**6. Assessing the dependence of HO-1-mediated cytoprotection on BVR**

Given the diverse abilities of BVR to regulate cellular processes, it has been postulated that the HO-1 cytoprotective response is completely dependent on BVR. The hypothesis proposes that the increased expression of HO-1 caused by the wide variety of inducing agents is mediated by the signaling and DNA binding activities of BVR. Furthermore, in a study examining the catalytic steps of the HO-1 reaction, it was determined that binding of BVR and HO-1 was required to increase the rate of release of biliverdin from the HO-1 active site. The study found that in the absence of BVR, biliverdin release was the ratelimiting step of the HO-1 reaction, and its presence dramatically increased turnover by HO-1. In showing that even the catalytic activity of the enzyme was dependent on BVR, the study supported the idea that the HO-1 cytoprotective response was totally dependent on BVR. However, finding that HO-1 activity was dependent on BVR was counter-intuitive because the level of HO-1 can be induced many-fold by cellular exposure to stress signals (Wright et al., 2006). In contrast, the expression level of BVR is typically unchanged after stress (although it may be inducible in kidney (Maines et al., 2001)). Thus, it would seem to be unproductive for the cell to induce HO-1 and have its activity be limited by the supply of BVR. Such a situation also would allow little opportunity for biliverdin to accumulate in cells. Yet, studies show that biliverdin has completely different cytoprotective roles than bilirubin (see above). Thus, the enzymatic data showing that BVR regulated HO-1 enzymatic activity were baffling.

More recent enzymatic studies measuring steady state metabolism by HO-1 showed that BVR had no effect on the rate of HO-1-mediated catalysis (Reed et al., 2010). In fact, this study showed that the rates of HO-1-mediated heme catabolism measured by the formation of both biliverdin and a ferrozene:ferrous iron complex in the absence of BVR were slightly higher than that measured by bilirubin formation in the presence of BVR. The earlier results finding that HO-1 activity was dependent on BVR were attributed to the unusual conditions required to monitor the individual catalytic steps (namely anaerobic with limiting concentrations of NADPH).

The question still remains whether the HO-1-related gene response following exposure to stress signals is completely dependent on BVR. The HO-1 promoter contains a variety of responsive elements that includes two AP-1 sites, a CRE, a Maf recognition domain (binding partner to Nrf2), and a partial site for NFκB binding (Alam and Cook, 2007). Understanding how these responsive elements recruit transcription factors to influence gene transcription is complicated by the fact that many of the response elements overlap and thus, may inhibit or enhance DNA binding by factors to adjacent elements. The transcription factors that activate the HO-1 promoter will vary with the types of stress to which the cell is exposed (reviewed in (Alam and Cook, 2007). Furthermore, studies have shown that the signaling pathways activated by the same stress signals also can vary in different cell types (Ryter et al., 2006). As a result, the transcription factors mediating a specific cytoprotective response can vary in different cell types. Thus, a type of HO-1-mediated cytoprotection may depend on BVR in some cell types but not others. Because of these considerations, the discussion pertaining to the dependence of HO-1 expression on BVR will be a generalization based on the typical roles of the implicated transcription factors in cellular processes.

#### **6.1 Anti-oxidant protection**

550 Pharmacology

(Converso et al., 2006), and even as a shortened, soluble component in the nucleus have been reported (Lin et al., 2007). The significance of these findings has yet to be elucidated, but they may indicate that HO-1 also can transport heme to the nucleus (discussed below) and may be involved in targeting BVR to different organelles to mediate signaling or the biliverdin/BVR redox cycle to scavenge ROS from the mitochondria (discussed above).

Although the kinase-dependent activity described above ultimately results in the modulation of gene transcription, BVR also has the capacity to bind to DNA and directly influence gene expression through a basic leucine zipper-binding motif. BVR binds to AP-1/CRE sites in DNA which are typically bound by c-Jun/fos heterodimers to mediate the stress response (Ahmad et al., 2002). In this capacity, BVR binds as homo- and hetero-dimers to recruit or block the binding of other transcription factors and thus, BVR can enhance or inhibit gene expression directly. At this point, BVR-mediated gene transcription has been shown to induce ATF-2 (Kravets et al., 2004). ATF-2 has also been shown to be capable of

Thus, with regards to the role of BVR in recruiting NFκB to the HO-1 promoter, BVR can facilitate its binding directly or through the upregulation and activation of ATF-2. However, it is likely that NFκB would bind to the promoter in different 5'-flanking regions when bound to BVR and ATF-2. Most of the specific effects of BVR-DNA binding on gene expression have not been elucidated. But the enzyme has been shown to translocate to the nucleus of HeLa cells (Tudor et al., 2008) after hemin induction and in rat kidney after exposure of animals to nephrotoxins (Maines et al., 2001). In fact, HeLa, heme-mediated induction of HO-1 expression

**6. Assessing the dependence of HO-1-mediated cytoprotection on BVR** 

Given the diverse abilities of BVR to regulate cellular processes, it has been postulated that the HO-1 cytoprotective response is completely dependent on BVR. The hypothesis proposes that the increased expression of HO-1 caused by the wide variety of inducing agents is mediated by the signaling and DNA binding activities of BVR. Furthermore, in a study examining the catalytic steps of the HO-1 reaction, it was determined that binding of BVR and HO-1 was required to increase the rate of release of biliverdin from the HO-1 active site. The study found that in the absence of BVR, biliverdin release was the ratelimiting step of the HO-1 reaction, and its presence dramatically increased turnover by HO-1. In showing that even the catalytic activity of the enzyme was dependent on BVR, the study supported the idea that the HO-1 cytoprotective response was totally dependent on BVR. However, finding that HO-1 activity was dependent on BVR was counter-intuitive because the level of HO-1 can be induced many-fold by cellular exposure to stress signals (Wright et al., 2006). In contrast, the expression level of BVR is typically unchanged after stress (although it may be inducible in kidney (Maines et al., 2001)). Thus, it would seem to be unproductive for the cell to induce HO-1 and have its activity be limited by the supply of BVR. Such a situation also would allow little opportunity for biliverdin to accumulate in cells. Yet, studies show that biliverdin has completely different cytoprotective roles than bilirubin (see above). Thus, the enzymatic data showing that BVR regulated HO-1

**5.3 BVR-mediated modulation of gene transcription through DNA binding** 

binding to and activating NFκB (Kaszubska et al., 1993).

was shown to be dependent on BVR expression (Tudor et al., 2008).

enzymatic activity were baffling.

With this perspective in mind, the various protective roles of cell signaling and gene transcription associated with HO-1 induction are mediated largely through activation of either Nrf2 or NFκB. Certainly one of the first evolutionary roles needed for survival of a cell would have been to develop a defense system for the oxidative stress associated with the activity of heme enzymes. Heme oxygenase is the only enzyme that uses its heme cofactor as a substrate. Furthermore, as discussed below, HO-1 can be induced directly by heme interaction with the HO-1 gene repressor Bach-1. Therefore, it is the only known eukaryotic enzyme that has a substrate that regulates a transcription factor needed for its expression. This scenario would suggest that heme oxygenase represents a very old link in the evolutionary chain. By the same line of reasoning used above to rationalize the evolutionary role of HO-1 function, Nrf2/anti-oxidant response must represent an early evolutionary adaptation to allow living organisms to survive oxidative stress. Interestingly, Bach-1 is an effective repressor of the HO-1 gene because it blocks Nrf2 from binding to the HO-1 promoter. Because heme metabolism is intimately connected to reductive/oxidative homeostasis in the cell, it seems plausible that Nrf2 can mediate HO-1 expression independently from BVR as a response (initially at least) to oxidative stress.

One caveat to the hypothesis that the anti-oxidative response is independent of BVR is dictated by whether or not the shuttling of heme to the nucleus by BVR is the only mechanism by which this can occur (discussed above). As alluded to above, Bach 1 blocks the binding of Nrf2 to the antioxidant response element when Bach 1 is not bound to heme

Elucidating the Role of Biliverdin Reductase in

**6.2 Anti-apoptotic/proliferation protection** 

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 553

promoter. In fact, genetic polymorphisms affecting this region have been shown to

Thus, the right panel of Figure 2 is drawn to indicate the BVR-independent activation of HO-1 gene transcription after exposure to oxidative stress. ROS can activate Nrf2 directly by oxidizing Keap-1 which is responsible for binding Nrf2 in the cytoplasm and facilitating its degradation (Itoh et al., 2003). Oxidation of critical cysteine residues in Keap-1 releases Nrf2, allowing it to translocate to the nucleus and activate gene transcription (reviewed in (Kwak et al., 2004)). In addition, CO inhibits mitochondrial electron transport resulting in ROS production which inhibit phosphatases needed to inactivate PI3K. Prolonged activation of PI3K results in stimulation of Akt which can phosphorylate Nrf2 (Piantadosi, 2008). Phosphorylation causes Keap-1 to release and activate Nrf2. ROS can also lead to activation of JNK MAPK. It has been shown that JNK target, c-Jun, can bind to Nrf2 (Shen et al., 2005). Thus, c-Jun/fos dimers binding to the AP-1 site facilitate the recruitment of Nrf2 to bind to the overlapping Maf-recognition element and induce HO-1 transcription. Previous binding by the c-Jun/fos dimer may help facilitate the binding of Nrf2 if Nrf2 can exchange with fos for binding to c-Jun. BVR is shown in this panel to serve as a heme transporter to the nucleus. That putative role is also indicated for the shortened form of HO-1 that has been identified in the nucleus (Lin et al., 2007) and for Nrf2. One additional role that BVR could play in the prolonged anti-oxidant response is to modulate the initial response by increasing

correspond to susceptibility to oxidative stress-related diseases (Ishii et al., 2000).

or decreasing the anti-oxidant response depending on the needs of the cell.

different cytoprotective responses mediated by HO-1 induction.

For the reasons below, it is postulated that BVR plays an integral role in the signaling responsible for anti-proliferative, anti-inflammatory, and anti-apoptotic responses by facilitating the interaction of NFκB with the HO-1 promoter and by modulating the activity of the transcription factor. As mentioned above, NFκB regulates the expression of cytokines, growth factors, and cell cycle effector proteins (Du et al., 1993;Hayden and Ghosh, 2011;Peng et al., 1995). As a result, the factor regulates important physiological processes such as immune response and apoptosis, and overstimulation or dysregulation of this factor is associated with inflammation, transformation, and proliferation of cells (Bubici et al., 2006). Thus, the cytoprotective role of HO-1 induction for processes such as inflammation, apoptosis, and proliferation would logically involve mechanisms affecting NFκB function. Scientific studies have shown that NFκB and Nrf2 are oppositely and variably regulated by different types of cellular stress (Bellezza et al., 2010). Nrf2 is typically activated by low or moderate levels oxidative stress, whereas NFκB is turned on by inflammatory signals or very high levels of oxidative stress. Thus, with regards to the HO-1 promoter and various cytoprotective responses mediated by enzyme induction, stress signals will specifically alter the relative levels of cellular transcription factors and in turn, will determine whether Nrf2 or NFκB binds to activate transcription of the HO-1 gene. The JNK MAPK target, c-Jun has been shown to bind to Nrf2 (Shen et al., 2005), and inhibit NFκB (Tan et al., 2009). However, the P38 MAPK target, ATF-2 has been shown to bind to NFκB (Kaszubska et al., 1993). The model below proposes that the relative levels of c-Jun and ATF-2 play a major role in

One essential role that BVR may play in modulating cytoprotective gene expression associated with HO-1 induction is the activation and recruitment of NFκB to the HO-1

(Sun et al., 2002). In support of the idea that an anti-oxidant response can be mediated directly by Nrf2 without involvement of BVR, HO-1 was found to be constitutively expressed in Bach 1 knockout mice (Sun et al., 2002). Thus, it is proposed that nuclear heme localization and functional Nrf2 are the essential components of the initial gene response to oxidative stress. In some instances, BVR has been shown to be critical for heme translocation to the nucleus. However, the results are mixed and may be cell type-specific. For instance, the same laboratory has shown that inhibition of BVR expression with small interference (antisense) RNA blocked the hemin-mediated induction of HO-1 cells in HeLa cells (Tudor et al., 2008) but had no effect in COS cells (Ahmad et al., 2002).

Recent studies showing that shortened HO-1 also translocates to the nucleus may serve as another mechanism by which hemin is transported to the nucleus to directly influence gene transcription (Lin et al., 2007). This would allow the shuttling role of BVR to be bypassed in some cells and would explain results showing that increased expression of a catalytically inactive mutant was also able to up-regulate HO-1 expression (Lin et al., 2008). Another way that cells could possibly bypass BVR-mediated heme shuttling can be postulated from the results of a study showing that heme bound to and stabilized Nrf2 (Alam et al., 2003). This led to activation of Nrf2 and the heme-responsive element in the HO-1 promoter. Thus, Nrf2 could also transport heme to the nucleus to influence gene transcription during oxidative stress. Therefore, it does seem necessary to require BVR to transport heme to the nucleus for most cell types.

There also are several general experimental findings that support this inutitive argument. First, Nrf2 is believed to be the primary transcription factor activated directly by oxidative stress, and its activation is associated with induction of phase II and antioxidant enzymes (Bellezza et al., 2010;Ishii et al., 2002;Shen et al., 2005). A multitude of studies have implicated Nrf2 in the induction of HO-1 during oxidative stress (Alam et al., 2003;Gong and Cederbaum, 2006a;Gong and Cederbaum, 2006b;Sun Jang et al., 2009). Finally, in two studies looking at the effects of oxidative stress (by hydrogen peroxide treatment), it was found that cell viability was only marginally affected (Baranano et al., 2002) or not affected at all (Maghzal et al., 2009) by silencing BVR (with interference RNA). The latter study also showed that BVR induction and over-expression also did not provide protection to the cells exposed to hydrogen peroxide. Thus, it may not be a coincidence that studies have not yet implicated a connection between BVR actions and Nrf2. In fact, the binding site for Nrf2 in the HO-1 promoter overlaps with one of the AP-1 sites to which BVR can bind (Alam and Cook, 2007). Differences between antioxidant response elements and AP-1 binding sites have been distinguished previously (Yoshioka et al., 1995). Thus, it appears Nrf2 binding and AP-1 binding by BVR might be somewhat competitive with Nrf2 binding occurring initially after oxidative stress and BVR replacing the transcription factor from the antioxidant response element after prolonged oxidative stress.

In addition, to the demonstration of constitutive HO-1 expression in Bach-1 knockout mice, there appears to be a unique aspect of the HO-1 promoter which allows Nrf2 to initiate transcription without recruitment of other transcription factors. It has been found that the chromatin remodeling protein, BRG1, interacts with Nrf2 to form a Z-DNA structure which permits access for RNA polymerase II to initiate transcription of the HO-1 gene (Zhang et al., 2006). This interaction between BRG-1 and Nrf2 was exclusive to the HO-1 gene (but not other Nrf2–regulated genes) by virtue of a series of TG repeats that are present in the HO-1

(Sun et al., 2002). In support of the idea that an anti-oxidant response can be mediated directly by Nrf2 without involvement of BVR, HO-1 was found to be constitutively expressed in Bach 1 knockout mice (Sun et al., 2002). Thus, it is proposed that nuclear heme localization and functional Nrf2 are the essential components of the initial gene response to oxidative stress. In some instances, BVR has been shown to be critical for heme translocation to the nucleus. However, the results are mixed and may be cell type-specific. For instance, the same laboratory has shown that inhibition of BVR expression with small interference (antisense) RNA blocked the hemin-mediated induction of HO-1 cells in HeLa cells (Tudor

Recent studies showing that shortened HO-1 also translocates to the nucleus may serve as another mechanism by which hemin is transported to the nucleus to directly influence gene transcription (Lin et al., 2007). This would allow the shuttling role of BVR to be bypassed in some cells and would explain results showing that increased expression of a catalytically inactive mutant was also able to up-regulate HO-1 expression (Lin et al., 2008). Another way that cells could possibly bypass BVR-mediated heme shuttling can be postulated from the results of a study showing that heme bound to and stabilized Nrf2 (Alam et al., 2003). This led to activation of Nrf2 and the heme-responsive element in the HO-1 promoter. Thus, Nrf2 could also transport heme to the nucleus to influence gene transcription during oxidative stress. Therefore, it does seem necessary to require BVR to transport heme to the nucleus for

There also are several general experimental findings that support this inutitive argument. First, Nrf2 is believed to be the primary transcription factor activated directly by oxidative stress, and its activation is associated with induction of phase II and antioxidant enzymes (Bellezza et al., 2010;Ishii et al., 2002;Shen et al., 2005). A multitude of studies have implicated Nrf2 in the induction of HO-1 during oxidative stress (Alam et al., 2003;Gong and Cederbaum, 2006a;Gong and Cederbaum, 2006b;Sun Jang et al., 2009). Finally, in two studies looking at the effects of oxidative stress (by hydrogen peroxide treatment), it was found that cell viability was only marginally affected (Baranano et al., 2002) or not affected at all (Maghzal et al., 2009) by silencing BVR (with interference RNA). The latter study also showed that BVR induction and over-expression also did not provide protection to the cells exposed to hydrogen peroxide. Thus, it may not be a coincidence that studies have not yet implicated a connection between BVR actions and Nrf2. In fact, the binding site for Nrf2 in the HO-1 promoter overlaps with one of the AP-1 sites to which BVR can bind (Alam and Cook, 2007). Differences between antioxidant response elements and AP-1 binding sites have been distinguished previously (Yoshioka et al., 1995). Thus, it appears Nrf2 binding and AP-1 binding by BVR might be somewhat competitive with Nrf2 binding occurring initially after oxidative stress and BVR replacing the transcription factor from the

In addition, to the demonstration of constitutive HO-1 expression in Bach-1 knockout mice, there appears to be a unique aspect of the HO-1 promoter which allows Nrf2 to initiate transcription without recruitment of other transcription factors. It has been found that the chromatin remodeling protein, BRG1, interacts with Nrf2 to form a Z-DNA structure which permits access for RNA polymerase II to initiate transcription of the HO-1 gene (Zhang et al., 2006). This interaction between BRG-1 and Nrf2 was exclusive to the HO-1 gene (but not other Nrf2–regulated genes) by virtue of a series of TG repeats that are present in the HO-1

et al., 2008) but had no effect in COS cells (Ahmad et al., 2002).

antioxidant response element after prolonged oxidative stress.

most cell types.

promoter. In fact, genetic polymorphisms affecting this region have been shown to correspond to susceptibility to oxidative stress-related diseases (Ishii et al., 2000).

Thus, the right panel of Figure 2 is drawn to indicate the BVR-independent activation of HO-1 gene transcription after exposure to oxidative stress. ROS can activate Nrf2 directly by oxidizing Keap-1 which is responsible for binding Nrf2 in the cytoplasm and facilitating its degradation (Itoh et al., 2003). Oxidation of critical cysteine residues in Keap-1 releases Nrf2, allowing it to translocate to the nucleus and activate gene transcription (reviewed in (Kwak et al., 2004)). In addition, CO inhibits mitochondrial electron transport resulting in ROS production which inhibit phosphatases needed to inactivate PI3K. Prolonged activation of PI3K results in stimulation of Akt which can phosphorylate Nrf2 (Piantadosi, 2008). Phosphorylation causes Keap-1 to release and activate Nrf2. ROS can also lead to activation of JNK MAPK. It has been shown that JNK target, c-Jun, can bind to Nrf2 (Shen et al., 2005). Thus, c-Jun/fos dimers binding to the AP-1 site facilitate the recruitment of Nrf2 to bind to the overlapping Maf-recognition element and induce HO-1 transcription. Previous binding by the c-Jun/fos dimer may help facilitate the binding of Nrf2 if Nrf2 can exchange with fos for binding to c-Jun. BVR is shown in this panel to serve as a heme transporter to the nucleus. That putative role is also indicated for the shortened form of HO-1 that has been identified in the nucleus (Lin et al., 2007) and for Nrf2. One additional role that BVR could play in the prolonged anti-oxidant response is to modulate the initial response by increasing or decreasing the anti-oxidant response depending on the needs of the cell.

#### **6.2 Anti-apoptotic/proliferation protection**

For the reasons below, it is postulated that BVR plays an integral role in the signaling responsible for anti-proliferative, anti-inflammatory, and anti-apoptotic responses by facilitating the interaction of NFκB with the HO-1 promoter and by modulating the activity of the transcription factor. As mentioned above, NFκB regulates the expression of cytokines, growth factors, and cell cycle effector proteins (Du et al., 1993;Hayden and Ghosh, 2011;Peng et al., 1995). As a result, the factor regulates important physiological processes such as immune response and apoptosis, and overstimulation or dysregulation of this factor is associated with inflammation, transformation, and proliferation of cells (Bubici et al., 2006). Thus, the cytoprotective role of HO-1 induction for processes such as inflammation, apoptosis, and proliferation would logically involve mechanisms affecting NFκB function. Scientific studies have shown that NFκB and Nrf2 are oppositely and variably regulated by different types of cellular stress (Bellezza et al., 2010). Nrf2 is typically activated by low or moderate levels oxidative stress, whereas NFκB is turned on by inflammatory signals or very high levels of oxidative stress. Thus, with regards to the HO-1 promoter and various cytoprotective responses mediated by enzyme induction, stress signals will specifically alter the relative levels of cellular transcription factors and in turn, will determine whether Nrf2 or NFκB binds to activate transcription of the HO-1 gene. The JNK MAPK target, c-Jun has been shown to bind to Nrf2 (Shen et al., 2005), and inhibit NFκB (Tan et al., 2009). However, the P38 MAPK target, ATF-2 has been shown to bind to NFκB (Kaszubska et al., 1993). The model below proposes that the relative levels of c-Jun and ATF-2 play a major role in different cytoprotective responses mediated by HO-1 induction.

One essential role that BVR may play in modulating cytoprotective gene expression associated with HO-1 induction is the activation and recruitment of NFκB to the HO-1

Elucidating the Role of Biliverdin Reductase in

panel) which serves to activate all three arms of MAPK signaling.

near the CRE.

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 555

amplification of P38 MAPK relative to JNK and ERK MAPKs would be consistent with the effects of CO as the molecule activates P38 but inhibits JNK/ERK MAPKs (summarized above). In addition, biliverdin has been shown to be a potent inhibitor of JNK MAPK (Tang et al., 2007). Furthermore, c-Jun activation has been linked to cellular proliferation (Yoshioka et al., 1995) so switching from c-Jun-driven to ATF-2-driven transcription would be protective against proliferation/transformation. To show attenuation of the JNK MAPK pathway, the JNK arm of MAPK is shown as a dashed arrow in the figure panel to show that its activation is attenuated relative to that of P38 kinase. Because BVR has been shown to activate ERK MAPK (Lerner-Marmarosh et al., 2008), the arrow from ERK is a mixed dash/dot symbol to show moderate activation. Activation of ERK MAPK has been shown to facilitate anti-apoptotic responses (Wada and Penninger, 2004), and this might be related to the ability of ERK proteins to catalyze phosphorylation of the NFκB-inhibitory protein that keeps NFκB in the cytosol (for review of NFκB activation see (Shen et al., 2005)). As described above, signaling and DNA transcription mediated by BVR also lead to activation and increased expression of the P38 target, ATF-2, so this is another factor that increases the relative activation and concentration of ATF-2 (note the arrow from BVR to ATF-2 in the panel). Furthermore, BVR activates PKCβII (the latter can also activate BVR so a double headed arrow connects the two kinases in the

As ATF-2 concentrations and its level of activation increases relative to c-Jun and fos, c-Jun/fos hetero-dimerization would be replaced with c-Jun/ATF-2 dimerization at the AP-1 site. Because a P38-mediated pathway leading to activation of Nrf2 has been reported as a cytoprotective response in a cell line derived from human bronchial epithelial cells that were exposed to CeO2 nanoparticles, it is possible that the c-Jun/ATF-2 dimer serves as a more potent transcription factor in the recruitment of Nrf2 to the HO-1 promoter (Eom and Choi, 2009). Further increases in the concentration of ATF-2 would favor homo-dimerization of the transcription factor at the CRE site instead of the AP-1 site. ATF-2 has been shown to bind to NFκB (Kaszubska et al., 1993), and it has been shown that P38-mediated phosphorylation of Nrf2 promotes its association with the inhibitory protein, Keap1 (Keum et al., 2006). Both of these aspects of P38 pathway activation would favor activation of NFκB over Nrf2. Thus, it is proposed that the ATF-2 dimerization is the key signal that recruits NFκB to bind to the HO-1 promoter to induce expression of the gene. As proposed in the anti-oxidant response with the c-Jun/fos dimer facilitating recruitment of Nrf2 to the promoter, the ATF-2 dimer would allow NFκB to bind to the promoter as it exchanges with one of the ATF-2 units of the dimer. Another consistent aspect of the transition from the binding of c-Jun to that of ATF-2 in the recruitment of NFκB is the finding that c-Jun has been shown to inhibit NFκB activation (Tan et al., 2009).Thus, in the left panel of figure 2, the role of ATF-2 is represented by having its arrow point towards that for NFκB in the nucleus. Consistent with studies showing that Nrf2 and NFκB are co-regulated in opposite directions in response to stress signals (Bellezza et al., 2010), binding by NFκB is proposed to displace Nrf2 from the HO-1 promoter. The change in binding to the CRE site also may be critical in this regard because the AP-1 site overlaps more with the Nrf2 binding site than the CRE site. Thus, there would be less competition between Nrf2 and NFκB for binding to the HO-1 promoter. Because BVR can bind to both CRE and NFκB (Gibbs and Maines, 2007;Kravets et al., 2004), it may also serve to recruit NFκB to bind

In addition to activating NFκB, BVR also binds to the transcription factor in a manner that modulates its activity to favor protective gene expression relative to harmful pro-apoptotic,

promoter (Gibbs and Maines, 2007). It is proposed that this function is the main determinant in mediating the anti-apoptotic response associated with HO-1 induction. NFκB activation has been associated with prevention of apoptosis following treatment with cytokines and tumor promoters (Papa et al., 2006;Sen et al., 1996). Biliverdin inhibits activation of NFκB (Gibbs and Maines, 2007), and BVR reverses this affect by metabolizing biliverdin to bilirubin and by promoting PKCζ-mediated phosphorylation and activation of the transcription factor (Lerner-Marmarosh et al., 2007).

The importance of BVR in mediating the apoptotic response has been demonstrated repeatedly. When BVR was over-expressed in HEK 293 and MCF-7 cells, the cells were arrested in G1/Go stage (Gibbs and Maines, 2007). Furthermore, over-expression of BVR also protected cells from NFκB-mediated proliferation after stimulation with TNF-α (Gibbs et al., 2010). These findings are consistent with a number of studies that have used various cell types (HEK293 (Miralem et al., 2005), HeLa (Ahmad et al., 2002), cardiomyocytes (Pachori et al., 2007) and renal epithelial cells (Young et al., 2009)) to show that the inhibition of BVR expression using interference RNA resulted in apoptosis after the cells were challenged with arsenite, hydrogen peroxide, hypoxia/reoxygenation, and angiotensin II, respectively. Thus, repression of BVR expression consistently leads to apoptosis after cells are challenged with various types of stressors. Most importantly, with regards to protection from apoptosis, NFκB has been shown to be a necessary factor for the activation of the tumor suppressor protein, P53 (Ryan et al., 2000). Consistent with the role of BVR in preventing apoptosis, bilirubin also has been shown to have anti-apoptotic effects in a variety of studies (Bulmer et al., 2008;Kim et al., 2006;Parfenova et al., 2006). In addition, activation of ERK MAP kinases (mediated by BVR (Lerner-Marmarosh et al., 2008)) has been shown to favor anti-apoptotic responses (Wada and Penninger, 2004) which would also be consistent with the antiapoptotic role of BVR. For these reasons, it seems probable that the BVR-mediated recruitment and activation of NFκB are essential for anti-apoptotic HO-1 induction.

It is speculated that BVR also plays another important role in the anti-apoptotic signal. Studies have shown that BVR induces expression of P38 MAPK target, ATF-2 (Kravets et al., 2004). ATF-2 is constitutively expressed and not induced by environmental stimuli (unlike c-Jun) (Angel and Karin, 1991;Herdegen and Leah, 1998). Furthermore, it can form mixed dimers with c-Jun, and this hetero-dimer binds with much tighter affinity to AP-1 sites than c-Jun/fos dimers (Benbrook and Jones, 1990). Furthermore, ATF-2 dimerizes with itself and binds to the CRE response element instead of the AP-1 stress response element. Most importantly with respect to anti-proliferative, anti-inflammatory, and anti-apoptotic cytoprotection, ATF-2 also has been shown to dimerize with NFκB (Kaszubska et al., 1993). It is proposed in the mechanism below that these functions of ATF-2 along with the participation of BVR mediate HO-1-related cytoprotection from apoptotic, inflammatory, and hyper-proliferative stimuli.

The left panel of Figure 2 shows cell signaling cascades that may mediate the general antiapoptotic HO-1 response. It seems likely that apoptotic/anti-apoptotic specificity involves a change in gene expression mediated by c-Jun/fos dimerization to one mediated by ATF-2 homo-dimerization. The critical events in this transition are BVR-mediated activation of NFκB (by direct phosphorylation and metabolism of biliverdin (BV in figure) to bilirubin (BR) and amplification of P38 MAPK relative to the c-Jun arm of MAPK). As mentioned above, biliverdin has been shown to be an inhibitor of NFκB (Gibbs and Maines, 2007). The

promoter (Gibbs and Maines, 2007). It is proposed that this function is the main determinant in mediating the anti-apoptotic response associated with HO-1 induction. NFκB activation has been associated with prevention of apoptosis following treatment with cytokines and tumor promoters (Papa et al., 2006;Sen et al., 1996). Biliverdin inhibits activation of NFκB (Gibbs and Maines, 2007), and BVR reverses this affect by metabolizing biliverdin to bilirubin and by promoting PKCζ-mediated phosphorylation and activation of the

The importance of BVR in mediating the apoptotic response has been demonstrated repeatedly. When BVR was over-expressed in HEK 293 and MCF-7 cells, the cells were arrested in G1/Go stage (Gibbs and Maines, 2007). Furthermore, over-expression of BVR also protected cells from NFκB-mediated proliferation after stimulation with TNF-α (Gibbs et al., 2010). These findings are consistent with a number of studies that have used various cell types (HEK293 (Miralem et al., 2005), HeLa (Ahmad et al., 2002), cardiomyocytes (Pachori et al., 2007) and renal epithelial cells (Young et al., 2009)) to show that the inhibition of BVR expression using interference RNA resulted in apoptosis after the cells were challenged with arsenite, hydrogen peroxide, hypoxia/reoxygenation, and angiotensin II, respectively. Thus, repression of BVR expression consistently leads to apoptosis after cells are challenged with various types of stressors. Most importantly, with regards to protection from apoptosis, NFκB has been shown to be a necessary factor for the activation of the tumor suppressor protein, P53 (Ryan et al., 2000). Consistent with the role of BVR in preventing apoptosis, bilirubin also has been shown to have anti-apoptotic effects in a variety of studies (Bulmer et al., 2008;Kim et al., 2006;Parfenova et al., 2006). In addition, activation of ERK MAP kinases (mediated by BVR (Lerner-Marmarosh et al., 2008)) has been shown to favor anti-apoptotic responses (Wada and Penninger, 2004) which would also be consistent with the antiapoptotic role of BVR. For these reasons, it seems probable that the BVR-mediated

recruitment and activation of NFκB are essential for anti-apoptotic HO-1 induction.

It is speculated that BVR also plays another important role in the anti-apoptotic signal. Studies have shown that BVR induces expression of P38 MAPK target, ATF-2 (Kravets et al., 2004). ATF-2 is constitutively expressed and not induced by environmental stimuli (unlike c-Jun) (Angel and Karin, 1991;Herdegen and Leah, 1998). Furthermore, it can form mixed dimers with c-Jun, and this hetero-dimer binds with much tighter affinity to AP-1 sites than c-Jun/fos dimers (Benbrook and Jones, 1990). Furthermore, ATF-2 dimerizes with itself and binds to the CRE response element instead of the AP-1 stress response element. Most importantly with respect to anti-proliferative, anti-inflammatory, and anti-apoptotic cytoprotection, ATF-2 also has been shown to dimerize with NFκB (Kaszubska et al., 1993). It is proposed in the mechanism below that these functions of ATF-2 along with the participation of BVR mediate HO-1-related cytoprotection from apoptotic, inflammatory,

The left panel of Figure 2 shows cell signaling cascades that may mediate the general antiapoptotic HO-1 response. It seems likely that apoptotic/anti-apoptotic specificity involves a change in gene expression mediated by c-Jun/fos dimerization to one mediated by ATF-2 homo-dimerization. The critical events in this transition are BVR-mediated activation of NFκB (by direct phosphorylation and metabolism of biliverdin (BV in figure) to bilirubin (BR) and amplification of P38 MAPK relative to the c-Jun arm of MAPK). As mentioned above, biliverdin has been shown to be an inhibitor of NFκB (Gibbs and Maines, 2007). The

transcription factor (Lerner-Marmarosh et al., 2007).

and hyper-proliferative stimuli.

amplification of P38 MAPK relative to JNK and ERK MAPKs would be consistent with the effects of CO as the molecule activates P38 but inhibits JNK/ERK MAPKs (summarized above). In addition, biliverdin has been shown to be a potent inhibitor of JNK MAPK (Tang et al., 2007). Furthermore, c-Jun activation has been linked to cellular proliferation (Yoshioka et al., 1995) so switching from c-Jun-driven to ATF-2-driven transcription would be protective against proliferation/transformation. To show attenuation of the JNK MAPK pathway, the JNK arm of MAPK is shown as a dashed arrow in the figure panel to show that its activation is attenuated relative to that of P38 kinase. Because BVR has been shown to activate ERK MAPK (Lerner-Marmarosh et al., 2008), the arrow from ERK is a mixed dash/dot symbol to show moderate activation. Activation of ERK MAPK has been shown to facilitate anti-apoptotic responses (Wada and Penninger, 2004), and this might be related to the ability of ERK proteins to catalyze phosphorylation of the NFκB-inhibitory protein that keeps NFκB in the cytosol (for review of NFκB activation see (Shen et al., 2005)). As described above, signaling and DNA transcription mediated by BVR also lead to activation and increased expression of the P38 target, ATF-2, so this is another factor that increases the relative activation and concentration of ATF-2 (note the arrow from BVR to ATF-2 in the panel). Furthermore, BVR activates PKCβII (the latter can also activate BVR so a double headed arrow connects the two kinases in the panel) which serves to activate all three arms of MAPK signaling.

As ATF-2 concentrations and its level of activation increases relative to c-Jun and fos, c-Jun/fos hetero-dimerization would be replaced with c-Jun/ATF-2 dimerization at the AP-1 site. Because a P38-mediated pathway leading to activation of Nrf2 has been reported as a cytoprotective response in a cell line derived from human bronchial epithelial cells that were exposed to CeO2 nanoparticles, it is possible that the c-Jun/ATF-2 dimer serves as a more potent transcription factor in the recruitment of Nrf2 to the HO-1 promoter (Eom and Choi, 2009). Further increases in the concentration of ATF-2 would favor homo-dimerization of the transcription factor at the CRE site instead of the AP-1 site. ATF-2 has been shown to bind to NFκB (Kaszubska et al., 1993), and it has been shown that P38-mediated phosphorylation of Nrf2 promotes its association with the inhibitory protein, Keap1 (Keum et al., 2006). Both of these aspects of P38 pathway activation would favor activation of NFκB over Nrf2. Thus, it is proposed that the ATF-2 dimerization is the key signal that recruits NFκB to bind to the HO-1 promoter to induce expression of the gene. As proposed in the anti-oxidant response with the c-Jun/fos dimer facilitating recruitment of Nrf2 to the promoter, the ATF-2 dimer would allow NFκB to bind to the promoter as it exchanges with one of the ATF-2 units of the dimer. Another consistent aspect of the transition from the binding of c-Jun to that of ATF-2 in the recruitment of NFκB is the finding that c-Jun has been shown to inhibit NFκB activation (Tan et al., 2009).Thus, in the left panel of figure 2, the role of ATF-2 is represented by having its arrow point towards that for NFκB in the nucleus. Consistent with studies showing that Nrf2 and NFκB are co-regulated in opposite directions in response to stress signals (Bellezza et al., 2010), binding by NFκB is proposed to displace Nrf2 from the HO-1 promoter. The change in binding to the CRE site also may be critical in this regard because the AP-1 site overlaps more with the Nrf2 binding site than the CRE site. Thus, there would be less competition between Nrf2 and NFκB for binding to the HO-1 promoter. Because BVR can bind to both CRE and NFκB (Gibbs and Maines, 2007;Kravets et al., 2004), it may also serve to recruit NFκB to bind near the CRE.

In addition to activating NFκB, BVR also binds to the transcription factor in a manner that modulates its activity to favor protective gene expression relative to harmful pro-apoptotic,

Elucidating the Role of Biliverdin Reductase in

Cytokines/ Growth factors

MAPK

PKCβII

Signaling Receptor

> JNK ERK

Anti-apoptotic/ Anti-proliferative Response

Cytoplasm

HO-1

BV

BVR

BR

Heme

PKCζ

NFκB

CO

ATF-2

P38

Nucleus

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 557

In the middle panel, the anti-inflammatory signaling that results in the later-staged recruitment of NFκB to the HO-1 promoter is represented. The scheme for Nrf2 activation preceding NFκB activation is shown in the far right panel. In the anti-inflammatory response, CO formed by HO-1 inhibits the JNK and ERK MAPK pathways. The COmediated inhibition of c-Jun protects against uncontrolled proliferation. However, BVR modulates ERK to protect against apoptosis through activation of NFκB. Initially, c-Jun inhibits NFκB activation which is shown as the red block line. Eventually, ERK activity and ATF-2 dimerization at the HO-1 promoter will favor gene transcription mediated by NFκB. BVR also modulates NFκB activity to favor protective gene expression, so the arrow from NFκB is dashed. In addition, as mentioned above, diversion of NFκB to stimulate the protective induction of HO-1 limits its ability to stimulate inflammatory gene expression

> Immune Receptor

> > PKCβII

MAPK

c-jun

ATF-2

P38 JNK ERK

Elk1

Nrf2

Anti-oxidative stress

PI3K

HO-1

Heme

CO

H2O2

MAPK

JNK

c-jun

BVR :Heme

Mito.

Bach-1 X Bach-1 :Heme

BRG-1

sHO-1 :Heme

?

Akt

H2O2

Cytokines, Interleukins

ROS ROS

CO

and allows more Nrf2 to activate transcription of other anti-oxidant genes.

Anti-inflammatory Response

BVR

NFκB

See anti-oxidative pathway for ROSmediated activation of Nrf2

Fig. 2. Signal transduction regulation of the HO-1 protective response to cellular stress signals. The schematic diagram shows the putative signaling pathways responsible for gene

induction/repression following different types of cellular stress (apoptotic signals, inflammatory stimuli, and oxidative stress). Arrows ending in the cytoplasm point at downstream kinases or transcription factors activated by the stimuli at the base of the arrow. Arrows ending in the nuclei represent the binding of transcription factors to gene promoters to affect expression and mediate the cytoprotective responses. Double-headed arrows represent kinase reactions that can occur in both directions (see text for details). Dashed arrows indicate attenuated activation, and arrows directed to the same point either have a role in binding together at the promoter or modulating the activity of one another.

HO-1

Heme

Nrf2

pro-inflammatory and pro-proliferative targets (Gibbs et al., 2010). Consistent with the possibility that BVR regulates the activity of NFκB, TNF-α-mediated stimulation caused NFκB to act as a repressor of BVR expression which demonstrates that BVR is competitive with the inflammation process mediated by TNF-α through activation of NFκB. Because of the BVRmediated modulation of NFκB, the arrow from NFκB is drawn as a dash in the panel.

#### **6.3 Anti-inflammatory protection**

It appears that the anti-inflammatory effects associated with HO-1 induction (middle panel of figure 2) might be mediated by both BVR-dependent and BVR-independent processes. ROS formation (produced by immune cells) is a big component of inflammation. Thus, it seems unlikely that the anti-inflammatory response is totally regulated by BVR (middle panel of figure 2). Thus, for the reasons given in the preceding paragraph, Nrf2 will be activated independently of BVR, probably as an initial response to inflammatory stimuli. Another implication of the role of Nrf2 in the HO-1-related anti-inflammatory response has come from studies with plant-derived, phenolic diterpenes that elicit both anti-oxidant and anti-inflammatory responses. Not coincidentally, these compounds also mediate HO-1 induction through Nrf2 following stimulation of phosphatidylinositol 3-kinase (PI3 kinase)/Akt signaling (Martin et al., 2004;Pugazhenthi et al., 2007). The Akt protein kinase activated by PI3K is distinct from the PKCζ that is known to be activated by BVR (Lerner-Marmarosh et al., 2007). As described above, CO has been implicated in mediating signaling through Akt (Piantadosi and Zhang, 1996). Thus, this pathway of Nrf2 activation appears to be related directly to the catalytic activity of HO-1. These mechanisms activating Nrf2 contribute to the anti-inflammatory effects associated with HO-1 induction.

An appealing hypothesis, that seems consistent with research findings, proposes that BVRindependent processes activate Nrf2 at the early stages of inflammation, whereas BVRmediated MAPK and NFκB activation play critical roles in the cellular response at later stages of inflammation. In support of this idea, NFκB signaling typically opposes Nrf2 mediated signaling as a later event in response to many stress events (Bellezza et al., 2010). The putative ability of BVR to bind and modulate the activity of NFκB is important in the latter response because agonist binding to immune receptors cause potent activation of NFκB. It is important to emphasize that this modulating role is not the only way BVR would protect cells from NFκB-mediated stress. By replacing NFκB with Nrf2 at the HO-1 promoter, BVR would be diverting NFκB from promoting harmful gene expression while freeing up Nrf2 to activate protective gene expression. The anti-inflammatory effects of CO generated by HO-1 activity also would act independently of BVR (described above). A recent study demonstrated that HO-1 activity (as opposed to merely BVR-related cell signaling and DNA binding) was essential for the anti-inflammatory effects following treatment with endotoxin (Tamion et al., 2006). The anti-inflammatory response demonstrated by the treated animals was explained by both the inhibition of tumor necrosis factor-α production and the elevation of interleukin-10. Because the anti-inflammatory effects required catalytic activity by the induced HO-1 (activity was inhibited by treatment with tin mesoporphyrin), it can be assumed that either BVR-mediated signaling/DNAbinding was dependent on HO-1 activity or the effects were caused by bilirubin/CO. Either premise dictates that BVR effects occurred secondary to those mediated by HO-1 activity. For these reasons, it seems that the anti-inflammatory response is pleiotropic and depends on both BVR-dependent and BVR-independent signaling and gene transcription.

pro-inflammatory and pro-proliferative targets (Gibbs et al., 2010). Consistent with the possibility that BVR regulates the activity of NFκB, TNF-α-mediated stimulation caused NFκB to act as a repressor of BVR expression which demonstrates that BVR is competitive with the inflammation process mediated by TNF-α through activation of NFκB. Because of the BVR-

It appears that the anti-inflammatory effects associated with HO-1 induction (middle panel of figure 2) might be mediated by both BVR-dependent and BVR-independent processes. ROS formation (produced by immune cells) is a big component of inflammation. Thus, it seems unlikely that the anti-inflammatory response is totally regulated by BVR (middle panel of figure 2). Thus, for the reasons given in the preceding paragraph, Nrf2 will be activated independently of BVR, probably as an initial response to inflammatory stimuli. Another implication of the role of Nrf2 in the HO-1-related anti-inflammatory response has come from studies with plant-derived, phenolic diterpenes that elicit both anti-oxidant and anti-inflammatory responses. Not coincidentally, these compounds also mediate HO-1 induction through Nrf2 following stimulation of phosphatidylinositol 3-kinase (PI3 kinase)/Akt signaling (Martin et al., 2004;Pugazhenthi et al., 2007). The Akt protein kinase activated by PI3K is distinct from the PKCζ that is known to be activated by BVR (Lerner-Marmarosh et al., 2007). As described above, CO has been implicated in mediating signaling through Akt (Piantadosi and Zhang, 1996). Thus, this pathway of Nrf2 activation appears to be related directly to the catalytic activity of HO-1. These mechanisms activating Nrf2

mediated modulation of NFκB, the arrow from NFκB is drawn as a dash in the panel.

contribute to the anti-inflammatory effects associated with HO-1 induction.

on both BVR-dependent and BVR-independent signaling and gene transcription.

An appealing hypothesis, that seems consistent with research findings, proposes that BVRindependent processes activate Nrf2 at the early stages of inflammation, whereas BVRmediated MAPK and NFκB activation play critical roles in the cellular response at later stages of inflammation. In support of this idea, NFκB signaling typically opposes Nrf2 mediated signaling as a later event in response to many stress events (Bellezza et al., 2010). The putative ability of BVR to bind and modulate the activity of NFκB is important in the latter response because agonist binding to immune receptors cause potent activation of NFκB. It is important to emphasize that this modulating role is not the only way BVR would protect cells from NFκB-mediated stress. By replacing NFκB with Nrf2 at the HO-1 promoter, BVR would be diverting NFκB from promoting harmful gene expression while freeing up Nrf2 to activate protective gene expression. The anti-inflammatory effects of CO generated by HO-1 activity also would act independently of BVR (described above). A recent study demonstrated that HO-1 activity (as opposed to merely BVR-related cell signaling and DNA binding) was essential for the anti-inflammatory effects following treatment with endotoxin (Tamion et al., 2006). The anti-inflammatory response demonstrated by the treated animals was explained by both the inhibition of tumor necrosis factor-α production and the elevation of interleukin-10. Because the anti-inflammatory effects required catalytic activity by the induced HO-1 (activity was inhibited by treatment with tin mesoporphyrin), it can be assumed that either BVR-mediated signaling/DNAbinding was dependent on HO-1 activity or the effects were caused by bilirubin/CO. Either premise dictates that BVR effects occurred secondary to those mediated by HO-1 activity. For these reasons, it seems that the anti-inflammatory response is pleiotropic and depends

**6.3 Anti-inflammatory protection** 

In the middle panel, the anti-inflammatory signaling that results in the later-staged recruitment of NFκB to the HO-1 promoter is represented. The scheme for Nrf2 activation preceding NFκB activation is shown in the far right panel. In the anti-inflammatory response, CO formed by HO-1 inhibits the JNK and ERK MAPK pathways. The COmediated inhibition of c-Jun protects against uncontrolled proliferation. However, BVR modulates ERK to protect against apoptosis through activation of NFκB. Initially, c-Jun inhibits NFκB activation which is shown as the red block line. Eventually, ERK activity and ATF-2 dimerization at the HO-1 promoter will favor gene transcription mediated by NFκB. BVR also modulates NFκB activity to favor protective gene expression, so the arrow from NFκB is dashed. In addition, as mentioned above, diversion of NFκB to stimulate the protective induction of HO-1 limits its ability to stimulate inflammatory gene expression and allows more Nrf2 to activate transcription of other anti-oxidant genes.

Fig. 2. Signal transduction regulation of the HO-1 protective response to cellular stress signals. The schematic diagram shows the putative signaling pathways responsible for gene induction/repression following different types of cellular stress (apoptotic signals, inflammatory stimuli, and oxidative stress). Arrows ending in the cytoplasm point at downstream kinases or transcription factors activated by the stimuli at the base of the arrow. Arrows ending in the nuclei represent the binding of transcription factors to gene promoters to affect expression and mediate the cytoprotective responses. Double-headed arrows represent kinase reactions that can occur in both directions (see text for details). Dashed arrows indicate attenuated activation, and arrows directed to the same point either have a role in binding together at the promoter or modulating the activity of one another.

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#### **7. Conclusions**

Hemin is an essential cofactor for heme proteins that carry out a multitude of vital oxidative and oxygen transport-related functions in the cell. Unfortunately, hemin is also reactive and extremely harmful to cells when accumulated in the free form. A highly regulated system has evolved to control the levels of cellular heme. HO-1 and BVR catalyze the steps involved in heme catabolism. Interestingly, the enzymes are also involved in a host of cytoprotective functions mediating anti-oxidant, anti-apoptotic, anti-proliferative, and anti-inflammatory responses that have been proven to be therapeutic in many clinical disease models. HO-1 directly mediates most anti-oxidant effects through the following mechanisms: 1) the removal of heme in coordination with the up-regulation of the iron storage protein, ferritin; 2) the production of a lipophilic, anti-oxidant, biliverdin; 3) the regulation of both cell signaling and gene expression; and 4) the regulation of the cytochrome P450 system. Gene transcription mediated by Nrf2 largely mediates the cellular response to oxidative stress. BVR contributes to the antioxidant response by catalyzing a redox cycle that involves the BVR-mediated conversion of the potent antioxidant, bilirubin. In addition to catalyzing the second step of heme catabolism, BVR also acts as an upstream activator of MAPK and phosphatidylinositol-3-kinase pathway; directly binds to DNA; and participates in the transactivation of AP-1 sites in the HO-1 promoter. The anti-apoptotic effects associated with HO-1 induction are most often caused by BVR-mediated cell signaling and DNAbinding that leads to NFκB activation, but signaling effects related to HO-1-catalyzed CO production work in concert with the effects of BVR to protect against inflammation. Much remains to be learned about the specifics of cytoprotection via BVR-mediated signaling and gene transcription in addition to the roles of biliverdin and bilirubin in altering gene transcription. Similarly, because most in vitro studies of the enzymology of HO-1 have used a shortened mutant that does not bind to membrane or interact with membrane binding partners in the same manner as full length HO-1, almost nothing is known about how interactions between HO-1, BVR, and cytochrome P450 reductase are regulated to influence cell signaling, gene expression, the metabolism by HO-1, and oxidative stress related to P450-mediated, metabolism.

#### **8. References**


Hemin is an essential cofactor for heme proteins that carry out a multitude of vital oxidative and oxygen transport-related functions in the cell. Unfortunately, hemin is also reactive and extremely harmful to cells when accumulated in the free form. A highly regulated system has evolved to control the levels of cellular heme. HO-1 and BVR catalyze the steps involved in heme catabolism. Interestingly, the enzymes are also involved in a host of cytoprotective functions mediating anti-oxidant, anti-apoptotic, anti-proliferative, and anti-inflammatory responses that have been proven to be therapeutic in many clinical disease models. HO-1 directly mediates most anti-oxidant effects through the following mechanisms: 1) the removal of heme in coordination with the up-regulation of the iron storage protein, ferritin; 2) the production of a lipophilic, anti-oxidant, biliverdin; 3) the regulation of both cell signaling and gene expression; and 4) the regulation of the cytochrome P450 system. Gene transcription mediated by Nrf2 largely mediates the cellular response to oxidative stress. BVR contributes to the antioxidant response by catalyzing a redox cycle that involves the BVR-mediated conversion of the potent antioxidant, bilirubin. In addition to catalyzing the second step of heme catabolism, BVR also acts as an upstream activator of MAPK and phosphatidylinositol-3-kinase pathway; directly binds to DNA; and participates in the transactivation of AP-1 sites in the HO-1 promoter. The anti-apoptotic effects associated with HO-1 induction are most often caused by BVR-mediated cell signaling and DNAbinding that leads to NFκB activation, but signaling effects related to HO-1-catalyzed CO production work in concert with the effects of BVR to protect against inflammation. Much remains to be learned about the specifics of cytoprotection via BVR-mediated signaling and gene transcription in addition to the roles of biliverdin and bilirubin in altering gene transcription. Similarly, because most in vitro studies of the enzymology of HO-1 have used a shortened mutant that does not bind to membrane or interact with membrane binding partners in the same manner as full length HO-1, almost nothing is known about how interactions between HO-1, BVR, and cytochrome P450 reductase are regulated to influence cell signaling, gene expression, the metabolism by HO-1, and oxidative stress related to

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**7. Conclusions** 

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**25** 

*Brazil* 

**Antibothropic Action of** *Camellia sinensis*

**by** *Bothrops jararacussu* **Snake Venom** 

**and Its Main Toxin, Bothropstoxin-I** 

Yoko Oshima-Franco et al.\* *University of Sorocaba/UNISO,* 

**Extract Against the Neuromuscular Blockade** 

Snake bite envenoming, a serious public health problem in rural areas of tropical and subtropical countries, was included in 2007 as a neglected disease by the World Health Organization (WHO, 2007). Under this geographical perspective Africa, Asia, Oceania and Latin America are the most vulnerable countries to this kind of accident, but also shared by many developing countries (Harrison et al*.*, 2009; Warrel, 2010). An excellent meta-analytic approach about the subject was described by Chippaux (2011), who analysed more than 3,000 references for estimating the burden of snakebites in sub-Saharan Africa. Brazil encloses both requirements, as a developing and a tropical country, and needs to strengthen measures against venomous snake accidents, since, according to Lima et al*.* (2009), it is the country with the major number of accidents (about 20,000 cases/year), followed by Peru (4,500), Venezuela (2,500-3,000), Colombia (2,675), Ecuador (1,200-1,400) and Argentina

As mentioned by Nicoleti et al*.* (2010), venomous snakes in Brazil are represented by *Bothrops, Bothropoides, Bothriopsis, Bothrocophias, Rhinocerophis, Crotalus*, *Lachesis, Leptomicrurus* and *Micrurus* (see the new taxonomic arrangement proposed by Fenwick et al*.*, 2009). Envenoming by the first five genera produce similar toxic manifestations and treatment assessment are quite the same. They represent 86.9% of accidents, whereas 8.7% were caused by *Crotalus*, 3.6%

*Bothrops jararacussu* snake belongs to the Viperidae family and its venom is able to induce severe signs of local and systemic envenoming, such as necrosis, shock, spontaneous

Luana de Jesus Reis Rosa1, Gleidy Ana Araujo Silva1, Jorge Amaral Filho1, Magali Glauzer Silva1, Patricia Santos Lopes2, José Carlos Cogo3, Adélia Cristina Oliveira Cintra4 and Maria Alice da Cruz-Höfling5

*Lachesis* and 0.8% by *Leptomicrurus* and *Micrurus* (Ministério da Saúde, 2004).

**1. Introduction** 

(1,150-1.250) (Warrel, 2004).

<sup>1</sup>*University of Sorocaba/UNISO, Brazil*

*4University of São Paulo/USP, Brazil* 

<sup>2</sup>*Federal University of São Paulo/UNIFESP, Brazil 3University of Vale do Paraiba/UNIVAP, Brazil* 

*5University of Campinas/UNICAMP/I.B./D.H.E., Brazil*

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