Signaling pathways for heme oxygenase-1 protein expression by LCY-2-CHO, cyclooxygenase inhibitors and AMPK activator.

• Main Authors: Hao-Cheng Kang, 康皓程
• Other Authors: Wan-Wan Lin
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/06206950260167022536

碩士 === 國立臺灣大學 === 藥理學研究所 === 93 === 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.