Compound C stimulates heme oxygenase-1 gene expression via the Nrf2-ARE pathway to preserve human endothelial cell survival
Graphical abstract
Introduction
Compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine) is a cell-permeable pyrrazolopyrimidine derivative that functions as a potent ATP-competitive inhibitor of adenosine monophosphate-activated protein kinase (AMPK) [1]. Although compound C is frequently employed to determine the role for AMPK in various biological processes, several AMPK-independent actions of compound C have been discovered. Recently, compound C has been found to inhibit the activation of hypoxia-inducible factor-1, bone morphogenetic protein type I receptors, and several kinases in an AMPK-independent fashion [2], [3], [4]. In addition, compound C exerts potent anti-proliferative and pro-apoptotic effects in various cell lines through AMPK-independent mechanisms [5], [6], [7], [8]. Interestingly, we recently identified AMPK as a novel inducer of HO-1 in endothelial cells and surprisingly found that compound C could likewise stimulate the expression of HO-1 in these cells, suggesting another AMPK-independent action of this compound [9].
Heme oxygenase-1 (HO-1) is an inducible enzyme that oxidatively degrades heme to generate equivalent molar amounts of carbon monoxide (CO), biliverdin, and ferrous iron [10]. Subsequently, biliverdin is metabolized to bilirubin by biliverdin reductase while iron is sequestered by ferritin and either secreted by cells or recycled for heme biosynthesis. HO-1 is a highly inducible gene that is activated by a number of transcription factors; however, NF-E2-related factor 2 (Nrf2) has emerged as the predominant mediator of HO-1 gene transcription [11]. Activation of Nrf2 is regulated by the cytosolic protein Kelch-like ECH-associated protein 1 (Keap1) that negatively modulates the nuclear translocation of Nrf2 and facilitates degradation of Nrf2 via the proteasome. Upon activation, Nrf2 enters the nucleus where it binds to the antioxidant responsive element (ARE) of the HO-1 promoter to trigger gene expression. Nrf2 contributes to the induction of HO-1 in response to various forms of cellular stress, including oxidative, nitrosative, hemodynamic, and endoplasmic reticulum stress [12], [13], [14], [15]. The induction of HO-1 in response to stress provides a crucial defense mechanism against cell injury [16], [17], [18]. The protective functions of HO-1 are attributable to its ability to degrade the pro-oxidant and pro-inflammatory molecule heme and to its enzymatic reaction products. In particular, the bile pigments biliverdin and bilirubin are potent anti-oxidants that are capable of scavenging various oxidants while CO is a diatomic gas that prevents cell death in response to a large number of inimical stimuli.
In the present study, we investigated the mechanism by which compound C stimulates the expression of HO-1 in human endothelial cells. Furthermore, we determined the functional significance of the induction of HO-1 by compound C in vascular endothelium.
Section snippets
Materials
Penicillin, gelatin, uric acid, methyl-l-arginine, streptomycin, Nonidet P40, rotenone, antimycin A, allopurinol, dithiothreitol, sodium dodecyl sulfate (SDS), NaCl, EDTA, ferrous iron, glycerol, ethidium bromide, Triton X-100, heparin, trypan blue, N-acetyl-l-cysteine, Tris, Hepes, glutathione, tricarbonyldichlororuthenium (II) dimer (CORM2), and compound C were from Sigma–Aldrich (St. Louis, MO). Phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and pepstatin A were from Roche Applied
Results
Treatment of endothelial cells with compound C stimulated a concentration- and time-dependent increase in HO-1 protein. The induction of HO-1 protein by compound C was delayed, with a significant increase in HO-1 protein appearing 8 h after compound C administration, and levels remained elevated following 24 h of treatment (Fig. 1A). An increase in HO-1 protein was detected after 24 h with 2 μM of compound C and higher concentrations of compound C resulted in a progressive increase in HO-1 protein (
Discussion
The present study identifies compound C as a potent inducer of HO-1 gene expression in human endothelial cells. The induction of HO-1 is mediated by the Nrf2-ARE signaling pathway and is dependent on the production of reactive oxygen species. In addition, the induction of HO-1 functions to limit the cytotoxic effect of compound C through the generation of biliverdin, bilirubin, and CO. The ability of compound C to stimulate HO-1 expression may contribute to the pleiotropic actions of this agent
Acknowledgements
This work was supported by the National Institutes of Health Grants HL74966, HL59976, and HL77288.
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