Analysis of the specific function of EGF family in epidermal keratinocytes using siRNA transgenic mouse

• Project/Area Number: 15390343
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Dermatology
• Research Institution: Ehime University
• Principal Investigator: SAYAMA Koji Ehime University, School of Medicine, Associate Professor, 医学部, 助教授 (80187286)
• Co-Investigator(Kenkyū-buntansha): HASHIMOTO Koji Ehime University, School of Medicine, Professor, 医学部, 教授 (00110784) ; SHIRAKATA Yuji Ehime University, School of Medicine, Instructor, 医学部, 助手 (50226320) ; 山崎 研志 愛媛大学, 医学部, 助手 (40294798)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥15,000,000 (Direct Cost: ¥15,000,000); Fiscal Year 2004: ¥5,000,000 (Direct Cost: ¥5,000,000); Fiscal Year 2003: ¥10,000,000 (Direct Cost: ¥10,000,000)
• Keywords: siRNA / HB-EGF / TGF-alpha / amphiregulin / epiregulin / knockout mouse / keratinocyte / cre recombinase / knockout mouse / cre recombinase / TGF-α

Research Abstract

The purpose of this study is to clarify a specific function of each factor in epidermis, using a siRNA(small interfering RNA) transgenic mouse which defects four kinds of EGF family growth factor (TGF-alpha, HB-EGF, amphiregulin, epiregulin) having the most important work in the epidermis. Several kinds determined a candidate siRNA arrangement (19 sequence) using software for siRNA choice and we composed siRNA by in vitro transcription and, using siRNA construction kit, refined it in a column. Transfection did candidate siRNA to keratinocyte to be next and reviewed mRNA manifestation and determined a best siRNA arrangement. We reviewed manifestation of each mRNA in EGF stimulation by real time PCR, RNase protection assay. We tried to generate each siRNA transgenic mice, however we could not get in the research periods. Therefore, using keratinocyte specific knockout mouse of HB-EGF, we analyzed a function of HB-EGF in a skin healing of wound process. Migration of keratinocyte was inhibited when we used neutralizing antibody of HB-EGF with the in vitro wound healing model whereas HB-EGF addition promoted wound healing. In addition, the mRNA of HB-EGF was induced immediately and conspicuously among EGF families by scraping. Finally we performed wound healing assay using keratinocyte-specific HB-EGF knockout mice. A delay of healing of wound was accepted in HB-EGF knockout mouse. We conclude that HB-EGF plays an important role in skin wound healing.

Research Products

• [Journal Article] SOCS3/CIS3 negative regulation of STAT3 in HGF-induced keratinocyte migration. (2005)
  • Author(s): Tokumaru S, Sayama K, Yamasaki K, Shirakata Y, et al.
  • Journal Title: Biochem Biophys Res Commun 327 Pages: 100-105
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Cre/loxP mediated gene transfer into living skin equivalent model (2005)
  • Author(s): Hanakawa Y, Shirakata Y, et al.
  • Journal Title: British Journal of Dermatology (in press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] SOCS3/CIS3 negative regulation of STAT3 in HGF-induced keratinocyte migration. (2005)
  • Author(s): Tokumaru S, Sayama K, Yamasaki K, Shirakata Y, Hanakawa Y, Yahata Y, Dai X, Tohyama M, Yang L, Yoshimura A, Hashimoto K.
  • Journal Title: Biochem Biophys Res Commun. 327 Pages: 100-105
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Keratinocyte G2/M growth arrest by 1,25-dihydroxyvitamin D3 is caused by Cdc2 phosphorylation through Wee1 and Myt1 regulation. (2004)
  • Author(s): Dai X, Yamasaki K, Yang L, Sayama K, Shirakata Y, et al.
  • Journal Title: Journal of Investigative Dermatology 122 Pages: 1356-1364
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] TGF-beta is not involved in early phase growth inhibition of keratinocytes by 1alpha25(OH)2vitamin D3. (2004)
  • Author(s): Shirakata Y, Ueno H, Hanakawa Y, et al.
  • Journal Title: Journal of Dermatological Science 36 Pages: 41-50
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] All-trans-retinoic acid induces interleukin-8 via the nuclear factor-kappaB and p38 mitogen-activated protein kinase pathways in normal human keratinocyte (2004)
  • Author(s): Dai X, Yamasaki K, Shirakata Y, et al.
  • Journal Title: Journal of Investigative Dermatology 123 Pages: 1078-1085
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Keratinocyte G2/M growth arrest by 1,25-dihydroxyvitamin D3 is caused by Cdc2 phosphorylation through Weel and Mytl regulation. (2004)
  • Author(s): Dai X, Yamasaki K, Yang L, Sayama K, Shirakata Y, Tokumara S, Yahata Y, Tohyama M, Hashimoto K.
  • Journal Title: J Invest Dermatol. 122 Pages: 1356-1364
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] TGF-beta is not involved in early phase growth inhibition of keratinocytes by lalpha,25(OH)2vitamin D3. (2004)
  • Author(s): Shirakata Y, Ueno H, Hanakawa Y, Kameda K, Yamasaki K, Tokumaru S, Yahata Y, Tohyama M, Sayama K, Hashimoto K.
  • Journal Title: J Dermatol Sci. 36 Pages: 41-50
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] All-trans-retinoic acid induces interleukin-8 via the nuclear factor-kappaB and p38 mitogen-activated protein kinase pathways in normal human keratinocytes. (2004)
  • Author(s): Dai X, Yamasaki K, Shirakata Y, Sayama K, Hashimoto K.
  • Journal Title: J Invest Dermatol. 123 Pages: 1078-1085
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Keratinocyte G2/M growth arrest by 1.25-dihydroxyvitamin D3 is caused by Cdc2 phosphorylation through Weel and Mytl regulation. (2004)
  • Author(s): Dai X, Yamasaki K, Yang L, Sayam K, Shirakata Y. et al.
  • Journal Title: Journal of Investigative Dermatology 122 Pages: 1356-1364
• [Journal Article] TGF-beta is not involved in early phase growth inhibition of keratinocytes by lalpha 25(OH)2vitamin D3. (2004)
  • Author(s): Shirakata Y, Ueno H, Hanakawa Y, et al.
  • Journal Title: Journal of Dermatological Science 36 Pages: 41-50
• [Journal Article] Cre-loxP adenovirus-mediated foreign gene expression in skin-equivalent keratinocytes.
  • Author(s): Hanakawa Y, Shirakata Y, Nagai H, Yahata Y, Tokumaru S, Yamasaki K, Tohyama M, Sayama K, Hashimoto K.
  • Journal Title: Br J Dermatol. (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Yahata Y, Shirakata Y, Tokumaru S, Yamasaki K, Sayama K, et al.: "Nuclear translocation of phosphorylated STAT3 is essential for VEGF-induced human dermal microvascular endothelial cell migration and tube formation"The Journal of Biological Chemistry. 278. 40026-40031 (2003)
• [Publications] Shirakata Y, Tamai K, Nakaoka H, Tokumaru S, Sayama K, et al.: "Severe Palmo-plantar Hyperkeratosis in Koebner Epidermolysis Bullosa Simplex"The Journal of Dermatology. 30. 135-140 (2003)
• [Publications] Tsuda T, Thoyama M, Yamasaki K, Shirakata Y, Yahata Y, Sayama K et al.: "Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro"The Journal of Dermatological Science. 31. 37-42 (2003)
• [Publications] Yamasaki K, Hanakawa Y, Tokumaru S, Shirakata Y, Sayama K, et al.: "SOCS1/JAB and SOCS3/CIS3 negatively regulate the STATs signaling pathway in normal human epidermal keratinocytes"The Journal of Investigative Dermatology. 120. 571-580 (2003)
• [Publications] Yamasaki K, Toriu N, Hanakawa Y, Shirakata Y, Sayama K, et al.: "Keratinocyte growth inhibition by high-dose epidermal growth factor is mediated by transforming growth factor β autoinduction : A negative feedback mechanism for keratinocyte growth"The Journal of Investigative Dermatology. 120. 1030-1037 (2003)
• [Publications] Shirakata Y, Tokumaru S, Yamasaki K, Sayama K, Hashimoto K: "So-called biological dressing effects of cultured epidermal sheets are mediated by the production of EGF family, TGF-β and VEGF"The Journal of Dermatological Science. 32. 209-215 (2003)
• [Publications] 佐山浩二: "皮膚疾患最新の治療"本郷 允彦. 2 (2003)