探討oxLDL在人類臍帶靜脈內皮細胞ECV304經由調控PI3K/Akt 和Ras/ERK途徑來活化 hypoxia-inducible factor-1α的作用機轉

Abstract

在粥狀動脈硬化初期，氧化態低密度脂蛋白 (oxidized low density lipoprotein，oxLDL) 和內皮細胞之反應扮演著重要的角色。轉錄因子HIF-1的活性增加會導致下游調控的基因增加，這些基因可以參與在能量代謝適應、細胞存活和增生、血管新生、細胞侵入和轉移以及血管張力等病理疾病的過程中。 在本篇實驗中，我們發現到oxLDL (200μg/ml) 會誘使內皮細胞株ECV304中HIF-1α蛋白質增加。由luciferase reporter gene assay中，我們也確定了oxLDL會使HIF-1活性增加的現象。然而，本篇實驗中我們也發現到oxLDL並不會影響HIF-1α的轉錄作用，也無影響pVHL蛋白質表現和與ubiquitin結合的HIF-1α的分解層面中。 當我們投與激酶的抑制劑，譬如wortmannin (100 nM)、rapamycin (100 nM)、manumycin A (1μM) 和PD98059 (50μM)，皆可以有效地減少oxLDL誘導HIF-1活性增加的現象。由西方點墨法，我們看到oxLDL可以活化Akt和ERK以及它們下游的激酶蛋白，如mTOR、S6K、4EBP 和eIF-4E，由這些實驗結果可以知道oxLDL是有參與在HIF-1α蛋白質形成的轉譯作用中。更進一步地，我們利用dominant-negative (DN) transfection的技術，送入DN-Akt 和DN-Ras 發現都能有效的抑制由oxLDL所造成HIF-1α蛋白質增加的結果。綜合上述的實驗結果，在人類臍靜脈內皮細胞株ECV304中，oxLDL可以增加轉錄因子HIF-1的活性和HIF-1α蛋白質合成，主要是經由調控PI3K/Akt和 Ras/ERK這兩條訊息傳遞途徑而來。因此，轉錄因子HIF-1在oxLDL所造成的粥狀動脈硬化病程中，扮演著重要的角色。

Oxidized low density lipoprotein (oxLDL) and endothelial cells play central roles in the early stage of atherosclerosis. Increased HIF-1 activity leads to upregulation of genes that are involved in many aspects of pathological diseases, including metabolic adaptation, cell survival/proliferation, angiogenesis, invasion/metastasis and vascular tone. In this study, we demonstrated that oxLDL (200μg/ml) induced hypoxia-inducible factor-1α(HIF-1α) protein accumulation in human endothelial cell line (ECV304) under normoxic condition. HIF-1-dependent luciferase reporter gene analysis underscored HIF-1 transactivation. OxLDL has no effect on HIF-1α transcriptional and degradational levels, including pVHL protein expression and association with ubiquitin. OxLDL-induced HIF-1αactivity was attenuated by the pretreatment with wortmannin (100 nM), rapamycin (100 nM), manumycin A (1 μM) and PD98059 (50 μM). Western blotting analysis showed that oxLDL induced the phosphorylation of Akt , ERK and their downstream protein kinases, including mTOR, S6K, 4EBP and eIF-4E, which served to regulate HIF-1α expression at translational step. Moreover, transfection of a dominant-negative vector of Akt or Ras resulted in abolishing the HIF-1αproduction triggered by oxLDL. These results indicated that oxLDL induced HIF-1-dependent reporter gene activation and HIF-1α accumulation in ECV304 via PI3K/Akt and Ras/ERK pathways. These findings may suggest a role of HIF-1 in atherosclerosis and oxLDL-induced pathogenesis.