O-GlcNAcase Expression Research Articles

Evidence of a compensatory regulation of colonic O-GlcNAc transferase and O-GlcNAcase expression in response to disruption of O-GlcNAc homeostasis

O-GlcNAcylation is a post-translational modification of thousands of intracellular proteins that dynamically regulates many fundamental cellular processes. Cellular O-GlcNAcylation levels are regulated by a unique couple of enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which adds and removes the GlcNAc residue, respectively. Maintenance of O-GlcNAc homeostasis is essential to ensure optimal cellular function and disruption of this homeostasis has been linked to the etiology of several human diseases including cancer. The mechanisms through which the cell maintains O-GlcNAc homeostasis are not fully understood but several studies have suggested that a reciprocal regulation of OGT and OGA expression could be one of them. In this study, we investigated the putative regulation of OGT and OGA expression in response to disruption in O-GlcNAc homeostasis in colon. We provide in vitro and in vivo evidences that in colon cells, modulation of O-GlcNAcylation levels leads to a compensatory regulation of OGT and OGA expression in an attempt to restore basal O-GlcNAcylation levels. Our results also suggests that the regulation of colonic OGA expression in response to changes in O-GlcNAc homeostasis occurs mostly at the transcriptional level whereas OGT regulation seems to rely mainly on post-transcriptional mechanisms.

E2f1 deletion attenuates infarct-induced ventricular remodeling without affecting O-GlcNAcylation.

Several post-translational modifications figure prominently in ventricular remodeling. The beta-O-linkage of N-acetylglucosamine (O-GlcNAc) to proteins has emerged as an important signal in the cardiovascular system. Although there are limited insights about the regulation of the biosynthetic pathway that gives rise to the O-GlcNAc post-translational modification, much remains to be elucidated regarding the enzymes, such as O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which regulate the presence/absence of O-GlcNAcylation. Recently, we showed that the transcription factor, E2F1, could negatively regulate OGT and OGA expression in vitro. The present study sought to determine whether E2f1 deletion would improve post-infarct ventricular function by de-repressing expression of OGT and OGA. Male and female mice were subjected to non-reperfused myocardial infarction (MI) and followed for 1 or 4week. MI significantly increased E2F1 expression. Deletion of E2f1 alone was not sufficient to alter OGT or OGA expression in a naïve setting. Cardiac dysfunction was significantly attenuated at 1-week post-MI in E2f1-ablated mice. During chronic heart failure, E2f1 deletion also attenuated cardiac dysfunction. Despite the improvement in function, OGT and OGA expression was not normalized and protein O-GlcNAcyltion was not changed at 1-week post-MI. OGA expression was significantly upregulated at 4-week post-MI but overall protein O-GlcNAcylation was not changed. As an alternative explanation, we also performed guided transcriptional profiling of predicted targets of E2F1, which indicated potential differences in cardiac metabolism, angiogenesis, and apoptosis. E2f1 ablation increased heart size and preserved remote zone capillary density at 1-week post-MI. During chronic heart failure, cardiomyocytes in the remote zone of E2f1-deleted hearts were larger than wildtype. These data indicate that, overall, E2f1 exerts a deleterious effect on ventricular remodeling. Thus, E2f1 deletion improves ventricular remodeling with limited impact on enzymes regulating O-GlcNAcylation.

O-GlcNacylation Links TxNIP to Inflammasome Activation in Pancreatic β Cells.

Thioredoxin interacting protein (TxNIP), which strongly responds to glucose, has emerged as a central mediator of glucotoxicity in pancreatic β cells. TxNIP is a scaffold protein interacting with target proteins to inhibit or stimulate their activity. Recent studies reported that high glucose stimulates the interaction of TxNIP with the inflammasome protein NLRP3 (NLR family, pyrin domain containing 3) to increase interleukin-1 β (IL1β) secretion by pancreatic β cells. To better understand the regulation of TxNIP by glucose in pancreatic β cells, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAcylation) in regulating TxNIP at the posttranslational level. O-GlcNAcylation of proteins is controlled by two enzymes: the O-GlcNAc transferase (OGT), which transfers a monosaccharide to serine/threonine residues on target proteins, and the O-GlcNAcase (OGA), which removes it. Our study shows that TxNIP is subjected to O-GlcNAcylation in response to high glucose concentrations in β cell lines. Modification of the O-GlcNAcylation pathway through manipulation of OGT or OGA expression or activity significantly modulates TxNIP O-GlcNAcylation in INS1 832/13 cells. Interestingly, expression and O-GlcNAcylation of TxNIP appeared to be increased in islets of diabetic rodents. At the mechanistic level, the induction of the O-GlcNAcylation pathway in human and rat islets promotes inflammasome activation as evidenced by enhanced cleaved IL1β. Overexpression of OGT in HEK293 or INS1 832/13 cells stimulates TxNIP and NLRP3 interaction, while reducing TxNIP O-GlcNAcylation through OGA overexpression destabilizes this interaction. Altogether, our study reveals that O-GlcNAcylation represents an important regulatory mechanism for TxNIP activity in β cells.

Hyperglycemia-Associated Dysregulation of O-GlcNAcylation and HIF1A Reduces Anticancer Action of Metformin in Ovarian Cancer Cells (SKOV-3).

Although cancer cells need more glucose than normal cells to maintain energy demand, chronic hyperglycemia induces metabolic alteration that may dysregulate signaling pathways, including the O-GlcNAcylation and HIF1A (Hypoxia-inducible factor 1-alpha) pathways. Metformin was demonstrated to evoke metabolic stress and induce cancer cell death. The aim of this study was to determine the cytotoxic efficiency of metformin on SKOV-3 cells cultured in hyperglycemia and normoglycemia. To identify the potential mechanism, we assessed the expression of O-linked β-N-acetlyglucosamine transferase (OGT) and glycoside hydrolase O-GlcNAcase (OGA), as well as hypoxia-inducible factor 1-alpha (HIF1A) and glucose transporters (GLUT1, GLUT3). SKOV-3 cells were cultured in normoglycaemia (NG, 5 mM) and hyperglycemia (HG, 25 mM) with and without 10 mM metformin for 24, 48, and 72 h. The proliferation rate, apoptotic and necrotic SKOV-3 cell death were evaluated. Real-Time qPCR was employed to determine mRNA expression of OGT, OGA, GLUT1, GLUT3, and HIF1A. Metformin significantly reduced the proliferation of SKOV-3 cells under normal glucose conditions. Whereas, the efficacy of metformin to induce SKOV-3 cell death was reduced in hyperglycemia. Both hyperglycemia and metformin induced changes in the expression of genes involved in the O-GlcNAcylation status and HIF1A pathway. The obtained results suggest that dysregulation of O-GlcNAcylation, and the related HIF1A pathway, via hyperglycemia, is responsible for the decreased cytotoxic efficiency of metformin in human ovarian cancer cells.

Evaluation of a PET Radioligand to Image O-GlcNAcase in Brain and Periphery of Rhesus Monkey and Knock-Out Mouse.

Accumulation of hyperphosphorylated tau, a microtubule-associated protein, plays an important role in the progression of Alzheimer disease. Animal studies suggest that one strategy for treating Alzheimer disease and related tauopathies may be inhibition of O-GlcNAcase (OGA), which may subsequently decrease pathologic tau phosphorylation. Here, we report the pharmacokinetics of a novel PET radioligand, 18F-LSN3316612, which binds with high affinity and selectivity to OGA. Methods: PET imaging was performed on rhesus monkeys at baseline and after administration of either thiamet-G, a potent OGA inhibitor, or nonradioactive LSN3316612. The density of the enzyme was calculated as distribution volume using a 2-tissue-compartment model and serial concentrations of parent radioligand in arterial plasma. The radiation burden for future studies was based on whole-body imaging of monkeys. Oga∆Br, a mouse brain-specific knockout of Oga, was also scanned to assess the specificity of the radioligand for its target enzyme. Results: Uptake of radioactivity in monkey brain was high (∼5 SUV) and followed by slow washout. The highest uptake was in the amygdala, followed by striatum and hippocampus. Pretreatment with thiamet-G or nonradioactive LSN3316612 reduced brain uptake to a low and uniform concentration in all regions, corresponding to an approximately 90% decrease in distribution volume. Whole-body imaging of rhesus monkeys showed high uptake in kidney, spleen, liver, and testes. In Oga∆Br mice, brain uptake of 18F-LSN3316612 was reduced by 82% compared with control mice. Peripheral organs were unaffected in Oga∆Br mice, consistent with loss of OGA expression exclusively in the brain. The effective dose of 18F-LSN3316612 in humans was calculated to be 22 μSv/MBq, which is typical for 18F-labeled radioligands. Conclusion: These results show that 18F-LSN3316612 is an excellent radioligand for imaging and quantifying OGA in rhesus monkeys and mice. On the basis of these data, 18F-LSN3316612 merits evaluation in humans.

Transcriptional regulation of O-GlcNAc homeostasis is disrupted in pancreatic cancer

Many intracellular proteins are reversibly modified by O-linked GlcNAc (O-GlcNAc), a post-translational modification that dynamically regulates fundamental cellular processes in response to diverse environmental cues. Accumulating evidence indicates that both excess and deficiency of protein O-GlcNAcylation can have deleterious effects on the cell, suggesting that maintenance of O-GlcNAc homeostasis is essential for proper cellular function. However, the mechanisms through which O-GlcNAc homeostasis is maintained in the physiologic state and altered in the disease state have not yet been investigated. Here, we demonstrate the existence of a homeostatic mechanism involving mutual regulation of the O-GlcNAc-cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) at the transcriptional level. Specifically, we found that OGA promotes Ogt transcription through cooperation with the histone acetyltransferase p300 and transcription factor CCAAT/enhancer-binding protein β (C/EBPβ). To examine the role of mutual regulation of OGT and OGA in the disease state, we analyzed gene expression data from human cancer data sets, which revealed that OGT and OGA expression levels are highly correlated in numerous human cancers, particularly in pancreatic adenocarcinoma. Using a KrasG12D -driven primary mouse pancreatic ductal adenocarcinoma (PDAC) cell line, we found that inhibition of extracellular signal-regulated kinase (ERK) signaling decreases OGA glycosidase activity and reduces OGT mRNA and protein levels, suggesting that ERK signaling may alter O-GlcNAc homeostasis in PDAC by modulating OGA-mediated Ogt transcription. Our study elucidates a transcriptional mechanism that regulates cellular O-GlcNAc homeostasis, which may lay a foundation for exploring O-GlcNAc signaling as a therapeutic target for human disease.

F199. O-GLCNAC DYSREGULATION IN SCHIZOPHRENIA CORTEX

BackgroundO-linked β-linked N-acetylglucosamine (O-GlcNAc) is a post-translational glycosylation modification with ubiquitous functions in cell biology. The attachment of GlcNAc to serine or threonine (S/T) residues transiently adorns thousands of nuclear, cytosolic, and mitochondrial proteins and modulates protein function and localization via dynamic cooperation with kinase and phosphatase enzymes. O-GlcNAc transferase (OGT) exists in complex with S/T phosphatase subunits PP1β and PP1γ and, along with the activity of O-GlcNAcase (OGA), facilitates rapid cycling between O-GlcNAcylation and phosphorylation states to serve as an “on/off switch” for substrate activation. Altered levels of OGT, OGA, and/or O-GlcNAc have been shown to influence many pathways pertinent to schizophrenia (SZ) pathophysiology. Notably, elevated O-GlcNAc and enhanced O-GlcNAcylation mediate glucose tolerance and insulin resistance which can lead to diabetes, an illness often found comorbid with SZ. Elevated O-GlcNAcylation can also produce mitochondrial abnormalities consistent with those identified in SZ. In an exploratory study of glycosylation enzyme transcript expression, our lab found OGT mRNA levels 253% higher in SZ than non-psychiatrically ill comparison (COMP) subjects (p < 0.0001). Based on this evidence, we hypothesized that OGT protein levels or the ratio of OGT:OGA enzymes are elevated in SZ brain.MethodsExpression of OGT and OGA were measured using western blots of superior temporal gyrus (STG; Brodmann Area 22) homogenates from sex- and age-matched pairs of SZ and COMP subjects (N = 17). Standard immunoblotting methods and commercially available antibodies were used to detect the targets of interest and protein levels were normalized to intralane valosin containing protein (VCP) expression; VCP expression has previously been found to be unchanged in SZ STG.ResultsIn the current study, we found OGA protein levels reduced 18% in SZ (p < 0.01) and SZ subjects demonstrate a trend toward increased ratios of OGT:OGA (p = 0.05). OGT was not different between groups (p = 0.77).DiscussionOur current results partially support our original hypothesis that an altered ratio of OGT:OGA may contribute to abnormalities of O-GlcNAcylation and consequent cellular metabolic abnormalities in SZ. A trend toward increased OGT:OGA along with decreased expression of OGA would produce the same functional outcome as the originally predicted OGT increase: upregulation of protein O-GlcNAcylation. Given that the mRNA study used samples of dorsolateral prefrontal cortex (DLPFC) while our protein-level measures were from STG, it is not inconceivable that potential O-GlcNAc dysregulation could arise from upregulated OGT in one brain region, but downregulated OGA in another. To elaborate on these findings, we will investigate OGT and OGA expression in the DLPFC and will assess total O-GlcNAcylation in both brain regions to determine functional consequences of altered enzyme expression.

Down-regulation of OGT promotes cisplatin resistance by inducing autophagy in ovarian cancer.

Cisplatin resistance significantly affects the survival rate of patients with ovarian cancer. However, the main mechanism underlying cisplatin resistance in ovarian cancer remains unclear.Methods: Immunohistochemistry was used to determine the expression of OGT, OGA and O-GlcNAc in chemoresistant and chemosensitive ovarian cancer tissues. Functional analyses (in vitro and in vivo) were performed to confirm the role of OGT in cisplatin resistance. Autophagy-related proteins were tested by western blot. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus reporter were used for autophagy flux analysis. Immunoprecipitation assay was utilized to detect protein-protein interactions.Results: We found that O-GlcNAc and O-GlcNAc transferase (OGT) levels were significantly lower in chemoresistant ovarian cancer tissues than in chemosensitive tissues, whereas O-GlcNAcase (OGA) levels did not differ. The down-regulation of OGT increased cisplatin resistance in ovarian cancer cells but had no effect on the efficacy of paclitaxel. The down-regulation of OGT improved tumor resistance to cisplatin in a mouse xenograft tumor model. OGT knockdown enhanced cisplatin-induced autophagy, which reduced apoptotic cell death induced by cisplatin, and promoted autolysosome formation. A reduction in O-GlcNAcylated SNAP-29 levels caused by the down-regulation of OGT promoted the formation of the SNARE complex and autophagic flux.Conclusion: Our findings suggest that down-regulation of OGT enhances cisplatin-induced autophagy via SNAP-29, resulting in cisplatin-resistant ovarian cancer. OGT may represent a novel target for overcoming cisplatin resistance in ovarian cancer.

Mutations in N-acetylglucosamine (O-GlcNAc) transferase in patients with X-linked intellectual disability

N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans. Here, we report on two hemizygous mutations in OGT in individuals with X-linked intellectual disability (XLID) and dysmorphic features: one missense mutation (p.Arg284Pro) and one mutation leading to a splicing defect (c.463–6T>G). Both mutations reside in the tetratricopeptide repeats of OGT that are essential for substrate recognition. We observed slightly reduced levels of OGT protein and reduced levels of its opposing enzyme O-GlcNAcase in both patient-derived fibroblasts, but global O-GlcNAc levels appeared to be unaffected. Our data suggest that mutant cells attempt to maintain global O-GlcNAcylation by down-regulating O-GlcNAcase expression. We also found that the c.463–6T>G mutation leads to aberrant mRNA splicing, but no stable truncated protein was detected in the corresponding patient-derived fibroblasts. Recombinant OGT bearing the p.Arg284Pro mutation was prone to unfolding and exhibited reduced glycosylation activity against a complex array of glycosylation substrates and proteolytic processing of the transcription factor host cell factor 1, which is also encoded by an XLID-associated gene. We conclude that defects in O-GlcNAc homeostasis and host cell factor 1 proteolysis may play roles in mediation of XLID in individuals with OGT mutations.

Ketogenic diet leads to O-GlcNAc modification in the BTBRT + tf/j mouse model of autism

BackgroundProtein O-linked-β-N-acetyl glucosamine (O-GlcNAc) is a post-translational modification to Ser/Thr residues that integrates energy supply with demand. Abnormal O-GlcNAc patterning is evident in several neurological disease states including epilepsy, Alzheimer's disease and autism spectrum disorder (ASD). A potential treatment option for these disorders includes the high-fat, low-carbohydrate, ketogenic diet (KD). The goal of this study was to determine whether the KD induces changes in O-GlcNAc in the BTBRT+tf/j (BTBR) mouse model of ASD. MethodsJuvenile male (5weeks), age-matched C57 or BTBR mice consumed a chow diet (13% kcal fat) or KD (75% kcal fat) for 10–14days. Following these diets, brain (prefrontal cortex) and liver were examined for gene expression levels of key O-GlcNAc mediators, global and protein specific O-GlcNAc as well as indicators of energy status. ResultsThe KD reduced global O-GlcNAc in the livers of all animals (p<0.05). Reductions were likely mediated by lower protein levels of O-GlcNAc transferase (OGT) and increased O-GlcNAcase (OGA) (p<0.05). In contrast, no differences in global O-GlcNAc were noted in the brain (p>0.05), yet OGT and OGA expression (mRNA) were elevated in both C57 and BTBR animals (p<0.05). ConclusionsThe KD has tissue specific impacts on O-GlcNAc. Although levels of O-GlcNAc play an important role in neurodevelopment, levels of this modification in the juvenile mouse brain were stable with the KD despite large fluctuations in energy status. This suggests that it is unlikely that the KD exerts it therapeutic benefit in the BTBR model of ASD by O-GlcNAc related pathways.

Fatty acid synthase inhibits the O-GlcNAcase during oxidative stress

The dynamic post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increased O-GlcNAcylation of numerous proteins, and this increase is thought to promote cell survival. The mechanisms by which the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), the enzymes that add and remove O-GlcNAc, respectively, are regulated during oxidative stress to alter O-GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevated O-GlcNAc levels in U2OS cells but has little impact on the activity of OGT. In contrast, the expression and activity of OGA are enhanced. We hypothesized that this seeming paradox could be explained by proteins that bind to and control the local activity or substrate targeting of OGA, thereby resulting in the observed stress-induced elevations of O-GlcNAc. To identify potential protein partners, we utilized BioID proximity biotinylation in combination with stable isotopic labeling of amino acids in cell culture (SILAC). This analysis revealed 90 OGA-interacting partners, many of which exhibited increased binding to OGA upon stress. The associations of OGA with fatty acid synthase (FAS), filamin-A, heat shock cognate 70-kDa protein, and OGT were confirmed by co-immunoprecipitation. The pool of OGA bound to FAS demonstrated a substantial (∼85%) reduction in specific activity, suggesting that FAS inhibits OGA. Consistent with this observation, FAS overexpression augmented stress-induced O-GlcNAcylation. Although the mechanism by which FAS sequesters OGA remains unknown, these data suggest that FAS fine-tunes the cell's response to stress and injury by remodeling cellular O-GlcNAcylation.

Yeast cells as an assay system for in vivo O-GlcNAc modification

BackgroundO-GlcNAcylation is a reversible protein post-translational modification, where O-GlcNAc moiety is attached to nucleocytoplasmic protein by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Although O-GlcNAc modification widely occurs in eukaryotic cells, the budding yeast Saccharomyces cerevisiae notably lacks this protein modification and the genes for the GlcNAc transferase and hydrolase. MethodsHuman OGT isoforms and OGA were ectopically expressed in S. cerevisiae, and the effects of their expressions on yeast growth and O-GlcNAc modification levels were assessed. ResultsExpression of sOGT, in S. cerevisiae catalyzes the O-GlcNAc modification of proteins in vivo; conversely, the expression of OGA mediates the hydrolysis of these sugars. sOGT expression causes a severe growth defect in yeast cells, which is remediated by the co-expression of OGA. The direct analysis of yeast proteins demonstrates protein O-GlcNAcylation is dependent on sOGT expression; conversely, the hydrolysis of these sugar modifications is induced by co-expression of OGA. Protein O-GlcNAcylation and the growth defects of yeast cells are caused by the O-GlcNAc transferase activity because catalytically inactive sOGT does not exhibit toxicity in yeast cells. Expression of another OGT isoform, ncOGT, also results in a growth defect in yeast cells. However, its toxicity is largely attributed to the TPR domain rather than the O-GlcNAc transferase activity. ConclusionsO-GlcNAc cycling can occur in yeast cells, and OGT and OGA activities can be monitored via yeast growth. General significanceYeast cells may be used to assess OGT and OGA.

Dexamethasone-Induced Skeletal Muscle Atrophy Increases O-GlcNAcylation in C2C12 Cells.

Skeletal muscle atrophy is a well-known adverse effect of chronic treatment with glucocorticoids and it also occurs when stress conditions such as sepsis and cachexia increase the release of endogenous glucocorticoids. Although the mechanisms of action of these hormones have been elucidated, the possible molecular mechanisms causing atrophy are not yet fully understood. The involvement of the O-GlcNAcylation process has recently been reported in disuse atrophy. O-GlcNAcylation, a regulatory post-translational modification of nuclear and cytoplasmic proteins consists in the attachment of O-GlcNAc residues on cell proteins and is regulated by two enzymes: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays a crucial role in many cellular processes and it seems to be related to skeletal muscle physiological function. The aim of this study is to investigate the involvement of O-GlcNAcylation in glucocorticoid-induced atrophy by using an "in vitro" model, achieved by treatment of C2C12 with 10 μM dexamethasone for 48 h. In atrophic condition, we observed that O-GlcNAc levels in cell proteins increased and concomitantly protein phosphorylation on serine and threonine residues decreased. Analysis of OGA expression at mRNA and protein levels showed a reduction in this enzyme in atrophic myotubes, whereas no significant changes of OGT expression were found. Furthermore, inhibition of OGA activity by Thiamet G induced atrophy marker expression. Our current findings suggest that O-GlcNAcylation is involved in dexamethasone-induced atrophy. In particular, we propose that the decrease in OGA content causes an excessive and mostly durable level of O-GlcNAc residues on sarcomeric proteins that might modify their function and stability. J. Cell. Biochem. 117: 1833-1842, 2016. © 2016 Wiley Periodicals, Inc.

E2F1 Transcription Factor Regulates O-linked N-acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Expression

Protein O-GlcNAcylation, which is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), has emerged as an important posttranslational modification that may factor in multiple diseases. Until recently, it was assumed that OGT/OGA protein expression was relatively constant. Several groups, including ours, have shown that OGT and/or OGA expression changes in several pathologic contexts, yet the cis and trans elements that regulate the expression of these enzymes remain essentially unexplored. Here, we used a reporter-based assay to analyze minimal promoters and leveraged in silico modeling to nominate several candidate transcription factor binding sites in both Ogt (i.e. the gene for OGT protein) and Mgea5 (i.e. the gene for OGA protein). We noted multiple E2F binding site consensus sequences in both promoters. We performed chromatin immunoprecipitation in both human and mouse cells and found that E2F1 bound to candidate E2F binding sites in both promoters. In HEK293 cells, we overexpressed E2F1, which significantly reduced OGT and MGEA5 expression. Conversely, E2F1-deficient mouse fibroblasts had increased Ogt and Mgea5 expression. Of the known binding partners for E2F1, we queried whether retinoblastoma 1 (Rb1) might be involved. Rb1-deficient mouse embryonic fibroblasts showed increased levels of Ogt and Mgea5 expression, yet overexpression of E2F1 in the Rb1-deficient cells did not alter Ogt and Mgea5 expression, suggesting that Rb1 is required for E2F1-mediated suppression. In conclusion, this work identifies and validates some of the promoter elements for mouse Ogt and Mgea5 genes. Specifically, E2F1 negatively regulates both Ogt and Mgea5 expression in an Rb1 protein-dependent manner.

30 Years Old: O-GlcNAc Reaches the Age of Reason - Regulation of Cell Signaling and Metabolism by O-GlcNAcylation.

30 Years Old: O-GlcNAc Reaches the Age of Reason - Regulation of Cell Signaling and Metabolism by O-GlcNAcylation.

O-GlcNAcase Expression is Sensitive to Changes in O-GlcNAc Homeostasis.

O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification involving an attachment of a single β-N-acetylglucosamine moiety to serine or threonine residues in nuclear and cytoplasmic proteins. Cellular O-GlcNAc levels are regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the modification, respectively. The levels of O-GlcNAc can rapidly change in response to fluctuations in the extracellular environment; however, O-GlcNAcylation returns to a baseline level quickly after stimulus removal. This process termed O-GlcNAc homeostasis appears to be critical to the regulation of many cellular functions including cell cycle progress, stress response, and gene transcription. Disruptions in O-GlcNAc homeostasis are proposed to lead to the development of diseases, such as cancer, diabetes, and Alzheimer’s disease. O-GlcNAc homeostasis is correlated with the expression of OGT and OGA. We reason that alterations in O-GlcNAc levels affect OGA and OGT transcription. We treated several human cell lines with Thiamet-G (TMG, an OGA inhibitor) to increase overall O-GlcNAc levels resulting in decreased OGT protein expression and increased OGA protein expression. OGT transcript levels slightly declined with TMG treatment, but OGA transcript levels were significantly increased. Pretreating cells with protein translation inhibitor cycloheximide did not stabilize OGT or OGA protein expression in the presence of TMG; nor did TMG stabilize OGT and OGA mRNA levels when cells were treated with RNA transcription inhibitor actinomycin D. Finally, we performed RNA Polymerase II chromatin immunoprecipitation at the OGA promoter and found that RNA Pol II occupancy at the transcription start site was lower after prolonged TMG treatment. Together, these data suggest that OGA transcription was sensitive to changes in O-GlcNAc homeostasis and was potentially regulated by O-GlcNAc.

MicroRNA-539 Is Up-regulated in Failing Heart, and Suppresses O-GlcNAcase Expression

Derangements in metabolism and related signaling pathways characterize the failing heart. One such signal, O-linked β-N-acetylglucosamine (O-GlcNAc), is an essential post-translational modification regulated by two enzymes, O-GlcNAc transferase and O-GlcNAcase (OGA), which modulate the function of many nuclear and cytoplasmic proteins. We recently reported reduced OGA expression in the failing heart, which is consistent with the pro-adaptive role of increased O-GlcNAcylation during heart failure; however, molecular mechanisms regulating these enzymes during heart failure remain unknown. Using miRNA microarray analysis, we observed acute and chronic changes in expression of several miRNAs. Here, we focused on miR-539 because it was predicted to target OGA mRNA. Indeed, co-transfection of the OGA-3'UTR containing reporter plasmid and miR-539 overexpression plasmid significantly reduced reporter activity. Overexpression of miR-539 in neonatal rat cardiomyocytes significantly suppressed OGA expression and consequently increased O-GlcNAcylation; conversely, the miR-539 inhibitor rescued OGA protein expression and restored O-GlcNAcylation. In conclusion, this work identifies the first target of miR-539 in the heart and the first miRNA that regulates OGA. Manipulation of miR-539 may represent a novel therapeutic target in the treatment of heart failure and other metabolic diseases.

OGA heterozygosity suppresses intestinal tumorigenesis in Apc(min/+) mice.

Emerging evidence suggests that aberrant O-GlcNAcylation is associated with tumorigenesis. Many oncogenic factors are O-GlcNAcylated, which modulates their functions. However, it remains unclear how O-GlcNAcylation and O-GlcNAc cycling enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), affect the development of cancer in animal models. In this study, we show that reduced level of OGA attenuates colorectal tumorigenesis induced by Adenomatous polyposis coli (Apc) mutation. The levels of O-GlcNAcylation and O-GlcNAc cycling enzymes were simultaneously upregulated in intestinal adenomas from mice, and in human patients. In two independent microarray data sets, the expression of OGA and OGT was significantly associated with poor cancer-specific survival of colorectal cancer (CRC) patients. In addition, OGA heterozygosity, which results in increased levels of O-GlcNAcylation, attenuated intestinal tumor formation in the Apcmin/+ background. Apcmin/+ OGA+/− mice exhibited a significantly increased survival rate compared with Apcmin/+ mice. Consistent with this, Apcmin/+ OGA+/− mice expressed lower levels of Wnt target genes than Apcmin/+. However, the knockout of OGA did not affect Wnt/β-catenin signaling. Overall, these findings suggest that OGA is crucial for tumor growth in CRC independently of Wnt/β-catenin signaling.

Altering O-Linked β-N-Acetylglucosamine Cycling Disrupts Mitochondrial Function

Mitochondrial impairment is commonly found in many diseases such as diabetes, cancer, and Alzheimer disease. We demonstrate that the enzymes responsible for the addition or removal of the O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, are critical regulators of mitochondrial function. Using a SILAC (stable isotope labeling of amino acids in cell culture)-based proteomics screen, we quantified the changes in mitochondrial protein expression in OGT- and OGA-overexpressing cells. Strikingly, overexpression of OGT or OGA showed significant decreases in mitochondria-localized proteins involved in the respiratory chain and the tricarboxylic acid cycle. Furthermore, mitochondrial morphology was altered in these cells. Both cellular respiration and glycolysis were reduced in OGT/OGA-overexpressing cells. These data demonstrate that alterations in O-GlcNAc cycling profoundly affect energy and metabolite production.

Transcriptional regulation of O‐GlcNAc homeostasis (607.8)

A vast number of nuclear and cytoplasmic proteins are modified by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc), a dynamic post‐translational modification that has emerged as a key regulator of diverse cellular processes. The addition and removal of O‐GlcNAc are catalyzed by O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA), respectively. Accumulating evidence indicates that both excess and deficiency of O‐GlcNAc in the cell can have deleterious effects, suggesting that maintenance of O‐GlcNAc homeostasis is critical for cellular function. However, the mechanism by which O‐GlcNAc homeostasis is maintained in the normal state and disrupted in the disease state has not yet been investigated. Here, we demonstrate that Ogt expression is upregulated by OGA, creating a feedback mechanism that serves to maintain a balance between OGT and OGA expression levels in the cell. We found that OGA promotes Ogt transcription through cooperation with the histone acetyltransferase p300 and the transcription factor CCAAT‐enhancer‐binding protein β (C/EBP‐β). In this protein complex, OGA and p300 act as transcriptional coactivators of C/EBP‐β through their glycosidase and acetyltransferase activities, respectively. Since O‐GlcNAc homeostasis is often disrupted in cancer cells, we also sought to determine whether classical oncoproteins are involved in regulating Ogt expression. We found that extracellular signal‐regulated kinase (ERK) increases OGA glycosidase activity and promotes Ogt transcription, suggesting that chronic growth factor stimulation may be responsible for disrupted O‐GlcNAc homeostasis in cancer. Our study elucidates a transcriptional mechanism that controls cellular O‐GlcNAc homeostasis, which should lay a foundation for exploring O‐GlcNAc signaling as a therapeutic target for human disease.Grant Funding Source: Supported by NIH R01‐DK089098