**5. Mechanisms of cellular protection by BVR**

#### **5.1 BVR-catalyzed redox cycle with lipid peroxides**

The anti-oxidant effects and other positive health benefits of bilirubin, that are attributable to catalysis by BVR, are obviously important means of BVR-mediated cytoprotection. The antioxidant effects of this compound were discussed above, but the reason for its potency involves metabolism by BVR. In a study examining the cytoprotective effects of bilirubin in neuronal cells, it was found that as little as 10 nM bilirubin (physiologic levels) protected against 10,000-fold higher concentrations of hydrogen peroxide (Baranano et al., 2002). From these results, it was postulated that BVR participates in a redox cycle with lipid peroxides in which bilirubin is oxidized by the lipid peroxides to biliverdin which, in turn, is reduced by BVR to reform bilirubin (Figure 1). Thus, BVR may have an important role in extending the antioxidant potency of bilirubin. It should be noted that a recent study concluded that the cytoprotective role of this redox cycle was limited as BVR overexpression and inhibition of BVR expression with antisense RNA did not seem to influence hydrogen peroxide-mediated cytotoxicity (Maghzal et al., 2009).

Fig. 1. BVR-catalyzed redox cycle with lipid peroxides. See text for details

#### **5.2 BVR-mediated modulation of cell signaling**

BVR may be the most versatile protein known. Research over the last decade has revealed new functions for the enzyme that have broadened its role in the cytoprotective response to

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

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

The anti-oxidant effects and other positive health benefits of bilirubin, that are attributable to catalysis by BVR, are obviously important means of BVR-mediated cytoprotection. The antioxidant effects of this compound were discussed above, but the reason for its potency involves metabolism by BVR. In a study examining the cytoprotective effects of bilirubin in neuronal cells, it was found that as little as 10 nM bilirubin (physiologic levels) protected against 10,000-fold higher concentrations of hydrogen peroxide (Baranano et al., 2002). From these results, it was postulated that BVR participates in a redox cycle with lipid peroxides in which bilirubin is oxidized by the lipid peroxides to biliverdin which, in turn, is reduced by BVR to reform bilirubin (Figure 1). Thus, BVR may have an important role in extending the antioxidant potency of bilirubin. It should be noted that a recent study concluded that the cytoprotective role of this redox cycle was limited as BVR overexpression and inhibition of BVR expression with antisense RNA did not seem to influence hydrogen peroxide-mediated

iron overload from catalytically active HO-1.

protective way to recycle iron and manage its levels in the cell.

Fig. 1. BVR-catalyzed redox cycle with lipid peroxides. See text for details

BVR may be the most versatile protein known. Research over the last decade has revealed new functions for the enzyme that have broadened its role in the cytoprotective response to

**5.2 BVR-mediated modulation of cell signaling** 

**5. Mechanisms of cellular protection by BVR 5.1 BVR-catalyzed redox cycle with lipid peroxides** 

cytotoxicity (Maghzal et al., 2009).

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

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 the activation of this pathway by BVR.

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 effects associated with expression of HO-1.

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 to influence HO-1 gene transcription.

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

Elucidating the Role of Biliverdin Reductase in

concentrations of NADPH).

**6.1 Anti-oxidant protection** 

the Expression of Heme Oxygenase-1 as a Cytoprotective Response to Stress 551

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

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

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

roles of the implicated transcription factors in cellular processes.

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

(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).

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

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

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