AMPK訊息傳遞路徑在發炎反應中所扮演的角色

Abstract

AMPK是一個調節能量恆定的關鍵訊息分子，它也呈現了雙向調控發炎反應的功能，但它造成發炎的作用機制仍然不是很清楚。我們利用生化的技術將AICAR當成是AMPK的活化劑來更進一步闡述它在動脈血管平滑肌(VSMCs)、老鼠巨噬細胞(J774)和人類臍帶平滑肌(HUVECs)中分子間的交互作用。我們在幾種細胞測試中發現AICAR會誘導COX-2的表現。這個作用是藉由COX-2基因轉錄而來，而這個現象會被AMPK抑制劑:compound C和5’-iodotubercidin所移除。初步推測了AMPK在誘導COX-2的作用上所扮演的角色。在藥理學上和生化學上的研究證實，在這三種細胞中COX-2的產生，p38 MAPK的活化是位於AMPK的下游。進一步地，我們也發現TAK1會和AMPKα2產生交互作用，而且這二個分子彼此之間是需要各自的kinase區域來進行交互作用。更進一步的實驗結果顯示，在體內或體外實驗中，TAK1的磷酸化現象推測了其活化的階段會加強AMPK活化作用。我們的實驗結果首次證實TAK1在AMPK訊息軸上的重要地位，如此的交互作用賦予了AMPK在調節細胞能量的功能之外，又多了另一條路徑。在許多的細胞中，經由一個下游p38 MAPK的訊息路徑，再透過AMPK使得TAK1活化而致使COX-2基因的表現。 PKC在單核球貼附作用扮演了很重要的角色，目前對於PKC所傳遞細胞貼附作用的機制仍然不是很清楚。在這個研究中，我們探討了PMA所引起人類單核球貼附作用的訊息網路。我們利用藥理學上的抑制劑，其結果顯示AMPK、Syk、Src和ERK參與了PKC所傳遞的人類單核球貼附到培養盤的過程。生化學上的分析更加確定PMA活化了這些激酶的能力以及AMPK-Syk-Src這條訊息路徑參與其中。在PMA的刺激之下，AMPK和Syk兩個蛋白質直接進行交互作用；AMPK是需要kinase區域，而Syk則是需要linker region的區域。值得注意的是，我們鑑別出一個AMPK新的下游目標，而且AICAR可以誘導Syk在Ser 178的位置磷酸化並使其活化。然而，即使在AICAR刺激之下或是藉由蛋白質過量的表現，AMPK的單獨活化仍不足以使單核球產生貼附的作用。因此，在我們更進一步的研究中顯示：PKC所傳遞ERK這一條路徑(不經由AMPK活化的路徑)也參與了細胞的貼附作用。此外，在PMA誘導人類單核球和人類初級單核球貼附到內皮細胞是需要AMPK、Syk、Src和ERK的訊息。總而言之，我們推測有兩條路徑參與PMA所傳遞的單核球貼附作用。首先，PKC可以藉由活化LKB1/AMPK而使Syk磷酸化和活化，進而使Src與FAK活化。另外，PKC所傳遞ERK活化的路徑也呈現共同促使細胞骨架重新組合與細胞貼附作用。這是我們第一次證實Syk是一個AMPK新穎的受質，同時也闡述AMPK除了擁有能量恆定的功能外，在單核球貼附作用上也扮演了亮眼的角色。

AMP-activated protein kinase (AMPK), a critical signaling molecule for regulating energy homeostasis, might bi-directionally regulate inflammation, and its action mechanism leading to inflammation is not fully understood. We utilized 5-aminoimidazole-4-carboxamide riboside (AICAR) as a pharmacological activator of AMPK to unveil the effects of and signaling cascades mediated by AMPK on cyclooxygenase (COX)-2 gene expression in rat aortic vascular smooth muscle cells (VSMCs), murine macrophage cell line (J774), and human umbilical vein endothelial cells (HUVECs). Biochemical approaches were further conducted to elucidate interactions among signaling molecules. We found that AICAR could induce COX-2 protein expression in the cell types tested. This event was mediated by COX-2 gene transcription, and abrogated by compound C and 5’-iodotubercidin, suggesting the essential role of AMPK in COX-2 induction. Pharmacological and biochemical studies indicated that p38 mitogen activated protein kinase (MAPK) activation is the common downstream signal of AMPK in COX-2 expression in all three cell types. Furthermore, we also found that TAK1 is associated with AMPKα2, and this binding requires an interaction between the kinase domains of both molecules. Notably data of TAK1 phosphorylation indicate that the activating state is enhanced upon AMPK activation in vivo and in vitro. Our data for the first time prove a pivotal role of TAK1 in the AMPK signaling axis. Such interaction gives AMPK an additional pathway for regulating cellular functions. Via a downstream p38 MAPK signaling cascade, AMPK-dependent TAK1 activation leads to the expression of the inflammatory COX-2 gene in various cell types. PKC plays a pivotal role in mediating monocyte adhesion; however, the underlying mechanisms of PKC-mediated cell adhesion are still unclear. In this study, we elucidated the signaling network of phorbol ester PMA-stimulated human monocyte adhesion. Our results with pharmacological inhibitors suggested the involvement of AMPK, Syk, Src and ERK in PKC-dependent adhesion of THP-1 monocytes to culture plates. Biochemical analysis further confirmed the ability of PMA to activate these kinases, as well as the involvement of AMPK-Syk-Src signaling in this event. Direct protein interaction between AMPK and Syk, which requires the kinase domain of AMPK and linker region of Syk, was observed following PMA stimulation. Notably, we identified Syk as a novel downstream target of AMPK; AICAR can induce Syk phosphorylation at Ser178 and activation of this kinase. However, activation of AMPK alone, either by stimulation with AICAR or by overexpression, is not sufficient to induce monocyte adhesion. Studies further demonstrated that PKC-mediated ERK signaling independent of AMPK activation is also involved in cell adhesion. Moreover, AMPK, Syk and ERK signaling were also required for PMA to induce THP-1 cell adhesion to endothelial cells as well as to induce adhesion response of human primary monocytes. Taken together, we propose a bifurcated kinase signaling pathway involved in PMA-mediated adhesion of monocytes. PKC can activate LKB1/AMPK, leading to phosphorylation and activation of Syk, and subsequent activation of Src and FAK. In addition, PKC-dependent ERK activation induces a coordinated signal for cytoskeleton rearrangement and cell adhesion. For the first time we demonstrate Syk as a novel substrate target of AMPK, and shed new light on the role of AMPK in monocyte adhesion, in addition to its well identified functions in energy homeostasis.