGF。紫外线照射后，生物素化的膜锚定的HB-EGF几乎完全消失了。通过生物测定法测量了培养基中EGFR配体的固体形式。紫外线照射后5分钟，可溶性EGFR配体在5分钟内提高了1.5倍。抑制测定法将77％的固体EGFR配体为HB-EGF。此外，R8301是一种特异性HB-EGF脱落的抑制剂，在1μm下完全抑制了紫外线诱导的HB-EGF脱落。我们还证实了HB-EGF抑制剂，例如R8301，抗HB-EGF阻断抗体和CRM197（一种固体HB-EGF的特异性抑制剂）完全抑制了UV诱导的EGFR磷酸化。最后，我们使用角质形成细胞特异性的HB-EGF敲除小鼠研究了HB-EGF参与UV诱导的表皮增生，CRE/LOXP技术产生的小鼠。在HB-EGF敲除小鼠中，紫外线诱导的表皮增生完全抑制。总之，我们清楚地证明了紫外线诱导的EGFR磷酸化和表皮增生是通过HB-EGF脱落介导的