探討第八型岩藻醣轉移酶在乾癬角質細胞增生的角色

Abstract

背景： 乾癬是一個常見、遺傳性的慢性發炎皮膚疾病，其重要特徵為表皮角質細胞異常增生及分化，而導致表皮增厚。第八型岩藻醣轉移酶(α1,6-fucosyltransferase , Fut8)，是一個將岩藻醣基從GDP轉移到核心醣胜肽N-linked acetylglucosamine (GlcNAc)的酵素，此反應被稱作核心岩藻醣化(core fucosylation)。許多研究已證實，核心岩藻醣化參與許多病理生理過程，例如在癌細胞轉移及細胞不正常增生扮演很重要的角色。本研究探討Fut8在角質細胞的表達與乾癬嚴重程度的相關性，並證明Fut8會促進皮膚角質細胞的增生。 方法： 做法上，我們從Taiwanese Skin Inflammatory Disease Consortium收集乾癬病患的皮膚檢體整合成生物樣本庫，比較皮膚組織切片中Fut8的表達量與乾癬疾病嚴重程度的相關性；本研究同時建立FUT8變異的轉植皮膚角質細胞株，並利用WST-1 assay 以及活細胞長時間影像，探討FUT8的過度表達或基因抑制是否會影響角質細胞增生。最後，以胸苷細胞週期同步化 (double thymidine block cell cycle synchronization)，探討FUT8過度表達與基因抑制如何影響細胞週期，以及細胞週期調控蛋白的表現。 結果： 本研究納入七位乾癬患者，發現在較嚴重的乾癬患者中，Fut8在患部的表現大於非患部。免疫染色顯示Fut8在乾癬患部表皮的表現與角蛋白14 (keratin 14)的表現區域重疊。本研究另在細胞實驗上證實FUT8過度表達會促進角質細胞的增生，而抑制FUT8基因會降低角質細胞的增生；胸苷細胞周期同步化結果顯示，FUT8過度表達會促進角質細胞的細胞週期S期，進而加速細胞分裂，並促進週期蛋白D1、週期蛋白A2以及週期蛋白B1的表現；相反地，抑制FUT8基因會造成G2/M期細胞週期遲滯，並抑制週期蛋白B1的表現。 本研究證實Fut8與乾癬嚴重程度的相關性及Fut8調控角質細胞的增生，Fut8可望為治療乾癬，以及其他角質細胞異常增生的皮膚疾病的分子標靶。

Background: Psoriasis is a common, heritable, and chronic inflammatory skin disorder. Epidermal thickness is an important index of psoriasis severity and is directly caused by keratinocyte hyperproliferation. Fucosyltransferase 8 (Fut8) is the only enzyme catalyzing α1,6-fucosylation in mammal and has been observed to regulate cell proliferaton in various malignant cancer. This study aimed to investigate the relationship between Fut8 and psoriasis severity as well as further elucidate how Fut8 regulates keratinocyte proliferation. Methods: Our skin tissue bio-bank, Taiwanese Skin Inflammatory Disease Consortium, included skin biopsy samples collected from psoriasis patients. Fut8 expression in skin biopsy was examined with immunohistochemistry and immunofluorescence. The correlations between clinical features of psoriasis patients and Fut8 expression levels in skin biopsy were analyzed. In addition, stable cell clones of FUT8 overexpression or knockdown were established in HaCaT cells, an immortalized human keratinocyte cell line. WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt) cell proliferation assay and time-lapse microscopy were used to investigate whether Fut8 overexpression or knockdown affects keratinocyte proliferation. Double thymidine block was applied to synchronize cell cycle and expression of cell cycle regulators was examined. Results: Among the seven psoriasis patients recruited in this study, FUT8 expression tended to be upregulated in lesional epidermis from patients with more severe psoriasis. Fut8 was upregulated in psoriasis lesional epidermis and colocalized with keratin 14. Overexpressing FUT8 in HaCaT cells promoted cell proliferation, while FUT8 knockdown inhibited HaCaT cell proliferation. Under double-thymidine block cell cycle synchronization, overexpressing FUT8 in HaCaT cells accelerated cell proliferation by promoting G1-to-S phase transition and inducing cyclin A2, cyclin D and cyclin B1 expression. On the contrary, FUT8 knockdown caused cell cycle retardation by G2/M phase arrest through down-regulation of cyclin B1. Our study revealed the potential roles of Fut8 in psoriasis severity and keratinocyte proliferation. We believe that Fut8 may be a promising therapeutic target for psoriasis and possibly other skin inflammation diseases which involve dysregulated keratinocyte proliferation.