Research on inhibitors for autocrine and cross-induction mechanism in human keratinocytes

已有研究表明，正常人角质形成细胞（NHEK）中存在多个EGF家族的自分泌生长因子，并能诱导另一个EGF家族的生长，称为交叉诱导机制。为了阐明自分泌和交叉诱导机制，我们研究了HB-EGF自分泌和交叉诱导的信号转导途径。HB-EGF磷酸化EGFR 5分钟后，和HB-EGF的诱导抑制AG 1483，EGFR抑制剂的加入。ERK，p38和JNK被磷酸化的HB-EGF后10分钟，这表明所有三个途径的激活HB-EGF。用不同的信号转导抑制剂处理NHEK，然后用RPA检测EGF家族mRNA的诱导。HB-EGF的自诱导和交叉诱导被姜黄素（一种天然JNK抑制剂）完全阻断，但未被p38或MEK抑制剂阻断。这些结果表明，HB-EGF的自身和交叉诱导机制是由EGFR和JNK途径介导的。对于皮肤W ...更多信息 皮肤修复中最重要的生长因子是EGF家族。HB-EGF是NHEK中的一种自分泌生长因子，被认为在皮肤伤口愈合中起重要作用，尽管还没有使用小鼠模型研究HB-EGF参与体内皮肤伤口愈合。由于HB-EGF基因的生殖系靶向导致胚胎致死，我们使用Cre/loxP技术与角蛋白5启动子组合产生角质形成细胞特异性HB-EGF缺陷小鼠（KS-HB-EGF-/-小鼠）。使用6-mm穿刺活检在小鼠背部皮肤上制造皮肤伤口，并通过抗角蛋白抗体染色的表皮边缘的迁移来测量伤口闭合。在第9天，与野生型小鼠相比，KS-HB-EGF-/-小鼠的伤口闭合明显延迟。为了进一步阐明HB-EGF在皮肤伤口愈合中的作用，我们使用NHEK进行刮伤测定。HB-EGF mRNA在刮伤后2 h迅速诱导，尽管观察到TGF-α、双调蛋白和表皮调节蛋白mRNA的轻微增加。总的来说，HB-EGF通过加速角质形成细胞迁移在皮肤伤口愈合中起着重要作用。少

It has been reported that several EGF family are autocrine gowth factors in normal human keratinocytes (NHEK) and can induce the other EGF family known as cross-induction mechanism. To elucidate autocrine and cross-induction mechanism, we investigated the signal transduction pathways of auto- and cross-induction by HB-EGF. HB-EGF phosphorylated EGFR after 5 min, and induction of HB-EGF was inhibited by the addition of AG1483, an EGFR inhibitor. ERK, p38, and JNK were phosphorylated by the addition of HB-EGF after 10 min, suggesting the activation of all three pathways by HB-EGF. NHEK were treated with various signal transduction inhibitors ; then, the induction of EGF family mRNA was examined by RPA. The auto- and cross-induction of HB-EGF was completely blocked by treatment with curcumin, a natural JNK inhibitor, but not by p38 or MEK inhibitors. These results demonstrate that the auto- and cross-induction mechanisms of HB-EGF are mediated by the EGFR and JNK pathways. For cutaneous w … More ound healing, the most important growth factor is EGF family. HB-EGF, an autocrine growth factor in NHEK, is thought to play an important role in skin wound healing, although there have been no study about an involvement of HB-EGF in in vivo skin wound healing using mouse model. Since germline targeting of the HB-EGF gene resulted in embryonic lethality, we generated keratinocyte specific HB-EGF deficient mice (KS-HB-EGF-/-mice) using Cre/loxP technology in combination with keratin 5 promoter. A cutaneous wound was made on the mouse back skin using 6-mm punch biopsy and wound closure was measured by migration of epidermal edge stained by anti-keratin antibody. Wound closure was markedly retarded in KS-HB-EGF-/-mice compared to wild type mice on day 9. To further clarify the role of HB-EGF in cutaneous wound healing, we performed scraping wound assay using NHEK. HB-EGF mRNA was rapidly induced at 2 h after scraping, although the slight increase of TGF-alpha, amphiregulin and epiregulin m RNA was observed. Taking together, HB-EGF plays an important role in skin wound healing by accelerating keratinocyte migration. Less