Heme oxygenase-1 (HO-1), a ubiquitous inducible stress-response protein, serves a major metabolic function in heme turnover. HO activity cleaves heme to form biliverdin-IXα, carbon monoxide (CO), and iron. Genetic experiments have revealed a central role for HO-1 in tissue homeostasis, protection against oxidative stress, and in the pathogenesis of disease. Four decades of research have witnessed not only progress in elucidating the molecular mechanisms underlying the regulation and function of this illustrious enzyme, but also have opened remarkable translational applications for HO-1 and its reaction products. CO, once regarded as a metabolic waste, can act as an endogenous mediator of cellular signaling and vascular function. Exogenous application of CO by inhalation or pharmacologic delivery can confer cytoprotection in preclinical models of lung/vascular injury and disease, based on anti-apoptotic, anti-inflammatory, and anti-proliferative properties. The bile pigments, biliverdin and bilirubin, end products of heme degradation, have also shown potential as therapeutics in vascular disease based on anti-inflammatory and anti-proliferative activities. Further translational and clinical trials research will unveil whether the HO-1 system or any of its reaction products can be successfully applied as molecular medicine in human disease.
carbon monoxide; bilirubin; heme oxygenase-1; lung injury
Carbon monoxide (CO) is a biologically active molecule produced in the body by the stress-inducible enzyme, heme oxygenase. We have previously shown that CO suppresses fibrosis in a murine bleomycin model. To investigate the mechanisms by which CO opposes fibrogenesis, we performed gene expression profiling of fibroblasts treated with transforming growth factor-β1 and CO. The most highly differentially expressed categories of genes included those related to muscular system development and the small proline-rich family of proteins. We confirmed in vitro, and in an in vivo bleomycin model of lung fibrosis, that CO suppresses α–smooth muscle actin expression and enhances small proline-rich protein-1a expression. We further show that these effects of CO depend upon signaling via the extracellular signal–regulated kinase pathway. Our results demonstrate novel transcriptional targets for CO and further elucidate the mechanism by which CO suppresses fibrosis.
carbon monoxide; heme oxygenase-1; lung fibrosis; small proline-rich protein; α-smooth muscle actin
Acute lung injury (ALI) is a major cause of morbidity and mortality in critically ill patients. Hyperoxia causes lung injury in animals and humans, and is an established model of ALI. Caveolin-1, a major constituent of caveolae, regulates numerous biological processes, including cell death and proliferation. Here we demonstrate that caveolin-1–null mice (cav-1−/−) were resistant to hyperoxia-induced death and lung injury. Cav-1−/− mice sustained reduced lung injury after hyperoxia as determined by protein levels in bronchoalveolar lavage fluid and histologic analysis. Furthermore, cav-1−/− fibroblasts and endothelial cells and cav-1 knockdown epithelial cells resisted hyperoxia-induced cell death in vitro. Basal and inducible expression of the stress protein heme oxygenase-1 (HO-1) were markedly elevated in lung tissue or fibroblasts from cav-1−/− mice. Hyperoxia induced the physical interaction between cav-1 and HO-1 in fibroblasts assessed by co-immunoprecipitation studies, which resulted in attenuation of HO activity. Inhibition of HO activity with tin protoporphyrin-IX abolished the survival benefits of cav-1−/− cells and cav-1−/− mice exposed to hyperoxia. The cav-1−/− mice displayed elevated phospho-p38 mitogen-activated protein kinase (MAPK) and p38β expression in lung tissue/cells under basal conditions and during hyperoxia. Treatment with SB202190, an inhibitor of p38 MAPK, decreased hyperoxia-inducible HO-1 expression in wild-type and cav-1−/− fibroblasts. Taken together, our data demonstrated that cav-1 deletion protects against hyperoxia-induced lung injury, involving in part the modulation of the HO-1–cav-1 interaction, and the enhanced induction of HO-1 through a p38 MAPK–mediated pathway. These studies identify caveolin-1 as a novel component involved in hyperoxia-induced lung injury.
acute lung injury; acute respiratory distress syndrome; caveolin-1; heme oxygenase-1
Cigarette smoke–induced apoptosis and necrosis contribute to the pathogenesis of chronic obstructive pulmonary disease. The induction of heme oxygenase-1 provides cytoprotection against oxidative stress, and may protect in smoking-related disease. Since mitochondria regulate cellular death, we examined the functional expression and mitochondrial localization of heme oxygenase-1 in pulmonary epithelial cells exposed to cigarette smoke extract (CSE), and its role in modulating cell death. Heme oxygenase-1 expression increased dramatically in cytosolic and mitochondrial fractions of human alveolar (A549), or bronchial epithelial cells (Beas-2b) exposed to either hemin, lipopolysaccharide, or CSE. Mitochondrial localization of heme oxygenase-1 was also observed in a primary culture of human small airway epithelial cells. Furthermore, heme oxygenase activity increased dramatically in mitochondrial fractions, and in whole cell extracts of Beas-2b after exposure to hemin and CSE. The mitochondrial localization of heme oxygenase-1 in Beas-2b was confirmed using immunogold-electron microscopy and immunofluorescence labeling on confocal laser microscopy. CSE caused loss of cellular ATP and rapid depolarization of mitochondrial membrane potential. Apoptosis occurred in Beas-2b at low concentrations of cigarette smoke extract, whereas necrosis occurred at high concentrations. Overexpression of heme oxygenase-1 inhibited CSE-induced Beas-2b cell death and preserved cellular ATP levels. Finally, heme oxygenase-1 mRNA expression was elevated in the lungs of mice chronically exposed to cigarette smoke. We demonstrate the functional compartmentalization of heme oxygenase-1 in the mitochondria of lung epithelial cells, and its potential role in defense against mitochondria-mediated cell death during CSE exposure.
cigarette smoke; COPD; heme oxygenase-1; mitochondria
Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD). Recent reports of increased matrix metalloproteinase-2 (MMP-2) in lungs of patients with emphysema support the paradigm of proteinase/antiproteinase imbalance in the pathogenesis of COPD. We sought to define the signaling pathways activated by smoke and to identify molecules responsible for emphysema-associated MMP-2 expression. In this study, we show that cigarette smoke extract (CSE) induced MMP-2 protein expression and increased MMP-2 gelatinase activity of normal lung fibroblasts. We previously identified a transcription factor, early growth response 1 (EGR-1), with robust expression in the lung tissues of patients with COPD compared with control smokers. Here, the treatment of fibroblasts with CSE resulted in marked induction of EGR-1 mRNA and protein in a dose- and time-dependent manner, accompanied by increased EGR-1 binding activity. CSE-induced MMP-2 mRNA and protein expression and activity were significantly inhibited using EGR-1 small interfering RNA (siRNA) or in Egr-1–null−/− mouse fibroblasts. Furthermore, we observed induction of membrane type 1 matrix metalloproteinase (MT1-MMP), which has an EGR-1–binding site on its promoter, in CSE-treated primary normal lung fibroblasts. The concomitant MT1-MMP expression and MMP-2 activation by CSE are inhibited by EGR-1 siRNA. Rapid activation of mitogen-activated protein kinases was observed in CSE-treated fibroblasts. Chemical inhibitors of ERK1/2 MAPK, but not of p38 and JNK, decreased CSE-induced EGR-1 protein expression and MMP-2 activity of fibroblasts. The identification that induction of MMP-2 and MT1-MMP by CSE from lung fibroblasts is EGR-1–dependent reveals a molecular mechanism for matrix remodeling in cigarette smoke–related emphysema.
chronic obstructive pulmonary disease; EGR-1; cigarette smoke extract; MMP-2; MT1-MMP
Heme oxygenase (HO)-1, an inducible, low–molecular-weight stress protein, confers cellular and tissue protection in multiple models of injury and disease, including oxidative or inflammatory lung injury, ischemia/reperfusion (I/R) injuries, and vascular injury/disease. The tissue protection provided by HO-1 potentially relates to the endogenous production of the end products of its enzymatic activity: namely, biliverdin (BV)/bilirubin (BR), carbon monoxide (CO), and iron. Of these, CO and BV/BR show promise as possible therapeutic agents when applied exogenously in models of lung or vascular injury. CO activates intracellular signaling pathways that involve soluble guanylate cyclase and/or p38 mitogen-activated protein kinase. Although toxic at elevated concentrations, low concentrations of CO can confer antiinflammatory, antiapoptotic, antiproliferative, and vasodilatory effects. BV and BR are natural antioxidants that can provide protection against oxidative stress in cell culture and in plasma. Application of BV or BR protects against I/R injury in several organ models. Recent evidence has also demonstrated antiinflammatory and antiproliferative properties of these pigments. To date, evidence has accumulated for salutary effects of CO, BV, and/or BR in lung/vascular injury models, as well as in models of transplant-associated I/R injury. Thus, the exogenous application of HO end products may provide an alternative to pharmacologic or gene therapy approaches to harness the therapeutic potential of HO-1.
bilirubin; carbon monoxide; heme oxygenase-1; inflammation; ischemia/reperfusion
Cyclooxygenase-2 (COX-2) is a key enzyme involved in the inflammatory process that is rapidly induced in macrophages in response to LPS. Carbon monoxide (CO), a byproduct of heme oxygnease-1, can suppress proinflammatory response in various in vitro and in vivo models of inflammation. This study was undertaken to examine whether CO can regulate (and if so, to delineate the mechanism by which CO regulates) LPS-induced COX-2 expression in macrophages. RAW 264.7 murine macrophages were stimulated with LPS (0–10 ng/ml) with or without CO (500 ppm). Northern and Western blot analysis was done. Progstaglandin E2 and nitrite concentration was measured from cell culture supernatant. Electrophoretic mobility shift assay was performed to assess nuclear factor binding. CO downregulated LPS-induced COX-2 mRNA and protein expression. CO also inhibited LPS-induced prostaglandin E2 secretion (P < 0.05). CO also decreased LPS-induced CCAAT/enhancer-binding protein (C/EBP) β and δ protein expression in LPS-treated RAW 264.7 cells. Gel shift analysis revealed that CO treatment decreased LPS-induced activation of protein binding to C/EBP consensus oligonucleotides of murine cyclooxygenase-2 promoter. CO also decreased LPS-induced nitric oxide synthase-2 protein expression and nitrite production, and decreased LPS-induced activation of protein binding to C/EBP consensus oligonucleotides of murine nitric oxide synthase-2 promoter. CO may act as an important regulator of inflammation by virtue of its ability to regulate C/EBPs.
heme oxygenase; lipopolysaccharides; nitric oxide synthase
Caveolin-1 has been reported to regulate apoptosis, lipid metabolism, and endocytosis in macrophages. In the present study, we demonstrate that caveolin-1 can act as a potent immunomodulatory molecule. We first observed caveolin-1 expression in murine alveolar macrophages by Western blotting and immunofluorescence microscopy. Loss-of-function experiments using small interfering RNA showed that downregulating caveolin-1 expression in murine alveolar and peritoneal macrophages increased LPS-induced proinflammatory cytokine TNF-α and IL-6 production but decreased anti-inflammatory cytokine IL-10 production. Gain-of-function experiments demonstrated that overexpression of caveolin-1 in RAW264.7 cells decreased LPS-induced TNF-α and IL-6 production and augmented IL-10 production. p38 mitogen-activated protein kinase (MAPK) phosphorylation was increased by overexpressing caveolin-1 in RAW264.7 cells, whereas c-Jun N-terminal kinase, extracellular signal-regulated kinase MAPK, and Akt phosphorylation were inhibited. The antiinflammatory modulation of LPS-induced cytokine production by caveolin-1 was significantly abrogated by the administration of p38 inhibitor SB203580 in RAW264.7 cells. Peritoneal macrophages isolated from MKK3 null mice did not demonstrate any modulation of LPS-induced cytokine production by caveolin-1. LPS-induced activation of NF-κB and AP-1 determined by electrophoretic mobility shift assay were significantly reduced by overexpressing caveolin-1 in RAW264.7 cells. The reductions were attenuated by the administration of p38 inhibitor SB203580. Taken together, our data suggest that caveolin-1 acts as a potent immunomodulatory effecter molecule in immune cells and that the regulation of LPS-induced cytokine production by caveolin-1 involves the MKK3/p38 MAPK pathway.
caveolin-1; cytokines; inflammation; lipopolysaccharide; macrophages