Membrane-anchored growth factor shedding in tissue regeneration and repaire

角质细胞的增殖和迁移对皮肤伤口愈合至关重要，部分是由表皮生长因子受体(EGFR)-配体相互作用以自分泌方式介导的。EGFR配体最初以膜锚定形式合成，但可以作为可溶性形式加工和脱落。我们在这里提供了证据(1)，伤口刺激在体外诱导表皮生长因子受体配体的角质细胞脱落，特别是配体肝素结合的表皮生长因子样生长因子（HB-EGF）。由此产生的可溶性配体刺激了EGFR和信号转导分子Stat3（转录的信号传感器和激活因子3）的瞬时激活。OSU8-1是一种EGFR配体脱落抑制剂，在体外实验中可以消除伤口诱导的EGFR和Stat3的激活，抑制角质形成细胞的迁移。可溶性EGFR- fc能够中和所有EGFR配体，在体外也能抑制角质形成细胞的迁移。由于角化细胞迁移失败，OSU8-1应用于小鼠伤口部位大大延缓了再上皮化，但如果将重组可溶性HB-EGF与OSU8-1一起添加，则可以克服这种影响。这些发现表明，EGFR配体的脱落是角化细胞迁移的一个关键事件，并表明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)为了鉴定HB-EGF脱落酶，采用生物素化KB-R8301-Sepharose进行亲和层析。从人纤维肉瘤细胞系HT1080细胞的裂解液中纯化了3个rb - r8301结合蛋白。初步结构分析正在进行中。

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.