內皮細胞中凝血酶調節素TM及轉錄因子KLF2受剪力調控及其訊息傳遞路徑之探討

Abstract

動脈粥狀硬化的致病過程中，發炎反應 (inflammation) 扮演十分重要的角色，在血管內皮細胞中的凝血酶調節素 (thrombomodulin；簡稱TM)，具有抗凝血及抗發炎的功能；轉錄因子KLF2則是被發現具有藉調控TM及eNOS等多種基因表現，以調節凝血機制之特性，因此，本研究針對TM及KLF2的表現及調控作為目標。由於動脈粥狀硬化的初始及發展與血液流動產生之剪力作用於血管內皮細胞造成的各種反應密切相關，本研究使用人類臍帶靜脈內皮細胞 (HUVEC) 及牛動脈內皮細胞 (BAEC)，探討剪力刺激對細胞中TM及KLF2的表現及其調控之訊息傳遞路徑。 在高剪力 (25 dyn/cm2) 刺激不同時間下，TM mRNA表現量屬於暫態誘發，在大約四到六小時之間達到最高量，約為控制組的1.6倍左右，隨著時間增長，表現量會回到基礎值；而KLF2 mRNA則是屬於持續誘發，最高表現量可以達到約為控制組的85倍左右；但是就蛋白質表現而言，剪力作用對TM及KLF2的蛋白質量卻沒有明顯的增加。比較不同大小剪力對細胞的刺激，在高剪力 (25 dyn/cm2) 刺激狀態下，TM及KLF2 mRNA都有明顯的增加表現，而低剪力 (4.5 dyn/cm2) 的作用較不明顯，表示在高剪力的環境下對於細胞有較佳的保護作用；然而，TM及KLF2的蛋白質表現量似乎都沒有太大變化，推測可能剪力會對於TM及KLF2 mRNA的穩定性進行調控所造成。欲探討剪力對於兩者的調控路徑，使用NO donor (即NOC18) 及eNOS inhibitor (即L-NAME)，結果發現單獨剪力刺激時所生成的NO可能對於細胞中的TM及KLF2具有保護效果，若對eNOS進行抑制降低NO量，則有可能會造成TM啟動子活性表現降低；但是當NO的量超過正常狀態甚多，細胞中可能產生回饋抑制效應，降低TM及KLF2的表現。 此外，本研究亦針對在細胞激素刺激下來探討剪力對TM及KLF2的保護作用，當加入TNF-α會使得TM mRNA的表現量有明顯下降，若同時有剪力的刺激下，TNF-α對TM mRNA的抑制現象則可被逆轉而回復到正常表現量，證明了剪力對於細胞激素所造成的細胞傷害是具有保護作用的；相較之下，KLF2受到TNF-α的影響並不是十分明顯，因此，TNF-α似乎並不參與在剪力調控KLF2的訊息傳導路徑中。 綜合以上實驗結果，剪力對於TM及KLF2誘發其表現可能經由mRNA及蛋白質之穩定性調控的機制，推測剪力有可能是經由活化eNOS產生NO調控了細胞中的氧化還原狀態，進而對TM的表現產生調控；KLF2則是經由其他的路徑受到剪力調控；在TNF-α與剪力的雙重作用下，剪力對於TNF-α所造成的細胞傷害是具有保護功能的，它可以使TM mRNA表現量回復到正常範圍，達到保護心血管系統的功效。

During atherogenesis, inflammation plays an important role. Thrombomodulin (TM), in vascular endothelial cells, has anti-coagulation and anti-inflammation properties. Transcription factor, Kruppel-like factor 2 (KLF2) has been shown to participate in the regulation of the expression of TM and endothelial nitric oxide synthase and thus is important for regulating thrombotic function. Endothelial cells are constantly under the influence of flow-induced hear stress and the atherosclerotic lesions are closely related to this hemodynamic effect. In the present study, we focused on the study of gene expression of TM and KLF2 in endothelial cells exposed to shear stress. Human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) were used to probe into the signaling mechanisms involved in this shear-induced TM and KLF2 expression. ECs exposed to shear stress of 25 dyn/cm2 transiently induced the TM mRNA expression with peak induction about 1.6-folds after exposure to shear stress for 4 to 6 hrs before it returned to basal level. However, KLF2 mRNA expression in those ECs was found to be a sustained one with peak induction more than 85-folds. While mRNA expression of TM and KLF2 were significantly induced in shear-induced ECs, protein expression of TM and KLF2 had no obvious increase. Furthermore, TM mRNA and KLF2 mRNA were significantly increased in ECs exposed to higher shear stress (25 dyn/cm2) in contrast to those ECs exposed to low shear stress (4.5 dyn/cm2) indicating that TM mRNA induction is sensitive to shear force. However, the protein expression of TM and KLF2 from shear-treated ECs show no significant difference as compared to those control ECs. This indicates that protein stability may be involved in those shear-treated ECs. ECs under shear flow condition constantly release nitric oxide (NO). The role of NO in this shear-induced TM and KLF2 mRNA expression was examined. ECs exposed to a NO donor (NOC18) significantly suppressed the TM and KLF2 mRNA level with a dose-dependent manner. Consistently, shear stress to ECs increased the TM promoter activity. Interestingly, ECs pretreated with an eNOS inhibitor (L-NAME) did not enhance the shear-induced TM promoter activity. Furthermore, the protection effect of shear flow on cytokine-treated ECs was examined. ECs treated with tumor necrosis factor (TNF) greatly reduced the TM and KLF2 expression. ECs pretreated with TNF followed with shear flow, however, significantly attenuated the TNF-induced suppression of TM mRNA levels. In contrast, the basal KLF2 and the shear-induced KLF2 mRNA levels were not affected by TNF pretreatment. In summary, shear flow to ECs increases the TM and KLF2 mRNA expression in a dose-dependent manner. However, this induction can not be reflected by the TM protein levels indicating that the stability of TM mRNA and protein may be involved. Furthermore, shear flow exerts its protective effect by attenuating the TNF-induced suppression of TM mRNA expression. ECs treated with NO suppress the TM and KLF2 mRNA expression. Although the detailed up-regulation mechanism of TM remains unclear, this study suggests that shear flow plays an important role in regulating TM expression and consequently affects endothelial integrity.