2009 Fiscal Year Final Research Report
Elucidation of pathophysiology or loricrin keratoderma
| Project/Area Number |
18591250
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Dermatology
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| Research Institution | Kagawa University |
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Principal Investigator |
YONEDA Kozo Kagawa University, 医学部附属病院, 准教授 (60260626)
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| Co-Investigator(Kenkyū-buntansha) |
KUBOTA Yasuo 香川大学, 医学部, 教授 (10126047)
NAKAI Kozo 香川大学, 医学部附属病院, 助教 (40363204)
ARAKI Nobukazu 香川大学, 医学部, 教授 (10202748)
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| Project Period (FY) |
2006 – 2009
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| Keywords | ロリクリン / シグナル伝達 / 遺伝性皮膚疾患 |
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| Research Abstract |
Loricrin is a major constituent of the epidermal cornified cell envelope. Recently, heterozygous loricrin gene mutations have been indentified in loricrin keratoderma. We have previously shown that the wild loricrin construct, when transiently transfected into HaCaT cells, leads to the activation of caspases and positive TUNEL staining with features of apoptosis. The apparent transfection rate is low with loricrin construct, supporting its apoptotic role but hindering further study. To bypass this problem, we generated stable HaCaT cell lines that expressed wild and mutant loricrin using an ecdysone-inducible promoter system. The cell lines expressing mutant loricrin grew more rapidly than those expressing wild loricrin. HaCaT cells with mutant loricrin express phosphorylated forms of Akt. The confocal immunofluorescence microscopic observation reveals that phospho-Akt localizes at nucleolus. The activity of Akt kinase is about 9 times higher in HaCaT cells with mutant loricrin than those in the cell lines with mock or wild loricrin. ERK1/2, epidermal growth factor receptor, vascular endothelial cell growth factor receptor (VEGFR) II and Stat 3 are all phosphorylated in mutant loricrin cells. The docking proteins, Gab1 and c-Cbl, are also tyrosine-phosphorylated in mutant loricrin cells. Neither p38 MAP kinase nor SAPK/JNK is phosphorylated in both wild and mutant loricrin cells. Next we measured the concentration of VEGF in a culture medium. VEGF in the culture medium is about 3 times more abundant in HaCaT cells with mutant loricrin compared with wild loricrin. Furthermore, chromatin immunoprecipitation assays indicate that Stat3 protein binds to the VEGF promoter in HaCaT cells with mutant loricrin. Thus, VEGF release and the subsequent activation of the VEGFR II by an autocrine/paracrine pathway link loricrin gene mutation to rapid cell proliferation in the HaCaT LK cellular model.
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