The heme oxygenases, which consist of constitutive and inducible isozymes (HO-1, HO-2), catalyze the rate-limiting step in the metabolic conversion of heme to the bile pigments (i.e., biliverdin and bilirubin) and thus constitute a major intracellular source of iron and carbon monoxide (CO). In recent years, endogenously produced CO has been shown to possess intriguing signaling properties affecting numerous critical cellular functions including but not limited to inflammation, cellular proliferation, and apoptotic cell death. The era of gaseous molecules in biomedical research and human diseases initiated with the discovery that the endothelial cell-derived relaxing factor was identical to the gaseous molecule nitric oxide (NO). The discovery that endogenously produced gaseous molecules such as NO and now CO can impart potent physiological and biological effector functions truly represented a paradigm shift and unraveled new avenues of intense investigations. This review covers the molecular and biochemical characterization of HOs, with a discussion on the mechanisms of signal transduction and gene regulation that mediate the induction of HO-1 by environmental stress. Furthermore, the current understanding of the functional significance of HO shall be discussed from the perspective of each of the metabolic by-products, with a special emphasis on CO. Finally, this presentation aspires to lay a foundation for potential future clinical applications of these systems.

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress. The anti-inflammatory properties of heme oxygenase-1 may serve as a basis for this cytoprotection. We demonstrate here that carbon monoxide, a by-product of heme catabolism by heme oxygenase, mediates potent anti-inflammatory effects. Both in vivo and in vitro, carbon monoxide at low concentrations differentially and selectively inhibited the expression of lipopolysaccharide-induced pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta, and macrophage inflammatory protein-1beta and increased the lipopolysaccharide-induced expression of the anti-inflammatory cytokine interleukin-10. Carbon monoxide mediated these anti-inflammatory effects not through a guanylyl cyclase-cGMP or nitric oxide pathway, but instead through a pathway involving the mitogen-activated protein kinases. These data indicate the possibility that carbon monoxide may have an important protective function in inflammatory disease states and thus has potential therapeutic uses.

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Accumulating evidence suggests that oxidative stress plays a central role in the pathogenesis of many pulmonary diseases including adult respiratory distress syndrome, emphysema, asthma, bronchopulmonary dysplasia, and interstitial pulmonary fibrosis. The morbidity and mortality of these diseases remain high even with optimal medical management. In our attempts to devise new therapies for these disorders, it is crucial to improve our understanding of the basic mechanism(s) of oxidant-induced lung injury. A major line of investigation seeks to characterize the cellular and molecular responses of the lung to oxidant insults. Much progress has been made in our understanding of the role of the "classic" antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione peroxidase) in mediating the lung's resistance against oxidant lung injury. However, it is becoming clear that other oxidant-induced gene products may also play vital roles in the lung's adaptive and/or protective response to oxidative stress. One such stress-response protein is heme oxygenase-1, HO-1. Since the identification of HO-1 in 1968, many of the studies involving this enzyme were understandably focused on the regulation and function of HO-1 in heme metabolism. This emphasis is self-evident as HO-1 catalyzes the first and rate-limiting step in heme degradation. Interestingly, however, evidence accumulated over the past 25 years demonstrates that HO-1 is induced not only by the substrate heme but also by a variety of non-heme inducers such as heavy metals, endotoxin, heat shock, inflammatory cytokines, and prostaglandins. The chemical diversity of HO-1 inducers led to the speculation that HO-1, besides its role in heme degradation, may also play a vital function in maintaining cellular homeostasis. Further support for this hypothesis was provided by Tyrrell and colleagues who showed in 1989 that HO-1 is also highly induced by a variety of agents causing oxidative stress. Subsequently, many investigators have focused their attention on the function and regulation of HO-1 in various in vitro and in vivo models of oxidant-mediated cellular and tissue injury. The magnitude of HO-1 induction after oxidative stress and the wide distribution of this enzyme in systemic tissues coupled with the intriguing biological activities of the catalytic byproducts, carbon monoxide, iron, and bilirubin, makes HO-1 a highly attractive and interesting candidate stress-response protein which may play key role(s) in mediating protection against oxidant-mediated lung injury. This review will focus on the current understanding of the physiological significance of HO-1 induction and the molecular regulation of HO-1 gene expression in response to oxidative stress. We hope that this discussion will stimulate interest and investigations into a field which is still largely uncharted in the pulmonary research community.

The transcription factor Nrf2, which normally exists in an inactive state as a consequence of binding to a cytoskeleton-associated protein Keap1, can be activated by redox-dependent stimuli. Alteration of the Nrf2-Keap1 interaction enables Nrf2 to translocate to the nucleus, bind to the antioxidant-responsive element (ARE) and initiate the transcription of genes coding for detoxifying enzymes and cytoprotective proteins. This response is also triggered by a class of electrophilic compounds including polyphenols and plant-derived constituents. Recently, the natural antioxidants curcumin and caffeic acid phenethyl ester (CAPE) have been identified as potent inducers of haem oxygenase-1 (HO-1), a redox-sensitive inducible protein that provides protection against various forms of stress. Here, we show that in renal epithelial cells both curcumin and CAPE stimulate the expression of Nrf2 in a concentration- and time-dependent manner. This effect was associated with a significant increase in HO-1 protein expression and haem oxygenase activity. From several lines of investigation we also report that curcumin (and, by inference, CAPE) stimulates ho-1 gene activity by promoting inactivation of the Nrf2-Keap1 complex, leading to increased Nrf2 binding to the resident ho-1 AREs. Moreover, using antibodies and specific inhibitors of the mitogen-activated protein kinase (MAPK) pathways, we provide data implicating p38 MAPK in curcumin-mediated ho-1 induction. Taken together, these results demonstrate that induction of HO-1 by curcumin and CAPE requires the activation of the Nrf2/ARE pathway.

Heme oxygenase-1 (HO-1) protects cells from various insults including oxidative stress. Transcriptional activators, including the Nrf2/Maf heterodimer, have been the focus of studies on the inducible expression of ho-1. Here we show that a heme-binding factor, Bach1, is a critical physiological repressor of ho-1. Bach1 bound to the multiple Maf recognition elements (MAREs) of ho-1 enhancers with MafK in vitro and repressed their activity in vivo, while heme abrogated this repressor function of Bach1 by inhibiting its binding to the ho-1 enhancers. Gene targeting experiments in mice revealed that, in the absence of Bach1, ho-1 became expressed constitutively at high levels in various tissues under normal physiological conditions. By analyzing bach1/nrf2 compound-deficient mice, we documented antagonistic activities of Bach1 and Nrf2 in several tissues. Chromatin immunoprecipitation revealed that small Maf proteins participate in both repression and activation of ho-1. Thus, regulation of ho-1 involves a direct sensing of heme levels by Bach1 (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate effects repressor-activator antagonism.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway elicits a survival signal against multiple apoptotic insults. In addition, phase II enzymes such as heme oxygenase-1 (HO-1) protect cells against diverse toxins and oxidative stress. In this work, we describe a link between these defense systems at the level of transcriptional regulation of the antioxidant enzyme HO-1. The herb-derived phenol carnosol induced HO-1 expression at both mRNA and protein levels. Luciferase reporter assays indicated that carnosol targeted the mouse ho1 promoter at two enhancer regions comprising the antioxidant response elements (AREs). Moreover, carnosol increased the nuclear levels of Nrf2, a transcription factor governing AREs. Electrophoretic mobility shift assays and luciferase reporter assays with a dominantnegative Nrf2 mutant indicated that carnosol increased the binding of Nrf2 to ARE and induced Nrf2-dependent activation of the ho1 promoter. While investigating the signaling pathways responsible for HO-1 induction, we observed that carnosol activated the ERK, p38, and JNK pathways as well as the survival pathway driven by PI3K. Inhibition of PI3K reduced the increase in Nrf2 protein levels and activation of the ho1 promoter. Expression of active PI3K-CAAX (where A is aliphatic amino acid) was sufficient to activate AREs. The use of dominant-negative mutants of protein kinase C and Akt1, two kinases downstream from PI3K, demonstrated a requirement for active Akt1, but not protein kinase C. Moreover, the long-term antioxidant effect of carnosol was partially blocked by PI3K or HO-1 inhibitors, further demonstrating that carnosol attenuates oxidative stress through a pathway that involves PI3K and HO-1.High levels of reactive oxygen species cause damage to cells and are involved in several human pathologies, including neurodegenerative disorders and cancer (1, 2). Therefore, the use of compounds with antioxidant properties may help prevent or alleviate diseases in which oxidative stress is a primary cause (3). Carnosol, a diterpene derived from the herb rosemary, is a representative member of a family of plant-derived phenols, which also include curcumin, carnosic acid, phenylethyl isothiocyanate, epigallocatechin gallate, and other green tea polyphenols. These bioactive phytochemicals exhibit Michael acceptor function and therefore behave as antioxidants (4). In addition, being themselves xenobiotic compounds, they activate a xenobiotic response in the target cells affecting the expression of phase II enzymes such as NAD(P)H:quinone oxidoreductase, aldoketoreductase, glutathione S-transferase, ␥-glutamylcysteine synthetase, glutathione synthetase, and heme oxygenase-1 (HO-1) 1 (5-7). Heme oxygenase isozymes (HO-1 and HO-2) catalyze the stepwise degradation of heme to release free iron and equimolar concentrations of carbon monoxide and the linear tetrapyrrole biliverdin, which is converted to bilirubin by the enzyme biliverdin reductase (8). The HO-1 isozyme is a phase II enzyme that is transcriptionally regulated by a large...

Exposure of rats to hypoxia (7% O2Low cellular oxygen tension is a feature of both physiological conditions, such as adaptations to high altitude and physical endurance exercise (1), and pathophysiological conditions including ischemia, fibrosis (2), and neoplasia (3). Mammalian cells respond to hypoxia in part by increased expression of several genes that encode both tissue-specific and ubiquitous proteins (4). These proteins participate in diverse biological processes including erythropoiesis, which enhances the oxygen carrying capacity of the blood; angiogenesis, which permits delivery of oxygen carrying blood to hypoxic sites; glycolysis, as a means of energy production; xenobiotic detoxification; and cellular adaptation to stress. Hypoxia-inducible proteins within these respective categories include erythropoietin (EPO) 1 (5), vascular endothelial growth factor (6), glycolytic enzymes (7-9), NAD(P)H:quinone oxidoreductase (10), and heat shock proteins (11,12). Where examined, increased expression of specific proteins in response to hypoxia is regulated primarily at the level of gene transcription (although posttranscriptional mechanisms have also been characterized).Another stress-associated protein whose expression is stimulated by hypoxia is heme oxygenase-1 (HO-1) (13, 14). HO-1, a microsomal membrane enzyme, catalyzes the first and ratelimiting reaction in heme catabolism, the oxidative cleavage of b-type heme molecules to yield equimolar quantities of biliverdin, carbon monoxide (CO), and iron. Biliverdin is subsequently converted to bilirubin by the action of biliverdin reductase. The expression of HO-1 is dramatically induced not only by the substrate, heme, but a variety of stress-associated agents, including heavy metals, hyperthermia, and UV irradiation (reviewed in Maines (15)). A common feature among these inducers, including heme, is that they generate reactive oxygen species and/or diminish glutathione levels. This correlation and the observation that bilirubin functions as an antioxidant (16) has led to the hypothesis that induction of HO-1 is part of a general response to oxidant stress and that this enzyme plays a protective role during such conditions (17)(18)(19).Stimulation of HO-1 expression by most if not all inducers is controlled primarily at the level of gene transcription and in our studies on the regulation of the mouse HO-1 gene, we have identified two 5Ј distal enhancer regions, SX2 and AB1, that mediate gene activation by a variety of pro-oxidants including heme, heavy metals, TPA, hydrogen peroxide, and LPS (20 -23). The mechanism of HO-1 induction by hypoxia has not been investigated and because this induction has been proposed to occur as a consequence of oxidative stress (13), we examined the role of the SX2 and AB1 enhancers in hypoxia-dependent gene activation. In this report we show that these enhancers do not mediate transcriptional activation of the HO-1 gene in response to hypoxia. Rather, this induction is mediated by a 163-bp fragment located directly downstream of ...