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Membrane-anchored growth factor shedding in tissue regeneration and repaire

组织再生和修复中膜锚定生长因子的脱落

基本信息

批准号：
11670141
负责人：
HIGASHIYAMA Shigeki
金额：
$ 2.3万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670141/
关键词：
wound healing HB-EGF shedding EGFR keratinocyte cell migration wound healing EGFR Keratinocyte cell migration Shedding keratinocyte

项目摘要

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are in part mediated in an autocrine fashion by *idermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here (1) that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR and the signal transduction molecule Stat3 (signal transducer and activator of transcription 3). OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and Stat3, and caused suppression of keratinocyte migration in vitro. Soluble EGFR-Fc, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing. 2) In oreder to identify the HB-EGF shedding enzyme, affinity cplumn *romatography was carried using biotinylated KB-R8301-Sepharose. Three RB-R8301binding proteins were purified homogeneously from the lysate of a human fibrosarcoma cell line, HT1080 cells. Primary structure analyses of them are under going.

角质形成细胞的增殖和迁移对皮肤伤口愈合至关重要，部分是通过表皮生长因子受体(EGFR)-配体相互作用以自分泌方式介导的。EGFR配体最初是以膜锚定形式合成的，但可以加工和脱落为可溶性形式。我们在这里提供了证据：(1)创伤刺激在体外诱导角质形成细胞脱落EGFR配体，特别是配体肝素结合的EGF样生长因子(HB-EGF)。由此产生的可溶性配体刺激了EGFR和信号转导分子STAT3(信号转导和转录激活因子3)的瞬时激活。EGFR配体脱落抑制剂OSU8-1可阻断创伤诱导的EGFR和STAT3的激活，抑制角质形成细胞在体外的迁移。能够中和所有EGFR配体的可溶性EGFR-Fc在体外也能抑制角质形成细胞的迁移。OSU8-1应用于小鼠伤口处可显著延缓角质形成细胞迁移失败所致的再上皮化，但重组可溶性HB-EGF与OSU8-1联合应用可克服这一影响。这些发现表明，EGFR配体的脱落是角质形成细胞迁移的关键事件，并提示它们可能在伤口愈合的早期阶段作为一种有效的临床治疗方法。2)用生物素标记的KB-R8301-Sepharose亲和层析法对HB-EGF脱落酶进行鉴定。从人纤维肉瘤细胞株HT1080的裂解物中纯化了三种RB-R8301结合蛋白。对它们的一级结构分析正在进行中。