血管內皮細胞與平滑肌細胞共同培養下透過控制內皮細胞之beta-catenin在酪胺酸142上之磷酸化對於內皮細胞發炎反應之影響

EC/SMC Co-culture Modulates EC Inflammation by Regulating the Phosphorylation of beta-catenin at Tyrosine142

謝鎔澤(Rong-Ze Hsieh)

指導教授 ： 裘正健 ； 陳令儀

國立清華大學/生命科學院/分子醫學研究所/碩士(2012年)

https://doi.org/10.6843/NTHU.2012.00124

摘要

血管管壁是一種相當複雜的組織，在生理的功能上，被各種細胞和細胞間的交互作用所調控，例如細胞激素(cytokine)的分泌或者細胞和細胞之間的直接接觸(cell-cell direct contact)。在以下的研究裡，我們以血管細胞共同培養(vascular cell co-culture)的方式，試圖釐清內皮細胞(endothelial cells)和平滑肌細胞(smooth muscle cells)經由direct contact後對血管生理環境所帶來的影響。在共同培養的模式下，我們發現內皮細胞和平滑肌細胞間的交互作用造成內皮細胞的-catenin蛋白在tyrosine 142和serine 45/threonine 41兩種位置的磷酸化現象。而平滑肌細胞conditional medium的刺激只能造成Ser45/Thr41的活化，且將平滑肌細胞替換為纖維母細胞(fibroblast)的結果無法活化beta-catenin的磷酸化。另外，利用阻斷性胜肽(blocking peptide)和小干擾RNA (small interfering RNA, siRNA)技術我們證實connexin 43經由影響Fer kinase的活化調節beta-catenin的Tyr142的磷酸化。經由Cx43 siRNA和beta-catenin mutant plasmid的實驗，我們發現內皮細胞的vascular adhesion molecule-1 (VCAM-1)的基因表現和內皮細胞對於單核球的吸附(monocyte adhesion)能力也受到此訊息途徑的調控。本論文的研究結果證實內皮細胞和平滑肌細胞的直接接觸經由Cx43, Fer kinase和beta-catenin-Tyr142的磷酸化進而調節內皮細胞的發炎反應。關於解釋beta-catenin磷酸化現象，我們的研究也提出了其在調節血管細胞生理中其嶄新的腳色。

關鍵字

內皮細胞 ； 平滑肌細胞 ； 共同培養 ； 動脈硬化

並列摘要

The blood vessel wall is a complex structure and is highly modulated by homo- or heterocellular communications, such as cytokine release or cell-cell direct contact. In this study, I aim to elucidate the influence of direct heterocellular contact between endothelial cells (ECs) and smooth muscle cells (SMCs) by using a vascular cell co-culture model that is composed of monolayers of ECs and SMCs on opposite sides of a membrane. Under this co-culture system, the interaction between ECs and SMCs resulted in the phosphorylation of beta-catenin at tyrosine 142 (Tyr142) and serine 45/threonine 41 (Ser45/Thr41). ECs treated with SMC conditional medium induced beta-catenin phosphorylation at Ser45/Thr41 but not at Tyr142. The co-culture with fibroblasts (FBs) did not induce any beta-catenin phosphorylation. Moreover, by pre-treating cells with blocking peptides and using specific siRNA transfection, I demonstrated that connexin 43 modulated the phosphorylatin of beta-catenin-Tyr142 via Fer kinase. Transfecting ECs with siRNA specific for Cx43 or beta-catenin or beta-catenin-Tyr142 constitutive negative mutants inhibited vascular adhesion molecule-1 (VCAM-1) gene expression and thus modulated EC monocyte adhesion. These results indicate that vascular heterocellular communication regulates EC inflammation by inducing beta-catenin-Tyr142 phosphorylation through Cx43/Fer signaling via direct contact effects. My findings provide insights into the complexities of beta-catenin phosphorylation signaling in vascular cells, the mechanisms underlying the EC-SMC cross interaction in modulating signaling and gene expression in ECs and the consequent modulation of their inflammatory functions.

並列關鍵字

endothelial cells ； smooth muscle cells ； co-culture ； atherosclerosis ； beta-catenin

參考文獻

1. Hadi NR, Al-Amran F, Hussein MA, Rezeg FA. (2012) Evaluation of the effects of glimepiride (Amaryl) and repaglinide (novoNorm) on atherosclerosis progression in high cholesterol-fed male rabbits. J Cardiovasc Dis Res. 3:5-11.

2. Burnier L, Fontana P, Angelillo-Scherrer A, Kwak BR. (2009) Intercellular communication in atherosclerosis. Physiology (Bethesda). 24:36-44.

3. Chiu JJ, Chen LJ, Lee CI, Lee PL, Lee DY, Tsai MC, Lin CW, Usami S, Chien S. (2007) Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress. Blood. 110:519-28.

4. Shi W, Haberland ME, Jien ML, Shih DM, Lusis AJ. (2000) Endothelial responses to oxidized lipoproteins determine genetic susceptibility to atherosclerosis in mice. Circulation. 102:75-81.

5. Brisset AC, Isakson BE, Kwak BR. (2009) Connexins in vascular physiology and pathology. Antioxid Redox Signal. 11:267-82.