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 ...

Carbon monoxide (CO), which is produced endogenously in the breakdown of heme, has been recognized as an important physiological second messenger similar to NO. Additionally, pharmacological delivery of CO is protective in numerous models of injury, including ischemia/reperfusion, transplantation, hemorrhagic shock, and endotoxemia. However, the mechanism of action of CO is only partially elucidated focused primarily on how it modulates the cellular response to stress. The purpose of these investigations is to test the hypothesis that CO acts via inhibition of cytochrome c oxidase leading to the generation of low levels of reactive oxygen species (ROS) that in turn mediate subsequent adaptive signaling. We show here that CO increases ROS generation in RAW 264.7 cells, which is inhibited by antimycin A and is absent in respiration-deficient rho0 cells. CO inhibits cytochrome c oxidase, while maintaining cellular ATP levels and increasing mitochondrial membrane potential. The addition of antioxidants or inhibition of complex III of the electron transport chain by antimycin A attenuates the inhibitory effects of CO on lipopolysaccharide (LPS)-induced TNF-alpha and blocked CO-induced p38 MAPK phosphorylation, which we previously have shown to be important in the anti-inflammatory effects of CO.

Carbon monoxide (CO), a gaseous second messenger, arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (HO) enzymes. Many biological functions of HO, such as regulation of vessel tone, smooth muscle cell proliferation, neurotransmission, and platelet aggregation, and anti-inflammatory and antiapoptotic effects have been attributed to its enzymatic product, CO. How can such diverse actions be achieved by a simple diatomic gas; can its protective effects be explained via regulation of a common signaling pathway? A number of the known signaling effects of CO depend on stimulation of soluble guanylate cyclase and/or activation of mitogen-activated protein kinases. The consequences of this activation remain unknown but appear to differ depending on cell type and circumstances. The majority of studies reporting a protective role of CO focus on pathways initiated by the pathological stimulus (e.g., lipopolysaccharide, hypoxia, balloon injury, tumor necrosis factor alpha, etc.) and its consequential modulation by CO. What has been less studied is the manner in which CO exposure alone modulates the molecular machinery of the cell so that a subsequent stress stimulus will elicit a homeostatic response as opposed to one that is chaotic and disordered. CO potentially interacts with other intracellular hemoprotein targets, although little is known about the functional significance of such interactions other then the known targets including mitochondrial oxidases, oxygen sensors, and nitric oxide synthases. The earliest response of a cell exposed to low concentrations of CO is clearly an increase in reactive oxygen species formation that we define as oxidative conditioning. This has important consequences for inflammation, proliferation, mitochondria biogenesis, and apoptosis. Within this review, we will highlight recent research on the molecular events underlying the physiologic effects of CO-which lead to cytoprotective conditioning.

Activated macrophages play a critical role in controlling chronic tissue inflammation through the release of a variety of mediators including cytokines, chemokines, growth factors, active lipids, reactive oxygen, and nitrogen species. The mechanisms that regulate macrophage activation in chronic inflammation are poorly understood. A hallmark of chronic inflammation is the turnover of extracellular matrix components, and recent work has suggested that interactions with the extracellular matrix can exert important influences on macrophage effector functions. We have examined the effect of low molecular weight fragments of the extracellular matrix glycosaminoglycan hyaluronan (HA) on the induction of nitric-oxide synthase (iNOS) in macrophages. We found that HA fragments induce iNOS mRNA, protein and activity alone, and markedly synergize with interferon-␥ to induce iNOS gene expression in murine macrophages. In addition, we found that resident tissue alveolar macrophages respond minimally, but inflammatory alveolar macrophages exhibit a marked induction in iNOS expression in response to HA fragments. Finally, we demonstrate that the mechanism of HA fragment-induced expression of iNOS requires activation of the transcriptional regulator nuclear factor B. These data support the hypothesis that HA may be an important regulator of macrophage activation at sites of chronic tissue inflammation. Nitric oxide (NO ⅐ )1 mediates a number of the host defense functions of activated macrophages, including antimicrobial and tumoricidal activity (1-7). NO ⅐ and its metabolites have also been implicated in the pathogenesis of the tissue damage associated with acute and chronic inflammation (8 -12). Macrophages generate NO ⅐ from the guanido moiety of L-arginine through a reaction catalyzed by the inducible form of nitricoxide synthase (2). In contrast to the constitutive, calcium-dependent form of the enzyme found in the central nervous system and endothelial cells, iNOS can be induced by numerous immune stimuli. Maximal, synergistic iNOS induction occurs in response to the combination of a priming stimulus, such as IFN␥, and a triggering stimulus, examples of which include LPS, tumor necrosis ␣, and interleukin-2 (6, 13, 14).Hallmarks of chronic inflammation include the accumulation of activated macrophages and of macrophage-derived mediators. However, the mechanisms of macrophage activation in this setting have not been clearly defined. The ECM undergoes increased degradation and turnover during inflammation, and fragments of ECM molecules have been found to possess biological activities distinct from their parent compounds (15-17). It has therefore been proposed that ECM fragments may be responsible for activating macrophages that infiltrate chronically inflamed tissues (18). We have recently demonstrated that fragments of the ECM component HA can bind to macrophages and induce the expression of a number of inflammatory genes (32), suggesting that HA fragments may be capable of activating macrophages at non-infectious sites of...