LCY-2-CHO、環氧化酵素抑制劑及AMPK活化劑誘導甲型血基質氧化酵素表現之訊息傳導路徑

Abstract

許多形式的細胞壓力都會誘導Heme oxygenase-1 (HO-1)的表現以執行細胞層級的防衛機制來對抗組織性傷害，也因為具有這種細胞保護功能，使得HO-1的調控被認為是一個極有可能的切入點以求應用到臨床來治療一些由細胞凋亡、發炎機制、氧化性壓力或動脈粥狀硬化所造成的疾病。 LCY-2-CHO為一咔唑類化合物，目前已被證明在巨噬細胞中可以抑制由LPS所誘發的促發炎介質及蛋白的表現。此外，LCY-2-CHO亦可減少嗜中性白血球的顆粒性發炎介質的釋放以及超氧陰離子自由基的產生。在本篇論文，我們證明了LCY-2-CHO在血管平滑肌細胞可經由活化p38 MAPK、PKC、PI3K-Akt和eNOS以及Nrf2轉錄因子的細胞核轉位作用來增加HO-1基因的表現。LCY-2-CHO亦可藉由減少IKKα的磷酸化以及抑制IκBα的降解來減少由刺激劑所誘發的促發炎介質及蛋白(如iNOS、COX-2、GRO-α和IL-8)的表現。另外，LCY-2-CHO也會造成PAI-1蛋白表現大量減少。綜合上述結果，我們確定了LCY-2-CHO在血管平滑肌細胞的抗發炎作用以及其應用於動脈粥狀硬化治療的可能性。 COX-2是前列腺素此種發炎介質生合成過程中的重要速率決定酵素，因此在發炎作用中扮演了中樞角色。非固醇類抗發炎藥物(NSAIDs)的主要藥理作用即是藉由抑制COX的活性來減少eicosanoids的合成。我們的實驗結果顯示無論是在老鼠血管平滑肌細胞、人類臍靜脈內皮細胞或是老鼠巨噬細胞(J774)，許多COX抑制劑都可以誘導HO-1蛋白的表現。以celecoxib為例，我們發現其增加HO-1 mRNA及蛋白表現的作用會被外加的PGE2所抑制，然而若單獨外加PGE2卻亦會誘導HO-1的表現。相反地，外加15dPGJ2反而會和COX抑制劑一起產生加成效果。因此，內生性適度的PGE2釋放對於HO-1的恆定是扮演負向調控的角色。我們亦觀察到celecoxib 可以活化HO-1基因轉錄過中所必需的轉錄因子，Nrf2和HIF-1α。此外，因為celecoxib誘導HO-1蛋白表現的作用可被MEK、p38 MAPK 及PI3K抑制劑所抑制，再加上celecoxib的確可以活化ERK、p38 MAPK及Akt，因此我們認為藉由COX抑制劑減少內生性PGE2生成的作用會影響細胞中訊息傳遞路徑的平衡，進而啟動ERK、p38 MAPK及PI3K-Akt的訊息傳導路徑，來造成Nrf2及HIF-1α的活化及核轉移作用以誘發HO-1基因的表現。這些發現將可提供COX抑制劑其抗發炎作用機轉一個新的方向。 AMPK是一種可以感知能量變化的酵素，尤其是細胞面臨能量缺乏的壓力時，如細胞內ATP量的減少或AMP量的增加時，AMPK就會被活化而調控一連串的訊息傳導路徑，來保護生物體免於代謝失衡的疾病。在各種不同的細胞，AMPK與發炎反應之間的關係仍然是備受爭議的，因此我們利用平滑肌細胞來探討AMPK在發炎過程中所可能扮演的角色。我們發現AICAR(AMPK致活劑)會誘導iNOS、COX-2及HO-1蛋白的表現。對於iNOS而言，AICAR活化AMPK後，可進一步的活化PKC、p38 MAPK及Akt，其中又主要以PKC的活化來造成IKK的磷酸化，進一步使p65轉錄因子轉位至核內，形成NF-κB與同源DNA片段結合以啟動iNOS基因的轉錄。對於COX-2而言，AICAR透過活化AMPK可進一步的活化PKC及p38，造成C/EBPβ的核轉位作用，而增加COX-2 mRNA及蛋白的表現。此外，對於HO-1來說，除了AMPK所活化的PKC、p38 MAPK及Akt之外，PKA亦傳遞訊息來造成HO-1蛋白表現的增加。雖然AICAR增加HO-1來保護細胞的現象與其增加iNOS與COX-2所造成的促發炎作用似乎存在有矛盾之處，然而，我們認為AICAR具有雙面的能力而以極為精細的方式來適當的調控細胞的發炎狀態。

Heme oxygenase-1 (HO-1) is induced by several forms of cellular stress, and exerts multiple cellular defense mechanisms against tissue injury. This important cytoprotective action makes it conceivable to target HO-1 induction as a promising therapeutic intervention in treating a variety of disorders related to cell apoptosis, inflammation, oxidation and atherosclerosis. A synthetic carbazole, LCY-2-CHO, has been proved to inhibit the expression of LPS-induced pro-inflammatory mediators and proteins production in macrophages. In addition, it also attenuates neutrophil degranulation and superoxide anion generation, responsible for directly down-regulating leukocyte functions. In the present study, we demonstrate that in rat VSMC, LCY-2-CHO, via the ability to activate p38 MAPK, PKC, PI3K-Akt, and eNOS as well as the nuclear translocation of Nrf2, increases the gene transcription of HO-1. LCY-2-CHO also can reduce the increase of stimuli-induced pro-inflammatory mediators and proteins, including iNOS, COX-2, GRO-α, and IL-8. In addition, incubation of VSMC with LCY-2-CHO leads to the decrease of PAI-1 protein expression. Inhibitory effect on NF-κB activation was evidenced by the diminishment of IKKα phosphorylation and IκBα degradation. Taken together, given anti-inflammatory properties in VSMC, these results highlight the therapeutic potential of LCY-2-CHO in atherosclerosis. COX-2 is a key rate-limiting enzyme in the biosynthesis of inflammatory mediators prostaglandins and plays pivotal roles in inflammatory situations. The reduction of eicosanoids by inhibitory COX activity has been the primary therapeutic action of nonsteroid anti-inflammatory drugs (NSAIDs). Our results performed in rat VSMC, HUVEC and murine J774 macrophages indicate the ability of several COX inhibitors to induce HO-1 protein expression. Taken celecoxib as an example, we found HO-1 mRNA level was concomitantly increased. NSAIDs induced HO-1 protein expression was arrested by exogenous PGE2, despite that PGE2 itself also possessed HO-1 inducing capability. In contrast, 15dPGJ2 treatment leads to an additive response to COX inhibitors, suggesting that tonic releasing PGE2 maybe a negative player to control HO-1 homeostasis. We also observed that essential transcription factors for HO-1 gene transcription, Nrf2 and HIF-1α, can be activated by celecoxib. Moreover, celecoxib effect is attenuated by MEK, p38 MAPK and PI3K inhibitors. Concomitantly celecoxib is able to activate p38 MAPK and Akt with more prominence than ERK. We suggest that removal endogenous PGE2 production through COX inhibition triggers signaling pathways of p38, PI3K and ERK, leading to activate Nrf2 and HIF-1α, and in turn HO-1 gene induction. These findings provide new light on the action mechanism of COX inhibitors for wide implication in anti-inflammation. AMP-activated protein kinase (AMPK), an energy-sensing enzyme that is activated in response to cellular stress causing ATP depletion, is a critical signaling molecule for the regulation of energy homeostasis and might play a part in protecting the body from metabolic diseases. The relation between AMPK and inflammation is still controversial in different cell types, thus we utilized rat VSMC to investigate the possible role of AMPK in the inflammatory process. In this study, we found AICAR, a pharmacological activator of AMPK, induces iNOS, COX-2 and HO-1 protein expression. For the iNOS induction, AMPK, activated by AICAR, may be able to activate PKC, p38, and Akt, and among them, activated PKC is the major factor responsible for the AICAR-induced IKK phosphorylation, which can further cause p65 nuclear translocation, essential for NFκB binding to cognate DNA element and then iNOS geneexpression. For the COX-2 induction, AICAR, through the activation of AMPK, may cause PKC and p38 activation, which can further promote the C/EBPβ nuclear translocation, and then up-regulate COX-2 mRNA and protein expression. In addition, for HO-1 mRNA and protein induction, AMPK-mediated p38, PKC and Akt play a major role. In summary, AICAR is suggested to have the dual-side ability to finely well-regulate the cellular inflammation state. AICAR-stimulated induction of HO-1, a cytoprotective protein seems to run counter to the pro-inflammatory effect in terms of iNOS and COX-2 protein expression.