鞘氨醇 1-磷酸鹽引發人類內皮細胞表面之血小板/內皮細胞附著因子-1磷酸化機制之研究

Studies of the Mechanisms of Sphingosine 1-phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Phosphorylation in Human Endothelial Cells

黃鈺婷(Yu-Ting Huang)

指導教授 ： 李心予

國立臺灣大學/生命科學院/動物學研究研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00178

摘要

鞘氨醇 1-磷酸鹽 (Sphingosine 1-phosphate; S1P) 是一個具有多種生物功能性的小型磷脂鹽類，S1P可以和隸屬於G型蛋白耦合受器的專屬膜上受器結合，傳遞訊息並藉此廣泛地調控各種細胞的行為。目前已知在內皮細胞中受到S1P影響的生物功能包括細胞增生、附著、移行、傷口癒合、細胞基質切割、管狀結構的生成、黏著因子表現、細胞素與趨化因子的合成與分泌等等。血小板/內皮細胞附著因子-1 (PECAM-1) 是一個細胞膜上的醣蛋白，主要表現在內皮細胞、血小板及一些血液細胞上。內皮細胞層上的PECAM-1分子大多聚集在細胞側邊的邊界上，這些PECAM-1會利用其胞外的結構和鄰近細胞的PECAM-1形成連結，藉此協助細胞之間的聯繫並控制細胞上下兩側的通透性。除了結構上的功能外，PECAM-1在胞內的分子結構組成包含了兩個特殊的免疫受體酪氨酸抑制基序 (immunoreceptor tyrosine inhibitory motifs; ITIM)，當細胞遭遇到某些特定的外來刺激後，會使基序內含的酪氨酸產生磷酸化，磷酸化後的區位結構即會引發下游訊息分子的接合及活化，並引發後續的訊息傳遞及細胞生理活性的改變，然而在不同的來源細胞與外來刺激下，會分別經由不同種類的磷酸激酶來負責PECAM-1的磷酸化。目前已知PECAM-1在內皮細胞的移行、生存、管狀結構的生成、及免疫細胞對內皮細胞層的穿透過程中都扮演了很重要的角色。本篇研究目的在探討S1P引發人類臍靜脈內皮細胞上PECAM-1磷酸化的機制，首先使用免疫沉澱與西方墨點法來偵測蛋白質磷酸化態與總量的比例，觀察到S1P可以直接引發人類臍靜脈內皮細胞中PECAM-1的磷酸化，並確定這個磷酸化的現象與S1P的處理時間及處理濃度有關。在前處理了Gi與Src家族磷酸激酶的化學抑制劑後，可以確定這兩種訊息傳遞分子參與了這個磷酸化的過程。此外，我們利用西方墨點法與共軛焦顯微鏡驗證了在S1P處理後，位於側邊細胞邊界的活化態Src磷酸激酶呈現明顯的增加。為了確定是哪一個Src家族的磷酸激酶參與了PECAM-1磷酸化的過程，我們把目標鎖定在cSrc和Fyn這兩種細胞內主要的Src磷酸激酶成員上，將針對cSrc和Fyn的小型干擾核酸使用電破法轉殖入臍靜脈內皮細胞來截斷cSrc和Fyn的蛋白質表現，轉殖結束後持續培養細胞48小時再進行S1P的處理並比較不同的干擾核酸對S1P誘發PECAM-1磷酸化的效果，結果顯示cSrc和Fyn同時具有調控S1P所引發之PECAM-1磷酸化的能力。

關鍵字

鞘氨醇 1-磷酸鹽 ； 內皮細胞 ； 血小板/內皮細胞附著因子 ； 磷酸化

並列摘要

Sphingosine 1-phosphate (S1P) is a multifunctional bioactive lipid. Through a family of specific G protein-coupled receptors encoded by endothelial differentiation gene (edg), S1P regulates various cellular functions in different cell types. In endothelial cells, S1P has been demonstrated to modulate cell proliferation, adhesion, migration, wound healing, matix remodeling, tube formation, adhesion molecule expression, cytokine and chemokine releasing. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein which forms trans-homophilic bindings at cell-cell border. PECAM-1 performs both structural and signaling mediatory functions. Its cytoplasmic immunoreceptor tyrosine inhibitory motifs (ITIM) and tyrosine residues, when being phosphorylated, provide docking sites for various signaling molecules and modulate following cellular functions, such as cell migration, cell survival, tube formation, and transendothelial migration. However, the effect of S1P on endothelial PECAM-1 is still unknown, and the identification of the kinases responsible for PECAM-1 phosphorylation varies in different cell types upon different stimulations. In this study, we confirmed that S1P treatment induced PECAM-1 phosphorylation in human umbilical cord vein cells (HUVECs) by immunoprecipitation and following immunoblotting. The induction occurred immediately in less than three minutes. The induction could be block by pertussis toxin (PTx) and PP2 pretreatment, indicating the participation of Gi and Src family kinases. Results from immunoblotting and confocal microscopy proved that S1P also triggered the activation of Src family kinases at cell border. Finally, to identify the Src member responsible for PECAM-1 phosphorylation, specific siRNA was transfected to HUVECs. The obtained PECAM-1 phosphorylation ratio revealed that both cSrc and Fyn participated in this S1P-induced PECAM-1 phosphorylation.

並列關鍵字

sphingosine 1-phosphate ； endothelial cells ； PECAM-1 ； phosphorylation ； Src family kinases

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