Abstract
The upregulation of heme oxygenase-1 (HO-1) by the carbon monoxide-releasing molecule (CORM)-2 may be mediated through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [Nox] and reactive oxygen species (ROS) generation, which could provide cytoprotection against various cellular injuries. However, the detailed mechanisms of CORM-2-induced HO-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain largely unknown. Therefore, we dissected the mechanisms underlying CORM-2-induced HO-1 expression in HPAEpiCs. We found that the administration of mice with CORM-2 attenuated the tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule-1 (ICAM-1) expression and leukocyte count as revealed by immunohistochemical staining, western blot, real-time polymerase chain reaction (PCR), and cell count. Furthermore, TNF-α-induced ICAM-1 expression associated with monocyte adhesion to HPAEpiCs was attenuated by infection with adenovirus (adv)-HO-1 or incubation with CORM-2. These inhibitory effects of HO-1 were reversed by pretreatment with hemoglobin (Hb). Moreover, CORM-2-induced HO-1 expression was mediated via the phosphorylation of p47phox, c-Src, epidermal growth factor receptor (EGFR), Akt, and NF-E2-related factor 2 (Nrf2), which were inhibited by their pharmacological inhibitors, including diphenyleneiodonium (DPI) or apocynin (APO), ROS [N-acetyl-L-cysteine (NAC)], PP1, AG1478, PI3K (LY294002), or Akt (SH-5), and small interfering RNAs (siRNAs). CORM-2-enhanced Nrf2 expression, and anti-oxidant response element (ARE) promoter activity was also inhibited by these pharmacological inhibitors. The interaction between Nrf2 and AREs was confirmed with a chromatin immunoprecipitation (ChIP) assay. These findings suggest that CORM-2 increases the formation of the Nrf2 and AREs complex and binds with ARE-binding sites via Src, EGFR, and PI3K/Akt, which further induces HO-1 expression in HPAEpiCs. Thus, the HO-1/CO system might suppress TNF-α-mediated inflammatory responses and exert a potential therapeutic strategy in pulmonary diseases.
Highlights
Heme oxygenase (HO), a rate-limiting enzyme, metabolizes heme into biliverdin-IXα, ferrous iron, and carbon monoxide (CO)
These inhibitory effects of carbon monoxide-releasing molecule (CORM)-2 were mediated via the upregulation of heme oxygenase-1 (HO-1), which were reversed by pretreatment with the inhibitor of HO-1 activity (ZnPP IX) (Figure 1B), suggesting that HO-1 induction by CORM-2 protects against inflammatory responses in mice challenged with TNF-α
Our results showed that CORM-2-induced reactive oxygen species (ROS) generation and HO-1 expression were inhibited by DPI or APO, suggesting that NADPH oxidase (Nox) plays an important role in these responses
Summary
Heme oxygenase (HO), a rate-limiting enzyme, metabolizes heme into biliverdin-IXα, ferrous iron, and carbon monoxide (CO). The roles of Nox/ROS, c-Src, EGFR, PI3K/Akt, and Nrf2/AREs in CORM-2-induced HO-1 expression were investigated in HPAEpiCs. several reports have shown that when the exogenous application of the HO-1 end-product CO is administered at low concentrations, or alternatively, by pharmacological application of CORMs, it can confer protective effects in models of inflammatory response. The experiments in this study were performed to dissect the mechanisms by which CORM-2 induces HO-1 expression mediated via Nrf2/AREs activation in HPAEpiCs and suppressed TNF-α-mediated inflammatory responses. These findings suggested that in HPAEpiCs, CORM-2-induced HO-1 expression is, at least in part, mediated through a Nox2/ROS/c-Src/EGFR/ PI3K/Akt-dependent Nrf2/AREs pathway
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