The Biology of Bilirubin Production

Original Article &&&&&&&&&&&&&& The Biology of Bilirubin Production

Phyllis A. Dennery, MD evolving that would allow for better purification and character- Yi-Hao Weng, MD ization of the 32-kDa HO-1 protein. With the synthesis of David K. Stevenson, MD metalloporphyrins, HO activity could be inhibited leading to further Guang Yang, PhD understanding of the cellular consequences of HO activity and to the development of strategies for the prevention of neonatal hyperbilirubinemia. Later, characterization of the constitutive form, HO-2 was achieved.4 In the late 1980s, the regulation of the HO-1 5 Heme oxygenase (HO), the rate-limiting enzyme in bilirubin production, gene was addressed. Shibahara et al. showed the induction of HO-1 has been identified from the late 1960s. This enzyme has been shown to at the transcriptional level. As the field advanced, HO-1 gene 6 have many other roles in recent years. The inducible form is regulated by induction was noted to be a generalized marker of oxidative stress. oxidative stress, inflammation, and heavy metals, among others, and is This ubiquitous nature of the HO-1 inducible response is now clear. cytoprotective in many instance. Nonetheless, there are instances when As molecular biology and molecular genetics techniques improve, HO-1 can be deleterious due to the release of iron from the reaction. the biochemistry, role, and regulation of HO are becoming more Another important by-product, carbon monoxide, is a vasodilator and a clearly understood. neurotransmitter and has been implicated in signal transduction pathways. More recently, nonenzymatic, signaling roles of HO have been REGULATION OF HO suggested. This may serve to regulate the endogenous activity of this enzyme when cellular heme levels are low. The organization of the HO-2 gene remains relatively obscure. Journal of Perinatology 2001; 21:S17–S20. Although HO-2 is referred to as the constitutive isoform of HO, it has been shown to be upregulated by a few factors to date such as with corticosterone in fetal rat brain,7 and with the NOS inhibitor, N(G)- nitro- L -arginine, and the HO inhibitor, zinc mesoporphyrin HISTORICAL PERSPECTIVE (FIGURE 1) (ZnMP), in cultured rat aortic cell.8 A consensus sequence of the In the late 1960s, heme oxygenase (HO) was identified as the first glucocorticoid response element (GRE) in the promoter region of the and rate-limiting enzyme in the formation of bilirubin (Figure 1). HO-2 gene has been documented and specific binding of This enzyme allows for the degradation of heme from hemoglobin glucocorticoid receptor protein to the GRE was also observed.7 It is not or other heme-containing proteins to form biliverdin. This process known whether other factors can induce HO-2 but preliminary is energy requiring because NADPH donates electrons through the observations in the HO-1 knockout mice reveal increased lung HO-2 cytochrome cP450 system and molecular oxygen is consumed for mRNA at baseline,9 suggesting a compensatory upregulation of HO-2 the liberation of iron from the porphyrin ring of heme, the release in the absence of HO-1. of carbon monoxide (CO), as well as the formation of biliverdin (Figure 2). Biliverdin reductase, a microsomal enzyme, allows for the reduction of biliverdin to bilirubin. With the description of an Purification Cloning of of HO-1 HO-1cDNA Protein-protein assay for microsomal HO activity, this was seen to increase in the and protein-DNA 1 Enzymatic Cloning of interractions of HO presence of hemin, the substrate for the reaction. Thereafter, other assay for HO HO-2 cDNA compounds such as cobalt chloride and various heavy metals were found to upregulate HO activity.1,2 Maines and Kappas 3 then 1960’s 1970’s1980’s 1990’s 2000’s identified a developmental pattern of heme catabolic enzymes, HO is a HO HO-1 Non-enzymatic catalytic activity is mRNA is Physiologic roles for roles for HO-1 and thereby suggesting an explanation for the overproduction of enzyme inducible inducible HO-1 and HO-2 HO-2 Developmental bilirubin in neonatal jaundice. In addition, techniques were regulation of HO-1 is a generalized HO response to oxidative stress

Department of Pediatrics, Stanford University, Stanford, CA. Figure 1. Schematic representation of the advances in the field of HO. The

Address correspondence to Phyllis A. Dennery, MD, Department of Pediatrics, Stanford numbers within the arrow represent the decade in which the discoveries were University, 750 Welch Road #315, Palo Alto, CA 94304. made.

HEME antioxidant response element ARE with the consensus sequence 12 NADPH GCnnnGTA as with other antioxidant enzymes. Heme treatment O 2 HEME OXYGENASE (HO) results in a specific and marked increase in the NF-kappa B and Cytochrome c P450 Reductase AP-2 transcription factors, a sequence upstream between positions NADP À3.5 and 12.5 kbp is required for the induction by cadmium H O 2 (CdRE)10 and the distal enhancer regions are important in CO + Fe 2+ regulation of HO-1 in inflammation.13 This demonstrates that Bilirubin Biliverdin HO-1 is readily inducible.

NADP NADPH

Figure 2. Pathway of heme degradation. Heme is metabolized by HO to CYTOPROTECTIVE ROLE OF HEME OXYGENASE 2+ form CO and iron (Fe ). This reaction is energy requiring, as the reduced Because of its oxidative regulation, HO-1 is not merely catalytic but a form of nicotinamide adenine dinucleotide phosphate (NADPH) and synthetic enzyme for the formation of potent antioxidant bile molecular oxygen (O ) are metabolized by cytochrome cP450 reductase to 2 pigments and the important neurotransmitter CO. Due to the the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP) production of bilirubin, a potent antioxidant surpassing vitamin E,14 and water (H2O2 ). Biliverdin is further reduced to bilirubin by biliverdin reductase, and NADPH is also oxidized to NADP in this step. and the sequestration of heme, a pro-oxidant molecule, the overall effect of the HO-1 reaction may be cytoprotective. Nonetheless, recent evidence suggests that HO-1 is not always cytoprotective and that there may be a beneficial threshold of HO-1 induction in cells. In a Much more is known about the regulation of the HO-1 gene model system allowing for regulation of HO-1 in a dose-dependent and its promoter. The inducibility of HO-1 is explained by the fashion, HO was a pro-oxidant at higher levels (greater than configuration of the HO-1 gene. The 6.8-kilobase (kb) HO-1 five-fold increased activity) owing to the associated release of gene is organized into four introns and five exons. A promoter reactive iron as a consequence of increased HO activity.15 sequence (not TATAA) is located approximately 28 base pairs Furthermore, the association of HO-1 protein with neurons in upstream from the transcription initiating site. There are several degenerative brain diseases and the protection of these neurons by transcriptional enhancer elements in the 50 flanking region, HO inhibitors 16,17 suggest a detrimental role for HO-1 over- including heat shock elements and metal regulatory elements.10 expression in certain disease processes. Inducer responsive sequences have been identified in the proximal With the availability of HO knockouts, the understanding of the enhancer directly upstream of the promoter. Two or more distal function of HO has improved. Null mutants for HO-2 have enhancers located 4 and 10 kb upstream of the transcription increased susceptibility to hyperoxia despite induction of HO-1 9 initiation site also exist 11 (Figure 3). The many factors that affect and have ejaculatory abnormalities.18 The neurons of these HO-1 gene regulation do so through different binding regions on animals are also more susceptible to oxidative injury.19 As to HO-1 the HO-1 gene. The HO-1 promoter region contains an null mutants, these animals age faster than their wild-type

Figure 3. The HO-1 gene. The HO-1 gene contains five exons (white boxes) and four introns (black boxes between introns). There is a promoter (P), a proximal enhancer (PE), and two distal enhancers (DE1 and DE2). Each of these regions contain transcription factor binding sites (patterned boxes within the brackets). C/EBP, CCAT enhancer binding protein; AP-1 activator protein-1, SP-1, stress protein-1; CDRE, cadmium response element IL-GRE, glucocorticoid response element; HSE, heat shock element; MTRE, metal response element; AP-2, activator protein-2; STAT-3, signal transducer and activator of transcription; USF, upstream stimulatory factor).

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counterparts and demonstrate anemia and iron deposition in the HO-1 HO-2 kidneys and liver.20 Additionally, these animals are more likely to show cardiac xenograft rejection,21 have abnormal inflammatory responses 20 and increased renal injury in response to an ischemic HO-2 insult.22 Despite the strong evidence to suggest a protective role for HO-1 HO-1 in many situations, HO-1 disruption in the lung was not associated with increased susceptibility to hyperoxia,23 nor was lung HO-1 overexpression.24 The mechanisms mediating the cytoprotective or toxic effects of HO are not clearly understood. Specifically, it is not known whether disruption of HO-1 or HO-2 alters bilirubin production in the knockout mice but the HO-2 HO-2 knockouts infused with low doses of bilirubin have decreased HO-1 DNA ischemia-mediated neurotoxicity.25 HO-1

ROLE OF CO IN CELLULAR FUNCTION HO-1 mRNA Another by-product of the HO reaction that has received considerable attention is CO. This diatomic gas is known to be HO-1 extremely toxic at high concentrations as it interferes with the delivery of oxygen to tissues by binding tightly to hemoglobin.26 Figure 4. Proposed pathway for HO-1 and HO-2. HO-1 binds to HO-2 Nonetheless, CO has recently been shown, like nitric oxide, to be a and this complex migrates to the nucleus. Binding of the protein complex to physiologic regulator guanosine 30,50-monophosphate (cGMP) the promoter of the HO-1 DNA results in the formation of HO-1 mRNA and resulting in both vasodilatory effects and altered neurotransmis- HO-1 protein. sion.27 In addition, CO at low concentrations both in vivo and in vitro selectively inhibited the expression of pro-inflammatory cytokines and increased the expression of the anti-inflammatory there appears to be an interaction of HO-1 and HO-2 protein within cytokine interleukin-10 through a pathway involving the cells and a binding site for the HO-1/HO-2 protein complex on the mitogen-activated protein kinases (MAPK) rather than through a HO-1 gene.32 Therefore, HO-1 may be involved in the regulation of guanylyl cyclase-cGMP or nitric oxide pathway. These investigators its own gene transcription (Figure 4). HO-1 degrades heme; yet, in have also demonstrated that expression of HO-1 or exposure of the absence of heme, a fair amount of HO-1 is still present in tissues. endothelial cells to exogenous CO enhanced p38 MAPK activation Perhaps a pathway that allowed for the upregulation of HO-1 in the by tumor necrosis factor-alpha resulting in decreased apoptosis. absence of heme would permit constitutive tissue expression of HO-1 Furthermore, specific inhibition of p38 MAPK activation abrogated when no stress is present. Others suggest that HO-1 may be involved the antiapoptotic effect of HO-1.28 Phosphorylation of this kinase in the regulation of other genes such as superoxide dismutase.34 This and subsequent downstream events occur early in response to could also allude to a role for HO-1 as a signaling molecule. cellular stress. Some have demonstrated a clearly cytotoxic effect of From its humble beginnings as a catabolic enzyme, HO appears to this event 29,30 whereas others demonstrate a protective role for this be an important molecule with roles in oxidative stress, kinase.28,31 These contradictory findings further illustrate the inflammation, immunity, and cellular regulation and signaling. complexity of understanding the physiologic role of the HO/CO system. References 1. Maines MD, Kappas A. Cobalt induction of hepatic heme oxygenase; with NONENZYMATIC ROLES FOR HO evidence that cytochrome P-450 is not essential for this enzyme activity. Proc It had been suggested that HO was found only in the microsomal Natl Acad Sci USA 1974;71:4293–7. fraction attached to the smooth endoplasmic reticulum. However, 2. Kappas A, Maines MD. Tin: a potent inducer of heme oxygenase in kidney. recent investigations have shown that HO can be found in the nuclear Science 1976;192:60–2. 3. Maines MD, Kappas A. Study of the developmental pattern of heme fraction suggesting that HO may play a signaling role. In fact, we catabolism in liver and the effects of cobalt on cytochrome P-450 and the have recently demonstrated that transfection of HO-2 into NIH 3T3 rate of heme oxidation during the neonatal period. J Exp Med 1975;141: cells expressing an HO-1 promoter attached to the luciferase gene 1400–10. resulted in increased HO-1 gene expression.32 Others also suggest a 4. Maines MD, Trakshel GM, Kutty RK. Characterization of two constitutive signaling role for HO-2 as they demonstrate that this enzyme can be forms of rat liver microsomal heme oxygenase. Only one molecular species of phosphorylated through the protein kinase C pathway.33 Furthermore, the enzyme is inducible. J Biol Chem 1986;261:411–9.

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5. Shibahara S, Muller R, Taguchi H, Yoshida T. Cloning and expression of 20. Poss KD, Tonegawa S. Heme oxygenase 1 is required for mammalian iron cDNA for rat heme oxygenase. Proc Natl Acad Sci USA 1985;82:7865–9. reutilization. PNAS 1997;94:10919–24. 6. Applegate LA, Luscher P, Tyrrell RM. Induction of heme oxygenase: a general 21. Soares MP, Lin Y, Anrather J, et al. Expression of heme oxygenase-1 can response to oxidant stress in cultured mammalian cells. Cancer Res determine cardiac xenograft survival. Nat Med 1998;4:1073–7. 1991;51:974–8. 22. Nath KA, Vercellotti GM, Grande JP, et al. Heme protein-induced chronic 7. Maines MD, Eke BC, Zhao X. Corticosterone promotes increased heme renal inflammation: suppressive effect of induced heme oxygenase-1. Kidney oxygenase-2 protein and transcript expression in the newborn rat brain. Int 2001;59:106–17. Brain Res 1996;722:83–94. 23. Dennery P, Weng YH, Yang G, Tatarov A, Spitz DR, Poss K. Improved 8. Tozer GM, Prise VE, Motterlini R, Poole BA, Wilson J, Chaplin DJ. The resistance to oxygen toxicity associated with absence of HO-1 in mice; role of ! comparative effects of the NOS inhibitor, N -nitro- L -arginine, and the iron. Pediatr Res 1998;43:280A. haemoxygenase inhibitor, zinc protoporphyrin IX, on tumour blood flow. Int 24. Welty SE, Rogers LK, Tom DJ, et al. Hyperoxic lung injury is potentiated by J Radiat Oncol Biol Phys 1998;42:849–53. SP-C promoter driven expression of an HO-1 transgene in mice. Am Rev 9. Dennery PA, Spitz DR, Yang G, et al. Oxygen toxicity and iron accumulation Respir Crit Care Med 1999;159:A218 (Abstract). in the lungs of mice lacking heme oxygenase-2. J Clin Invest 25. Dore S, Sampei K, Goto S, et al. Heme oxygenase-2 is neuroprotective in 1998;101:1001–11. cerebral ischemia [In Process Citation]. Mol Med 1999;5:656–63. 10. Alam J, Cai J, Smith A. Isolation and characterization of the mouse heme 26. Zimmerman SS, Truxal B. Carbon monoxide poisoning. Pediatrics 1981; oxygenase-1 gene. Distal 50 sequences are required for induction by heme or 68:215–24. heavy metals. J Biol Chem 1994;269:1001–9. 27. Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snyder SH. Carbon monoxide: a 11. Choi AM, Knobil K, Otterbein SL, Eastman DA, Jacoby DB. Oxidant stress putative neural messenger. Science 1993;259:381–4. responses in influenza virus pneumonia: gene expression and transcription 28. Brouard S, Otterbein LE, Anrather J, et al. Carbon monoxide generated by factor activation. Am J Physiol 1996;271:L383–91. heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med 12. Choi AM, Alam J. Heme oxygenase-1: function, regulation, and implication 2000;192:1015–26. of a novel stress-inducible protein in oxidant-induced lung injury. Am J 29. Galan A, Garcia-Bermejo ML, Troyano A, et al. Stimulation of p38 mitogen- Respir Cell Mol Biol 1996;15:9–19. activated protein kinase is an early regulatory event for the cadmium- 13. Terry CM, Clikeman JA, Hoidal JR, Callahan KS. Effect of tumor necrosis induced apoptosis in human promonocytic cells. J Biol Chem 2000;275: factor-alpha and interleukin-1 alpha on heme oxygenase-1 expression in 11418–24. human endothelial cells. Am J Physiol 1998;274:H883–91. 30. Valladares A, Alvarez AM, Ventura JJ, Roncero C, Benito M, Porras A. p38 14. Stocker R, Yamamoto Y, McDonagh AF, Glazzer AN, Ames BN. Bilirubin is an mitogen-activated protein kinase mediates tumor necrosis factor-alpha– antioxidant of possible physiologic importance. Science 1987;235:1043–6. induced apoptosis in rat fetal brown adipocytes. Endocrinology 2000;141: 15. Suttner DM, Dennery PA. Reversal of HO-1 related cytoprotection with 4383–95. increased expression is due to reactive iron. FASEB J 1999;13:1800–8. 31. Karahashi H, Nagata K, Ishii K, Amano F. A selective inhibitor of p38 MAP 16. Schipper HM, Bernier L, Mehindate K, Frankel D. Mitochondrial iron kinase, SB202190, induced apoptotic cell death of a lipopolysaccharide- sequestration in dopamine-challenged astroglia: role of heme oxygenase-1 treated macrophage-like cell line, J774.1. Biochim Biophys Acta and the permeability transition pore. J Neurochem 1999;72:1802–11. 2000;1502:207–23. 17. Ham D, Schipper HM. Heme oxygenase-1 induction and mitochondrial iron 32. Weng YH, Dennery PA. Possible functional implication of heme oxygenase sequestration in astroglia exposed to amyloid peptides. Cell Mol Biol (Paris) (HO)-1 protein and HO-2 protein binding. Am J Resp Crit Care Med 2000;46:587–96. 2001;165:578A. 18. Burnett AL, Johns DG, Kriegsfeld LJ, et al. Ejaculatory abnormalities in mice 33. Dore S, Takahashi M, Ferris CD, et al. Bilirubin, formed by activation of with targeted disruption of the gene for heme oxygenase-2. Nat Med heme oxygenase-2, protects neurons against oxidative stress injury. Proc Natl 1998;4:84–7. Acad Sci USA 1999;96:2445–50. 19. Dore S, Goto S, Sampei K, et al. Heme oxygenase-2 acts to prevent neuronal 34. Frankel D, Mehindate K, Schipper HM. Role of heme oxygenase-1 in the death in brain cultures and following transient cerebral ischemia. regulation of manganese superoxide dismutase gene expression in Neuroscience 2000;99:587–92. oxidatively-challenged astroglia. J Cell Physiol 2000;185:80–6.

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