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Department of Pharmacology and Toxicology, School of Pharmacy, Martin Luther University, Halle, Germany
Received February 10, 2006; accepted February 13, 2006
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), the use of SNP as an experimental tool to mimic intracellular effects of NO should be avoided in the future. This work not only helps revise concepts in NO and HO-1 research but also may direct future efforts to the role of iron and reactive oxygen species in the regulation of adenylyl cyclase.
; Wagener et al., 2003; Maines, 2004). Thus and in addition to its general anti-inflammatory function, HO-1 prevents long-term rejection or arteriosclerosis of transplants (Soares et al., 1998) and enhances the resistance of pancreatic islet cells to cytokine-mediated injury (Ye and Laychock, 1998). Moreover, the first human case of HO-1 deficiency, which occurs secondary to a genetic disorder, shows severe, persistent endothelial damage and increased tissue vulnerability to oxidant injury besides growth retardation and anemia (Yachie et al., 1999).
The reaction catalyzed by HO-1 results in the degradation of heme and yields biliverdin and carbon monoxide (CO), two metabolites that were initially considered mere waste products of heme catabolism without physiological function. This view has dramatically changed in recent years. Biliverdin is subsequently transformed to bilirubin by biliverdin reductase. Bilirubin, which is formed from biliverdin by biliverdin reductase, exerts strong antioxidant effects at physiological plasma concentrations. High-normal plasma levels of bilirubin were reported to be inversely related to atherogenic risk and to provide protection against endothelial damage (Hopkins et al., 1996; Mayer, 2000). Risk reduction by bilirubin was comparable with that of HDL cholesterol (Hopkins et al., 1996). CO is likewise known to produce antiapoptotic and cytoprotective actions. In addition, CO activates the soluble guanylyl cyclase/cGMP system and is thought to function, through this signaling pathway and in close resemblance to nitric oxide (NO), as a smooth muscle relaxing agent and neurotransmitter (Ryter et al., 2002; Wu and Wang, 2005).
A third component generated during heme catabolism is free iron, which in turn activates translational expression of ferritin. Ferritin has been shown to provide marked antioxidant cellular protection by rapidly sequestering free cytosolic iron, the crucial catalyst of oxygen-centered radical formation via the Fenton reaction in biological systems (Balla et al., 1992). Thus, in addition to HO-1, ferritin plays an important role as a fast acting endogenous cytoprotectant in cellular antioxidant defense mechanisms.
The unique combination of tissue protective and smooth muscle relaxing properties makes HO-1 an interesting target for treatment of cardiovascular diseases, including atherosclerosis, and other inflammatory disorders, among them neurodegenerative processes such as Alzheimer's and Parkinson's (Ryter et al., 2002; Maines, 2004). Support for the importance of HO-1 in human disease comes from clinical investigations demonstrating that HO-1 promoter polymorphisms with longer (GT)n repeats are associated with lower transcriptional activity as well as diminished vascular protection from atherogenic insults (Alam et al., 2004; Exner et al., 2004). In addition, HO-1 seems crucial for keeping the human uterus in a relaxed state during pregnancy and a reduced expression of placental HO-1 has been associated with a higher risk for pre-eclampsia (Bainbridge and Smith, 2005). Thus, therapeutic strategies aimed at moderately increasing tissue expression of HO-1 are potentially beneficial in a number of disease states. However, until recently, known inducers of HO-1, such as cadmium chloride and other heavy metals, did not have great promise for eventual therapeutic use in humans.
NO and NO donors were the first reported "benign" inducers of HO-1; these agents included long-established drugs in cardiovascular therapy, such as pentaerythritol tetranitrate. Those agents were shown to enhance, via cGMP-dependent pathways, expression of HO-1 at the mRNA, protein and catalytic level and, in addition, to prolong the half-life of HO-1 mRNA through cGMP-independent interactions of NO with the transcript (Yee et al., 1996; Durante et al., 1997; Bouton and Demple, 2000; Polte et al., 2000; Oberle et al., 2002). Meanwhile, it has become clear that other known HO-1 inducers, such as aspirin, can act via increasing endogenous NO formation from L-arginine (Grosser and Schröder, 2003; Grosser et al., 2003; Bach, 2005).
Sodium nitroprusside is a "spontaneous" NO donor, releasing NO (and NO+) upon dissolution in aqueous solvents and, in contrast to nitric acid esters, does not require enzymatic reduction or hydrolysis for this process. However, degradation of SNP generates two other biologically active products besides NO, notably cyanide and free iron. Therefore, the use of SNP as experimental tool for investigation of the effects of NO in biological systems is problematic, to say the least. Despite being such a "messy" NO donor, SNP has been used (and still is) in studies where it is supposed to act solely as donor of NO (which it does not) and where the effects observed are interpreted as being the consequence of NO release from SNP, which, based on the number of active mediators generated from SNP, has only a 
33% chance of being the correct explanation.
Using RAW 264.7 murine macrophages, Kim et al. (2006) clearly demonstrate that, in the case of HO-1 induction by SNP, NO has, at best, a negligible role as a mediator of this action of SNP. In a number of well designed experiments and by using appropriate controls such as the "true" donor of NO, SNAP, Kim et al. (2006) provide compelling evidence that SNP's NO or NO+ contribute minimally to HO-1 induction. Thus, SNP turns out to be a much stronger HO-1 inducer than SNAP although it possesses only 13% of SNAP's NO donating capacity under these conditions. In addition, scavengers of NO as well as inhibitors of its downstream signaling target, soluble guanylyl cyclase, are without effect on HO-1 induction by SNP in RAW macrophages.
Kim et al. (2006) conclude that free iron and not NO is the driving force behind the observed increases in HO-1 protein after exposure to SNP. Induction of HO-1 by SNP but not SNAP is blocked upon coincubation with the iron binding compound deferoxamine, and the authors are able to mimic the effect of SNP on HO-1 by use of exogenous iron (ferricyanide or ferric ammonium citrate). Cyanide, the third component released during SNP dissociation, does not function as a HO-1 inducer, because a cyanide inhibitor does not alter the SNP effect on HO-1. This is in agreement with earlier observations showing that in contrast to other NO donors, SNP promotes protein degradation and cytotoxicity because of the release of catalytic iron (Motterlini et al., 1996; (Wang et al., 2006).
How does iron induce HO-1? The answer given by Kim et al. (2006) is a very simple and unexpected one, based on the generally accepted concept of iron working through iron regulatory and binding proteins such IRP1 and IRP2. Using an inhibitor approach, Kim et al. (2006) demonstrate that iron released from SNP induces HO-1 via adenylyl cyclase activation and enhanced formation of cAMP; exogenous iron as well as SNP increase both intracellular cAMP and HO-1 levels. HO-1 induction by both SNP and iron is almost completely blocked by inhibitors of protein kinase A (PKA). This series of experiments also reveals that the NO/cGMP system tends to play a minor role because inhibitors of protein kinase G cause a small, nonsignificant reduction of HO-1 induction by SNP. Ultimately, however, this secondary signaling pathway seems to converge with the adenylyl cyclase/cAMP cascade because cGMP can activate expression of cAMP-sensitive cytoprotective pathways by inhibiting cAMP breakdown (Polte and Schröder 1998). The role of cAMP in mediating the effect of SNP/iron on HO-1 becomes even more convincing in the light of another series of experiments aimed at assessing the phosphorylation of PKA targets within the group of mitogen-activated protein kinases (MAPK). In RAW macrophages, extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) are responsive to SNP and iron, whereas p38 MAPK seems to be of no relevance for the induction of HO-1. SNP and 8-bromo-cAMP produced the same pattern of MAPK phosphorylation, notably of extracellular regulated kinase and JNK, which, in the case of SNP, is antagonized by both H89 (a PKA inhibitor) and deferoxamine (an iron chelator). Of various MAPK inhibitors employed by the authors, only those interfering with ERK and JNK are able to reverse HO-1 induction by SNP. The sheer number of control experiments at different levels within this signaling cascade is impressive and provides a very solid basis for this novel SNP-iron-cAMP-PKA-MAPK-HO-1 pathway proposed by Kim et al. (2006).
There is one step in this pathway, however, where the authors remain remarkably vague about potential bridging mechanisms: the link between iron release from SNP and increased cAMP levels. Although they acknowledge that iron is known to be involved in the regulation of genes and proteins, particularly those with pro- or anti-inflammatory functions, no attempt is made to explain the stimulatory effect of iron on the adenylyl cyclase/cAMP system.
However, considerable evidence has accumulated over the past few decades that free iron can increase adenylyl cyclase activity, either alone or in synergy with other stimuli (Baba et al., 1981; Tan et al., 1995; Sponsel et al., 1996). The underlying signaling mechanisms apparently involve the formation of reactive oxygen species (ROS) via iron-catalyzed free radical formation (Fenton reaction). Oxygen-derived free radicals are thought to enhance cAMP formation via tyrosine kinase-mediated effects on the catalytic subunit of adenylyl cyclase (Tan et al., 1995).
Based on such results, one could ask whether the inhibitor approach chosen by the authors to elucidate signaling events downstream of adenylyl cyclase would have been equally successful to explore the missing upstream link connecting iron and adenylyl cyclase. Based on the ROS concept of adenylyl cyclase regulation, antioxidants such as N-acetylcysteine should prevent the iron-dependent increases in cAMP observed by the authors. For now, there is enough evidence from the literature to add a hypothetical iron/ROS link to the upper part of the authors' elaborate signaling scheme (Fig. 1). It may take only one or two experiments to make the question mark disappear!
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Based on the data by Kim et al. (2006), the use of SNP as an experimental tool to mimic intracellular effects of NO should once and for all be avoided, particularly because numerous "clean" NO donors are available as alternatives. The importance and value of this work lies in the detailed and extensive approach by which the authors establish a clear causal link between iron (released from SNP) and its downstream effects on cAMP, MAPK, and HO-1, effects that were previously, and apparently mistakenly, attributed to SNP-derived NO. Perhaps just as important is that this article may not only lead to revised concepts in NO and HO-1 research but could redirect attention to the not fully explored role of iron and free radicals in the regulation of adenylyl cyclase.
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Footnotes |
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ABBREVIATIONS: HO-1, heme oxygenase-1; NO, nitric oxide; CO, carbon monoxide; SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetyl-DL-penicillamine; PKA, protein kinase A; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated kinase; JNK, c-Jun N-terminal kinase; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; ROS, reactive oxygen species.
Address correspondence to: Henning Schröder, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5208. E-mail: henning{at}stanford.edu
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TopAbstractReferences |
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Baba A, Lee E, Ohta A, Tatsuno T, and Iwata H (1981) Activation of adenylate cyclase of rat brain by lipid peroxidation. J Biol Chem 256: 3679-3684.
Bach F (2005) Heme oxygenase-1: a therapeutic amplification funnel. FASEB J 19: 1216-1219.
Bainbridge SA and Smith GN (2005) HO in pregnancy. Free Radic Biol Med 38: 979-988.[CrossRef][Medline]
Balla G, Jacob HS, Balla J, Rosenberg M, Nath K, Apple F, Eaton JW, and Vercellotti GM (1992) Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem 267: 18148-18153.
Bouton C and Demple B (2000) Nitric oxide-inducible expression of heme oxygenase-1 in human cells: translation-independent stabilization of the mRNA and evidence for direct action of nitric oxide. J Biol Chem 275: 32688-32693.
Durante W, Kroll MH, Christodoulides N, Peyton KJ, and Schafer AI (1997) Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells. Circ Res 80: 557-564.
Exner M, Minar E, Wagner O, and Schillinger M (2004) The role of heme oxygenase-1 promoter polymorphisms in human disease. Free Radic Biol Med 37: 1097-1104.[CrossRef][Medline]
Grosser N, Abate A, Oberle S, Vreman HJ, Dennery PA, Becker JC, Pohle T, Seidman DS, and Schröder H (2003) Heme oxygenase-1 induction may explain the antioxidant profile of aspirin. Biochem Biophys Res Commun 308: 956-960.[CrossRef][Medline]
Grosser N and Schröder H (2003) Aspirin protects endothelial cells from oxidant damage via the nitric oxide-cGMP pathway. Arterioscler Thromb Vasc Biol 23: 1345-1351.
Hopkins PN, Wu LL, Hunt SC, James BC, Vincent GM, and Williams RR (1996) Higher serum bilirubin is associated with decreased risk for early familial coronary artery disease. Arterioscler Thromb Vasc Biol 16: 250-255.
Kim HJ, Tsoy I, Park MK, Lee YS, Lee JH, Seo HG, and Chang KC (2006) Iron released by sodium nitroprusside contributes to heme oxygenase-1 induction via the cAMP-protein kinase A-mitogen-activated protein kinase pathway in RAW 264.7 cells. Mol Pharmacol 69: 1633-1640.
Maines MD (2004) The heme oxygenase system: past, present and future. Antioxid Redox Signal 6: 797-801.[Medline]
Mayer M (2000) Association of serum bilirubin concentration with risk of coronary artery disease. Clin Chem 46: 1723-1727.
Motterlini R, Foresti R, Intaglietta M, and Winslow RM (1996) NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am J Physiol 270: H107-H114.
Oberle S, Abate A, Grosser N, Vreman HJ, Dennery PA, Schneider HT, Stalleicken D, and Schröder H (2002) Heme oxygenase-1 induction may explain the antioxidant profile of pentaerithrityl trinitrate. Biochem Biophys Res Commun 290: 1539-1544.[CrossRef][Medline]
Polte T, Abate A, Dennery PA, and Schröder H (2000) Heme oxygenase-1 is a cyclic GMP-inducible endothelial protein and mediates the cytoprotective action of nitric oxide. Arterioscler Thromb Vasc Biol 20: 1209-1215.
Polte T and Schröder H (1998) Cyclic AMP mediates endothelial protection by nitric oxide. Biochem Biophys Res Commun 251: 460-465.[CrossRef][Medline]
Ryter SW, Otterbein LE, Morse D, and Choi AMK (2002) Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance. Mol Cell Biochem 234-235: 249-263.
Soares MP, Lin Y, Anrather J, Csizmadia E, Takigami K, Sato K, Grey ST, Colvin RB, Choi AM, Poss KD, et al. (1998) Expression of heme oxygenase-1 can determine cardiac xenograft survival. Nat Med 4: 1073-1077.[CrossRef][Medline]
Sponsel HT, Alfrey AC, Hammond WS, Durr JA, Ray C, and Anderson RJ (1996) Effect of iron on renal tubular epithelial cells. Kidney Int 50: 436-444.[Medline]
Tan CM, Xenoyannis S, and Feldman RD (1995) Oxidant stress enhances adenylyl cyclase activation. Circ Res 77: 710-717.
Wagener FA, Volk HD, Willis D, Abraham NG, Soares MP, Adema GJ, and Figdor CG (2003) Different faces of the heme-heme oxygenase system in inflammation. Pharmacol Rev 55: 551-557.
Wang J, Fillebeen C, Chen G, Andriopoulos B, and Pantopoulos K (2006) Sodium nitroprusside promotes IRP2 degradation via an increase in intracellular iron and in the absence of S nitrosylation at C178. Mol Cell Biol 26:1948-1954.
Wu L and Wang R (2005) Carbon monoxide: endogenous production, physiological functions, and pharmacological applications. Pharmacol Rev 57: 585-630.
Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, and Koizumi S (1999) Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin Investig 103: 129-135.[Medline]
Ye J and Laychock SG (1998) A protective role for heme oxygenase expression in pancreatic islets exposed to interleukin-1 beta. Endocrinology 139: 4155-4163.
Yee EL, Pitt BR, Billiar TR, and Kim YM (1996) Effect of nitric oxide on heme metabolism in pulmonary artery endothelial cells. Am J Physiol 271: L512-L518.
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