在人類臍靜脈內皮細胞中NF-κB在黃體素誘導p53向上調節的角色

Abstract

內皮細胞參與多樣化的生理過程，包括血管生成、止血及發炎。在本實驗室先前研究指出，黃體素在生理濃度下(500 nM)會抑制人類臍靜脈內皮細胞的增生，此種抑制作用是透過增加p53的表現來達成。在給予黃體素處理後的十分鐘內就可觀察到細胞內磷酸化的ERK蛋白質量的增加，但是黃體素處理後所誘導的p53增加則在給藥後二十三小時才會出現。使用NCBI資料庫分析，發現p53基因啟動子上面有NF-κB、HLH及EBPβ等轉錄因子結合位置，在許多細胞內轉錄因子當中，NF-κB被認為與血管細胞血管增生功能有密切關係，本篇的研究主題是觀察NF-κB在黃體素誘導p53向上調節所扮演的角色。經由分析細胞中蛋白質表現量發現，黃體素並不會刺激細胞內NF-κB的總量增加，但是在黃體素誘導p53蛋白表現量增加之前的時間點可觀察到IκBα有被磷酸化的現象，之後NF-κB經由細胞質進入細胞核，增加NF-κB與p53基因的結合，影響p53的表現。使用NF-κB抑制劑Bay11-7082可以減少黃體素增加p53的表現。本實驗室的先前研究結果顯示黃體素是透過活化ERK路徑，增加p53蛋白的表現。本研究給予ERK-1/2抑制劑 U0126或PD98059會減少IκBα的磷酸化作用，顯示黃體素增加IκBα的磷酸化作用是透過活化ERK。利用基因序列刪除法，我們的實驗結果顯示NF-κB結合位置對於黃體素誘導p53啟動子活性具有決定性的角色，而HLH及EBPβ結合位置對於p53基因啟動子也提供部份調節的作用。我們的研究亦發現黃體素受器PR-A，在經過黃體素刺激後會進入細胞核內，而PR-B沒有出現這種現象；從免疫共沈澱的實驗結果中，發現到NF-κB與PR-A的結合有增加的現象，而這二者因子結合增加的現象，暗示PR-A可能會調節NF-κB的作用來調控p53的表現，進而抑制血管內皮細胞的增生。

Endothelial cells are involved in a diversity of physiologic processes including angiogenesis, hemostasis, and inflammation. Previously, our laboratory has demonstrated that progesterone at a physiological concentration (500 nM) inhibited proliferation of human umbilical venous endothelial cells (HUVEC) through a p53-dependent pathway and the ERK-mediated pathway is involved in the progesterone-induced increase of p53. The progesterone-induced increase of the level of phosphorylated ERK was observed at 10 min after treatment, while the increased p53 level was not observed until 23 h after treatment. Based on the NCBI database, the human p53 gene promoter contains three transcription factor binding sites including NF-κB, HLH, and EBPβ. It has been indicated that NF-κB might be involved in the regulation of angiogenesis. Accordingly, the aim of this study is to investigate how NF-κB regulates p53 in HUVEC. Our data showed that the total protein levels of NF-κB in progesterone-treated HUVEC were not changed significantly. However, progesterone increased IκBα phosphorylation as well as NF-κB nuclear translocation and binding onto the p53 promoter. Treatment of HUVEC with NF-κB inhibitor, Bay11-7082, reduced the progesterone-induced increase of p53, suggesting that progesterone induced increase of p53 through increase of IκBα phosphorylation, which in turn increased NF-κB nuclear translocation and binding onto the p53 promoter. Treatment of HUVEC with an ERK inhibitor, U0126 or PD98059, reduced the progesterone-induced increase of IκBα phosphorylation, suggesting that progesterone increased IκBα phosphorylation through the ERK pathway. Using gene deletion, our data suggest that the NF-κB binding site on the p53 gene promoter is essential for progesterone-induced p53 up-regulation, wherease the HLH and EBPβ binding sites might also play important roles in regulating the p53 promoter activity. Progesterone receptor-A (PR-A), but not PR-B, translocated into the nucleus after treatment with progesterone. Immunoprecipitation showed that progesterone increased the binding between NF-κB and PR-A. These results suggested that PR-A might participate in NF-κB-regulated p53 expression and further inhibited the proliferation of vascular endothelial cells.