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

人角质形成细胞自分泌及交叉诱导机制抑制剂的研究

• 批准号: 13557071
• 负责人: HASHIMOTO Koji
• 金额: $ 7.87万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557071/
• 关键词: keratinocytes; autocrine; HB-EGF; ADAM; MMP; conditional knockout mouse; adenovirus vector; real time PCR; adenovirus vector; adnovirus vector

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

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

项目成果

Iwamoto R, Yamazaki S, Asakura M, Takashima S, Hasuwa H, Miyado K, Adachi S, Kitakaze M, Haahimoto K, Raab G, Klagsbrun M, Nanba D, Higashiyama S, Hori M, Mekada E: "HB-EGF and ErbB signaling is essential for heart function"Proc Natl Acad Sci. (in press).

Iwamoto R、Yamazaki S、Asakura M、Takashima S、Hasuwa H、Miyado K、Adachi S、Kitakaze M、Haahimoto K、Raab G、Klagsbrun M、Nanba D、Higashiyama S、Hori M、Mekada E：“HB-EGF和

Shouda T, Yoshida T, Hanada T, Wakioka T, Oishi M, Miyoshi K, Komiya S, Kosai K, Hanakawa Y, Hashimoto K, Nagata K, Yoshimura A: "Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis"J Clin In

Shouda T、Yoshida T、Hanada T、Wakioka T、Oishi M、Miyoshi K、Komiya S、Kosai K、Hanakawa Y、Hashimoto K、Nagata K、Yoshimura A：“诱导细胞因子信号调节剂 SOCS3/CIS3 作为治疗策略

Hattori N, Komine M, Yano S, Kaneko T, Hanakawa Y, Hashimoto K, Tamaki K: "Interferon-gamma, a strong suppressor of cell proliferation, induces upregulation of keratin K6, one of the inflammatory- and proliferation-associated keratins"J Invest Dermatol. 1

Hattori N、Komine M、Yano S、Kaneko T、Hanakawa Y、Hashimoto K、Tamaki K：“干扰素-gamma 是细胞增殖的强抑制剂，可诱导角蛋白 K6 的上调，角蛋白 K6 是与炎症和增殖相关的角蛋白之一”

Shirakata Y, Tamai K, Nakaoka H, Tokumaru S, Sayama K, Murakami S, Hashimoto K: "Severe Palmo-plantar Hyperkeratosis in Koebner Epidermolysis Bullosa Simplex"J Dermatol. (In press).

Shirakata Y、Tamai K、Nakaoka H、Tokumaru S、Sayama K、Murakami S、Hashimoto K：“Koebner 单纯性大疱性表皮松解症中的严重掌跖角化过度”J Dermatol。

Hamada K, Shinomiya H, Asano Y, Kihana T, Iwamoto M, Hanakawa Y, Hashimoto K, Hirose S, Kyo S, Ito M.: "Molecular cloning of human squamous cell carcinoma antigen 1 gene and characterization of its promoter"Biochim Biophys Acta. 19. 124-131 (2001)

Hamada K、Shinomiya H、Asano Y、Kihana T、Iwamoto M、Hanakawa Y、Hashimoto K、Hirose S、Kyo S、Ito M.：“人鳞状细胞癌抗原 1 基因的分子克隆及其启动子的表征”Biochim Biophys