Protein O-GlcNAcylation: A critical regulator of the cellular response to stress

Abstract

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of chronic disease processes. However, there is increasing evidence that O-GlcNAc levels are increased in response to stress and that acute augmentation of this response is cytoprotective, at least in the short term. Conversely, a reduction in O-GlcNAc levels appears to be associated with decreased cell survival in response to an acute stress. Here we summarize our current understanding of protein O-GlcNAcylation on the cellular response to stress and in mediating cellular protective mechanisms focusing primarily on the cardiovascular system as an example. We consider the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis and explore the parallels between O-GlcNAc signaling and redox signaling. We also discuss the apparent paradox between the reported adverse effects of increased O-GlcNAcylation with its recently reported role in mediating cell survival mechanisms.

Introduction

The post-translational modification (PTM) of proteins is a common mechanism for regulating protein function, location and turnover. Protein phosphorylation is probably the most widely studied form of PTM; however there are many other PTMs, including acylation, ubiquitylation, methylation, acetylation, thiolation, nitration, and glycosylation [1]. The PTM of serine and threonine residues of nuclear and cytoplasmic proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc), first identified in 1984 [2], is increasing recognized as a highly dynamic PTM that contributes to the regulation of numerous biological processes, including nuclear transport, transcription and translation, cytoskeletal organization, signal transduction, proteasomal function and cell survival [3-14]. The majority of our understanding of the contribution of altered O-GlcNAcylation to pathophysiology is in the context of diseases such as aging [15-17], cancer [18-20], neurodegenerative disorders [6, 10, 21, 22] and diabetes and diabetic complications [23, 24]. Glucose metabolism via the hexosamine biosynthesis pathway (HBP) is essential for the synthesis of O-GlcNAc and there is considerable evidence supporting a role for sustained increases of O-GlcNAc in contributing to glucose toxicity and insulin resistance [23, 24].

O-GlcNAc modified proteins have been found in all metazoans studied thus far [25] and O-GlcNAc transferase (OGT), which catalyzes the final step in O-GlcNAc synthesis, has been identified in plants, human, rat, mouse, and the nematode Caenorhabditis elegans [26]. Given that the genes for OGT are highly conserved [27] and that OGT gene deletion is embryonically lethal [28], O-GlcNAc and the pathways involved in regulating O-GlcNAc levels must provide some evolutionary advantage to cells and organisms. Substantive evidence of this was first reported in 2004, when O-GlcNAc levels were shown to increase in response to a diverse array of stress stimuli, and inhibition of this response resulted in reduced cell survival [29]. Conversely, augmentation of O-GlcNAc levels conferred increased tolerance to stress. Subsequently, there have been a number of studies that have examined the role of this pathway in mediating the cell survival response particularly in the setting of the cardiovascular system. Thus, the goal of this review is to summarize our current understanding of the role of the HBP and O-GlcNAc on the regulation of cell function and survival in the cardiovascular system. In addition we will explore the apparent paradox between the beneficial pro-survival aspects of increased O-GlcNAc levels and the adverse effects associated with pathophysiological conditions such as diabetes.

Stress, the hexosamine biosynthesis pathway and O-GlcNAcylation

Cells sense stress primarily by two pathways: 1) damage to macromolecules and 2) changes in cellular redox potential, including an increase in reactive oxygen species (ROS) [30]. ROS frequently modify protein function by oxidation of susceptible cysteine residues [31]; within the glucose metabolic pathway, the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), is particularly sensitive to inhibition by ROS and it has been proposed to play a critical role as a sensor of intracellular oxidative stress [32]. GAPDH plays a regulatory role for the entry of glucose into glycolysis and oxidative phosphorylation; thus, its inhibition results in glucose units being channeled into other pathways, such as the pentose phosphate pathway and, of particular relevance here the HBP. Another common stress response is activation of glucose metabolizing pathways; at the cellular level this is reflected in increased glycogenolysis in response to an ischemic or hypoxic stress. At the organismal level this is seen as stress-induced hyperglycemic state, which occurs rapidly in response to severe injury including surgical trauma, hemorrhage, shock, sepsis and burns. In both cases this increase in glucose utilization is typically thought to be a valuable survival response, either by rapidly increasing available fuel for energy production or by facilitating mobilization of interstitial fluid reserves by increasing osmolarity [33-39]. However, the increased intracellular availability of glucose also provides additional substrate for the HBP, and if combined with ROS-mediated inhibition of GAPDH [40], would result in a further increase in flux through the HBP.

The HBP is estimated to consumes ~2-5% of the total glucose entering the cell [41]. It should be noted however, that this is based on a single study in cultured adipocytes, and a quantitative assessment of glucose flux through this pathway has yet to be determined in any intact organ system. The enzyme glutamine-fructose-6-phosphate transaminase (EC 2.6.1.16), also know as l-glutamine-d-fructose 6-phosphate amidotransferase (GFAT), regulates the entry of glucose into the HBP and converts fructose 6-phosphate to glucosamine 6-phosphate with glutamine as the amine donor [42]. Uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) is the primary end product of the HBP and is a substrate for multiple glycosylation reactions resulting in the synthesis of glycoproteins, glycolipids and proteoglycans. UDP-GlcNAc is also the essential sugar nucleotide donor for the synthesis of O-GlcNAc modified proteins catalyzed by a unique glycosyl transferase - UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase (OGT, O-GlcNAc transferase) [43, 44]. OGT is distinct from all other protein glycosyltransferases because it is exists in the nucleocytoplasmic compartment, not the secretory pathway, and it catalyzes the formation of a reversible attachment of GlcNAc via an O-linkage to specific serine and threonine residues on numerous nuclear and cytoplasmic protein [2, 45, 46].

Our understanding of the regulation of OGT activity is complex and incomplete; however, its activity is reported to be sensitive to changes in UDP-GlcNAc concentration [47]. Thus, a stress-induced increase in glucose metabolism via the HBP will lead to an increase in UDP-GlcNAc levels, which in turn will increase the flux through OGT and lead to an elevation of O-GlcNAc levels on nuclear and cytoplasmic proteins [48]. Hypoxic stress has also been shown to increase GFAT expression [49], which would potentially further increase glucose entry into the HBP. However, it should be noted that our understanding of the acute regulation of GFAT activity is remarkably limited. There two isoforms - GFAT1 and GFAT2 – and both are subject to phosphorylation by cAMP-dependent protein kinase. However, phosphorylation of GFAT1 decreases its activity [50], whereas phosphorylation of GFAT2 leads to increased activity [51]; the importance of the differential effects of phosphorylation on GFAT activity is unclear. Some studies indicate that GFAT expression and HBP flux in the heart are subject to regulation in response to chronic stress [17, 52]. Acute ischemic stress also increases UDP-GlcNAc [48, 53]; however, the relative importance of increased substrate availability compared to increased GFAT activity to overall HBP flux is unknown.

In addition to the HBP, GFAT, UDP-GlcNAc and OGT, the level of protein OGlcNAcylation is also regulated by a ß-N-acetylglucosaminidase (O-GlcNAcase; also known as meningioma expressed antigen 5 (mgea5) and nuclear cytoplasmic O-GlcNAcase and acetyltransferase (NCOAT)), which catalyzes the removal of the sugar moiety from the proteins [54, 55]. Like OGT, O-GlcNAcase is also highly conserved in mammals, and is expressed in all tissue types examined with a similar tissue distribution to OGT [55]. O-GlcNAcase activity is specific for N-acetyl-ß-D-glucosaminides and unlike lysosomal hexosaminidases has a pH optimum near neutral [54, 55]. The catalytic domain of O-GlcNAcase is in the N-terminus [56], while the C-terminus has been reported in vitro to have a histone acetyl-transferase activity [57]. Acute inhibition of O-GlcNAcase leads to relatively rapid increases in overall O-GlcNAc levels, which are associated with increased tolerance to stress; however, little is known about effects of ischemic or oxidative stress on O-GlcNAcase activity. It has been shown that O-GlcNAcase contains a caspase-3 cleavage site, and thus it may be regulated in response to apoptosis [58]; however, this may have other regulatory roles such as stabilization, localization, targeting, since enzyme activity is unaffected by cleavage [57-59].

Thus, while O-GlcNAcylation is essential for cell survival and activation of pathways leading to O-GlcNAc synthesis are associated with increased tolerance to stress, the mechanism(s) involved in regulating O-GlcNAc cycling have not been well defined. Furthermore, our knowledge regarding the regulation of GFAT, OGT and O-GlcNAcase activities under either normal physiological conditions or in response to stress remain surprisingly limited.

Hexosamine biosynthesis, O-GlcNAcylation and cell survival

Even though our understanding of the regulatory mechanisms involved in mediating O-GlcNAc levels is limited, there is a relatively extensive literature implicating increased O-GlcNAcylation as a mediator of the effects of a range of chronic disease states including insulin resistance, diabetes; cancer and neurodegenerative disorders, including Alzheimer's disease [6, 10, 18, 19, 21, 57, 60-70]. However, in 2004 it was demonstrated for the first time that activation of pathways leading to increased O-GlcNAc levels appeared to be an endogenous stress response that was linked to cell survival [29]. Since OGT catalytic activity is highly sensitive to changes in UDP-GlcNAc concentrations [47], an acute increase in UDP-GlcNAc concentrations should be an effective means for increasing O-GlcNAc levels. One approach to acutely increase UDP-GlcNAc levels is the addition of exogenous glucosamine, which is readily transported into cells via the glucose transporter system and subsequently phosphorylated to glucosamine-6-phosphate by hexokinase. Thus, glucosamine bypasses GFAT, which regulates glucose entry into the HBP, resulting in a rapid increase in UDP-GlcNAc levels [71].

Therefore, we postulated that acute pre-treatment of the isolated perfused heart with glucosamine, would lead to increased O-GlcNAc levels and that this would protect the heart against ischemic injury. Perfusion of an isolated rat heart with 5mM glucosamine resulted in a marked increase in O-GlcNAc levels in the heart within 10 min [72], and glucosamine treated hearts exhibited significantly improved functional recovery following ischemia reperfusion compared to untreated hearts [72]. Alloxan, which inhibits OGT [73], both prevented the increase in O-GlcNAc and blocked the protection afforded by glucosamine treatment [72]. While alloxan has significant limitations as an inhibitor of OGT [74], at the time of these studies no other inhibitors were available. Nevertheless, this was the first report that supported the notion that acutely activating the pathways leading to O-GlcNAc synthesis could be an effective cardioprotective strategy. At the cellular level the tolerance of cardiomyocytes to hypoxia and reoxygenation was also improved by increasing O-GlcNAc levels with glucosamine or by increasing glucose concentration [75]. Importantly, the protection observed with high glucose was prevented by inhibiting GFAT, as was the associated increase in O-GlcNAc, demonstrating the role of the HBP in mediating this response. Interestingly, in untreated cardiomyocytes, there was a marked increase in O-GlcNAc early during the reoxygenation period, which was abolished in the absence of glucose [75]. This lends further support to the importance of glucose metabolism via the HBP in mediating the stress induced increase in O-GlcNAc.

Another commonly used approach for increasing O-GlcNAc levels independent of the HBP is to prevent removal of O-GlcNAc from proteins by inhibiting O-GlcNAcase. PUGNAc [O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate] is an O-GlcNAc analog that is a competitive inhibitor of O-GlcNAcase and has been widely used to increase O-GlcNAc levels [76, 77]. PUGNAc treatment had a similar effect on improving cardiomyocyte viability and reducing necrosis as glucosamine treatment [75]. These cardiomyocyte studies confirmed the findings from the isolated heart, demonstrating that increasing O-GlcNAc levels before the ischemic or hypoxic stress enhanced the tolerance to that stress. However, cardioprotective treatment strategies that have been shown to be effective when used prior to ischemia often prove to be ineffective when used only during reperfusion [78]. Therefore, we examined the effectiveness of both glucosamine and PUGNAc in mediating functional recovery when they were administered only during reperfusion [79]. Following 20min of zero-flow ischemia hearts were reperfused for 60 min with no additional treatment or with the addition of glucosamine or PUGNAc for the first 20 min of reperfusion. Both glucosamine and PUGNAc significantly improved functional recovery and decreased tissue injury as indicated by lower cardiac troponin I (cTnI) release during reperfusion [79]. Of note there was a significant correlation between increased O-GlcNAc levels and lower cTnI release further supporting a link between O-GlcNAcylation and improved tolerance to ischemia reperfusion injury [79]. Furthermore the fact that both glucosamine and PUGNAc, which increase O-GlcNAc levels by entirely different mechanisms, provided similar protection against ischemic/hypoxic injury, lends credence to the notion that the increase in O-GlcNAcylation plays a causative role in mediating the protective response.

Jones et al. demonstrated that administration of PUGNAc in vivo significantly reduced infarct size following ischemia/reperfusion in mice [80]. This indicated that the protection associated with augmentation of O-GlcNAc levels was not limited to the in vitro models described above, but was also effective in more clinically relevant in vivo model of myocardial infarction. Interestingly they also reported that ischemic preconditioning, a widely studied and powerful protective strategy, was also a potent stimulus for increasing O-GlcNAcylation. They also demonstrated that PUGNAc and ischemic preconditioning reduced infarct size to a similar extent [80]. These observations raise the intriguing possibility that activation of O-GlcNAc formation may be one mechanism underlying ischemic preconditioning; however, this hypothesis has yet to tested.

Further evidence that in vivo augmentation of O-GlcNAc levels afforded protection was provided by Yang et al. [81], who demonstrated that administration of glucosamine during resuscitation following trauma-hemorrhage improved cardiac output 2-fold compared to vehicle-treated controls. They also found that glucosamine treatment improved perfusion of various organs systems, including kidney and brain, and attenuated the trauma-hemorrhage-induced increase in serum levels of the inflammatory cytokines IL-6 and TNF-α. Similarly, PUGNAc administration following trauma-hemorrhage also improved cardiac output, decreased total peripheral resistance and increased perfusion of critical organs systems compared to vehicle treated controls [82]. Similar to glucosamine, PUGNAc also attenuated the increase in plasma IL-6 and TNFα levels. Recent studies have demonstrated that both PUGNAc and glucosamine significantly increased 24 hr survival rates in a model of severe trauma hemorrhage and resuscitation. This was associated with decreased inflammatory cytokines and reduced liver injury, particularly in the PUGNAc treated group [83]. Both glucosamine and PUGNAc treatment significantly increased O-GlcNAc levels in multiple tissues compared to the untreated trauma-hemorrhage groups. Surprisingly, however, trauma-hemorrhage and resuscitation resulted in a marked loss of overall O-GlcNAcylation compared to sham surgery control group and treatment with glucosamine or PUGNAc appeared to prevent this loss of O-GlcNAcylation rather than increase levels above normal [82]. The trauma-hemorrhage and resuscitation induced loss of O-GlcNAcylation was present even 24hrs after resuscitation [83]. It is not known whether this reduction in overall O-GlcNAc levels is a result of decreased synthesis due to decreased flux through either GFAT or OGT or is a consequence of increased O-GlcNAcase activity resulting in increased degradation. These results highlight the need for a better understanding of mechanisms underlying the normal regulation of O-GlcNAc turnover and how stressors such as ischemia and reperfusion, hypoxia and reoxygenation or hemorrhagic shock and resuscitation affect the regulation of O-GlcNAc turnover.

The fact that both glucosamine and PUGNAc have similar protective effects at the cellular, isolated organ and whole animal level provide strong support for the fundamental concept that this protection is mediated via increased O-GlcNAcylation. However, glucosamine can modulate a number of pathways other than O-GlcNAc such as ganglioside [84] or cell surface N-glycan [85] synthesis and PUGNAc also inhibits other ß-hexosaminidases [86-88], which may alter processing of glycoconjugates in addition to O-GlcNAc. To provide a more definitive cause and effect relationship we used gain and loss of function studies in neonatal cardiomyocytes. We found that both glucosamine and OGT overexpression significantly increased O-GlcNAc levels and decreased hypoxia/reoxygenation injury to a similar extent [89]. Both interventions also attenuated the loss of mitochondrial membrane potential and the loss of mitochondrial cytochrome C induced by acute oxidative stress [89]. Conversely, decreased OGT expression attenuated the ischemia-induced increase in O-GlcNAc, and this was associated with increased necrosis, greater loss of cytochrome C and increased apoptosis following ischemia/reperfusion [89]. Others have also demonstrated that increasing or decreasing OGT levels increased or decreased cardiomyocyte survival respectively [90]. Lowering O-GlcNAc levels by overexpressing O-GlcNAcase also enhanced the sensitivity of cardiomyocytes to hypoxia/reoxygenation [91]. Taken together these studies demonstrate that strategies that augment O-GlcNAc levels are clearly pro-survival, whereas those that decrease O-GlcNAc levels reduce cell survival. In addition, it should be noted that a number of studies have reported potentially adverse effects of glucosamine including growth inhibition, cell cycle arrest and apoptosis, which may be related to effects other than increased O-GlcNAc levels [84, 92, 93]. However, in the setting of acute stress in cardiomyocytes we have typically found that glucosamine and OGT overexpression have remarkably similar protective effects [89, 94], suggesting that under such conditions the effect of glucosamine is mediated primarily via increased flux through OGT.

Although PUGNAc has been widely used pharmacological tool for increase O-GlcNAc levels via its inhibition of O-GlcNAcase. However, while PUGNAc, an O-GlcNAc analog. which is a potent competitive inhibitor of O-GlcNAcase, is not a selective O-GlcNAcase inhibitor (K_I=0.046 μM) as it also inhibits other glycoside hydrolases such as the lysosomal ß-hexosamindases (K_I=0.036 μM) and α-N-acetylglucosaminidases (K_I=6 μM) [88]. Such off target effects has the potential to complicate the interpretation of results obtained with PUGANc However, Macauley and coworkers [88], have recently developed novel O-GlcNAcase inhibitors, analogous derivatives of the parent NAG-thiazoline (1,2 dideoxy-2’-methyl-α-D-glycopyranoso-[2,1-d]-Δ2’-thiazoline), which have been shown to be good transition state mimics for the substrate-assisted catalytic mechanism used by O-GlcNAcase and equally effective but more selective inhibitors of eukaryotic O-GlcNAcase over the functionally related lysosomal ß-hexosamindases than PUGNAc. For example, NAG-Bt (1,2 dideoxy-2’-propyl-α-D-glycopyranoso-[2,1-d]-Δ2’-thiazoline) was found to be a potent O-GlcNAcase inhibitor (K_I=0.23 μM) and highly selective over the ß-hexosamindases (K_I=340 μM) with a ~1500-fold greater specificity than PUGNAc [88]. More recently van Aalten and colleagues have described other O-GlcNAcase inhibitors, “GlcNAcstatins”, which they have reported to be effective in the nanomolar range [95, 96]. The availability of such highly specific and effective O-GlcNAcase inhibitors will be especially valuable tools for improving our understanding of the role of O-GlcNAcylation in mediating cell function. On the other hand, while the more specific NAG-thiazolidine O-GlcNAcase inhibitor, NAG-Bt has been reported to have different effects of cell function than PUGNAc [97], in our studies we have found that PUGNAc and NAG-Bt to have similar protective effects against ischemia/reperfusion injury in both cardiomyocytes [75, 89] and the perfused heart [79, 98].

Mechanisms associated with O-GlcNAc mediated cell survival

A number of putative mechanisms have been proposed to account for the increased tolerance to stress and improved survival associated with increasing O-GlcNAcylation. Increased transcription of heat shock protein (HSP) 40 and HSP70 were associated with elevated O-GlcNAc levels, and HSP70 is known to be target for O-GlcNAc modification [99, 100]. Although we found no change in HSP70 expression in cardiomyocytes following glucosamine treatment [75], PUGNAc treatment resulted in augmented HSP70 levels in response to oxidative stress [80]. On the other hand Sohn et al., [101] found that O-GlcNAc mediated protection was not dependent on changes in HSP110, HSP90, HSP70 or HSP27 expression levels. The activity of the transcription factor Sp1 also increases when modified by O-GlcNAc [40, 102] and this may contribute to the increased synthesis of heat shock proteins [103] as well as pro-survival Bcl-2 family members [104].

However, in the isolated perfused heart, cardioprotection is conferred after a very brief (5 min) exposure to glucosamine [48, 72], suggesting that O-GlcNAc-related protection does not necessarily depends on de novo protein synthesis. Activation of O-GlcNAc formation attenuates the mPTP opening, a critical step in the initiation of apoptosis and cell death [80, 89, 90, 105]. Glucosamine, OGT overexpression and O-GlcNAcase inhibition all attenuate H₂O₂-induced loss of mitochondrial membrane potential and cyctochrome c release [89]. We found that this was associated with increased mitochondrial Bcl-2 levels; Bcl-2 inhibits the mitochondrial permeability transition pore (mPTP) opening possibly by direct interaction with VDAC (voltage dependant anion channel), one of the putative components of mPTP [106, 107]. Interestingly, cardiac mitochondria isolated from PUGNAc-treated mice and also OGT overexpressing neonatal rat cardiomyocytes are resistant to the Ca²⁺-induced mPTP formation, whereas inhibition of OGT increased sensitivity to Ca²⁺-induced mitochondrial swelling [80, 90, 105]. One of the O-GlcNAc modified proteins identified as a potential O-GlcNAc target is VDAC, and it has been hypothesized this modification of VDAC may preserve the mitochondrial integrity by interfering with mPTP formation [80].

Glucosamine-induced increase in O-GlcNAc levels also attenuated the ischemia-induced increase in p38 mitogen activated protein kinase (MAPK) phosphorylation, decreased ischemic contracture and reduced incidence of reperfusion-induced arrhythmias [48]. Paradoxically, at the end of reperfusion phosphorylated p38 levels were increased in response to glucosamine treatment. While p38 activation is frequently considered to be pro-apoptotic, it can also lead to activation of pro-survival pathways through downstream effectors, such as αB-crystallin and HSP27, both of which have been shown to play a role in ischemic protection [108-112] and are also targets for O-GlcNAc modification [10, 113, 114]. Preliminary studies have also shown that ischemia/reperfusion alters the level of O-GlcNAc modification of glycogen phosphorylase b, mitochondrial aconitase 2 and the cytoskeletal protein vinculin [53]. It remains to be determined whether changes in O-GlcNAcylation of these proteins alter the functional response to ischemia/reperfusion injury. Increased levels of O-GlcNAc have also been reported to inhibit protein degradation [5, 14], most likely due to inhibition of the proteasome [115], and this could contribute to enhanced cell survival.

Inflammation also plays an important role in the evolution of tissue injury in vivo, and a number of studies have demonstrated that acute increases in O-GlcNAc attenuate the inflammatory response induced by tissue injury and stress [81-83, 116-118]. Glucosamine and PUGNAc both decreased serum levels of the inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 following trauma-hemorrhage and resuscitation [81, 82]. Glucosamine treatment also significantly reduced the trauma-hemorrhage-induced increase in Inter-Cellular Adhesion Molecule (ICAM)-1 expression, IκB-α (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) phosphorylation, Nuclear factor-kB (NF-κB) expression and NF-κB DNA binding activity in the heart [116], suggesting that elevated O-GlcNAc levels may contribute to the down-regulation of the NF-κB pathway. Glucosamine-induced increase in O-GlcNAc levels in rat aortic smooth muscle cells was shown to inhibit tumor necrosis factor TNF-α stimulated chemokine and adhesion molecule (ICAM-1, vascular cell adhesion molecule (VCAM)-1) expression, IκB-α phosphorylation and NF-κB activation [117]. In isolated cardiomyocytes, glucosamine and OGT overexpression increased O-GlcNAc levels, attenuated lipopolysaccharide (LPS)-induced TNF-α and ICAM-1 expression and decreased IκB-α phosphorylation and nuclear NF-κB levels [119]. Conversely, knockdown of OGT decreased O-GlcNAc levels and enhanced the LPS-induced increase in IκB-α phosphorylation [119]. Glucosamine also increased O-GlcNAc levels in macrophages and attenuated NF-κB activation and iNOS expression[119]. Together, these results demonstrate that modulation of cellular O-GlcNAc signaling events alters NF-κB activation, indicating therefore that attenuation of NF-κB signaling may be a contributing factor to the protection associated with increased protein O-GlcNAc levels in vivo.

Thus, it is becoming increasingly evident that the protection associated with activation O-GlcNAcylation is multifactorial. Initial acute protection will likely involve transcriptionally independent mechanisms resulting from direct O-GlcNAc modification of proteins leading to changes in protein-protein interactions, modulation of stress-activated kinase pathways, activation of pro-survival pathways or inhibition of pro-apoptotic pathways. Longer-term cellular protection may be facilitated via increased transcription of pro-survival factors such as HSPs mediated in part by increased O-GlcNAcylation of transcription factors such as Sp1. The O-GlcNAc mediated attenuation of NF-κB signaling, indicates that augmentation of O-GlcNAc levels in vivo, may exert a protective effect not only at the level of the heart and other parenchymal tissues but also by attenuation of inflammatory responses in circulating mediators such as macrophages.

O-GlcNAcylation and calcium homeostasis

Maintenance of calcium homeostasis is critical for both normal cell signaling as well as for cell survival. When the concentration of cytoplasmic free Ca²⁺ ([Ca²⁺]_i) is excessive, Ca²⁺ overload occurs and cell death rapidly ensue. Thus, primary adaptations that likely evolved in response to stress are mechanisms designed to suppress Ca²⁺ overload. There are two general mechanisms that regulate cellular calcium homeostasis: voltage gated and non-voltage gated calcium entry. Voltage gated calcium channels are characteristic of excitable cells such as cardiomyocytes where they are activated by membrane depolarization, which leads to an increase in [Ca²⁺]_i which in the case of cardiomyocytes is responsible for muscle contraction. Non-voltage gated calcium channels are more commonly associated with non-excitable cells and are typically associated with agonist induced increase in [Ca²⁺]_I leading to the activation of a wide range of calcium-mediated signaling pathways. One such non-voltage gated calcium entry pathway is capacitative calcium entry (CCE), which refers to a Ca²⁺ influx pathway mediated by store-operated calcium channels (SOCs). Typically CCE is activated when endoplasmic (ER) or sarcoplasmic reticulum (SR) Ca²⁺ stores are depleted, usually by agonists leading to the activation of phospholipase C (PLC) and the generation of inositol-1,4,5-trisphosphate (IP₃) [120].

CCE characterizes all non-excitable cells except erythrocytes [121], and has been shown to co-exist with the voltage-gated Ca²⁺ channels in smooth [122] and skeletal muscle cells [123-127]. Although the presence of CCE in the heart is under explored, there has been a gradual accretion of data from us [128-130] and more recently other investigators [131, 132] clearly demonstrating the presence of CCE in cardiomyocytes. The development of links between the HBP and CCE has also evolved over the last decade, beginning with studies of CCE in response to IP₃-generating agonists in excitable cells. In 1995, Rivera et al. [133] were among the first groups to describe a CCE pathway in smooth muscle cells. Of particular relevance, in that study a marked diminution of CCE following exposure to hyperglycemia was observed; however, the specific metabolic pathway responsible was not described. Subsequently, Vemuri and Marchase [134] showed that glucosamine inhibited CCE in the J774 macrophage cell line without affecting ATP levels. As the primary pathway of glucosamine metabolism is via the HBP, this study provided the first evidence that CCE may be regulated by HBP activity and thus by extension O-GlcNAc levels.

The first report that CCE was an important attribute in mediating cardiomyocyte function was from Hunton et al. in 2002 [128]. They showed that neonatal cardiomyocytes exhibited an increase in [Ca²⁺]_i that was dependent on endoplasmic reticulum (ER) store depletion, which was blocked by agents known to inhibit CCE in other cells, such as SKF96365 but not by inhibitors of voltage-gated channels. Treatment of neonatal cardiomyocytes with IP₃ generating agonists, such as phenylephrine and angiotensin II, lead to a release of calcium from the SR and also caused [Ca²⁺]_i to increase via a CCE-like pathway. In addition, CCE inhibitors also negated the nuclear localization of nuclear factor of activated T-cells (NFAT), which occurs in response to both phenylephrine and angiotensin II. NFAT is a transcription factor critical in regulating cardiomyocyte hypertrophy; thus, CCE inhibitors also blunted the hypertrophic response of phenylephrine and angiotensin II. Of particular relevance to this discussion, all of these responses to CCE activation were rapidly and reversibly inhibited by both glucosamine [128] and hyperglycemia [130]; importantly, the inhibition of CCE-mediated responses by hyperglycemia was blocked by inhibition of the HBP [130].

Nagy et al. subsequently demonstrated that the effects of glucosamine on CCE in cardiomyocytes were a consequence of increased O-GlcNAc levels and that the O-GlcNAcase inhibitor PUGNAc mimicked the effects of glucosamine and attenuated CCE [135]. Subsequent investigations also demonstrated that CCE was not only present in adult cardiomyocytes, but also that it was inhibited by glucosamine [129]. Additional studies in the isolated perfused heart suggested that CCE contributed to the inotropic effects of phenylephrine and angiotensin II, which were also attenuate by glucosamine as well as an independent inhibitor of CCE, SKF96365 [136]. Taken together these studies demonstrated not only a role for CCE in mediating cardiomyocyte function, but also for the regulation of CCE by the HBP and O-GlcNAcylation. While there are multiple potential calcium entry mechanisms, which could contribute to calcium overload, the fact that increased O-GlcNAcylation specifically attenuates CCE led us to postulate that CCE may be an important mediator of stress-induced Ca²⁺ overload. This is supported by our observations that activation of the HBP and increased O-GlcNAc levels not only attenuates CCE in cardiomyocytes but also protects the isolated perfused heart against Ca²⁺ overload induced by the calcium paradox [72], and blunts ischemia/reperfusion-induced activation of Ca²⁺-mediated proteases[79]. This is further supported by reports demonstrating that in arterial myocytes hypoxia leads to store operated depletion and activates CCE [137-139]. Thus, one mechanism of protection associated with increased O-GlcNAc levels could be attenuation of the calcium overload mediated via an inhibition of CCE.

A major limitation underlying mechanistic studies into the regulation of CCE has been the lack of channel proteins responsible for mediating this process; however, there is a growing consensus that stromal interacting molecule (STIM)1 and Orai1 are essential for activation of CCE [127, 140, 141]. STIM1 contains an EF hand Ca²⁺-binding domain that serves as the sensor of Ca²⁺ within the ER or SR. When the intra-ER Ca²⁺ level drops, rapid clustering and translocation to ER near the plasma membrane takes place, where it interacts with of the components of the CCE channel including Orai1 leading to opening of the channel. In support of our notion that this process may be regulated by O-GlcNAc, STIM1 contains a proline-serine rich domain in its cytoplasmic tail that is not only appears to be a prime site for phosphorylation but also shares characteristics with the prime acceptor site recognized by OGT for the addition of O-GlcNAc [25]. Significantly, deletion of this same serine-proline rich region prevented the ability of STIM1 to activate Ca²⁺ influx [141]. It is also of note that in preliminary experiments we have found for the first time that both STIM1 and Orai1 proteins are present in cardiomyocytes (Chatham and Marchase, unpublished data). Thus we hypothesize that an increase in cellular O-GlcNAc levels leads to increased O-GlcNAcylation of the cytoplasmic domain of STIM1, thereby preventing the necessary clustering of STIM1 and blocking the interaction with Orai 1. This prevents the formation of channels required for CCE resulting in the diminution of calcium influx seen in response to IP₃-generating agonists as well as in response to stresses such as ischemia or the calcium paradox that otherwise results in calcium overload and cell damage.

Interaction between redox and O-GlcNAc signaling

It is well established that numerous stress stimuli lead to an increase in intracellular levels of reactive oxygen species (ROS) primarily as a result of increased ROS production from mitochondria [142]. Furthermore, it has been shown that inhibition of glyceraldehyde phosphate dehydrogenase (GAPDH) by mitochondrial superoxide was an important factor leading to the increase in O-GlcNAcylation associated with hyperglycemia [40, 65]. It is also noteworthy that in the original study where O-GlcNAcylation was shown to mediate cell survival [29], the stresses used to increase O-GlcNAc levels, such as ultraviolet (UV) light cobalt chloride, ethanol, and heat shock, are all associated with increased ROS production. We also found that there was a rapid, but transient increase in O-GlcNAc levels following reoxygenation of hypoxic cardiomyocytes, which was glucose dependent [75]. Further, recent studies have shown that augmentation of O-GlcNAc levels in cardiomyocytes attenuates hydrogen peroxide-induced loss of mitochondrial membrane potential [80, 89, 90] and that this may be due to O-GlcNAcylation of mitochondrial proteins such as VDAC [80, 90]. Taken together, these studies suggest that ROS and mitochondria may contribute to the regulation of O-GlcNAc synthesis, which could in turn modulate the response to oxidative stress. It is unclear however, whether, in response to acute stress the increase in O-GlcNAc levels is an indirect consequence of ROS mediated inhibition of GAPDH [40, 65], or whether the activities of GFAT, OGT, and O-GlcNAcase may directly modified in response to ROS.

Another example of the convergence of redox and O-GlcNAc signaling is in the modulation of nitric oxide (NO). NO is widely recognized as central reactive species involved in redox signaling, and nitric oxide synthase (NOS) is subject to both phosphorylation and O-GlcNAcylation. Endothelial NOS (eNOS) is activated by Akt-mediated phosphorylation, thereby increasing NO production. This Akt phosphorylation site (Ser1177) is also subject to O-GlcNAcylation, leading to lower activity and decreased NO production [65]. Thus, changes in O-GlcNAc levels can influence eNOS activity both via direct O-GlcNAc modification as well as indirectly via O-GlcNAcylation of its upstream kinase Akt. This also leads to the notion that the interaction of redox and O-GlcNAc mediating signaling may contribute to the regulation of an entire signaling pathway. For example, insulin signaling is regulated by NO the level of which, as noted above, can be modulated by changes O-GlcNAcylation. In addition a number of components of the insulin signaling pathway, such as IR-ß, IRS1/2, PI3K, Akt, and GSK3ß are all potential targets for O-GlcNAcylation, resulting in reduced activity and decreased insulin response [143].

The recent report demonstrating O-GlcNAcylation of the transcription factor FoxO1 [144], which functions as a nutrient and stress sensor, is yet another example of the intersection between redox and O-GlcNAc signaling. One of the consequence of FoxO1 activation is increased transcription of oxidative stress response enzymes such as catalase and MnSOD [144]. Thus, an ROS induced increase in O-GlcNAc formation, could lead to O-GlcNAc-induced activation of FoxO1 thereby increasing the levels of endogenous antioxidant proteins. Taken together these studies support the notion of a role of O-GlcNAc in modulating redox signaling as well as for ROS mediated regulation of O-GlcNAc signaling.

The paradox of bad versus good O-GlcNAcylation

The majority of literature regarding the pathophysiological role of O-GlcNAcylation is as a mediating factor in various chronic disease states, including cancer, neurodegenerative diseases and diabetes [6, 10, 15-24]. These adverse effects of increased O-GlcNAcylation would appear to be at odds with the recent literature demonstrating a cytoprotective effect. One possible explanation for the apparent paradox between the adverse and beneficial effects of increased O-GlcNAcylation could be that the protection is observed in response to acute increases in O-GlcNAc levels, whereas adverse effects are seen in response to chronic activation. For example, a short-term increase in overall O-GlcNAcylation may result in different target proteins being modified compared to those that are O-GlcNAcylated in response to a sustained increase in flux through OGT. However, to date there is no information regarding the temporal relationship between increased OGT flux and target protein specificity. It is also possible that in chronic pathophysiological conditions, there are changes in the activity or expression levels of GFAT, OGT and O-GlcNAcase, which result in an impaired or blunted response to otherwise normal physiological or stress stimuli.

Another explanation could be provided by the concept of allostasis, which suggests that the initial biological response to an acute stress is the activation of processes that are protective and improve survival. However, as the allostatic load increases and the stress becomes more frequent or continuous, activation of the same pathways results in the development of pathophysiology [145]. Thus, if increased O-GlcNAcylation is an acute survival response, the development of pathophysiology may be a consequence of chronic activation as seen in metabolic disease. Furthermore, as described by Zachara and Hart [4] an excessive elevation of O-GlcNAc may induce apoptosis; consequently, a stress induced increase in O-GlcNAc on top of an already elevated level of O-GlcNAc may lead to cell death.

Alternatively, it is possible that the effects of O-GlcNAc modification of the same pathway could contribute to both the adverse and beneficial effects of increased O-GlcNAc synthesis. As discussed above, there is considerable evidence supporting a role for O-GlcNAc in regulating Ca²⁺ entry into the cell via CCE, specifically, for elevated O-GlcNAc levels to inhibit the activation of CCE. CCE is known to play a critical role in regulating numerous Ca²⁺ mediated cell signaling pathways, particularly in non-excitable cells, but also more recently in excitable cells including cardiomyocytes [128, 130]. Sustained inhibition of these Ca²⁺ mediated cell signaling pathways, as might occur in the setting of a chronic increase in O-GlcNAc, could blunt the response to normal physiological stimuli, which would likely contribute to impaired cellular function. However, our data also suggest that O-GlcNAc mediated inhibition of CCE also attenuates Ca²⁺ overload, which plays an important role in protection associated with ischemia/reperfusion injury. Of note, we have shown that hearts from diabetic animals were resistant to Ca²⁺-overload [72] and also exhibited a blunted response to a-adrenergic stimulation[136]; both of which were reversed by acute inhibition of the HBP. However, in the same studies, we also showed that acute activation of the HBP with glucosamine also protected against Ca²⁺ overload and blunted α-adrenergic stimulation [72, 136]. Taken together these observations suggest that both the acute protection and adverse effects associated with increased O-GlcNAcylation may be mediated, at least in part, by inhibition of the same Ca²⁺ entry pathways.

Conclusion

It took approximately 20 years from the initial identification of O-GlcNAc modified proteins [2] until its beneficial role in mediating cell survival was clearly recognized [4]. It is becoming increasingly evident that O-GlcNAc modification of proteins is a metabolically modulated signaling pathway that regulates cell function and plays a particularly critical role in mediating the response of cells to stress. There is now an emerging consensus, at least in the cardiovascular system, that acute increases in O-GlcNAc synthesis or inhibition of O-GlcNAc degradation affords remarkable cardioprotection against oxidative stress including ischemia/reperfusion; however, the specific mechanisms underlying the protection associated with increased O-GlcNAc levels remains elusive. It should emphasized that it remains to be determined whether the protection against oxidative stress and ischemia/reperfusion injury, associated with short-term increases in O-GlcNAcylation is broadly applicable or whether it is specific to the heart.

In some biological systems, such as the heart, O-GlcNAc levels respond rapidly (i.e. within minutes) to activation of the HBP or inhibition of O-GlcNAcase [72, 79]. This suggests that the O-GlcNAc “on/off” cycle is a highly dynamic process that likely contributes to acute regulation of cell function in response to both physiological and pathophysiological stimuli. However, our knowledge regarding the regulation of O-GlcNAc turnover is remarkably limited. The increase in O-GlcNAc that occurs in response to stress such as ischemia could be due to a substrate driven increase in flux through the HBP, increased GFAT activity, or both. Alternatively, given the presence of such an active O-GlcNAc cycle, ischemia could indirectly or directly modulate the O-GlcNAc enzymes, such that activation of OGT and/or inhibition of O-GlcNAcase could lead to a rapid increase in O-GlcNAc levels independent of changes in the HBP.

At the cellular level gain and loss of function studies further demonstrate that OGT and O-GlcNAcase play critical role in regulating the response to acute oxidative stress [89-91]. At the transcriptional level, protection appears to be a result, at least in part of increased heat shock proteins synthesis [29]. On the other hand, the fact that protection can be afforded with very brief pre-treatment protocols, strongly suggests that transcriptionally independent mechanisms also play an important role. Potential mechanisms include activation of pro-survival kinase pathways [48], increased O-GlcNAcylation of small heat shock proteins or preventing loss of mitochondrial membrane potential [80, 89]. However, we also propose that O-GlcNAc mediated inhibition of CCE attenuates Ca²⁺ overload, may represent a key mechanism underlying the acute protective response associated with increased O-GlcNAcylation. The recent identification of key proteins responsible for mediating CCE, namely STIM1 and Orai1, provide a foundation for testing this hypothesis.

The potential interaction between O-GlcNAc signaling, ROS and mitochondrial function raises the possibility that O-GlcNAc signaling contribute to intracellular redox sensing and conversely that ROS may be involved in regulating cellular O-GlcNAc levels. Regardless, protein O-GlcNAcylation is clearly an integral component of complex signaling networks responsible for the regulation of cellular response to both physiological and pathophysiological stimuli, thereby playing a critical role in mediating cell survival. However, despite our extensive and growing knowledge regarding the influence of O-GlcNAc signaling on cellular function, our knowledge of the factors involved in regulating O-GlcNAc turnover remain substantially under developed. Thus, as we continue to evolve our understanding of role of O-GlcNAcylation in mediating the response to stress, additional efforts are required to better elucidate the mechanisms involved in regulating the activity and transcription of the proteins responsible for controlling O-GlcNAc synthesis and degradation.