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Intracellular and Extracellular Effects of S100B in the Cardiovascular Response to Disease

Intracellular and Extracellular Effects of S100B in the Cardiovascular Response to Disease Hindawi Publishing Corporation Cardiovascular Psychiatry and Neurology Volume 2010, Article ID 206073, 6 pages doi:10.1155/2010/206073 Review Article Intracellular and Extracellular Effects of S100B in the Cardiovascular Response to Disease James N. Tsoporis, Forough Mohammadzadeh, and Thomas G. Parker Division of Cardiology, Department of Medicine, Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada M5B 1W8 Correspondence should be addressed to Thomas G. Parker, parkertg@smh.toronto.on.ca Received 1 March 2010; Accepted 6 May 2010 Academic Editor: Rosario Donato Copyright © 2010 James N. Tsoporis et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. S100B, a calcium-binding protein of the EF-hand type, exerts both intracellular and extracellular functions. S100B is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. Forced expression of S100B in neonatal rat myocyte cultures and high level expression of S100B in transgenic mice hearts inhibit cardiac hypertrophy and the associated phenotype but augments myocyte apoptosis following myocardial infarction. By contrast, knocking out S100B, augments hypertrophy, decreases apoptosis and preserves cardiac function following myocardial infarction. Expression of S100B in aortic smooth muscle cells inhibits cell proliferation and the vascular response to adrenergic stimulation. S100B induces apoptosis by an extracellular mechanism via interaction with the receptor for advanced glycation end products and activating ERK1/2 and p53 signaling. The intracellular and extracellular roles of S100B are attractive therapeutic targets for the treatment of both cardiac and vascular diseases. 1. The Family of S100 Proteins their expression might be repressed in other cell types by negative regulatory factors which are controlled by envi- S100 proteins entail a multigenic family of calcium binding ronmental conditions. For instance induction of S100B in proteins of the EF-hand type (helix E-loop-helix F). These rat myocardium postinfarction [7] implies that transcription proteins are called S100 because of their solubility in regulation of these proteins is strongly controlled by negative a 100% -saturated solution with ammonium sulphate at and positive elements [8]. neutral pH. They are small acidic proteins, 10–12 KDa, and S100 proteins do not exhibit intrinsic catalytic activity. contain two distinct EF-hands, 4 α-helical segments, a central However, they are calcium sensor proteins and through hinge region of variable length, and the N- and C-terminal interaction with several intracellular effector proteins they contribute to the regulation of a broad range of functions variable domains. To date, 25 members of this family have been identified [1]. Of these, 21 family members (S100A1- such as contraction, motility, cell growth and differentiation, S100A18, trichohyalin, filagrin, and repetin) have genes cell cycle progression, organization of membrane-associated clustered on a 1.6-Mbp segment of human chromosome cytoskeleton elements, cell survival, apoptosis, protein phos- 1 (1q21) while other members are found at chromosome phorylation, and secretion [1, 3, 9]. In ordertomodulate loci 4q16 (S100P), 5q14 (S100Z), 21q22 (S100B), and Xp22 these types of activities, S100 proteins undergo conforma- (S100G) [2]. S100 proteins are widely expressed in a variety tional changes [10]. Upon calcium binding, the helices of of cell types and tissues. For example, S100A1 and S100A2 S100 proteins rearrange, revealing a hydrophobic cleft, which forms the target protein binding site [11]. Although target are found in the cytoplasm and nucleus, respectively, of smooth-muscle cells of skeletal muscle [3], S100P is located binding of S100 proteins is calcium-dependent, calcium in the cytoplasm of placental tissue [4, 5], and S100B in independent interactions have been reported [12]. Enzymes are the most common calcium independent target binding cytoplasm of astrocytes of nervous system [6]. However, 2 Cardiovascular Psychiatry and Neurology for the S100 proteins. For instance, S100B and S100A1 RAGE V C1 C2 bind with glycogen phosphorylase [13]. The most significant p53 Apoptosis ERK1/2 calcium-independent interactions of S100 proteins are their ability to bind to each other. Typically, they are homodimers, NO but heterodimerization adds to the complexity of this iNOS multiprotein family. Each subunit consists of two helix- S100B loop-helix motifs connected by a central linker or so-called ++ Ca hinge region. The C-terminal canonical EF-hand motif is S100B TEF-1 ++ composed of 12 amino acids, whereas the N-terminal S100- Ca specific EF-hand comprises 14 residues [3, 14]. ++ Ca NE TEF-1 Growing evidence indicates that in addition to intra- P (α -receptor) 1A cellular activities, some S100 proteins (e.g., S100B, S100A1, PKCβ S100A4, S100A8, and S100A9) exhibit extracellular functions [15]. However, secretion has been shown only for S100B, S100A8, and S100A9 [15]. The S100A8/A9 heterodimer is Figure 1: Schematic representation of proposed intracellular and secreted by a novel secretion pathway that depends on extracellular effects of S100B in cardiac myocytes. Norepinephrine an intact microtubule network and acts as a chemotactic (NE) activation of the calcium-dependent protein kinase C (PKC)- molecule in inflammation [16, 17]. The extracellular effects β, mediated by the α -adrenergic receptor, phosphorylates (P) of some S100 proteins require binding to the receptor transcriptional enhancer factor (1) TEF-1, resulting in DNA for advanced glycosylation end products (RAGE) [18–21]. binding and transactivation of the β-myosin heavy chain promoter. RAGE is a member of the immunoglobulin family of By contrast, S100B induction by NE and other hypertrophic cell surface molecules recognizing multiple ligands includ- signals (not shown) results in calcium-dependent block of PKC-β phosphorylation of TEF-1 and inhibition of β-MHC transcription. ing AGE, amphoterin, amyloid-β-peptide and β-fibrils, S100B can also induce apoptosis intracellularly via a inducible nitric S100A12, S100A6, and S100B [22]. The 45-kDa receptor oxide synthase (iNOS)-NO pathway or it can be secreted and via protein consists of 403 amino acids with an extracellular activation of the receptor for advanced glycation end products domain (1 variable and 2 constant Ig domains with disulfide (RAGE) (extracellular components V and CI), and induce apoptosis bridges), a single transmembrane region, and a short cytoso- via MEK-ERK1/2-p53 signaling. lic tail that triggers signal transduction [23]. RAGE ligands show selective binding to RAGE. S100B tetramer induces receptor dimerization by binding to RAGE [24]. S100B binds outgrowth; however, at micromolar concentrations it pro- to domains V and CI whereas the RAGE ligand S100A6 binds motes apoptosis [35, 36]. Such high extracellular levels are to domains CI and CII [23]. detected after brain injury or in neurodegenerative disorders like Down’s Syndrome, Alzheimer disease, or encephalitis [37, 38]. Both trophic and toxic effects of extracellular S100B 2. Noncardiovascular Actions of S100B are mediated in the brain by RAGE [36]. In addition to playing a major role in brain physiology [1], S100B has S100B is predominantly expressed in astrocytes, oligo- been implicated in cardiovascular development [39] and is dendrocytes, and schwann cells. S100B has intracellular considered a biochemical marker for brain injuries after and extracellular effects [1]. Intracellularly, S100B regulates bypass graft surgery [40] and dilated cardiomyopathy [41]. the cytoskeletal dynamics through disassembly of tubulin filaments, type III intermediate filaments [1], and bind- ing to fibrillary proteins such as CapZ [25] or inhibit- 3. Cardiovascular Actions of S100B ing GFAP phosphorylation when stimulated by cAMP or calcium/calmodulin [26]. S100B interacts in a calcium- 3.1. Intracellular S100B and Myocyte Hypertrophic Gene dependent manner with the cytoplasmic domain of myelin- Expression. The adult cardiac myocyte is terminally differen- associated glycoprotein and inhibits its phosphorylation by tiated and has lost the ability to proliferate. The myocardium protein kinase [27]. It is implicated in the phosphorylation therefore adapts to increasing workloads through hypertro- of tau protein [28], inhibition of Ndr kinase activity [29], phy of individual cells in response to hormonal, paracrine, inhibition of p53 phosphorylation [30], and regulation of and mechanical signals [42, 43]. This process is initially the activity of the GTPase Rac1 and Cdc effector, IQGAP compensatory but it can progress to irreversible enlargement [31]. S100B can also be secreted by a number of cell types and dilatation of the ventricle resulting in heart failure (e.g., astrocytes, glial cells) [32]. Astrocytes and glial cells [44]. Myocyte hypertrophy is accompanied by the down- secrete S100B, by a complex system involving alterations regulation of adult α-myosin heavy chain and a program of in intracellular calcium concentration [32]. S100B after fetal gene reexpression including the embryonic β-myosin secretion, or simply leakage from damaged cells, could heavy chain (MHC), the skeletal isoform of α actin (skACT), accumulate in the extracellular space and/or enter the blood and atrial natriuretic factor (ANF) [45, 46]. This response stream and cerebrospinal fluid [33, 34]. The action of S100B can be reproduced in vitro in cultured neonatal cardiac is strongly dependent on its extracellular concentration. myocytes by treatment with a number of trophic factors At nanomolar quantities, it has trophic effects on neurite including peptide growth factors and α -adrenergic agonists β-MHC promoter Cardiovascular Psychiatry and Neurology 3 [7]. Negative modulators of the hypertrophic response are pathway that initiates and sustains the hypertrophic response essential to maintain a balance between compensatory hyper- in cardiac myocytes by activating PKC signaling and which trophy and unchecked progression. Experimental evidence is subject to negative modulation by S100B also induces the suggests that S100B acts as an intrinsic negative regulator S100B gene. of the myocardial hypertrophic response [47–49]. S100B not normally expressed in the myocardium, is induced in 3.2. Intracellular S100B, Cardiovascular Hypertrophy and the peri-infarct region of the human heart after myocardial Apoptosis. To provide a physiologic model of S100B overex- infarction [47] and in rat heart commencing at day 7 pression effects, transgenic mice were created that contained following myocardial infarction as a result of experimental multiple copies of the human gene under the control of its coronary artery ligation [7]. In cultured neonatal rat cardiac own promoter. These animals demonstrate normal cardiac myocytes, transfection of an expression vector encoding the structure, and neuronal, but no basal cardiac expression human S100B protein inhibits the α -adrenergic induction of the transgene. In S100B transgenic mice, after chronic of the fetal genes β- MHC and the skACT [7]. The inhibition α -adrenergic agonist infusion, S100B is detected in the of α -adrenergic induction is selective as S100B does not heart and increased in the vasculature [49]. In addition, inhibit the capacity of thyroid hormone to induce α-myosin the myocyte hypertrophy and arterial smooth muscle cell heavy chain. To establish that S100B blocked α -adrenergic proliferation normally evoked in the heart and vasculature, induction of β -MHC and skACT by interrupting the PKC respectively, in response to α -adrenergic stimulation in signaling pathway, the interaction between forced S100B wild-type mice were abrogated in S100B transgenic mice expression and a constitutively activated mutant of PKCβ [49]. In knockout mice, α -adrenergic agonist infusion referred to as δPKCβ was tested [50]. δPKCβ transactivated provoked a potentiated myocyte hypertrophic response the β-MHC and skACT genes supporting the notion that the and augmented arterial smooth muscle cell proliferation. α -adrenergic induction of these genes is mediated by activa- Furthermore, in knockout mice, both the acute and chronic tion of the class-I PKC isoform β-PKC [7, 50]. Forced S100B increases in blood pressure in response to α -adrenergic expression could only block δPKCβ-induced transaction of agonist infusion were attenuated compared with wild-type β-MHC and skACT amidst concomitant treatment with an mice [49]. To determine whether this inhibition is general- α -adrenergic agonist or augmented extracellular calcium izable to other hypertrophic stimuli, transgenic and knock- suggesting that the capacity of S100B to modulate the out animals were subjected to descending aortic-banding hypertrophic phenotype is calcium dependent [7]. The tran- to produce pressure-overload. Aortic banding for 35 days scription factors TEF-1 (transcription factor-1) and related increased left ventricular (LV)/body weight (BW) ratio in TEF-1 (RTEF-1) upon phosphorylation by PKCβ bind to CD-1 controls (4.61 ± 0.06 g/kg versus 3.44 ± 0.16 g/kg MCAT elements on the skACT and β-MHC promoters and in sham operated, P< .05, n = 6) andproducedno activate transcription [51]. In cotransfection experiments, hypertrophy in S100B transgenic mice (3.37 ± 0.12 g/kg forced expression of S100B inhibited the activation of the versus 3.26 ± 0.11 g/kg in sham operated, P< .05, n = skACT and β-MHC promoters by overexpression of TEF- 8) and excessive hypertrophy in knock-out mice (5.12 ± 1 (unpublished observations). A direct interaction between 0.24 g/kg versus 3.19 ± 0.13 g/kg in sham operated, P< S100B and TEF-1 was demonstrated using a coimmuno- .05, n = 6). Similarly, thirty five days after experimental precipitation strategy (unpublished observations). These myocardial infarction, the S100B knockout mice mounted data suggest that S100B modulates the activation of the an augmented hypertrophic response compared to wild- fetal genes by direct binding to TEF-1. In addition to type mice [48]. Fetal gene expression was induced to a TEF-1, S100B interacts in a calcium-dependent manner greater magnitude in knockout mice compared to wild- with the giant phosphoprotein AHNAK/desmoyokin in type mice. The S100B transgenic mice did not develop cardiomyocytes and smooth muscle cells [49]. In cardiomy- the hypertrophic phenotype but demonstrated increased ocytes, AHNAK plays a role in cardiac calcium signaling apoptosis in the peri-infarct region compared to wild-type by modulating L-type calcium channels in response to β- and knockout mice. The postinfarct hypertrophic response adrenergic signaling [52, 53]. The S100B/AHNAK interac- in the myocardium is initiated by multiple trophic signals tion may participate in the S100B-mediated regulation of that include the state of local and systemic sympathetic cellular calcium homeostasis [53]. Whether there is any hyperactivity through α -adrenergic stimulation [54]. These relationship between S100B-mediated effects on calcium studies in S100B transgenic and knockout mice complement fluxes and S100B-dependent inhibition of the α -induction the culture data and support the hypothesis that S100B acts of the hypertrophic phenotype remains to be elucidated. both as an intrinsic negative regulator of hypertrophy and The function of the S100B/AHNAK interaction in smooth an apoptotic agent. Intracellular S100B may modulate the muscle cells is currently unknown. In the myocardium, apoptotic responses of postinfarct myocytes by activating S100B expression is transcriptionally controlled dependent a transcriptionally inducible form of nitric oxide synthase on positive (−782/−162 and −6,689/−4,463) and negative (iNOS) and production of nitric oxide (NO) [55]ashas been (4,463/−782) elements upstream of the transcription initi- described for astrocytes [35]. Forced expression of S100B ation site, selectively activated by α -adrenergic signaling may induce iNOS, NO production, and apoptosis. Thus 1A via PKCβ and inhibitory and stimulatory DNA binding by NO could be an intermediate pathway in the induction of transcription factors, TEF-1 and related RTEF-1, respectively apoptosis by intracellular S100B (Figure 1). Similar to S100B, [8] (Figure 1). This suggests that the same α -adrenergic S100A6 is upregulated in post-infarct myocardium and is 1 4 Cardiovascular Psychiatry and Neurology selectively induced by TNF-α and serves to limit myocyte subjects and experimental rodent models of myocardial apoptosis [56]. S100B colocalizes with S100A6 in cardiac infarction and in response to α -adrenergic stimulation. muscle [57], suggesting that heterodimerization may have S100B plays an important role in negative intrinsic regu- distinct phenotypic consequences. lation of aortic smooth muscle cell proliferation, cardiac myocyte hypertrophy, and, via RAGE ligation, apoptosis. The intracellular and extracellular roles of S100B are attractive 3.3. Extracellular S100B and Myocyte Apoptosis. Increasing therapeutic targets for the treatment of both cardiac and evidence suggests that S100B plays a role in the regulation vascular diseases. of apoptosis in post-MI myocardium by an extracellular mechanism after cellular release from damaged myocytes References and interaction with RAGE [58]. Exogenously administered S100B to neonatal rat cultures induced apoptosis in a dose- [1] R. Donato, G. Sorci, F. 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Intracellular and Extracellular Effects of S100B in the Cardiovascular Response to Disease

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Hindawi Publishing Corporation Cardiovascular Psychiatry and Neurology Volume 2010, Article ID 206073, 6 pages doi:10.1155/2010/206073 Review Article Intracellular and Extracellular Effects of S100B in the Cardiovascular Response to Disease James N. Tsoporis, Forough Mohammadzadeh, and Thomas G. Parker Division of Cardiology, Department of Medicine, Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada M5B 1W8 Correspondence should be addressed to Thomas G. Parker, parkertg@smh.toronto.on.ca Received 1 March 2010; Accepted 6 May 2010 Academic Editor: Rosario Donato Copyright © 2010 James N. Tsoporis et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. S100B, a calcium-binding protein of the EF-hand type, exerts both intracellular and extracellular functions. S100B is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. Forced expression of S100B in neonatal rat myocyte cultures and high level expression of S100B in transgenic mice hearts inhibit cardiac hypertrophy and the associated phenotype but augments myocyte apoptosis following myocardial infarction. By contrast, knocking out S100B, augments hypertrophy, decreases apoptosis and preserves cardiac function following myocardial infarction. Expression of S100B in aortic smooth muscle cells inhibits cell proliferation and the vascular response to adrenergic stimulation. S100B induces apoptosis by an extracellular mechanism via interaction with the receptor for advanced glycation end products and activating ERK1/2 and p53 signaling. The intracellular and extracellular roles of S100B are attractive therapeutic targets for the treatment of both cardiac and vascular diseases. 1. The Family of S100 Proteins their expression might be repressed in other cell types by negative regulatory factors which are controlled by envi- S100 proteins entail a multigenic family of calcium binding ronmental conditions. For instance induction of S100B in proteins of the EF-hand type (helix E-loop-helix F). These rat myocardium postinfarction [7] implies that transcription proteins are called S100 because of their solubility in regulation of these proteins is strongly controlled by negative a 100% -saturated solution with ammonium sulphate at and positive elements [8]. neutral pH. They are small acidic proteins, 10–12 KDa, and S100 proteins do not exhibit intrinsic catalytic activity. contain two distinct EF-hands, 4 α-helical segments, a central However, they are calcium sensor proteins and through hinge region of variable length, and the N- and C-terminal interaction with several intracellular effector proteins they contribute to the regulation of a broad range of functions variable domains. To date, 25 members of this family have been identified [1]. Of these, 21 family members (S100A1- such as contraction, motility, cell growth and differentiation, S100A18, trichohyalin, filagrin, and repetin) have genes cell cycle progression, organization of membrane-associated clustered on a 1.6-Mbp segment of human chromosome cytoskeleton elements, cell survival, apoptosis, protein phos- 1 (1q21) while other members are found at chromosome phorylation, and secretion [1, 3, 9]. In ordertomodulate loci 4q16 (S100P), 5q14 (S100Z), 21q22 (S100B), and Xp22 these types of activities, S100 proteins undergo conforma- (S100G) [2]. S100 proteins are widely expressed in a variety tional changes [10]. Upon calcium binding, the helices of of cell types and tissues. For example, S100A1 and S100A2 S100 proteins rearrange, revealing a hydrophobic cleft, which forms the target protein binding site [11]. Although target are found in the cytoplasm and nucleus, respectively, of smooth-muscle cells of skeletal muscle [3], S100P is located binding of S100 proteins is calcium-dependent, calcium in the cytoplasm of placental tissue [4, 5], and S100B in independent interactions have been reported [12]. Enzymes are the most common calcium independent target binding cytoplasm of astrocytes of nervous system [6]. However, 2 Cardiovascular Psychiatry and Neurology for the S100 proteins. For instance, S100B and S100A1 RAGE V C1 C2 bind with glycogen phosphorylase [13]. The most significant p53 Apoptosis ERK1/2 calcium-independent interactions of S100 proteins are their ability to bind to each other. Typically, they are homodimers, NO but heterodimerization adds to the complexity of this iNOS multiprotein family. Each subunit consists of two helix- S100B loop-helix motifs connected by a central linker or so-called ++ Ca hinge region. The C-terminal canonical EF-hand motif is S100B TEF-1 ++ composed of 12 amino acids, whereas the N-terminal S100- Ca specific EF-hand comprises 14 residues [3, 14]. ++ Ca NE TEF-1 Growing evidence indicates that in addition to intra- P (α -receptor) 1A cellular activities, some S100 proteins (e.g., S100B, S100A1, PKCβ S100A4, S100A8, and S100A9) exhibit extracellular functions [15]. However, secretion has been shown only for S100B, S100A8, and S100A9 [15]. The S100A8/A9 heterodimer is Figure 1: Schematic representation of proposed intracellular and secreted by a novel secretion pathway that depends on extracellular effects of S100B in cardiac myocytes. Norepinephrine an intact microtubule network and acts as a chemotactic (NE) activation of the calcium-dependent protein kinase C (PKC)- molecule in inflammation [16, 17]. The extracellular effects β, mediated by the α -adrenergic receptor, phosphorylates (P) of some S100 proteins require binding to the receptor transcriptional enhancer factor (1) TEF-1, resulting in DNA for advanced glycosylation end products (RAGE) [18–21]. binding and transactivation of the β-myosin heavy chain promoter. RAGE is a member of the immunoglobulin family of By contrast, S100B induction by NE and other hypertrophic cell surface molecules recognizing multiple ligands includ- signals (not shown) results in calcium-dependent block of PKC-β phosphorylation of TEF-1 and inhibition of β-MHC transcription. ing AGE, amphoterin, amyloid-β-peptide and β-fibrils, S100B can also induce apoptosis intracellularly via a inducible nitric S100A12, S100A6, and S100B [22]. The 45-kDa receptor oxide synthase (iNOS)-NO pathway or it can be secreted and via protein consists of 403 amino acids with an extracellular activation of the receptor for advanced glycation end products domain (1 variable and 2 constant Ig domains with disulfide (RAGE) (extracellular components V and CI), and induce apoptosis bridges), a single transmembrane region, and a short cytoso- via MEK-ERK1/2-p53 signaling. lic tail that triggers signal transduction [23]. RAGE ligands show selective binding to RAGE. S100B tetramer induces receptor dimerization by binding to RAGE [24]. S100B binds outgrowth; however, at micromolar concentrations it pro- to domains V and CI whereas the RAGE ligand S100A6 binds motes apoptosis [35, 36]. Such high extracellular levels are to domains CI and CII [23]. detected after brain injury or in neurodegenerative disorders like Down’s Syndrome, Alzheimer disease, or encephalitis [37, 38]. Both trophic and toxic effects of extracellular S100B 2. Noncardiovascular Actions of S100B are mediated in the brain by RAGE [36]. In addition to playing a major role in brain physiology [1], S100B has S100B is predominantly expressed in astrocytes, oligo- been implicated in cardiovascular development [39] and is dendrocytes, and schwann cells. S100B has intracellular considered a biochemical marker for brain injuries after and extracellular effects [1]. Intracellularly, S100B regulates bypass graft surgery [40] and dilated cardiomyopathy [41]. the cytoskeletal dynamics through disassembly of tubulin filaments, type III intermediate filaments [1], and bind- ing to fibrillary proteins such as CapZ [25] or inhibit- 3. Cardiovascular Actions of S100B ing GFAP phosphorylation when stimulated by cAMP or calcium/calmodulin [26]. S100B interacts in a calcium- 3.1. Intracellular S100B and Myocyte Hypertrophic Gene dependent manner with the cytoplasmic domain of myelin- Expression. The adult cardiac myocyte is terminally differen- associated glycoprotein and inhibits its phosphorylation by tiated and has lost the ability to proliferate. The myocardium protein kinase [27]. It is implicated in the phosphorylation therefore adapts to increasing workloads through hypertro- of tau protein [28], inhibition of Ndr kinase activity [29], phy of individual cells in response to hormonal, paracrine, inhibition of p53 phosphorylation [30], and regulation of and mechanical signals [42, 43]. This process is initially the activity of the GTPase Rac1 and Cdc effector, IQGAP compensatory but it can progress to irreversible enlargement [31]. S100B can also be secreted by a number of cell types and dilatation of the ventricle resulting in heart failure (e.g., astrocytes, glial cells) [32]. Astrocytes and glial cells [44]. Myocyte hypertrophy is accompanied by the down- secrete S100B, by a complex system involving alterations regulation of adult α-myosin heavy chain and a program of in intracellular calcium concentration [32]. S100B after fetal gene reexpression including the embryonic β-myosin secretion, or simply leakage from damaged cells, could heavy chain (MHC), the skeletal isoform of α actin (skACT), accumulate in the extracellular space and/or enter the blood and atrial natriuretic factor (ANF) [45, 46]. This response stream and cerebrospinal fluid [33, 34]. The action of S100B can be reproduced in vitro in cultured neonatal cardiac is strongly dependent on its extracellular concentration. myocytes by treatment with a number of trophic factors At nanomolar quantities, it has trophic effects on neurite including peptide growth factors and α -adrenergic agonists β-MHC promoter Cardiovascular Psychiatry and Neurology 3 [7]. Negative modulators of the hypertrophic response are pathway that initiates and sustains the hypertrophic response essential to maintain a balance between compensatory hyper- in cardiac myocytes by activating PKC signaling and which trophy and unchecked progression. Experimental evidence is subject to negative modulation by S100B also induces the suggests that S100B acts as an intrinsic negative regulator S100B gene. of the myocardial hypertrophic response [47–49]. S100B not normally expressed in the myocardium, is induced in 3.2. Intracellular S100B, Cardiovascular Hypertrophy and the peri-infarct region of the human heart after myocardial Apoptosis. To provide a physiologic model of S100B overex- infarction [47] and in rat heart commencing at day 7 pression effects, transgenic mice were created that contained following myocardial infarction as a result of experimental multiple copies of the human gene under the control of its coronary artery ligation [7]. In cultured neonatal rat cardiac own promoter. These animals demonstrate normal cardiac myocytes, transfection of an expression vector encoding the structure, and neuronal, but no basal cardiac expression human S100B protein inhibits the α -adrenergic induction of the transgene. In S100B transgenic mice, after chronic of the fetal genes β- MHC and the skACT [7]. The inhibition α -adrenergic agonist infusion, S100B is detected in the of α -adrenergic induction is selective as S100B does not heart and increased in the vasculature [49]. In addition, inhibit the capacity of thyroid hormone to induce α-myosin the myocyte hypertrophy and arterial smooth muscle cell heavy chain. To establish that S100B blocked α -adrenergic proliferation normally evoked in the heart and vasculature, induction of β -MHC and skACT by interrupting the PKC respectively, in response to α -adrenergic stimulation in signaling pathway, the interaction between forced S100B wild-type mice were abrogated in S100B transgenic mice expression and a constitutively activated mutant of PKCβ [49]. In knockout mice, α -adrenergic agonist infusion referred to as δPKCβ was tested [50]. δPKCβ transactivated provoked a potentiated myocyte hypertrophic response the β-MHC and skACT genes supporting the notion that the and augmented arterial smooth muscle cell proliferation. α -adrenergic induction of these genes is mediated by activa- Furthermore, in knockout mice, both the acute and chronic tion of the class-I PKC isoform β-PKC [7, 50]. Forced S100B increases in blood pressure in response to α -adrenergic expression could only block δPKCβ-induced transaction of agonist infusion were attenuated compared with wild-type β-MHC and skACT amidst concomitant treatment with an mice [49]. To determine whether this inhibition is general- α -adrenergic agonist or augmented extracellular calcium izable to other hypertrophic stimuli, transgenic and knock- suggesting that the capacity of S100B to modulate the out animals were subjected to descending aortic-banding hypertrophic phenotype is calcium dependent [7]. The tran- to produce pressure-overload. Aortic banding for 35 days scription factors TEF-1 (transcription factor-1) and related increased left ventricular (LV)/body weight (BW) ratio in TEF-1 (RTEF-1) upon phosphorylation by PKCβ bind to CD-1 controls (4.61 ± 0.06 g/kg versus 3.44 ± 0.16 g/kg MCAT elements on the skACT and β-MHC promoters and in sham operated, P< .05, n = 6) andproducedno activate transcription [51]. In cotransfection experiments, hypertrophy in S100B transgenic mice (3.37 ± 0.12 g/kg forced expression of S100B inhibited the activation of the versus 3.26 ± 0.11 g/kg in sham operated, P< .05, n = skACT and β-MHC promoters by overexpression of TEF- 8) and excessive hypertrophy in knock-out mice (5.12 ± 1 (unpublished observations). A direct interaction between 0.24 g/kg versus 3.19 ± 0.13 g/kg in sham operated, P< S100B and TEF-1 was demonstrated using a coimmuno- .05, n = 6). Similarly, thirty five days after experimental precipitation strategy (unpublished observations). These myocardial infarction, the S100B knockout mice mounted data suggest that S100B modulates the activation of the an augmented hypertrophic response compared to wild- fetal genes by direct binding to TEF-1. In addition to type mice [48]. Fetal gene expression was induced to a TEF-1, S100B interacts in a calcium-dependent manner greater magnitude in knockout mice compared to wild- with the giant phosphoprotein AHNAK/desmoyokin in type mice. The S100B transgenic mice did not develop cardiomyocytes and smooth muscle cells [49]. In cardiomy- the hypertrophic phenotype but demonstrated increased ocytes, AHNAK plays a role in cardiac calcium signaling apoptosis in the peri-infarct region compared to wild-type by modulating L-type calcium channels in response to β- and knockout mice. The postinfarct hypertrophic response adrenergic signaling [52, 53]. The S100B/AHNAK interac- in the myocardium is initiated by multiple trophic signals tion may participate in the S100B-mediated regulation of that include the state of local and systemic sympathetic cellular calcium homeostasis [53]. Whether there is any hyperactivity through α -adrenergic stimulation [54]. These relationship between S100B-mediated effects on calcium studies in S100B transgenic and knockout mice complement fluxes and S100B-dependent inhibition of the α -induction the culture data and support the hypothesis that S100B acts of the hypertrophic phenotype remains to be elucidated. both as an intrinsic negative regulator of hypertrophy and The function of the S100B/AHNAK interaction in smooth an apoptotic agent. Intracellular S100B may modulate the muscle cells is currently unknown. In the myocardium, apoptotic responses of postinfarct myocytes by activating S100B expression is transcriptionally controlled dependent a transcriptionally inducible form of nitric oxide synthase on positive (−782/−162 and −6,689/−4,463) and negative (iNOS) and production of nitric oxide (NO) [55]ashas been (4,463/−782) elements upstream of the transcription initi- described for astrocytes [35]. Forced expression of S100B ation site, selectively activated by α -adrenergic signaling may induce iNOS, NO production, and apoptosis. Thus 1A via PKCβ and inhibitory and stimulatory DNA binding by NO could be an intermediate pathway in the induction of transcription factors, TEF-1 and related RTEF-1, respectively apoptosis by intracellular S100B (Figure 1). Similar to S100B, [8] (Figure 1). This suggests that the same α -adrenergic S100A6 is upregulated in post-infarct myocardium and is 1 4 Cardiovascular Psychiatry and Neurology selectively induced by TNF-α and serves to limit myocyte subjects and experimental rodent models of myocardial apoptosis [56]. S100B colocalizes with S100A6 in cardiac infarction and in response to α -adrenergic stimulation. muscle [57], suggesting that heterodimerization may have S100B plays an important role in negative intrinsic regu- distinct phenotypic consequences. lation of aortic smooth muscle cell proliferation, cardiac myocyte hypertrophy, and, via RAGE ligation, apoptosis. The intracellular and extracellular roles of S100B are attractive 3.3. Extracellular S100B and Myocyte Apoptosis. Increasing therapeutic targets for the treatment of both cardiac and evidence suggests that S100B plays a role in the regulation vascular diseases. of apoptosis in post-MI myocardium by an extracellular mechanism after cellular release from damaged myocytes References and interaction with RAGE [58]. Exogenously administered S100B to neonatal rat cultures induced apoptosis in a dose- [1] R. Donato, G. Sorci, F. 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S100A1 knockout mice showed elevated systemic blood Proceedings of the National Academy of Sciences of the United pressure, reduced endothelium-dependent vasorelaxation, States of America, vol. 82, no. 20, pp. 7136–7139, 1985. and decreased survival after myocardial infarction [65, 66]. [7] J. N. Tsoporis, A. Marks, H. J. Kahn, et al., “S100β inhibits α1- Like our proposed mechanism for S100B release, S100A1 adrenergic induction of the hypertrophic phenotype in cardiac is released into the extracellular space in the setting of myocytes,” Journal of Biological Chemistry, vol. 272, no. 50, pp. myocardial injury and can bind RAGE [58]. Unlike S100B, 31915–31921, 1997. extracellular S100A1 inhibits apoptosis independent of [8] J. N. Tsoporis, A. Marks, L. J. Van Eldik, D. O’Hanlon, and T. G. 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