**Elucidating the Role of Biliverdin Reductase in the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress**

James R. Reed *Department of Pharmacology, Louisiana State University Health Sciences Center, New Orleans, LA, USA* 

#### **1. Introduction**

534 Pharmacology

Tyler, C. B.; K. A. (1982) Miczed: effects of phencyclidine on aggressive behavior in mice.

Valzelli, L. (1973) The isolation syndrome in mice. *Psychopharmacology 31:305-20.* 

*Pharmacol Biochem Behav* 17: 503-10.

Hemin is a cofactor in which an atom of iron is coordinated to the nitrogens of four pyrrole groups that make up the protoporphyrin IX ring (see figure below).

#### Hemin

Many types of enzymes in living systems use hemin as a prosthetic group to catalyze oxidation/reduction reactions or for the binding/transport of reactive molecules (e.g. oxygen). For instance, several cytochromes of the mitochondrial electron transport chain are "heme" enzymes as are the major drug/xenobiotic-metabolizing enzymes of the endoplasmic reticulum, the cytochromes P450 (CYP or P450). The heme group of the P450s allows these enzymes to use redox chemistry to bind molecular oxygen and cleave the O-O bond, thus forming a reactive, high-valent oxygen species that can insert oxygen into otherwise stable carbon-hydrogen bonds of drugs/xenobiotics (White and Coon, 1980). The unfavorable thermodynamics of this type of reaction has caused the P450s to be likened to "catalytic blowtorches" (Schlichting et al., 2000), and the process is essential for the elimination and clearance of many lipophilic compounds ingested from the environment. Catalase is an important protective heme enzyme that is responsible for degrading

Elucidating the Role of Biliverdin Reductase in

**1.2 The regulation of cellular heme levels** 

Montellano, 2000) (see figure below).

3 NADPH + 2 O2

HO O

(HO Reaction)

Hemin

O

HO

N N <sup>N</sup> <sup>N</sup>

conditions.

(BVR Reaction)

O N N N N O

Biliverdin IX

OH <sup>O</sup> OH <sup>O</sup>

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 537

Because of the harmful effects of free heme accumulation, the synthesis and catabolism of heme in living systems is highly regulated. The rate-limiting enzyme for heme synthesis is α-aminolevulinic acid synthetase, and its expression and activity is highly regulated by a variety of agents and stress signals (Ponka, 1997). Heme catabolism is carried out by two enzymatic reactions. The rate-limiting step of heme catabolism is catalyzed by heme oxygenase (HO). This enzyme catalyzes a complicated, multi-step reaction that uses molecular oxygen and electrons (received from a separate redox partner, the cytochrome P450 reductase) to cleave the α-meso bridge of the protoporphyrin IX ring and form ferrous iron, CO, and biliverdin in the process (Kikuchi et al., 2005;Liu et al., 1997;Liu and Ortiz de

The final step of heme catabolism involves the reduction of the biliverdin formed by HO to bilirubin. This enzymatic step (below) is catalyzed by the biliverdin reductase (BVR). BVR has dual cofactor specificity as both NADH and NADPH can provide electrons to the enzyme. NADPH is the preferred cofactor under basic conditions, whereas NADH is more favorable at lower pH (< 7.0) (Noguchi et al., 1979). The activities of both HO and BVR are highly regulated to effectively coordinate heme catabolism with its synthesis under different

3 NADPH+

+ CO + Fe+2

NAD(P)+

NAD(P)H

O N N N N O

O N N N N O

Biliverdin IX

OH <sup>O</sup> OH <sup>O</sup>

Bilirubin IX

OH <sup>O</sup> OH <sup>O</sup>

hydrogen peroxide. Furthermore, the heme enzymes, nitric oxide synthase and cyclooxygenase, have important signaling roles in the regulation of various cellular processes such as inflammation. However, in terms of the sheer abundance in higher living systems, hemoglobin is the most important heme enzyme as it uses the cofactor to transport oxygen in blood circulation to facilitate oxidative/phosphorylation and energy generation in distal tissues. Because the heme proteins interact with reactive oxygen species (ROS), they are susceptible to ROS-mediated damage, which in turn, results in the accumulation of free or unused heme.

#### **1.1 The toxicity of heme**

The reactive nature of hemin does not come without a cost. The free (not enzyme bound) form of the cofactor has been shown *in vitro* to increase the peroxidation of lipids and the fragmentation and cross-linking of DNA and protein resulting from oxidative stress (Kumar and Bandyopadhyay, 2005;Vincent, 1989). One likely explanation for these findings can be drawn from two of the basic reactions of reactive oxygen chemistry, the Fenton reaction and the Haber-Weiss reaction. In the Fenton reaction (below), superoxide anion reduces free molecular iron.

$$\text{O}\_2\text{'} + \text{Fe}^{\*3} \qquad \bigsqcup \begin{array}{c} \bigrightharpoonup \text{O}\_2\text{'} + \text{Fe}^{\*2} \end{array} \qquad \text{(Fenton Reaction)} \qquad \text{(} \text{Henton Reaction)}$$

In the Haber-Weiss reaction, the reduced iron can interact with hydrogen peroxide, resulting in cleavage of the O-O bond to form hydroxyl anion and hydroxyl radical (Vincent, 1989).

 Fe+2 + H2O2 Fe+3 + HO- + HO (Haber-Weiss Reaction)

Using the Haber-Weiss system as an analogy, it is likely that the free hemin functions in a manner similar to that of free iron as a means to produce hydroxyl radical. The hydroxyl radical has been shown to be much more destructive to proteins and DNA than both hydrogen peroxide and superoxide (Davies et al., 1987;Jackson et al., 1987).

It also has been proposed that hemin interacts with hydrogen peroxide to form a putative, hypervalent iron-oxygen species analogous to the reactive intermediate of peroxidase enzymes referred to as Compound I (Vincent, 1989).

Fe+2 + H2O2 Fe+3:O· + H2O (Peroxidase-like Reaction)

Because hemin is much more lipophilic than free iron, the oxidative stress associated with free hemin is more destructive to membrane lipids and organelles (Balla et al., 1991). In this respect, the putative iron-oxo species resulting from the reaction of hemin and hydrogen peroxide could be even more deleterious than hydroxyl radical as suggested by the fact that free radical scavengers of hydroxyl radical (e.g. dimethyl sulfoxide) did not protect lipids and proteins from damage when incubated with hemin and hydrogen peroxide (Vincent, 1989). Understandably, the propensity of hemin to damage lipid mixtures causes it to be extremely harmful to cellular membranes and organelles. Free hemin also has been shown to promote inflammatory reactions that have been associated with hepatic, renal, neuronal, and vascular injury (Kumar and Bandyopadhyay, 2005). In particular, several studies have demonstrated the contribution of hemin to the pathogenesis associated with atherosclerosis and ischemia/reperfusion (Wagener et al., 2001).

#### **1.2 The regulation of cellular heme levels**

Because of the harmful effects of free heme accumulation, the synthesis and catabolism of heme in living systems is highly regulated. The rate-limiting enzyme for heme synthesis is α-aminolevulinic acid synthetase, and its expression and activity is highly regulated by a variety of agents and stress signals (Ponka, 1997). Heme catabolism is carried out by two enzymatic reactions. The rate-limiting step of heme catabolism is catalyzed by heme oxygenase (HO). This enzyme catalyzes a complicated, multi-step reaction that uses molecular oxygen and electrons (received from a separate redox partner, the cytochrome P450 reductase) to cleave the α-meso bridge of the protoporphyrin IX ring and form ferrous iron, CO, and biliverdin in the process (Kikuchi et al., 2005;Liu et al., 1997;Liu and Ortiz de Montellano, 2000) (see figure below).

Hemin

536 Pharmacology

hydrogen peroxide. Furthermore, the heme enzymes, nitric oxide synthase and cyclooxygenase, have important signaling roles in the regulation of various cellular processes such as inflammation. However, in terms of the sheer abundance in higher living systems, hemoglobin is the most important heme enzyme as it uses the cofactor to transport oxygen in blood circulation to facilitate oxidative/phosphorylation and energy generation in distal tissues. Because the heme proteins interact with reactive oxygen species (ROS), they are susceptible to ROS-mediated damage, which in turn, results in the accumulation of

The reactive nature of hemin does not come without a cost. The free (not enzyme bound) form of the cofactor has been shown *in vitro* to increase the peroxidation of lipids and the fragmentation and cross-linking of DNA and protein resulting from oxidative stress (Kumar and Bandyopadhyay, 2005;Vincent, 1989). One likely explanation for these findings can be drawn from two of the basic reactions of reactive oxygen chemistry, the Fenton reaction and the Haber-Weiss reaction. In the Fenton reaction (below), superoxide anion reduces free

 O2- + Fe+3 O2 + Fe+2 (Fenton Reaction) In the Haber-Weiss reaction, the reduced iron can interact with hydrogen peroxide, resulting in cleavage of the O-O bond to form hydroxyl anion and hydroxyl radical

Using the Haber-Weiss system as an analogy, it is likely that the free hemin functions in a manner similar to that of free iron as a means to produce hydroxyl radical. The hydroxyl radical has been shown to be much more destructive to proteins and DNA than both

It also has been proposed that hemin interacts with hydrogen peroxide to form a putative, hypervalent iron-oxygen species analogous to the reactive intermediate of peroxidase

 Fe+2 + H2O2 Fe+3:O· + H2O (Peroxidase-like Reaction) Because hemin is much more lipophilic than free iron, the oxidative stress associated with free hemin is more destructive to membrane lipids and organelles (Balla et al., 1991). In this respect, the putative iron-oxo species resulting from the reaction of hemin and hydrogen peroxide could be even more deleterious than hydroxyl radical as suggested by the fact that free radical scavengers of hydroxyl radical (e.g. dimethyl sulfoxide) did not protect lipids and proteins from damage when incubated with hemin and hydrogen peroxide (Vincent, 1989). Understandably, the propensity of hemin to damage lipid mixtures causes it to be extremely harmful to cellular membranes and organelles. Free hemin also has been shown to promote inflammatory reactions that have been associated with hepatic, renal, neuronal, and vascular injury (Kumar and Bandyopadhyay, 2005). In particular, several studies have demonstrated the contribution of hemin to the pathogenesis associated with atherosclerosis

+ HO (Haber-Weiss Reaction)

Fe+2 + H2O2 Fe+3 + HO-

enzymes referred to as Compound I (Vincent, 1989).

and ischemia/reperfusion (Wagener et al., 2001).

hydrogen peroxide and superoxide (Davies et al., 1987;Jackson et al., 1987).

free or unused heme.

molecular iron.

(Vincent, 1989).

**1.1 The toxicity of heme** 

The final step of heme catabolism involves the reduction of the biliverdin formed by HO to bilirubin. This enzymatic step (below) is catalyzed by the biliverdin reductase (BVR). BVR has dual cofactor specificity as both NADH and NADPH can provide electrons to the enzyme. NADPH is the preferred cofactor under basic conditions, whereas NADH is more favorable at lower pH (< 7.0) (Noguchi et al., 1979). The activities of both HO and BVR are highly regulated to effectively coordinate heme catabolism with its synthesis under different conditions.

(BVR Reaction)

<sup>(</sup>HO Reaction)

Elucidating the Role of Biliverdin Reductase in

and Tonegawa, 1997b).

inhibition of enzyme activity.

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 539

hyperthermia, heavy metals, metal porphyrins, tumor factors, insulin, endotoxin, and sulfhydryl-reactive compounds (Keyse and Tyrrell, 1989) (reviewed in (Ryter et al., 2006)). Because most of the HO-1-inducing agents also cause elevated levels of oxidative stress, it has been postulated that HO-1 induction represents an early, "sentinel-type" response by the cell to counteract the deleterious effects of oxidative stress (Otterbein et al., 2000;Poss

Most of the in vitro/in vivo evidence for HO-1 playing a cytoprotective role has examined the effects of inducers and inhibitors of HO-1 when cells/animals are dosed with a stressor. These types of studies are described in the paragraphs below. More sophisticated lines of in vivo evidence using gene knockout/therapy to modulate HO-1 levels will be referred to separately. Metal porphyrins and heavy metals are often used alternately in studies to implicate a function of HO in a cellular process. Whereas in most cases, both types of compounds induce the HO-1 gene (a major exception is tin mesoporphyrin which inhibits HO-1 induction) and elevate protein expression, the metal porphyrins will often bind to the HO active site resulting in the

Both in vitro and in vivo studies have demonstrated that the elevated expression and activity of HO-1 is associated with a greater tolerance to various types of stress. It was already mentioned how an in vitro study was used to demonstrate that HO-1 was a 32 kDa heat shock protein which was induced by cellular stress and protected cells from toxicities related to these stressors (Keyse and Tyrrell, 1989). Another interesting in vitro study used HepG2 cells that were transfected to constitutively express CYP2E1 to demonstrate the protective role of HO-1 during CYP2E1-mediated metabolism and oxidative stress (Gong et al., 2004). Of the P450 enzymes, CYP2E1 is especially prone to the breakdown of its monoxygenase catalytic cycle with the concomitant release of superoxide, hydrogen peroxide, and excess water (Gorsky et al., 1984). A previous study by the same lab used these cells to show that the oxidative stress associated with CYP2E1-mediated metabolism could be cytotoxic, especially after prior cellular depletion of glutathione by treatment with L-buthionine-(*S*,*R*)-sulfoximine (Chen and Cederbaum, 1998). In the study examining the role of HO-1, the cytotoxicity associated with the CYP2E1-mediated metabolism of arachidonic acid was not observed when HO-1 expression was up-regulated by transfection of the cells with an adenovirus containing the cDNA for human HO-1 (Gong et al., 2004). Furthermore, when the cells were treated with chromium mesoporphyrin, which acts as an inhibitor of HO-1, the CYP2E1-related toxicity was potentiated. The in vitro study also implicated CO but not bilirubin in the protective effects of HO-1, probably through the CO-

In another in vitro study, the protective effect of the flavonoid, quercetin, on the hepatotoxicity of ethanol was attributed to its induction of HO-1 in hepatocytes because the effects of quercetin were abrogated by treatment with zinc mesoporphyrin (Yao et al., 2009). Addition of free iron increased the damage caused by ethanol, whereas CO treatment protected the cells from ethanol-induced toxicity. Thus, it was thought that the protection afforded by HO-1 induction was in part caused by the inhibition of P450-mediated

**3. Experimental evidence for the cytoprotective role of HO-1** 

**3.1 In vitro evidence for the cytoprotective role of HO-1** 

related inhibition of P450 activity (discussed below).

#### **2. Cellular functions of HO-1 and HO-2**

There are two isoforms of HO, known as HO-1 and HO-2 that are expressed through two different genes and are immunologically distinct (Maines et al., 1986;Maines, 1988). The two enzymes are approximately 40% homologous as both have in common a catalytic region of about 24 amino acids (Rotenberg and Maines, 1991) and a hydrophobic, C-terminal tail that serves to anchor the enzymes to the endoplasmic reticulum. HO-1, which is 33 kDa, is highly inducible by a multitude of stimuli and compounds and is constitutively expressed in liver and spleen. HO-2 is constitutively expressed in most tissues, and is highly expressed in brain, kidney, and testes. The HO-2 protein is 36 kDa as it contains additional regulatory sequences (e.g. extra heme-binding sites) which affect its activity in a tissue-specific manner and which might also be regulated by CO and NO binding (Ryter et al., 2006).

#### **2.1 Functions of HO-2**

It is generally believed that the main role of HO-2 might be to maintain homeostatic levels of heme during normal cellular metabolism. In one study, HO-2 knockout mice only displayed mild phenotypes and did not show evidence of altered iron maintenance (Poss et al., 1995). The study did show that the mice displayed ejaculation abnormalities (in males) and increased susceptibility to hyperoxic lung damage. Illustrating the importance of HO-2 to brain heme metabolism, these mice also showed dramatically reduced levels of HO activity in the brain. In addition, another study did demonstrate oxygen toxicity and iron accumulation in the lungs of HO-2 knockout mice (Dennery et al., 1998). Thus, HO-1 cannot completely compensate for the absence of HO-2 in terms of cellular function and the regulation of heme levels.

In brain, testes, and cardiovascular tissue, HO-2 activity plays a critical role in function by generating CO which functions as a tissue-specific signaling messenger that acts mainly through activation of guanylyl cyclase. In smooth muscle and endothelial tissue, CO mimics the effects of NO by causing relaxation and vasodilatation, respectively (Hangai-Hoger et al., 2007;Patel et al., 1993). CO also has anti-apoptotic and anti-inflammatory effects mediated through mitogen-activated protein kinases (MAPK) and not guanylyl cyclase (Piantadosi and Zhang, 1996). These signaling relationships will be discussed in more detail below in the chapter. CO also has been postulated to play a role in inhibiting P450 enzymes since the ferrous form of this type of heme protein forms a tight-binding complex with CO.

#### **2.2 Functions of HO-1**

Whereas HO-2 seems to be important in managing heme levels during normal cellular metabolism, HO-1 serves to maintain homeostatic levels of heme under conditions of cellular stress. Oxidative stress is associated with increased rates of heme protein damage and in turn, free heme accumulation. Early studies with HO-1 speculated on a cytoprotective role for this enzyme given the following: 1) It was identified as a 32 kDa heat shock protein (Keyse and Tyrrell, 1989) induced by a variety of stressors that included direct oxidative stress; 2) it metabolized a compound (hemin) that was known to be harmful to cells at high concentrations (Kutty and Maines, 1984); and 3) it was induced (greater than 40 fold in some instances (Wright et al., 2006)) in virtually all tissues following exposure to a variety of cellular stressors including oxidative stress, UV radiation, hyperoxia, hypoxia,

There are two isoforms of HO, known as HO-1 and HO-2 that are expressed through two different genes and are immunologically distinct (Maines et al., 1986;Maines, 1988). The two enzymes are approximately 40% homologous as both have in common a catalytic region of about 24 amino acids (Rotenberg and Maines, 1991) and a hydrophobic, C-terminal tail that serves to anchor the enzymes to the endoplasmic reticulum. HO-1, which is 33 kDa, is highly inducible by a multitude of stimuli and compounds and is constitutively expressed in liver and spleen. HO-2 is constitutively expressed in most tissues, and is highly expressed in brain, kidney, and testes. The HO-2 protein is 36 kDa as it contains additional regulatory sequences (e.g. extra heme-binding sites) which affect its activity in a tissue-specific manner

It is generally believed that the main role of HO-2 might be to maintain homeostatic levels of heme during normal cellular metabolism. In one study, HO-2 knockout mice only displayed mild phenotypes and did not show evidence of altered iron maintenance (Poss et al., 1995). The study did show that the mice displayed ejaculation abnormalities (in males) and increased susceptibility to hyperoxic lung damage. Illustrating the importance of HO-2 to brain heme metabolism, these mice also showed dramatically reduced levels of HO activity in the brain. In addition, another study did demonstrate oxygen toxicity and iron accumulation in the lungs of HO-2 knockout mice (Dennery et al., 1998). Thus, HO-1 cannot completely compensate for the absence of HO-2 in terms of cellular function and the

In brain, testes, and cardiovascular tissue, HO-2 activity plays a critical role in function by generating CO which functions as a tissue-specific signaling messenger that acts mainly through activation of guanylyl cyclase. In smooth muscle and endothelial tissue, CO mimics the effects of NO by causing relaxation and vasodilatation, respectively (Hangai-Hoger et al., 2007;Patel et al., 1993). CO also has anti-apoptotic and anti-inflammatory effects mediated through mitogen-activated protein kinases (MAPK) and not guanylyl cyclase (Piantadosi and Zhang, 1996). These signaling relationships will be discussed in more detail below in the chapter. CO also has been postulated to play a role in inhibiting P450 enzymes since the ferrous form of this type of heme protein forms a tight-binding complex with CO.

Whereas HO-2 seems to be important in managing heme levels during normal cellular metabolism, HO-1 serves to maintain homeostatic levels of heme under conditions of cellular stress. Oxidative stress is associated with increased rates of heme protein damage and in turn, free heme accumulation. Early studies with HO-1 speculated on a cytoprotective role for this enzyme given the following: 1) It was identified as a 32 kDa heat shock protein (Keyse and Tyrrell, 1989) induced by a variety of stressors that included direct oxidative stress; 2) it metabolized a compound (hemin) that was known to be harmful to cells at high concentrations (Kutty and Maines, 1984); and 3) it was induced (greater than 40 fold in some instances (Wright et al., 2006)) in virtually all tissues following exposure to a variety of cellular stressors including oxidative stress, UV radiation, hyperoxia, hypoxia,

and which might also be regulated by CO and NO binding (Ryter et al., 2006).

**2. Cellular functions of HO-1 and HO-2** 

**2.1 Functions of HO-2** 

regulation of heme levels.

**2.2 Functions of HO-1** 

hyperthermia, heavy metals, metal porphyrins, tumor factors, insulin, endotoxin, and sulfhydryl-reactive compounds (Keyse and Tyrrell, 1989) (reviewed in (Ryter et al., 2006)). Because most of the HO-1-inducing agents also cause elevated levels of oxidative stress, it has been postulated that HO-1 induction represents an early, "sentinel-type" response by the cell to counteract the deleterious effects of oxidative stress (Otterbein et al., 2000;Poss and Tonegawa, 1997b).
