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

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

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 inhibition of enzyme activity.

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 COrelated inhibition of P450 activity (discussed below).

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

Elucidating the Role of Biliverdin Reductase in

not prevent renal injury in the rats (Toda et al., 1995).

**3.3 Gene knockout/therapy evidence for the cytoprotective role of HO-1** 

hepatocytes. Iron also accumulated in renal proximal cortical tubules.

stannic mesoporphyrin indicating the role of HO-1.

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 541

In vivo studies also demonstrated the ability of HO-1 induction to protect against acute renal failure in rats following ischemia/reperfusion (Toda et al., 2002) and exposure to mercuric chloride (Yoneya et al., 2000). Ischemia/reperfusion involves exposing the tissue to a sequence of oxygen deprivation followed by reoxygenation. Reoxygenation is associated with high levels of oxidative stress. Thus, it is a good model to examine the protective role of HO-1. The kidney ischemia/reperfusion study used tin chloride to induce the HO-1. Tin chloride induces HO-1 in a tissue-specific manner and does not induce HO-1 in the liver but does induce it in the kidney, demonstrating the complicated regulation of the HO-1 gene (discussed below). The fundamental role of HO-1 in mediating renal protection was demonstrated by showing that treatment with tin mesoporphyrin, an inhibitor of HO-1, did

Over the last 10-15 years, novel research studies and interesting clinical findings have confirmed the cytoprotective role for HO-1. One of the seminal studies to demonstrate the protective role of HO-1 examined embryonic fibroblasts from HO-1 knockout mice and compared their attributes to those from normal wild-type animals (Poss and Tonegawa, 1997b). The cells from the knockout mice produced higher levels of ROS and also were less resistant to toxicity caused by hydrogen peroxide, paraquat, heavy metals, and heme exposure. The effects of HO-1 in the protection from free hemin exposure were quite dramatic offering 50% survival at a hemin concentration (200 μM) that was completely toxic to the cells from knockout mice. Another study from the same group, also compared the response of wild-type and HO-1 knockout mice to an intraperitoneal injection of endotoxin (Poss and Tonegawa, 1997a). Because the adult HO-1 knockout mice had a variety of health issues including anemia, iron-overloading, and chronic inflammation, younger mice (6 to 9 weeks) that did not display these phenotypes were used to study the effects of endotoxin. In terms of survival, the knockout mice were significantly more sensitive to endotoxin treatment and demonstrated higher levels of hepatic injury including increased serum liver enzyme levels and liver vacuolization. Interestingly, the hepatic injury seemed to be spatially and temporally related to iron loading malfunctions in both Kupffer cells and

Gene therapy studies to upregulate HO-1 have also been instrumental in proving that HO-1 is protective against cellular stress. In a study to demonstrate the role of HO-1 in vascular protection, a retroviral vector was used to transfect the human HO-1 gene into rat lung microvessel endothelium (Yang et al., 1999). Cells transfected with the retrovirus had over a 2-fold increase in HO-1 expression and activity. Furthermore, cGMP levels (probably regulated by CO activation of guanyly cyclase was almost 3-fold higher. These endothelial cells were significantly more resistant than untransfected cells to toxicity resulting from hydrogen peroxide and heme exposure. This protection was abolished upon treatment with

In another gene therapy study to investigate the ability of HO-1 to protect against the exposure of endotoxin in lung, an adenovirus encoding HO-1 was directly inoculated into rat trachea (Inoue et al., 2001). As a result, HO-1 was upregulated in both airway epithelium

activation of ethanol by CO. Another study by this group indicated that HO-1 was induced through the MAPK/Nrf2 pathways of signal transduction (Yao et al., 2007). The in vitro studies of course are critical in elucidating the signaling pathways involved in heme metabolism. These pathways are discussed more completely below. Interestingly, the second enzyme responsible for heme metabolism, BVR has a very active role in the signaling required to modulate HO-1 levels with the ever-changing levels of heme in the cell.

#### **3.2 In vivo evidence for the cytoprotective role of HO-1**

Many in vivo studies have also tested for the protective role of HO-1 after exposure to toxins. Acetominophen is a widely-used analgesic that unfortunately has a narrow therapeutic index, and overdosing results in liver failure. Cytochrome P450-mediated metabolism is responsible for the harmful effects of acetaminophen as it converts the compound to a reactive quinone-imine that alkylates cellular protein and DNA. Interestingly, cellular glutathione effectively scavenges the reactive intermediate and protects against cytotoxicity. However, when an overdose occurs, the intracellular glutathione gets depleted resulting in the destruction of critical proteins that are necessary for cell function (Gibson et al., 1996). Several studies have tested for the ability of HO-1 to protect against acetaminophen toxicity in rats. In one of these studies, acetaminophen treatment resulted in HO-1 induction. To test whether the HO-1 expression was cytoprotective, the rats were treated with hemin to induce HO-1 prior to exposure to acetaminophen. These rats were indeed protected from acetaminophen hepatotoxicity compared to animals that were not pretreated with hemin. The study also found that biliverdin pretreatment was able to protect the rats from acetaminophen-induced hepatotoxicity (Chiu et al., 2002).

HO-1 induction was also shown to be protective from liver damage caused by carbon tetrachloride (Nakahira et al., 2003) and halothane (Odaka et al., 2000). Both of these compounds can be activated to free radical species by P450-mediated metabolism. Treatment with hepatotoxic doses of these compounds resulted in the rapid accumulation of intracellular free heme which was followed by HO-1 induction. It was found that when the rats were pretreated with hemin (to induce HO-1) before halothane administration, hepatotoxicity was not observed. Similarly, when rats were treated with tin porphyrin 1 hour before administration of carbon tetrachloride to inhibit HO-1 activity, the carbon tetrachloride-induced liver injury was exacerbated (Nakahira et al., 2003). The findings of these studies suggest that free heme accumulation, presumably derived from the destruction of P450 enzymes, may be the main source of toxicity by these compounds. Thus, HO-1 induction was proposed to be an adaptive response that was critical for recovery from the toxic insults.

Many studies have investigated the ability of HO-1 to protect against endotoxin exposure. Endotoxin is a lipopolysaccharide produced by gram negative bacteria. Tissue exposure to endotoxin results in inflammatory injury and oxidative stress (Murphy et al., 1998) (McCord, 1993). In two separate studies, HO-1 induction in rats by hemin (Wen et al., 2007) and hemoglobin (Otterbein et al., 1995) pretreatment was protective against the deleterious effects of a subsequent (otherwise lethal) dose of endotoxin. In contrast, the rats were more susceptible to endotoxin toxicity, and the protective effects of HO-1 induction were ablated when the animals were treated with metal porphyrins that inhibited the HO-1 activity.

activation of ethanol by CO. Another study by this group indicated that HO-1 was induced through the MAPK/Nrf2 pathways of signal transduction (Yao et al., 2007). The in vitro studies of course are critical in elucidating the signaling pathways involved in heme metabolism. These pathways are discussed more completely below. Interestingly, the second enzyme responsible for heme metabolism, BVR has a very active role in the signaling

Many in vivo studies have also tested for the protective role of HO-1 after exposure to toxins. Acetominophen is a widely-used analgesic that unfortunately has a narrow therapeutic index, and overdosing results in liver failure. Cytochrome P450-mediated metabolism is responsible for the harmful effects of acetaminophen as it converts the compound to a reactive quinone-imine that alkylates cellular protein and DNA. Interestingly, cellular glutathione effectively scavenges the reactive intermediate and protects against cytotoxicity. However, when an overdose occurs, the intracellular glutathione gets depleted resulting in the destruction of critical proteins that are necessary for cell function (Gibson et al., 1996). Several studies have tested for the ability of HO-1 to protect against acetaminophen toxicity in rats. In one of these studies, acetaminophen treatment resulted in HO-1 induction. To test whether the HO-1 expression was cytoprotective, the rats were treated with hemin to induce HO-1 prior to exposure to acetaminophen. These rats were indeed protected from acetaminophen hepatotoxicity compared to animals that were not pretreated with hemin. The study also found that biliverdin pretreatment was able to protect the rats from acetaminophen-induced

HO-1 induction was also shown to be protective from liver damage caused by carbon tetrachloride (Nakahira et al., 2003) and halothane (Odaka et al., 2000). Both of these compounds can be activated to free radical species by P450-mediated metabolism. Treatment with hepatotoxic doses of these compounds resulted in the rapid accumulation of intracellular free heme which was followed by HO-1 induction. It was found that when the rats were pretreated with hemin (to induce HO-1) before halothane administration, hepatotoxicity was not observed. Similarly, when rats were treated with tin porphyrin 1 hour before administration of carbon tetrachloride to inhibit HO-1 activity, the carbon tetrachloride-induced liver injury was exacerbated (Nakahira et al., 2003). The findings of these studies suggest that free heme accumulation, presumably derived from the destruction of P450 enzymes, may be the main source of toxicity by these compounds. Thus, HO-1 induction was proposed to be an adaptive

Many studies have investigated the ability of HO-1 to protect against endotoxin exposure. Endotoxin is a lipopolysaccharide produced by gram negative bacteria. Tissue exposure to endotoxin results in inflammatory injury and oxidative stress (Murphy et al., 1998) (McCord, 1993). In two separate studies, HO-1 induction in rats by hemin (Wen et al., 2007) and hemoglobin (Otterbein et al., 1995) pretreatment was protective against the deleterious effects of a subsequent (otherwise lethal) dose of endotoxin. In contrast, the rats were more susceptible to endotoxin toxicity, and the protective effects of HO-1 induction were ablated when the animals were treated with metal porphyrins that inhibited the HO-1 activity.

required to modulate HO-1 levels with the ever-changing levels of heme in the cell.

**3.2 In vivo evidence for the cytoprotective role of HO-1** 

response that was critical for recovery from the toxic insults.

hepatotoxicity (Chiu et al., 2002).

In vivo studies also demonstrated the ability of HO-1 induction to protect against acute renal failure in rats following ischemia/reperfusion (Toda et al., 2002) and exposure to mercuric chloride (Yoneya et al., 2000). Ischemia/reperfusion involves exposing the tissue to a sequence of oxygen deprivation followed by reoxygenation. Reoxygenation is associated with high levels of oxidative stress. Thus, it is a good model to examine the protective role of HO-1. The kidney ischemia/reperfusion study used tin chloride to induce the HO-1. Tin chloride induces HO-1 in a tissue-specific manner and does not induce HO-1 in the liver but does induce it in the kidney, demonstrating the complicated regulation of the HO-1 gene (discussed below). The fundamental role of HO-1 in mediating renal protection was demonstrated by showing that treatment with tin mesoporphyrin, an inhibitor of HO-1, did not prevent renal injury in the rats (Toda et al., 1995).

## **3.3 Gene knockout/therapy evidence for the cytoprotective role of HO-1**

Over the last 10-15 years, novel research studies and interesting clinical findings have confirmed the cytoprotective role for HO-1. One of the seminal studies to demonstrate the protective role of HO-1 examined embryonic fibroblasts from HO-1 knockout mice and compared their attributes to those from normal wild-type animals (Poss and Tonegawa, 1997b). The cells from the knockout mice produced higher levels of ROS and also were less resistant to toxicity caused by hydrogen peroxide, paraquat, heavy metals, and heme exposure. The effects of HO-1 in the protection from free hemin exposure were quite dramatic offering 50% survival at a hemin concentration (200 μM) that was completely toxic to the cells from knockout mice. Another study from the same group, also compared the response of wild-type and HO-1 knockout mice to an intraperitoneal injection of endotoxin (Poss and Tonegawa, 1997a). Because the adult HO-1 knockout mice had a variety of health issues including anemia, iron-overloading, and chronic inflammation, younger mice (6 to 9 weeks) that did not display these phenotypes were used to study the effects of endotoxin. In terms of survival, the knockout mice were significantly more sensitive to endotoxin treatment and demonstrated higher levels of hepatic injury including increased serum liver enzyme levels and liver vacuolization. Interestingly, the hepatic injury seemed to be spatially and temporally related to iron loading malfunctions in both Kupffer cells and hepatocytes. Iron also accumulated in renal proximal cortical tubules.

Gene therapy studies to upregulate HO-1 have also been instrumental in proving that HO-1 is protective against cellular stress. In a study to demonstrate the role of HO-1 in vascular protection, a retroviral vector was used to transfect the human HO-1 gene into rat lung microvessel endothelium (Yang et al., 1999). Cells transfected with the retrovirus had over a 2-fold increase in HO-1 expression and activity. Furthermore, cGMP levels (probably regulated by CO activation of guanyly cyclase was almost 3-fold higher. These endothelial cells were significantly more resistant than untransfected cells to toxicity resulting from hydrogen peroxide and heme exposure. This protection was abolished upon treatment with stannic mesoporphyrin indicating the role of HO-1.

In another gene therapy study to investigate the ability of HO-1 to protect against the exposure of endotoxin in lung, an adenovirus encoding HO-1 was directly inoculated into rat trachea (Inoue et al., 2001). As a result, HO-1 was upregulated in both airway epithelium

Elucidating the Role of Biliverdin Reductase in

A (Immenschuh et al., 1998b).

**4.1 Protective roles of CO: Cell signaling mediated by CO** 

dependent signaling (Hangai-Hoger et al., 2007;Ishikawa et al., 2005).

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 543

The most definitive proof for the protective effects of CO has been derived from studies using CO-releasing molecules (Motterlini et al., 2003) as a surrogate for HO-1-derived CO. As indicated above, the CO formed by HO-1 has been shown to activate guanylyl cyclase to mediate the relaxation and dilation responses of smooth muscle and vascular endothelial cells, respectively (Cardell et al., 1998;Christodoulides et al., 1995). In vascular tissue, CO has also been shown to stimulate relaxation of endothelial smooth muscle cells by activation of calcium-dependent potassium channels (Williams et al., 2004) via a poorly-understood mechanism that does not involve guanylyl cyclase. CO has been shown to serve as a partial agonist to nitric oxide synthetase (NOS) and thus, may down-regulate the level of NOS-

Interestingly, it also was reported that cGMP-dependent signaling was able to induce HO-1 through the cAMP responsive element in its promoter by a mechanism that was not elucidated (Immenschuh et al., 1998a). Logically, these cGMP-related effects may be initiated by HO-1-generated CO. Of course, the cAMP promoter element also allows HO-1 to be induced directly through cAMP-dependent signaling and activation of protein kinase

Many of the details about other types of signaling involving CO are poorly understood as it appears to be both cell type- and stressor-specific (Song et al., 2003a){Song, 2003 2563 /id}. Most studies have implicated the ability of CO (and CO-releasing molecules) to activate the P38 MAPK pathway in carrying out its anti-apoptotic and anti-inflammatory effects

The MAPK pathways have been shown to regulate processes such as inflammation, differentiation, tumor promotion, proliferation, apoptosis, stress response, and ion channels (reviewed in (Shen et al., 2005;Wada and Penninger, 2004)). Two of the three arms of the MAPK pathway (JNK and p38) have been implicated in the cellular stress response. Downstream activation of the MAPK pathway, and specifically the JNK arm, leads to the dimerization and DNA binding of the stress response factors, c-Jun and c-fos. CO dramatically inhibited JNK MAPK signaling in murine macrophages exposed to endotoxin which resulted in lower production of inflammatory cytokine, IL-6 (Morse et al., 2003). Furthermore, c-Jun activation has been linked to cellular proliferation (Yoshioka et al., 1995) so this would explain part of the role of CO in mediating proliferation/transformation.

The p38 arm of the MAPK kinase activates ATF-2 which competes with c-fos for binding to c-Jun. This heterodimer binds with greater affinity to the HO-1 promoter than the c-fos/c-Jun heterodimer (Kravets et al., 2004). Furthermore, ATF-2 dimers can bind and directly activate the cyclic AMP responsive element (CRE) in the promoter region of HO-1 (Lee et al., 2002). ATF-2 activation may play a role in the activation of transcription factor, NFκB (Kaszubska et al., 1993). NFκB plays an essential role in the response to both apoptotic and inflammatory stimuli and regulates the expression of cytokines, growth factors, and cell cycle effector proteins (reviewed in (Bonizzi and Karin, 2004;Shen et al., 2005)). ATF-2 involvement in the activation of NFκB could explain the anti-apoptotic/anti-inflammatory affects of CO (see below for more details). In support of the idea that ATF-2 and c-Jun oppose one another in the protective gene expression associated with HO-1 induction, c-Jun

has been shown to inhibit activation of NFκB (Tan et al., 2009).

(Brouard et al., 2000;Dérijard et al., 1995;Keum et al., 2006;Otterbein et al., 2000).

and alveolar macrophages. This therapy was found to be as effective as HO-1 induction by hemin pretreatment in preventing the inflammatory reaction caused by aerosolized endotoxin exposure. Furthermore, the protection conferred by increased HO-1 expression seemed to be related to higher endogenous levels of Interleukin-10 production by the macrophages.

Gene therapy was also used to compare the oxidative stress resistance of cerebellar granular neurons isolated from wild type and transgenic, homozygous mice that were engineered to overexpress HO-1 (Chen et al., 2000) The transgenic mice overexpressing HO-1 generated lower levels of ROS and were more resistant to oxidative stress resulting from either glutamate or hydrogen peroxide treatment.

#### **3.4 Clinical evidence for the cytoprotective role of HO-1**

Finally, in a tragic clinical example, the protective role of HO-1 was profoundly demonstrated by an individual who did not have a functional HO-1 gene (Kawashima et al., 2002). The six-year old male patient presented with growth retardation, anemia, elevated levels of ferritin and heme in serum, low serum bilirubin, intravascular hemolysis, and hyperlipidemia. In contrast to the HO-1 knockout mice showing toxicity from iron overloading, the endothelial tissue of the human patient was more severely affected causing a spectrum of cardiovascular maladies. A lymphoblastoid-derived cell line from this patient was also extremely sensitive to hemin-induced oxidative stress (Yachie et al., 1999).

#### **3.5 Therapeutic potential of HO-1 modulation**

On the basis of these scientific and clinical findings, the role of HO-1 in maintaining homeostasis and protecting against cellular stress is now well established. In conjunction, the enzyme has been shown to be protective in various types of disease/injury models including the following: 1) inflammation (sepsis, atherosclerosis), 2) lung injury (pulmonary fibrosis, ventilator-induced injury), 3) cardiovascular injury/disease (myocardial infarction, hypertension), 4) ischemia/reperfusion, and 5) organ transplantation/rejection. There are now several excellent reviews that discuss the pharmacologic potential of HO-1 induction (Abraham and Kappas, 2008;Mancuso and Barone, 2009;Ryter et al., 2006). Unfortunately, this type of therapy is not straightforward as it has been observed that over-expression of HO-1 can be harmful from the accumulation of reactive iron (Suttner and Dennery, 1999) and the bilirubin that results from HO-1-mediated heme catabolism (Claireaux et al., 1953). Thus, HO-1 expression in this type of therapeutic treatment would need to be highly regulated to prevent over-expression of the enzyme.

#### **4. Mechanisms of cellular protection by HO-1**

Originally, it was thought that the sole mechanism by which HO-1 protected cells was through the catabolism of free heme and the elimination of its prooxidant activities (discussed at the beginning of the chapter). Ironically, it was originally thought that the other products of the HO-1 reaction were useless (or even toxic) by-products. Now, it is known that CO and biliverdin play multiple roles in protection and that there are actually several mechanisms by which HO-1 performs its cytoprotective functions. It is very likely that there are more mechanisms yet to be identified.

#### **4.1 Protective roles of CO: Cell signaling mediated by CO**

542 Pharmacology

and alveolar macrophages. This therapy was found to be as effective as HO-1 induction by hemin pretreatment in preventing the inflammatory reaction caused by aerosolized endotoxin exposure. Furthermore, the protection conferred by increased HO-1 expression seemed to be related to higher endogenous levels of Interleukin-10 production by the

Gene therapy was also used to compare the oxidative stress resistance of cerebellar granular neurons isolated from wild type and transgenic, homozygous mice that were engineered to overexpress HO-1 (Chen et al., 2000) The transgenic mice overexpressing HO-1 generated lower levels of ROS and were more resistant to oxidative stress resulting from either

Finally, in a tragic clinical example, the protective role of HO-1 was profoundly demonstrated by an individual who did not have a functional HO-1 gene (Kawashima et al., 2002). The six-year old male patient presented with growth retardation, anemia, elevated levels of ferritin and heme in serum, low serum bilirubin, intravascular hemolysis, and hyperlipidemia. In contrast to the HO-1 knockout mice showing toxicity from iron overloading, the endothelial tissue of the human patient was more severely affected causing a spectrum of cardiovascular maladies. A lymphoblastoid-derived cell line from this patient

On the basis of these scientific and clinical findings, the role of HO-1 in maintaining homeostasis and protecting against cellular stress is now well established. In conjunction, the enzyme has been shown to be protective in various types of disease/injury models including the following: 1) inflammation (sepsis, atherosclerosis), 2) lung injury (pulmonary fibrosis, ventilator-induced injury), 3) cardiovascular injury/disease (myocardial infarction, hypertension), 4) ischemia/reperfusion, and 5) organ transplantation/rejection. There are now several excellent reviews that discuss the pharmacologic potential of HO-1 induction (Abraham and Kappas, 2008;Mancuso and Barone, 2009;Ryter et al., 2006). Unfortunately, this type of therapy is not straightforward as it has been observed that over-expression of HO-1 can be harmful from the accumulation of reactive iron (Suttner and Dennery, 1999) and the bilirubin that results from HO-1-mediated heme catabolism (Claireaux et al., 1953). Thus, HO-1 expression in this type of therapeutic treatment would need to be highly

Originally, it was thought that the sole mechanism by which HO-1 protected cells was through the catabolism of free heme and the elimination of its prooxidant activities (discussed at the beginning of the chapter). Ironically, it was originally thought that the other products of the HO-1 reaction were useless (or even toxic) by-products. Now, it is known that CO and biliverdin play multiple roles in protection and that there are actually several mechanisms by which HO-1 performs its cytoprotective functions. It is very likely

was also extremely sensitive to hemin-induced oxidative stress (Yachie et al., 1999).

macrophages.

glutamate or hydrogen peroxide treatment.

**3.5 Therapeutic potential of HO-1 modulation** 

regulated to prevent over-expression of the enzyme.

**4. Mechanisms of cellular protection by HO-1** 

that there are more mechanisms yet to be identified.

**3.4 Clinical evidence for the cytoprotective role of HO-1** 

The most definitive proof for the protective effects of CO has been derived from studies using CO-releasing molecules (Motterlini et al., 2003) as a surrogate for HO-1-derived CO. As indicated above, the CO formed by HO-1 has been shown to activate guanylyl cyclase to mediate the relaxation and dilation responses of smooth muscle and vascular endothelial cells, respectively (Cardell et al., 1998;Christodoulides et al., 1995). In vascular tissue, CO has also been shown to stimulate relaxation of endothelial smooth muscle cells by activation of calcium-dependent potassium channels (Williams et al., 2004) via a poorly-understood mechanism that does not involve guanylyl cyclase. CO has been shown to serve as a partial agonist to nitric oxide synthetase (NOS) and thus, may down-regulate the level of NOSdependent signaling (Hangai-Hoger et al., 2007;Ishikawa et al., 2005).

Interestingly, it also was reported that cGMP-dependent signaling was able to induce HO-1 through the cAMP responsive element in its promoter by a mechanism that was not elucidated (Immenschuh et al., 1998a). Logically, these cGMP-related effects may be initiated by HO-1-generated CO. Of course, the cAMP promoter element also allows HO-1 to be induced directly through cAMP-dependent signaling and activation of protein kinase A (Immenschuh et al., 1998b).

Many of the details about other types of signaling involving CO are poorly understood as it appears to be both cell type- and stressor-specific (Song et al., 2003a){Song, 2003 2563 /id}. Most studies have implicated the ability of CO (and CO-releasing molecules) to activate the P38 MAPK pathway in carrying out its anti-apoptotic and anti-inflammatory effects (Brouard et al., 2000;Dérijard et al., 1995;Keum et al., 2006;Otterbein et al., 2000).

The MAPK pathways have been shown to regulate processes such as inflammation, differentiation, tumor promotion, proliferation, apoptosis, stress response, and ion channels (reviewed in (Shen et al., 2005;Wada and Penninger, 2004)). Two of the three arms of the MAPK pathway (JNK and p38) have been implicated in the cellular stress response. Downstream activation of the MAPK pathway, and specifically the JNK arm, leads to the dimerization and DNA binding of the stress response factors, c-Jun and c-fos. CO dramatically inhibited JNK MAPK signaling in murine macrophages exposed to endotoxin which resulted in lower production of inflammatory cytokine, IL-6 (Morse et al., 2003). Furthermore, c-Jun activation has been linked to cellular proliferation (Yoshioka et al., 1995) so this would explain part of the role of CO in mediating proliferation/transformation.

The p38 arm of the MAPK kinase activates ATF-2 which competes with c-fos for binding to c-Jun. This heterodimer binds with greater affinity to the HO-1 promoter than the c-fos/c-Jun heterodimer (Kravets et al., 2004). Furthermore, ATF-2 dimers can bind and directly activate the cyclic AMP responsive element (CRE) in the promoter region of HO-1 (Lee et al., 2002). ATF-2 activation may play a role in the activation of transcription factor, NFκB (Kaszubska et al., 1993). NFκB plays an essential role in the response to both apoptotic and inflammatory stimuli and regulates the expression of cytokines, growth factors, and cell cycle effector proteins (reviewed in (Bonizzi and Karin, 2004;Shen et al., 2005)). ATF-2 involvement in the activation of NFκB could explain the anti-apoptotic/anti-inflammatory affects of CO (see below for more details). In support of the idea that ATF-2 and c-Jun oppose one another in the protective gene expression associated with HO-1 induction, c-Jun has been shown to inhibit activation of NFκB (Tan et al., 2009).

Elucidating the Role of Biliverdin Reductase in

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 545

reductase. One of the odd aspects of the stoichiometry of these enzymes in the endoplasmic reticulum is that the amount of P450 enzymes far outnumber the amount of P450 reductase (with estimates as high as 25 P450s for every P450 reductase (Peterson et al., 1976). Thus, P450-mediated metabolism in the liver endoplasmic reticulum is extremely limited by the amount of available P450 reductase. Although the level of HO-1 in unstressed liver is very low, it can be induced to an amount that is comparable to that of P450s (Reed et al., 2011). Therefore, it seems likely that the induction of HO-1 would attenuate the rate of P450 mediated metabolism by limiting the ability of P450 to interact with P450 reductase. A preliminary investigation from our lab has provided support for this effect of HO-1 induction (Reed et al., 2011). Furthermore, recent studies in which cells were protected from oxidative stress by the transfection and induction of a mutant HO-1 which was not able to catalyze heme degradation also provides support for this type of indirect mechanism of cytoprotection (Lin et al., 2007;Lin et al., 2008). However as discussed below, the results with the shortened, inactive mutant could be explained if the mutant serves as a heme-carrier to

Although far from proven, HO-1 also might actually interact with P450 enzymes to accelerate the degradation of the P450s. Several studies, including a few that were cited above (Nakahira et al., 2003;Odaka et al., 2000), have postulated that HO-1 induction following a stress event coincides with a rapid accumulation of free heme which presumably originates from damaged P450 enzymes. Evidence also was derived by observing a dramatic increase in the rate of degradation of labeled heme from P450 enzymes after HO-1 was induced by either hemin or endotoxin treatment (Bissell and Hammaker, 1976). More specifically, the data suggested that HO-1 increased the degradation of the P450 and not just the catabolism of the heme released from the P450. Again, it is not possible to ascertain whether the damaged P450 releases the heme or the HO-1 binds to the P450 to scavenge and catabolize its heme group. More direct evidence of this putative effect of HO-1 was reported in a study finding that the incubation of purified P450s with either HO-1 and HO-2 caused the heme of the P450 enzymes to be degraded to biliverdin in essentially a 1 to 1 ratio (Kutty et al., 1988). The results also were consistent with the interaction of HO-1 and P450 causing one P450 (two were studied in the publication) to degrade to an inactive form. Surprisingly, the research supporting the idea that HO-1 facilitates degradation of P450s is decades old but has not been followed up on and confirmed. One reason for this is the fact that the full-length HO-1 is very unstable and susceptible to truncation that generates an inactive, soluble form (28 kDa). The C-terminal part of the protein that is cleaved causes the HO-1 to interact with membrane lipids, and its removal alters the manner by which the enzyme interacts with potential membrane binding partners (Huber, III et al., 2009;Huber, III and Backes, 2007). Most in vitro studies of HO-1 have expressed and purified a modified, but active, 30 kDa form of the enzyme that lacks the C-terminal membrane-binding sequence and is soluble as a result. Our lab has recently modified the amino acid sequence of full-length HO-1 to remove a thrombin cleavage site in the C-terminal tale of HO-1 (Huber, III and Backes, 2007). This mutant is full-length and binds to lipid vesicles. The fulllength HO-1 mutant also binds much tighter to the P450 reductase and has much higher catalytic efficiency than the active, soluble form of the enzyme (Huber, III et al., 2009). Thus, studies with this mutant will finally enable researchers to understand the enzymatic capability of HO-1 with respect to those of the other potential binding partners in the endoplasmic reticulum. The putative interaction of HO-1 with P450 may allow for very

shuttle heme to the nucleus in order to directly modulate gene transcription.

The ERK 1/2 MAPK regulates cellular growth and differentiation. Stimulation of the pathway has been shown to be protective against apoptosis (Wada and Penninger, 2004). However, overstimulation of ERK appears to be the major mechanism by which some oncogenes transforms cells (e.g. Ha-Ras (Hibi et al., 1993)). In one study of human airway smooth muscle cells, CO-mediated affects on guanylyl cyclase led to inhibition of ERK1/2 MAPK (Song et al., 2003b). Thus, this effect of CO on ERK 1/2 MAPK would serve to prevent overstimulation of this pathway and in turn, the uncontrolled proliferation of cells. ERK has been shown to phosphorylate the inhibitory protein of NFκB and thus facilitate activation of the transcription factor (Chun et al., 2003).

It has also been postulated that CO can activate transcription factors indirectly by mitochondrial-driven ROS production (Piantadosi, 2008). More specifically, it is known that CO is a potent inhibitor of the complex III-mediated terminal step of oxidative phosphorylation. The inhibition of this step of the mitochondrial electron transport chain results in excess ROS production which in turn, reacts with critical thiol groups of the phosphatases that turn off activated transcription factors. In this regard, CO-mediated, mitochondrial ROS production has been implicated in the prolonged activation of the phosphoinositide-3-kinase (PI3 kinase)/Akt pathway (Piantadosi, 2008;Pischke et al., 2005). Numerous studies have implicated this pathway in the protective effects of plant-derived antioxidants (that include induction of HO-1) by ultimately leading to the activation of the transcription factors, Nrf2 (Martin et al., 2004;Park et al., 2011;Pugazhenthi et al., 2007). Nrf2 is a member of the Cap-N-Collar/basic leucine zipper family of transcription factor that responds directly and indirectly to oxidative stress to mediate cytoprotective gene transcription through the antioxidant response elements (ARE) of gene promoters (reviewed in (Itoh et al., 1997;Itoh et al., 2003;Kwak et al., 2004)). Thus, CO-mediated activation of PI3 kinase/Akt provides anti-oxidative protection.

#### **4.2 Regulation of important enzymes by HO-1**

It was also discussed how the CO released from HO-1 can inhibit various enzyme activities. The ability of the molecule to inhibit cyclooxygenase may provide an anti-inflammatory effect by preventing the synthesis of inflammatory prostaglandins from arachidonic acid. CO also inhibits cytochromes P450. This action could be cytoprotective because unproductive P450 mediated metabolism results in the release of hydrogen peroxide and/or superoxide from the P450 active site. This activity is an unavoidable consequence of metabolism by these enzymes, and the amount of ROS produced in this manner is dependent on the substrate being metabolized and the specific type of P450 carrying out the reaction (Gorsky et al., 1984;Gruenke et al., 1995;Reed and Hollenberg, 2003). Furthermore, it has been reported that P450-mediated metabolism can generate destructive hydroxyl radicals under certain circumstances (Paller and Jacob, 1994;Terelius and Ingelman-Sundberg, 1988). The oxidative stress generated by P450-mediated metabolism is significant as it has been estimated that the rate of ROS formation by the endoplasmic reticulum can be as much as 30% of that by mitochondria during oxidative phosphorylation (Zangar et al., 2004).

In an idea originally proposed at the turn of the century, HO-1 may provide cytoprotection by indirectly inhibiting P450 activity (and its associated production of ROS) through its competition with P450 for binding to the P450 reductase (Emerson and LeVine, 2000). Both P450s and HO-1 obtain electrons needed for their respective reactions by binding to the P450

The ERK 1/2 MAPK regulates cellular growth and differentiation. Stimulation of the pathway has been shown to be protective against apoptosis (Wada and Penninger, 2004). However, overstimulation of ERK appears to be the major mechanism by which some oncogenes transforms cells (e.g. Ha-Ras (Hibi et al., 1993)). In one study of human airway smooth muscle cells, CO-mediated affects on guanylyl cyclase led to inhibition of ERK1/2 MAPK (Song et al., 2003b). Thus, this effect of CO on ERK 1/2 MAPK would serve to prevent overstimulation of this pathway and in turn, the uncontrolled proliferation of cells. ERK has been shown to phosphorylate the inhibitory protein of NFκB and thus facilitate

It has also been postulated that CO can activate transcription factors indirectly by mitochondrial-driven ROS production (Piantadosi, 2008). More specifically, it is known that CO is a potent inhibitor of the complex III-mediated terminal step of oxidative phosphorylation. The inhibition of this step of the mitochondrial electron transport chain results in excess ROS production which in turn, reacts with critical thiol groups of the phosphatases that turn off activated transcription factors. In this regard, CO-mediated, mitochondrial ROS production has been implicated in the prolonged activation of the phosphoinositide-3-kinase (PI3 kinase)/Akt pathway (Piantadosi, 2008;Pischke et al., 2005). Numerous studies have implicated this pathway in the protective effects of plant-derived antioxidants (that include induction of HO-1) by ultimately leading to the activation of the transcription factors, Nrf2 (Martin et al., 2004;Park et al., 2011;Pugazhenthi et al., 2007). Nrf2 is a member of the Cap-N-Collar/basic leucine zipper family of transcription factor that responds directly and indirectly to oxidative stress to mediate cytoprotective gene transcription through the antioxidant response elements (ARE) of gene promoters (reviewed in (Itoh et al., 1997;Itoh et al., 2003;Kwak et al., 2004)). Thus, CO-mediated activation of PI3

It was also discussed how the CO released from HO-1 can inhibit various enzyme activities. The ability of the molecule to inhibit cyclooxygenase may provide an anti-inflammatory effect by preventing the synthesis of inflammatory prostaglandins from arachidonic acid. CO also inhibits cytochromes P450. This action could be cytoprotective because unproductive P450 mediated metabolism results in the release of hydrogen peroxide and/or superoxide from the P450 active site. This activity is an unavoidable consequence of metabolism by these enzymes, and the amount of ROS produced in this manner is dependent on the substrate being metabolized and the specific type of P450 carrying out the reaction (Gorsky et al., 1984;Gruenke et al., 1995;Reed and Hollenberg, 2003). Furthermore, it has been reported that P450-mediated metabolism can generate destructive hydroxyl radicals under certain circumstances (Paller and Jacob, 1994;Terelius and Ingelman-Sundberg, 1988). The oxidative stress generated by P450-mediated metabolism is significant as it has been estimated that the rate of ROS formation by the endoplasmic reticulum can be as much as 30% of that by

In an idea originally proposed at the turn of the century, HO-1 may provide cytoprotection by indirectly inhibiting P450 activity (and its associated production of ROS) through its competition with P450 for binding to the P450 reductase (Emerson and LeVine, 2000). Both P450s and HO-1 obtain electrons needed for their respective reactions by binding to the P450

activation of the transcription factor (Chun et al., 2003).

kinase/Akt provides anti-oxidative protection.

**4.2 Regulation of important enzymes by HO-1** 

mitochondria during oxidative phosphorylation (Zangar et al., 2004).

reductase. One of the odd aspects of the stoichiometry of these enzymes in the endoplasmic reticulum is that the amount of P450 enzymes far outnumber the amount of P450 reductase (with estimates as high as 25 P450s for every P450 reductase (Peterson et al., 1976). Thus, P450-mediated metabolism in the liver endoplasmic reticulum is extremely limited by the amount of available P450 reductase. Although the level of HO-1 in unstressed liver is very low, it can be induced to an amount that is comparable to that of P450s (Reed et al., 2011). Therefore, it seems likely that the induction of HO-1 would attenuate the rate of P450 mediated metabolism by limiting the ability of P450 to interact with P450 reductase. A preliminary investigation from our lab has provided support for this effect of HO-1 induction (Reed et al., 2011). Furthermore, recent studies in which cells were protected from oxidative stress by the transfection and induction of a mutant HO-1 which was not able to catalyze heme degradation also provides support for this type of indirect mechanism of cytoprotection (Lin et al., 2007;Lin et al., 2008). However as discussed below, the results with the shortened, inactive mutant could be explained if the mutant serves as a heme-carrier to shuttle heme to the nucleus in order to directly modulate gene transcription.

Although far from proven, HO-1 also might actually interact with P450 enzymes to accelerate the degradation of the P450s. Several studies, including a few that were cited above (Nakahira et al., 2003;Odaka et al., 2000), have postulated that HO-1 induction following a stress event coincides with a rapid accumulation of free heme which presumably originates from damaged P450 enzymes. Evidence also was derived by observing a dramatic increase in the rate of degradation of labeled heme from P450 enzymes after HO-1 was induced by either hemin or endotoxin treatment (Bissell and Hammaker, 1976). More specifically, the data suggested that HO-1 increased the degradation of the P450 and not just the catabolism of the heme released from the P450. Again, it is not possible to ascertain whether the damaged P450 releases the heme or the HO-1 binds to the P450 to scavenge and catabolize its heme group. More direct evidence of this putative effect of HO-1 was reported in a study finding that the incubation of purified P450s with either HO-1 and HO-2 caused the heme of the P450 enzymes to be degraded to biliverdin in essentially a 1 to 1 ratio (Kutty et al., 1988). The results also were consistent with the interaction of HO-1 and P450 causing one P450 (two were studied in the publication) to degrade to an inactive form.

Surprisingly, the research supporting the idea that HO-1 facilitates degradation of P450s is decades old but has not been followed up on and confirmed. One reason for this is the fact that the full-length HO-1 is very unstable and susceptible to truncation that generates an inactive, soluble form (28 kDa). The C-terminal part of the protein that is cleaved causes the HO-1 to interact with membrane lipids, and its removal alters the manner by which the enzyme interacts with potential membrane binding partners (Huber, III et al., 2009;Huber, III and Backes, 2007). Most in vitro studies of HO-1 have expressed and purified a modified, but active, 30 kDa form of the enzyme that lacks the C-terminal membrane-binding sequence and is soluble as a result. Our lab has recently modified the amino acid sequence of full-length HO-1 to remove a thrombin cleavage site in the C-terminal tale of HO-1 (Huber, III and Backes, 2007). This mutant is full-length and binds to lipid vesicles. The fulllength HO-1 mutant also binds much tighter to the P450 reductase and has much higher catalytic efficiency than the active, soluble form of the enzyme (Huber, III et al., 2009). Thus, studies with this mutant will finally enable researchers to understand the enzymatic capability of HO-1 with respect to those of the other potential binding partners in the endoplasmic reticulum. The putative interaction of HO-1 with P450 may allow for very

Elucidating the Role of Biliverdin Reductase in

relate to cytoprotection.

levels, as described above.

metabolic needs of the cell (Ponka, 1997).

regulation.

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 547

Biliverdin also inhibits activation of NF-κB in HEK293 cells (Gibbs and Maines, 2007). The effect was observed to be specific for biliverdin and not bilirubin. In fact, overexpression of BVR, which converts the biliverdin to bilirubin, overcame the biliverdin-mediated inhibition of NF-κB. Thus, part of the anti-inflammatory effect of biliverdin may be caused by preventing activation of NF-κB. Biliverdin has also been shown to be a potent inhibitor of c-Jun N-terminal kinase and AP-1 pathway (Tang et al., 2007), and this effect has been associated with pro-inflammatory and pro-apoptotic responses. Bilirubin has been shown to modulate ERK1/2 signaling pathways (Taillé et al., 2003). Furthermore, it has now been shown that both biliverdin and bilirubin activate the aryl hydrocarbon receptor to induce expression of a spectrum of genes including CYP1A1 (Phelan et al., 1998). At this point, it is not fully appreciated how these cell-signaling effects mediated by biliverdin and bilirubin

In summary, the effects of biliverdin and bilirubin are complex and are poorly understood. Protection by these compounds seems to derive from antioxidant properties of the compounds as well as anti-nitrosative effects from scavenging NO. However, many more mechanisms may be involved to explain their effects. In fact, the effects of these compounds on cell signaling pathways are only beginning to be elucidated. It should also be mentioned that the balance between cytoprotection and toxicity is delicate in the case of bilirubin. High concentrations of this compound are neurotoxic and pro-oxidative (Claireaux et al., 1953;Stocker and Ames, 1987) adding to the complexity and importance of cellular heme

You can put a rose on a herring, but it will still stink and be red. The same analogy can be used when trying to argue the protective "benefits" of HO-1-mediated ferrous iron production. Free ferrous iron will be a "smelly, red herring" with regards to oxidative/reductive homeostasis in the cell. As mentioned at the beginning of the chapter, the metal is prone to generating highly destructive hydroxyl radicals. Thus, it is a powerful pro-oxidant which would cause it to potentiate oxidative stress. In fact, a study which overexpressed HO-1 through a tetracycline-inducible vector found that the mutant cells were much more prone to deleterious iron-overload (Suttner and Dennery, 1999). Thus, HO-1 expression had a negative effect on cell survival in this instance. Presumably, HO-1 overexpression also could have a negative health impact from the potential build-up of bilirubin

On the other hand, the metal is essential for the synthesis of heme and in turn, for all of the functions carried out by the heme proteins. Thus, HO-1 does provide a way for the iron in free heme or heme attached to damaged enzymes (which may act as even more potent prooxidants than free iron) to be recycled for future heme synthesis in the cell, so its activity does have a net positive effect on cellular health. Furthermore, heme induction also activates expression of the iron-storage protein, ferritin (Eisenstein et al., 1991). Thus, during hemerelated stress, both ferritin and HO-1 are coordinately regulated (Tsuji et al., 2000). Ferritin is an iron storage protein that allows for the controlled release of iron to coincide with the

**4.4 Ferrous iron release: The participation of ferritin and HO-1** 

efficient inhibition of the P450 by the HO-1-generated CO, providing a cytoprotective role by effectively removing the P450 as a contributor to cellular, oxidative stress.

#### **4.3 Protective role of biliverdin/bilirubin**

Originally biliverdin and the bilirubin formed by the BVR-catalyzed reduction of biliverdin were thought to be cellular waste products. However, it is apparent that both compounds have antioxidant properties and elicit various cytoprotective effects. Early studies implicated the antioxidant effects of these compounds by showing that they reacted with enzymatically generated superoxide in vitro (Galliani et al., 1985;Robertson, Jr. and Fridovich, 1982). Subsequent studies showed bilirubin to be a more potent antioxidant than α-tocopherol with respect to scavenging lipid peroxides (Neuzil and Stocker, 1993). In fact, both biliverdin and bilirubin were found to interact synergistically with vitamin E to prevent lipid peroxidation by an azo compound (Stocker and Peterhans, 1989). The fact that both of these bile pigments are lipophilic, especially bilirubin, makes them typically more effective than water soluble antioxidants in preventing the damage of membranes and organelles. Bilirubin bound to albumin was also shown to be an effective antioxidant in plasma by protecting the oxidation of low density lipoproteins (Stocker et al., 1987). In addition to its ability to scavenge ROS, bilirubin also inhibits the superoxide-generating NADPH oxidase (Kwak et al., 1991).

Evidence for cytoprotection mediated by the bile pigments comes from several studies. Bilirubin was shown to protect both neuronal cultures (Dore et al., 1999) and HeLa cells (Baranano et al., 2002) from hydrogen peroxide-induced toxicity. Furthermore, when cellular bilirubin was depleted by incubation of the cells with short antisense RNA to BVR, preventing the expression of BVR and in turn, its catalyzed conversion of biliverdin to bilirubin, intracellular levels of ROS increased and promoted apoptotic death of neuronal and HeLa cells (Baranano et al., 2002). It was found that the effects of bilirubin depletion had a greater pro-oxidant effect than depletion of cellular glutathione. In another study, pretreatment of cultured endothelial cells with bilirubin also protected cultured endothelial cells from pro-inflammatory responses after challenge by oxidized LDL and TNF-α (Kawamura et al., 2005). The level of protection of the endothelial cells was comparable to that achieved by preinduction of HO-1 with hemin. Interestingly, CO treatment of the cells did not protect them from these responses.

Bilirubin and biliverdin have also been shown to be protective in animal studies. Injection of bilirubin into rats prevented glutathione depletion following administration of cadmium chloride (Ossola and Tomaro, 1995). The two bile pigments also have been shown to be effective in various models of ischemia/reperfusion injury (Clark et al., 2000;Fondevila et al., 2004). Biliverdin treatment was as effective as hemin-mediated HO-1 induction in protecting rats from acetaminophen toxicity (Chiu et al., 2002). Bilirubin treatment also protected rats challenged with endotoxin by preventing an inflammatory response in the animals (Wang et al., 2004). Biliverdin and bilirubin also react with reactive nitrogen species such as nitric oxide and peroxynitrite. Thus, the compounds can attenuate NO signaling, and this was believed to be the cause of the anti-inflammatory effect in the endotoxin study (Wang et al., 2004). Another anti-nitrosative effect of HO-1 recently discovered is the finding that increased HO-1 expression was associated with induction of endothelial cell superoxide dismutase (Kruger et al., 2005). This, in turn, would lower the amount of superoxide available to react with NO to form peroxynitrite.

efficient inhibition of the P450 by the HO-1-generated CO, providing a cytoprotective role

Originally biliverdin and the bilirubin formed by the BVR-catalyzed reduction of biliverdin were thought to be cellular waste products. However, it is apparent that both compounds have antioxidant properties and elicit various cytoprotective effects. Early studies implicated the antioxidant effects of these compounds by showing that they reacted with enzymatically generated superoxide in vitro (Galliani et al., 1985;Robertson, Jr. and Fridovich, 1982). Subsequent studies showed bilirubin to be a more potent antioxidant than α-tocopherol with respect to scavenging lipid peroxides (Neuzil and Stocker, 1993). In fact, both biliverdin and bilirubin were found to interact synergistically with vitamin E to prevent lipid peroxidation by an azo compound (Stocker and Peterhans, 1989). The fact that both of these bile pigments are lipophilic, especially bilirubin, makes them typically more effective than water soluble antioxidants in preventing the damage of membranes and organelles. Bilirubin bound to albumin was also shown to be an effective antioxidant in plasma by protecting the oxidation of low density lipoproteins (Stocker et al., 1987). In addition to its ability to scavenge ROS, bilirubin also inhibits the superoxide-generating

Evidence for cytoprotection mediated by the bile pigments comes from several studies. Bilirubin was shown to protect both neuronal cultures (Dore et al., 1999) and HeLa cells (Baranano et al., 2002) from hydrogen peroxide-induced toxicity. Furthermore, when cellular bilirubin was depleted by incubation of the cells with short antisense RNA to BVR, preventing the expression of BVR and in turn, its catalyzed conversion of biliverdin to bilirubin, intracellular levels of ROS increased and promoted apoptotic death of neuronal and HeLa cells (Baranano et al., 2002). It was found that the effects of bilirubin depletion had a greater pro-oxidant effect than depletion of cellular glutathione. In another study, pretreatment of cultured endothelial cells with bilirubin also protected cultured endothelial cells from pro-inflammatory responses after challenge by oxidized LDL and TNF-α (Kawamura et al., 2005). The level of protection of the endothelial cells was comparable to that achieved by preinduction of HO-1 with hemin. Interestingly, CO treatment of the cells

Bilirubin and biliverdin have also been shown to be protective in animal studies. Injection of bilirubin into rats prevented glutathione depletion following administration of cadmium chloride (Ossola and Tomaro, 1995). The two bile pigments also have been shown to be effective in various models of ischemia/reperfusion injury (Clark et al., 2000;Fondevila et al., 2004). Biliverdin treatment was as effective as hemin-mediated HO-1 induction in protecting rats from acetaminophen toxicity (Chiu et al., 2002). Bilirubin treatment also protected rats challenged with endotoxin by preventing an inflammatory response in the animals (Wang et al., 2004). Biliverdin and bilirubin also react with reactive nitrogen species such as nitric oxide and peroxynitrite. Thus, the compounds can attenuate NO signaling, and this was believed to be the cause of the anti-inflammatory effect in the endotoxin study (Wang et al., 2004). Another anti-nitrosative effect of HO-1 recently discovered is the finding that increased HO-1 expression was associated with induction of endothelial cell superoxide dismutase (Kruger et al., 2005). This, in turn, would lower the amount of superoxide

by effectively removing the P450 as a contributor to cellular, oxidative stress.

**4.3 Protective role of biliverdin/bilirubin** 

NADPH oxidase (Kwak et al., 1991).

did not protect them from these responses.

available to react with NO to form peroxynitrite.

Biliverdin also inhibits activation of NF-κB in HEK293 cells (Gibbs and Maines, 2007). The effect was observed to be specific for biliverdin and not bilirubin. In fact, overexpression of BVR, which converts the biliverdin to bilirubin, overcame the biliverdin-mediated inhibition of NF-κB. Thus, part of the anti-inflammatory effect of biliverdin may be caused by preventing activation of NF-κB. Biliverdin has also been shown to be a potent inhibitor of c-Jun N-terminal kinase and AP-1 pathway (Tang et al., 2007), and this effect has been associated with pro-inflammatory and pro-apoptotic responses. Bilirubin has been shown to modulate ERK1/2 signaling pathways (Taillé et al., 2003). Furthermore, it has now been shown that both biliverdin and bilirubin activate the aryl hydrocarbon receptor to induce expression of a spectrum of genes including CYP1A1 (Phelan et al., 1998). At this point, it is not fully appreciated how these cell-signaling effects mediated by biliverdin and bilirubin relate to cytoprotection.

In summary, the effects of biliverdin and bilirubin are complex and are poorly understood. Protection by these compounds seems to derive from antioxidant properties of the compounds as well as anti-nitrosative effects from scavenging NO. However, many more mechanisms may be involved to explain their effects. In fact, the effects of these compounds on cell signaling pathways are only beginning to be elucidated. It should also be mentioned that the balance between cytoprotection and toxicity is delicate in the case of bilirubin. High concentrations of this compound are neurotoxic and pro-oxidative (Claireaux et al., 1953;Stocker and Ames, 1987) adding to the complexity and importance of cellular heme regulation.

#### **4.4 Ferrous iron release: The participation of ferritin and HO-1**

You can put a rose on a herring, but it will still stink and be red. The same analogy can be used when trying to argue the protective "benefits" of HO-1-mediated ferrous iron production. Free ferrous iron will be a "smelly, red herring" with regards to oxidative/reductive homeostasis in the cell. As mentioned at the beginning of the chapter, the metal is prone to generating highly destructive hydroxyl radicals. Thus, it is a powerful pro-oxidant which would cause it to potentiate oxidative stress. In fact, a study which overexpressed HO-1 through a tetracycline-inducible vector found that the mutant cells were much more prone to deleterious iron-overload (Suttner and Dennery, 1999). Thus, HO-1 expression had a negative effect on cell survival in this instance. Presumably, HO-1 overexpression also could have a negative health impact from the potential build-up of bilirubin levels, as described above.

On the other hand, the metal is essential for the synthesis of heme and in turn, for all of the functions carried out by the heme proteins. Thus, HO-1 does provide a way for the iron in free heme or heme attached to damaged enzymes (which may act as even more potent prooxidants than free iron) to be recycled for future heme synthesis in the cell, so its activity does have a net positive effect on cellular health. Furthermore, heme induction also activates expression of the iron-storage protein, ferritin (Eisenstein et al., 1991). Thus, during hemerelated stress, both ferritin and HO-1 are coordinately regulated (Tsuji et al., 2000). Ferritin is an iron storage protein that allows for the controlled release of iron to coincide with the metabolic needs of the cell (Ponka, 1997).

Elucidating the Role of Biliverdin Reductase in

the activation of this pathway by BVR.

effects associated with expression of HO-1.

to influence HO-1 gene transcription.

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 549

cellular stress signals. It is now known that BVR also functions as a dual-specific kinase of serine/threonine and tyrosine residues in proteins, and in this capacity, BVR affects the signaling and cellular responses to a variety of stimuli (Reviewed in (Kapitulnik and Maines, 2009)). Kinases capable of phosphorylating both threonine and tyrosine residues have been identified as those regulating upstream events in signal transduction pathways (Pawson and Scott, 2005). The discovery of this function of BVR was preceded by finding that its ability to metabolize biliverdin was dependent on protein phosphorylation and that

Subsequently, it was shown that BVR is regulated by insulin/insulin growth factor stimulation through receptor-mediated tyrosine phosphorylation (Lerner-Marmarosh et al., 2005). BVR binding to this receptor competes with insulin receptor substrates (IRS) 1 and 2 for binding to the receptor. BVR phosphorylates serine residues of the IRS which attenuates their affinity for the insulin receptor kinase, essentially inactivating them. Phosphorylated BVR can activate two protein kinase C proteins, βII and ζ, which are involved in cross-talk between the upstream components of the MAPK and phosphatidylinositol 3-kinase pathways, respectively. Protein kinase C βII also can activate BVR which partly contributes to the activation of BVR by stress signals (Maines et al., 2007). The activation of protein kinase C βII by BVR leads to activation of all three arms of the MAPK signaling. Thus, all of the effects of CO caused by its activation of the P38 MAPK (discussed above) also apply to

BVR-mediated signaling appears to play a critical role in the recruiting transcription factor, NFκB to the HO-1 promoter (Gibbs and Maines, 2007). Furthermore, NFκB has been shown to be activated by protein kinase Cζ which in turn, is directly activated by BVR (Lerner-Marmarosh et al., 2007). As described in detail below, the involvement of NFκB appears to be important in mediating the anti-apoptotic, anti-inflammatory, and anti-proliferative

BVR has the ability to form protein complexes with itself and other proteins, and serves to shuttle activated transcription factors to the nucleus. The ability of BVR to function as a dual cofactor enzyme with different pH optima expands its range of function in the cell (Kapitulnik and Maines, 2009). In addition to the activation of the ERK MAPK pathway by BVR through protein kinase C βII, BVR has been shown to play a critical role in shuttling the activated ERK to the nucleus to influence gene transcription (Lerner-Marmarosh et al., 2008). Interestingly, BVR also binds to NFκB (Gibbs and Maines, 2007). This is intriguing because the HO-1 promoter does not have a prototypical response element for NFκB, and it has been conjectured that it must be recruited to the promoter by other transcription factors (Alam and Cook, 2007). Thus, through this interaction, BVR also may play a role in allowing NFκB

In another transport capacity, BVR also complexes with heme and shuttles it to the nucleus where the heme can bind to regulatory elements that influence gene transcription. In fact, heme-mediated gene induction has been shown to be dependent on BVR in renal cells (Tudor et al., 2008). Interestingly, although HO-1 is most typically observed in the endoplasmic reticulum, instances of it being located in other parts of the cell including the plasma membrane (where it localizes to caveolae rafts (Kim et al., 2004)), mitochondria

the enzyme could catalyze autophosphorylation of this residue (Salim et al., 2001).

Interestingly, it has been suggested that there are *at least* two types of HO-1 inducing agents, heme-dependent and heme-independent (Bauer and Bauer, 2002). Subsequently, it was shown that heme-independent HO-1 induction did not necessarily induce ferritin (Sheftel et al., 2007). However, HO-1 will only produce excess free iron when there is an abundant supply of heme, so the ferritin will be induced in the cell when it is needed. In the study cited above (Suttner and Dennery, 1999) indicating iron over-load in cells overexpressing HO-1, the cells were transfected with an expression vector. Thus, hemin was not involved in the induction of HO-1 and consequently, ferritin was not induced enough to protect against iron overload from catalytically active HO-1.

Although it has not been proven definitively, it has been speculated that the location of HO-1 in the endoplasmic reticulum may facilitate the migration of free iron to the extracellular space and in turn, help maintain iron blood levels (Poss and Tonegawa, 1997a). Whether or not this putative role of HO-1 exists, the activity of the enzyme and the co-ordinate regulation of ferritin when HO-1 is induced through its cognate promoter give the cell a protective way to recycle iron and manage its levels in the cell.
