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On mechanism of superoxide signaling under physiological and pathophysiological conditions
Medical Hypotheses (2005) 64, 127–129
http://intl.elsevierhealth.com/journals/mehy
On mechanism of superoxide signaling under physiological and pathophysiological conditions I.B. Afanas’ev* Vitamin Research Institute, Nauchny pr.14A, Moscow 117820, Russia Received 27 November 2003; accepted 5 May 2004
Summary It has been demonstrated that in various physiological and pathophysiological processes superoxide functions as a signaling molecule by the way different from those mediated by hydrogen peroxide, hydroxyl radicals, or peroxynitrite. However, until now the mechanism of superoxide signaling remains obscure. A well known role of superoxide as a precursor of reactive hydroxyl radicals by the superoxide-dependent Fenton reaction or the formation of peroxynitrite must result in the damage of the target molecules and lead to pathological disorders. However, this mechanism is unlikely in such processes as the stimulation by superoxide of enzymatic phosphorylation and dephosphorylation. But, not being a “super-oxidant”, superoxide possesses the frequently forgotten “super”nucleophilic properties. Now, we propose a new mechanism for superoxide signaling depending on its nucleophilic reactions. Possible nucleophilic mechanisms of superoxide signaling in the hydrolysis of phosphatidylinositol to inositol 1,4,5-tris-phosphate and in the catalysis of phosphorylation by mitogen-activated protein kinases, phospholipase C and other enzymes are considered. c 2004 Elsevier Ltd. All rights reserved.
At present, an important role of superoxide signaling has been shown in a variety of physiological and pathophysiological responses such as trancriptional activation, cell proliferation, and apoptosis. Among them, new developments of superoxide participation in enzymatic phosphorylation/dephosphorylation reactions are of particular interest. These processes are mainly related to protein kinase cascade. Thus, as early as in 1995 Baas and Berk [1] have found that O2 activated mitogen-activated protein kinases in vascular smooth muscle cells by a way different from H2 O2 . Subsequent works demonstrated numerous examples of superoxide effects in similar processes. *
Tel./fax: +7-95-434-8219. E-mail address: [email protected].
Huang et al. [2] suggested that the PPARc (peroxisome proliferator activated receptor-cÞ agonists stimulation of EPK (extracellular signal-related protein kinase) phosphorylation in murine myoblasts was mediated by superoxide, because this process was inhibited by the superoxide scavengers (SOD) mimetic MnTBAP and glutathione. It is important that the superoxide producer DMNQ (2,3dimethyl-1,4-naphthoquinone) also stimulated ERK phosphorylation. Susa and Wakabayashi [3] proposed that the SOD-inhibitable ERK phosphorylation in rat aortic smooth muscle cells during extracellular alkalosis be initiated by superoxide produced by NADPH oxidase. Stimulation by superoxide of ERK1/2 phosphorylation was also showed in vascular smooth muscle cells subjected to cyclic stress [4]. Gurjar et al. [5] concluded that
0306-9877/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2004.05.009
128
Afanas’ev
superoxide-stimulated ERK activation mediated IL1b-dependent MMP-9 (matrix metalloproteinase-9) induction responsible for vascular injury. These processes were also inhibited by NAC treatment and MnSOD overexpression in vascular smooth muscle cells demonstrating important role of superoxide. Wang et al. [6] concluded that superoxide produced by eNO synthase activated the p42/44 MARK kinase pathway that increased TNF-a production; importantly, this pathway was different from the stimulation by hydrogen peroxide or NO. Superoxide produced by extracorporeal shock mediated ERK activation resulting in osteogenic cell growth and maturation [7]. Superoxide signaling in phosphorylation is not limited only to the mediation of protein kinase pathway. It was found that mitochondrial superoxide modulated nuclear CREB (cAMP-responsive element-binding protein) phosphorylation in hippocampal neurons [8]. Wu and de Champlain [9] have shown that superoxide enhanced the hydrolysis of phosphatidylinositol (PIP) to inositol 1,4,5tris-phosphate (IP3 ) in rat aortic smooth muscle cells PIP ) ðO 2 Þ ) IP3
ð1Þ
It is evident that the above examples demonstrate both physiological and pathophysiological effects of superoxide signaling. We should like to stress that although the other signaling molecules originated from superoxide (HO: , H2 O2 , and OONO) are able to stimulate the same processes, many studies explicitly show that superoxide signaling proceeds by its own way [1,6,10–12]. Until now the mechanisms of superoxide signaling remain obscure. It is usually accepted that these processes can proceed by the following routes The superoxide-depended iron-catalyzed decomposition of hydrogen peroxide (the Fenton reaction) H2 O 2
3þ O ) Fe2þ ) HO þ HO þ Fe3þ 2 þ Fe
ð2Þ
The reaction of superoxide with nitric oxide. þ O 2 þ NO þ H ) HOONO ) HO þ NO2
ð3Þ
Both processes lead to the formation of highly reactive hydroxyl radicals. Therefore, such a type of cell signaling had to be a nonselective process leading to the modification (destruction) of target molecules and the initiation of numerous pathophysiological disorders, including atherosclerotic, restenotic, and hypertensive cardiovascular diseases. However, the mediation of heterolytic reactions such as phosphorylation/dephosphorylation is dif-
ficult to explain by the formation of reactive hydroxyl or hydroxyl-like (for example, ferryl or ferroxyl) free radicals. Fortunately, in contrast to hydrogen peroxide, for which the mechanism of its signaling function in heterolytic processes is even more uncertain, superoxide, being both a radical and an anion can react with organic molecules by nucleophilic mechanism. Despite its famous name, superoxide is not a super-oxidant but a relatively moderate reductant. However, superoxide possesses another “super” quality – it is a “super-nucleophile”. Owing to this, superoxide is able to rapidly deprotonize alcohols, phenols, and thiols and hydrolyze esters [13]. O 2 þ ROH ) RO þ HOO
ð4Þ
HOO þ O 2 ) HOO þ O2
ð5Þ
0 0 O 2 þ RCOOR ) RCðOÞOO þ R O
ð6Þ
RCðOÞOO þ O 2 ) RCðOÞOO þ O2
ð7Þ
Does superoxide indeed mediate enzymatic phosphorylation/dephosphorylation reactions? Of course, it is purely hypothetical suggestion, but it seems that it does not contradict any experimental data including those quoted above. It should be noted that firstly the possibility of nucleophilic mechanism in the deesterification of phospholipids has been proposed by Niehaus [14] as early as in 1978, but later on this proposal has been forgotten. I should like also to mention that at present no other hypothetical mechanisms truthful from the chemical point of view were proposed for the explanation of superoxide role in phosphorylation/ dephosphorylation processes. It is obvious that in biological systems superoxide must react by nucleophilic mechanism with all molecules having ester or hydroxyl groups. However, taking into account an extremely low stationary concentration of superoxide in cells, its effect will be visible only in enzymatic catalytic processes. As the examples, we propose the nucleophilic mechanisms of superoxide mediation in following processes of dephosphorylation and phosphorylation. It is possible that the hydrolysis (i.e. dephosphorylation) of PIP to IP3 [9] may proceed by the following mechanism: PIP½OPðOÞðORÞ2 þ O 2 ) PIPO þ OOPðOÞðORÞ2
ð8Þ
OOPðOÞðORÞ2 þ O 2 ) OOPðOÞðORÞ2 þ O2
ð9Þ
On the other hand during enzymatic phosphorylation superoxide can deprotonize hydroxyl groups and accelerate by that the esterification of phos-
On mechanism of superoxide signaling under physiological and pathophysiological conditions phate. For example, the activation of ERK by PPARgamma agonists or DMNQ [2] can be presented as: PPARgamma
[4]
[5] O2.-
+
ERK(OH)
⇒
HOO.
+
ERK(O-)
DMNQ
ð10Þ ERKðO Þ þ HOPðOÞðORÞ2 ) ERK½OPðOÞðORÞ2
[6]
ð11Þ [7]
In conclusion we propose that the nucleophilic mechanism of superoxide signaling should be considered as an important possible route in physiological and pathophysiological processes mediated by superoxide. This proposal might be not only of theoretical but also practical importance because it highlights significance of suppressing the superoxide itself in cell damage by (SOD mimics or flavonoids) and not only widely used antioxidants such as vitamins E and C, which effectively react with hydroxyl and peroxyl radicals but are ineffective in the reactions with superoxide.
[8]
[9]
[10]
[11]
References [1] Baas AS, Berk BC. Differential activation of mitogenactivated protein kinases by H2 O2 and O2 .– in vascular smooth muscle cells. Circ Res 1995;77:29–36. [2] Huang WC, Chio CC, Chi KH, Wu HM, Lin WW. Superoxide anion-dependent Raf/MEK/ERK activation by peroxisome proliferator activated receptor gamma agonist 15-deoxydelta(12,14)-prostaglandin J(2), ciglitazone, and GW1929. Exp Cell Res 2002;277:192–200. [3] Susa S, Wakabayashi I. Extracellular alkalosis activates ERK mitogen-activated protein kinase of vascular smooth
[12]
[13]
[14]
129
muscle cells through NADPH-mediated formation of reactive oxygen species. FEBS Lett 2003;554:399–402. Chen Q, Li W, Quan Z, Sumpio BE. Modulation of vascular smooth muscle cell alignment by cyclic strain is dependent on reactive oxygen species and P38 mitogen-activated protein kinase. J Vasc Surg 2003;37: 660–8. Gurjar MV, Deleon J, Sharma RV, Bhalla RC. Role of reactive oxygen species in IL-1b-stimulated sustained ERK activation and MMP-9 induction. Am J Physiol Heart C 2001;281: H2568–74. Wang W, Wang S, Nishanian EV, Del Pilar Cintron A, Wesley RA, Danner RL. Signaling by eNOS through a superoxidedependent p42/44 mitogen-activated protein kinase pathway. Am J Physiol Cell Ph 2001;281:C544–54. Wang F-S, Wang C-J, Sheen-Chen S-M, Kuo Y-R, Chen R-F, Yang KD. Superoxide mediates shock wave induction of ERK-dependent osteogenetic transcription factor (CBFA1) and mesenchymal cell differentiation toward osteoprogenitors. J Biol Chem 2002;277:10931–7. Hongpaisan J, Winters CA, Brian Andrews S. Calciumdependent mitochondrial superoxide modulates nuclear CREB phosphorylation in hippocampal neurons. Mol Cell Neurosci 2003;24:1103–15. Wu L, de Champlain J. Effect of superoxide on signaling pathways in smooth muscle cells from rats. Hypertension 1999;34:1247–53. Li PF, Dietz R, von Harsdorf R. Differential effect of hydrogen peroxide and superoxide anion on apoptosis and proliferation of vascular smooth muscle cells. Circulation 1997;96:3602–9. Mendes AF, Caramona MM, Carvalho AP, Lopes MC. Differential roles of hydrogen peroxide and superoxide in mediating IL-1-induced NF-jB activation and iNOS expression in bovine articular chondrocytes. J Cell Biochem 2003;88: 783–93. Krauskopf A, Lhote P, Mutter M, Duffour J-F, Ruegg UT, Buetler TM. Vasopressin type 1A receptor up-regulation by cyclosporin A in vascular smooth muscle cells is mediated by superoxide. J Biol Chem 2003;278:41685–90. Afanas’ev IB. In: Superoxide Ion: chemistry and biological implications, vol. 1. Boca Raton, Florida: CRC Press; 1989 [Chapter 6]. Niehaus Jr WG. A proposed role of superoxide anion as a biological nucleophile in the deesterification of phospholipids. Bioorg Chem 1978;7:7–10.
http://intl.elsevierhealth.com/journals/mehy
On mechanism of superoxide signaling under physiological and pathophysiological conditions I.B. Afanas’ev* Vitamin Research Institute, Nauchny pr.14A, Moscow 117820, Russia Received 27 November 2003; accepted 5 May 2004
Summary It has been demonstrated that in various physiological and pathophysiological processes superoxide functions as a signaling molecule by the way different from those mediated by hydrogen peroxide, hydroxyl radicals, or peroxynitrite. However, until now the mechanism of superoxide signaling remains obscure. A well known role of superoxide as a precursor of reactive hydroxyl radicals by the superoxide-dependent Fenton reaction or the formation of peroxynitrite must result in the damage of the target molecules and lead to pathological disorders. However, this mechanism is unlikely in such processes as the stimulation by superoxide of enzymatic phosphorylation and dephosphorylation. But, not being a “super-oxidant”, superoxide possesses the frequently forgotten “super”nucleophilic properties. Now, we propose a new mechanism for superoxide signaling depending on its nucleophilic reactions. Possible nucleophilic mechanisms of superoxide signaling in the hydrolysis of phosphatidylinositol to inositol 1,4,5-tris-phosphate and in the catalysis of phosphorylation by mitogen-activated protein kinases, phospholipase C and other enzymes are considered. c 2004 Elsevier Ltd. All rights reserved.
At present, an important role of superoxide signaling has been shown in a variety of physiological and pathophysiological responses such as trancriptional activation, cell proliferation, and apoptosis. Among them, new developments of superoxide participation in enzymatic phosphorylation/dephosphorylation reactions are of particular interest. These processes are mainly related to protein kinase cascade. Thus, as early as in 1995 Baas and Berk [1] have found that O2 activated mitogen-activated protein kinases in vascular smooth muscle cells by a way different from H2 O2 . Subsequent works demonstrated numerous examples of superoxide effects in similar processes. *
Tel./fax: +7-95-434-8219. E-mail address: [email protected].
Huang et al. [2] suggested that the PPARc (peroxisome proliferator activated receptor-cÞ agonists stimulation of EPK (extracellular signal-related protein kinase) phosphorylation in murine myoblasts was mediated by superoxide, because this process was inhibited by the superoxide scavengers (SOD) mimetic MnTBAP and glutathione. It is important that the superoxide producer DMNQ (2,3dimethyl-1,4-naphthoquinone) also stimulated ERK phosphorylation. Susa and Wakabayashi [3] proposed that the SOD-inhibitable ERK phosphorylation in rat aortic smooth muscle cells during extracellular alkalosis be initiated by superoxide produced by NADPH oxidase. Stimulation by superoxide of ERK1/2 phosphorylation was also showed in vascular smooth muscle cells subjected to cyclic stress [4]. Gurjar et al. [5] concluded that
0306-9877/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2004.05.009
128
Afanas’ev
superoxide-stimulated ERK activation mediated IL1b-dependent MMP-9 (matrix metalloproteinase-9) induction responsible for vascular injury. These processes were also inhibited by NAC treatment and MnSOD overexpression in vascular smooth muscle cells demonstrating important role of superoxide. Wang et al. [6] concluded that superoxide produced by eNO synthase activated the p42/44 MARK kinase pathway that increased TNF-a production; importantly, this pathway was different from the stimulation by hydrogen peroxide or NO. Superoxide produced by extracorporeal shock mediated ERK activation resulting in osteogenic cell growth and maturation [7]. Superoxide signaling in phosphorylation is not limited only to the mediation of protein kinase pathway. It was found that mitochondrial superoxide modulated nuclear CREB (cAMP-responsive element-binding protein) phosphorylation in hippocampal neurons [8]. Wu and de Champlain [9] have shown that superoxide enhanced the hydrolysis of phosphatidylinositol (PIP) to inositol 1,4,5tris-phosphate (IP3 ) in rat aortic smooth muscle cells PIP ) ðO 2 Þ ) IP3
ð1Þ
It is evident that the above examples demonstrate both physiological and pathophysiological effects of superoxide signaling. We should like to stress that although the other signaling molecules originated from superoxide (HO: , H2 O2 , and OONO) are able to stimulate the same processes, many studies explicitly show that superoxide signaling proceeds by its own way [1,6,10–12]. Until now the mechanisms of superoxide signaling remain obscure. It is usually accepted that these processes can proceed by the following routes The superoxide-depended iron-catalyzed decomposition of hydrogen peroxide (the Fenton reaction) H2 O 2
3þ O ) Fe2þ ) HO þ HO þ Fe3þ 2 þ Fe
ð2Þ
The reaction of superoxide with nitric oxide. þ O 2 þ NO þ H ) HOONO ) HO þ NO2
ð3Þ
Both processes lead to the formation of highly reactive hydroxyl radicals. Therefore, such a type of cell signaling had to be a nonselective process leading to the modification (destruction) of target molecules and the initiation of numerous pathophysiological disorders, including atherosclerotic, restenotic, and hypertensive cardiovascular diseases. However, the mediation of heterolytic reactions such as phosphorylation/dephosphorylation is dif-
ficult to explain by the formation of reactive hydroxyl or hydroxyl-like (for example, ferryl or ferroxyl) free radicals. Fortunately, in contrast to hydrogen peroxide, for which the mechanism of its signaling function in heterolytic processes is even more uncertain, superoxide, being both a radical and an anion can react with organic molecules by nucleophilic mechanism. Despite its famous name, superoxide is not a super-oxidant but a relatively moderate reductant. However, superoxide possesses another “super” quality – it is a “super-nucleophile”. Owing to this, superoxide is able to rapidly deprotonize alcohols, phenols, and thiols and hydrolyze esters [13]. O 2 þ ROH ) RO þ HOO
ð4Þ
HOO þ O 2 ) HOO þ O2
ð5Þ
0 0 O 2 þ RCOOR ) RCðOÞOO þ R O
ð6Þ
RCðOÞOO þ O 2 ) RCðOÞOO þ O2
ð7Þ
Does superoxide indeed mediate enzymatic phosphorylation/dephosphorylation reactions? Of course, it is purely hypothetical suggestion, but it seems that it does not contradict any experimental data including those quoted above. It should be noted that firstly the possibility of nucleophilic mechanism in the deesterification of phospholipids has been proposed by Niehaus [14] as early as in 1978, but later on this proposal has been forgotten. I should like also to mention that at present no other hypothetical mechanisms truthful from the chemical point of view were proposed for the explanation of superoxide role in phosphorylation/ dephosphorylation processes. It is obvious that in biological systems superoxide must react by nucleophilic mechanism with all molecules having ester or hydroxyl groups. However, taking into account an extremely low stationary concentration of superoxide in cells, its effect will be visible only in enzymatic catalytic processes. As the examples, we propose the nucleophilic mechanisms of superoxide mediation in following processes of dephosphorylation and phosphorylation. It is possible that the hydrolysis (i.e. dephosphorylation) of PIP to IP3 [9] may proceed by the following mechanism: PIP½OPðOÞðORÞ2 þ O 2 ) PIPO þ OOPðOÞðORÞ2
ð8Þ
OOPðOÞðORÞ2 þ O 2 ) OOPðOÞðORÞ2 þ O2
ð9Þ
On the other hand during enzymatic phosphorylation superoxide can deprotonize hydroxyl groups and accelerate by that the esterification of phos-
On mechanism of superoxide signaling under physiological and pathophysiological conditions phate. For example, the activation of ERK by PPARgamma agonists or DMNQ [2] can be presented as: PPARgamma
[4]
[5] O2.-
+
ERK(OH)
⇒
HOO.
+
ERK(O-)
DMNQ
ð10Þ ERKðO Þ þ HOPðOÞðORÞ2 ) ERK½OPðOÞðORÞ2
[6]
ð11Þ [7]
In conclusion we propose that the nucleophilic mechanism of superoxide signaling should be considered as an important possible route in physiological and pathophysiological processes mediated by superoxide. This proposal might be not only of theoretical but also practical importance because it highlights significance of suppressing the superoxide itself in cell damage by (SOD mimics or flavonoids) and not only widely used antioxidants such as vitamins E and C, which effectively react with hydroxyl and peroxyl radicals but are ineffective in the reactions with superoxide.
[8]
[9]
[10]
[11]
References [1] Baas AS, Berk BC. Differential activation of mitogenactivated protein kinases by H2 O2 and O2 .– in vascular smooth muscle cells. Circ Res 1995;77:29–36. [2] Huang WC, Chio CC, Chi KH, Wu HM, Lin WW. Superoxide anion-dependent Raf/MEK/ERK activation by peroxisome proliferator activated receptor gamma agonist 15-deoxydelta(12,14)-prostaglandin J(2), ciglitazone, and GW1929. Exp Cell Res 2002;277:192–200. [3] Susa S, Wakabayashi I. Extracellular alkalosis activates ERK mitogen-activated protein kinase of vascular smooth
[12]
[13]
[14]
129
muscle cells through NADPH-mediated formation of reactive oxygen species. FEBS Lett 2003;554:399–402. Chen Q, Li W, Quan Z, Sumpio BE. Modulation of vascular smooth muscle cell alignment by cyclic strain is dependent on reactive oxygen species and P38 mitogen-activated protein kinase. J Vasc Surg 2003;37: 660–8. Gurjar MV, Deleon J, Sharma RV, Bhalla RC. Role of reactive oxygen species in IL-1b-stimulated sustained ERK activation and MMP-9 induction. Am J Physiol Heart C 2001;281: H2568–74. Wang W, Wang S, Nishanian EV, Del Pilar Cintron A, Wesley RA, Danner RL. Signaling by eNOS through a superoxidedependent p42/44 mitogen-activated protein kinase pathway. Am J Physiol Cell Ph 2001;281:C544–54. Wang F-S, Wang C-J, Sheen-Chen S-M, Kuo Y-R, Chen R-F, Yang KD. Superoxide mediates shock wave induction of ERK-dependent osteogenetic transcription factor (CBFA1) and mesenchymal cell differentiation toward osteoprogenitors. J Biol Chem 2002;277:10931–7. Hongpaisan J, Winters CA, Brian Andrews S. Calciumdependent mitochondrial superoxide modulates nuclear CREB phosphorylation in hippocampal neurons. Mol Cell Neurosci 2003;24:1103–15. Wu L, de Champlain J. Effect of superoxide on signaling pathways in smooth muscle cells from rats. Hypertension 1999;34:1247–53. Li PF, Dietz R, von Harsdorf R. Differential effect of hydrogen peroxide and superoxide anion on apoptosis and proliferation of vascular smooth muscle cells. Circulation 1997;96:3602–9. Mendes AF, Caramona MM, Carvalho AP, Lopes MC. Differential roles of hydrogen peroxide and superoxide in mediating IL-1-induced NF-jB activation and iNOS expression in bovine articular chondrocytes. J Cell Biochem 2003;88: 783–93. Krauskopf A, Lhote P, Mutter M, Duffour J-F, Ruegg UT, Buetler TM. Vasopressin type 1A receptor up-regulation by cyclosporin A in vascular smooth muscle cells is mediated by superoxide. J Biol Chem 2003;278:41685–90. Afanas’ev IB. In: Superoxide Ion: chemistry and biological implications, vol. 1. Boca Raton, Florida: CRC Press; 1989 [Chapter 6]. Niehaus Jr WG. A proposed role of superoxide anion as a biological nucleophile in the deesterification of phospholipids. Bioorg Chem 1978;7:7–10.