Usamah S Kayyali and 2Paul M Hassoun

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1Tufts-New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 2Johns Hopkins University School of Medicine, Baltimore, Maryland

Introduction

Xanthine oxidoreductase (XOR) is the terminal enzyme in purine metabolism (xanthine dehydrogenase). Because of its abundance in milk, XOR has been the subject of extensive biochemical characterization as the leading member of the family of molybdo-flavoenzymes. Another unique property of the enzyme is its ability to generate reactive oxygen species (ROS), especially when it is converted from the original translational product, xanthine dehydrogenase, to the oxidase form. This capacity to generate ROS brought XOR to the forefront in the research fields of oxidative stress, ROS signaling, and inflammation. However, after several decades of investigation, many unanswered questions remain about the specific roles XOR might play in health and disease. This chapter will review certain aspects of XOR with special emphasis on its physiology in microvascular endothelial cells. The reader is also encouraged to consult recent comprehensive reviews related to the general biology of XOR [1-3].

Biology and Function of Xanthine Oxidoreductase

Xanthine oxidase is derived from xanthine dehydrogenase, the translational product of the XOR gene, by post-translational modification. Biochemical characterization revealed that xanthine dehydrogenase (XDH), in its active form, exists as a homodimer of two protein molecules. Catalysis occurs by transfer of electrons along a chain of molybdo-pterin cofactor, two Fe2S2 sites, and an FAD site. XOR can be the target of oxidation and proteolysis, modifications that change the binding specificity of the enzyme and lead to the formation of xanthine oxidase (XO). Unlike XDH, which binds to NAD+ as its preferred electron acceptor leading to the formation of NADH, XO can no longer bind NAD+. However, the enzyme XO retains its activity in the conversion of hypoxanthine into xanthine and uric acid, and utilizes molecular oxygen as an electron acceptor, leading to the formation of the highly reactive oxygen species superoxide. The alteration in binding specificity for NAD+ is irreversible in the case of proteolysis, but can be reversed by reducing agents in the case of oxidation. However, it is important to point out that the dehydrogenase form of XOR can also utilize molecular oxygen and produce ROS, albeit less efficiently than the oxidase form. Furthermore, XOR can also function as an NADH oxidase, which generates ROS without metabolizing xanthine or hypoxanthine. The catalysis in this reaction occurs at the FAD site and does not involve the molybdenum cofactor, and therefore it is not prevented by XOR inhibitors, such as allopurinol, that target the molybdenum cofactor [2].

Structural discoveries followed the biochemical characterization of XOR and largely supported the model derived from biochemical characterization. The crystal structure of XOR from bovine milk was elucidated in an elegant study which demonstrated that the conformation of the Fe S

clusters, the FAD, and the moybdo cofactor domains corresponded to those characterized biochemically [4]. Furthermore, when XOR was subjected to limited proteolysis in vitro, it underwent a conformational change that resulted in changing of the electrostatic charge at the opening to the FAD site, thus blocking access of NAD+ to that site [4]. Such a conformational change supports the modifications in biochemical properties resulting from the conversion of xanthine dehydrogenase into the oxidase form.

Although its primary role is as the terminal limiting step in purine metabolism, XOR is known to metabolize other substrates as well, for example, ethanol. Of particular interest for endothelial physiology is the capacity of XOR to reduce nitrite resulting in the formation of NO. However, the physiological significance of this particular activity is not clear considering that the Km value for this reaction is much higher than the concentration of nitrite normally found in tissues. Nevertheless, XOR has been argued to be a potentially significant source of NO, particularly in ischemic tissues.

Xanthine Oxidoreductase in Endothelial Cells

Although XOR has been implicated in the pathogenesis of different types of injury, the source of XOR causing the injury is still under debate. The complexity stems from the differences in XOR distribution between species. Another source of difficulty comes from the particular method used to study distribution, such that the level of mRNA detected by in situ hybridization might not correspond to the amount of protein detected by immunocytochemistry, which in turn might not correspond to the amount of active enzyme in lysed tissue. In rats and mice, the highest levels of XOR mRNA, protein, and activity were found in the small intestine, followed by liver and lung. In bovine tissue, XOR was immunolocalized to the epithelium and endothelium of the mammary glands as well as in the liver, heart, and lungs. It is concentrated in capillary, but not in macrovascular, endothelial cells. In humans, XOR was immunolocalized to skeletal and cardiac muscles, and its activity was reported in brain autopsy samples. However, other reports claimed very low enzyme activity in human heart autopsy samples. In another study, while XOR mRNA was detected in the lung, heart, brain, and kidney, enzyme activity was very low. Lack of significant activity in the just-mentioned organs called into question the role of XOR in ischemic injury to these organs. However, the discrepancy has been explained either by dilution or loss of XOR activity during tissue isolation, or by involvement of XOR from a remote source (e.g., released from liver) in ischemic injury to organs such as lung. In addition, it is possible that although the basal level of XOR activity is low in these organs, it might be induced under disease conditions. For example, drastic changes in XOR activity are seen in mammary tissue under specific conditions (see later discussion).

XOR is found primarily in the cytoplasm of endothelial cells, but several other reports have localized it on the cell membrane. We and others have found XOR to be localized in discrete cytoplasmic vesicles in normoxic microvascular endothelial cells. However, XOR protein expression increases in response to hypoxia, and the enzyme assumes a diffuse cytoplasmic distribution (Figure 1). In mammary epithelial cells, XOR is also found as part of the milk fat globule complex associated with proteins such as butyro-phyllin. This localization, and the fact that XOR is normally undetectable in mammary epithelial cells but increases significantly in late pregnancy and during lactation, suggested a role for XOR in secretion. As previously mentioned, the fact that XOR can indeed be secreted in plasma has implicated the enzyme in damage caused to organs distant from the site of primary injury. The role of circulating XOR in disease is further discussed in a later section, "XOR and Models of Injury."

t * #

Normoxia

Hypoxia

Figure 1 XOR is localized in discrete vesicles in pulmonary microvascular endothelial cells (arrow) and undergoes redistribution in response to hypoxia. (see color insert)

Figure 1 XOR is localized in discrete vesicles in pulmonary microvascular endothelial cells (arrow) and undergoes redistribution in response to hypoxia. (see color insert)

Regulation of Xanthine Oxidoreductase in Health and Disease

The expression of XOR varies in response to several cytokines, hormones, and stresses such as hypoxia and exposure to tobacco smoke. Studies have shown that XOR expression can be altered at the activity, protein, mRNA, and gene promoter levels (Figure 2). However, it is important to point out that there are some differences in the XOR promoter region of human versus rodent. Unlike the rat and mouse, the human XOR promoter contains TATA-like elements, which are believed to play a role in repressing the basal level of XOR in humans compared to rats. The human XOR promoter region also contains sites responsive to cytokines, such as IL-6 responsive elements as well as potential TNF-a, IFN-g, and IL-1p responsive elements. Moreover, cytokines such as IFN-g, IL-1p, and TNF-a have been shown to upregulate XOR in human mammary epithelial cells. IFN also upregulates XOR in the L929 fibroblast cell line. Treatment of mice with endotoxin lipopolysaccharide (LPS) activates XOR in several tissues. In a rat model of acute lung injury, LPS in combination with IL-1 upregulated XOR in the lung. This effect was also observed in cultured rat pulmonary artery microvascular endothelial cells [5].

Upregulation of XOR by LPS was further enhanced by exposing rats or endothelial cells to hypoxia. Furthermore, hypoxia alone upregulates XOR expression in bovine pulmonary artery endothelial and smooth muscle cells [5], and rat epididymal fat pad [5], and pulmonary artery microvascular endothelial cells [6]. However, hypoxia and cytokines upregulate XOR at multiple levels of expression. For example, the fact that cytokines increase the XOR mRNA and protein severalfold, albeit not enough to explain the observed increase in activity, suggested that part of the upregulation is due to post-translational modification [7]. This also applies to hypoxia, which increases mRNA and protein levels only after days of exposure [5], while the enzyme activity can be upregulated as early as four hours [6]. Post-translational modification of the protein can account for acute enzymatic activation. XOR activity is reduced by removal of the molybdo-cofactor or by desulfuration [2]. Hence, addition of the molybdo-cofactor and sulfuration of the XOR protein have been proposed as mechanisms for post-translational activation. Post-translational modification (phosphorylation) of XOR, causing acute enzyme activation in response to hypoxia, is linked to activation of p38 MAP kinase-and CK2-dependent pathways in pulmonary artery endothelial cells [6].

Stimuli

Mechanisms

Levels of Regulation

Estrogen g-interferon

Tobacco smoke

LPS+IL-1

Prolactin

Corticosteroids

XOR promoter g-interferon Tobacco smoke Hypoxia LPS+IL-1

Hypoxia Prolactin Corticosteroids

p38,CKII

Binding to GAG

Binding to GAG

Active secretion

XOR mRNA

XOR protein post-translational activation, e.g., phosphorylation

Active secretion

Figure 2 XOR expression is regulated at multiple levels. (see color insert)

Hormones such as prolactin and glucocorticoids are also important in regulating XOR expression. Both hormones upregulate XOR in mammary epithelial cells. XOR upregu-lation by prolactin and cortisone (both important for lactation) is probably relevant to the mechanisms of secretion and activation in milk, a very rich source of this enzyme. However, upregulation of XOR by glucocorticoids has also been observed in human kidney epithelial cells [8] and rat pulmonary microvascular endothelial cells (Kayyali and Hassoun, unpublished data). Since corticosteroids are important in inflammation, the latter action might be related to the putative role of XOR in the acute phase response and inflammation as discussed later. Estrogens, on the other hand, inhibit the upregulation of XOR by hypoxia in pulmonary microvascular endothelial cells. However, the finding that the nonreceptor-binding a-estradiol also inhibits XOR upregulation suggests a novel antioxidant property for estrogen-related compounds. Although the mechanisms of action of estrogens in the regulation of XOR need to be further elucidated, a hope is that nonestrogenic compounds, such as a-estradiol and possibly phytoestrogens, might represent potential therapeutic drugs having cardioprotective effects (e.g., antioxidant properties) without the deleterious effects of the active hormone.

Tobacco smoke is another form of stress that upregulates XOR in tissues such as the stomach mucosa, striated muscle blood vessels, synaptosomes, and pulmonary microvascular endothelial cells. In addition XOR has been reported to be upregulated by other toxicants such as PMA and tetra-chlorodibenzodioxin (TCDD).

Signaling mechanisms that regulate the expression of XOR in different cells remain to be elucidated. One kinase that appears to be important for XOR regulation at different levels of expression is p38 MAP kinase. Inhibiting p38 and CK2 blocks the post-translational upregulation of endothelial XOR by hypoxia, a stimulus that has been shown to activate p38 [6]. In addition, inhibiting p38 blocks XOR gene promoter and transcriptional activation by tobacco smoke in pulmonary microvascular endothelial cells. Since p38 MAP kinase is a stress-activated kinase believed to be important in reactive oxygen signaling, its involvement in regulation of XOR expression merits further investigation.

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