O-GlcNAc Transferase Protein Expression Research Articles

Single cell and bulk RNA expression analyses identify enhanced hexosamine biosynthetic pathway and O-GlcNAcylation in acute myeloid leukemia blasts and stem cells.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry. We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation. Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.

The deubiquitinase EIF3H promotes hepatocellular carcinoma progression by stabilizing OGT and inhibiting ferroptosis

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal human malignancies, and with quite limited treatment alternatives. The proteasome is responsible for most of the protein degradation in eukaryotic cells and required for the maintenance of intracellular homeostasis. However, its potential role in HCC is largely unknown. In the current study, we identified eukaryotic translation initiation factor 3 subunit H (EIF3H), belonging to the JAB1/MPN/MOV34 (JAMM) superfamily, as a bona fide deubiquitylase of O-GlcNAc transferase (OGT) in HCC. We explored that EIF3H was positively associated with OGT in HCC and was related to the unfavorable prognosis. EIF3H could interact with, deubiquitylate, and stabilize OGT in a deubiquitylase-dependent manner. Specifically, EIF3H was associated with the GT domain of ERα via its JAB/MP domain, thus inhibiting the K48-linked ubiquitin chain on OGT. Besides, we demonstrated that the knockdown of EIF3H significantly reduced OGT protein expression, cell proliferation and invasion, and caused G1/S arrest of HCC. We also found that the deletion of EIF3H prompted ferroptosis in HCC cells. Finally, the effects of EIF3H depletion could be reversed by further OGT overexpression, implying that the OGT status is indispensable for EIF3H function in HCC carcinogenesis. In summary, our study described the oncogenic function of EIF3H and revealed an interesting post-translational mechanism between EIF3H, OGT, and ferroptosis in HCC. Targeting the EIF3H may be a promising approach in HCC.6aJiHquPswT5dhvMxQLovCVideo

MiRNA-424-5p Suppresses Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma and Attenuates Expression of O-GlcNAc-Transferase.

Simple SummaryThe identification of biomarkers that predict the metastatic potential of tumors is a current area of interest in cancer research. A previous study from our laboratory identified numerous microRNA (miRNA) biomarkers that are differentially expressed in pathologic stage I clear cell renal cell carcinoma (ccRCC) tumors that progress to metastatic disease. This study investigated the role of aberrant expression of one of these miRNA, miR-424-5p, and one of its associated protein targets, O-GlcNAc-transferase (OGT). We examined the influence of miR-424-5p and OGT expression on the proliferation, migration, and invasion of ccRCC cells, and confirmed the direct interaction between miR-424-5p and OGT. These findings suggest that the decrease in miR-424-5p expression observed in these small renal masses leads to an increase in OGT, which facilitates metastasis.MicroRNAs (miRNAs) are non-coding post-transcriptional regulators of gene expression that are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in tumor progression. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors, including miR-424-5p, that are associated with an aggressive phenotype. We investigate the impact of this dysregulated miRNA and its protein target O-GlcNAc-transferase (OGT) to better understand the mechanisms behind aggressive stage I ccRCC. The ccRCC cell lines 786-O and Caki-1 were used to assess the impact of miR-424-5p and OGT. Cells were transfected with pre-miR-424-5p, a lentiviral anti-OGT shRNA, or were treated with the demethylating agent 5-Aza-2′-deoxycytidine. Cell proliferation was measured via MT cell viability assay. Cell migration and invasion were analyzed using Transwell assays. The expression of miR-424-5p was determined through qRT-PCR, while OGT protein expression was evaluated through Western blotting. The interaction between miR-424-5p and OGT was confirmed via luciferase reporter assay. The transfection of ccRCC cells with pre-miR-424-5p or anti-OGT shRNA significantly inhibited cell proliferation, migration, and OGT expression, while miR-424-5p also attenuated cell invasion. Addition of the demethylating agent significantly reduced cell proliferation, migration, invasion, and OGT expression, while significantly increasing the expression of miR-424-5p. Altogether, these findings suggest that epigenetic downregulation of miR-424-5p, which in turn augments OGT expression, contributes to the creation of aggressive forms of stage I ccRCC.

O-GlcNAcylation inhibits hepatic stellate cell activation.

Protein O-GlcNAcylation is a critical post-translational modification regulating gene expression and fundamental cell functions. O-GlcNAc transferase (OGT) emerged as a key regulator of liver pathophysiology and disease. In this study, we aimed to evaluate the role of OGT in hepatic stellate cells (HSCs) and its consequent role in liver fibrosis. Primary HSCs were isolated from C57/B6 mice. Cell morphology and α-SMA immunofluorescence staining were observed under scanning confocal microscope. Transcriptomic profile was evaluated by RNAseq (Illumina). Promoter activity was examined by luciferase and β--Galactosidase reporter assays. Liver fibrosis mouse models were induced either by intraperitoneal injection of CCl4 at 3 times/week for 4 weeks or by feeding with methionine and choline deficient (MCD) diet for 4 weeks. OGT protein expression and protein O-GlcNAcylation were significantly decreased in CCl4 - or MCD diet-induced liver fibrosis as compared with normal liver in mice. OGT expression and protein O-GlcNAcylation were also decreased in primary HSCs isolated from liver with CCl4 -induced fibrosis compared with those from normal liver. RNA-seq showed that OGT knockdown in HSCs modulated key signaling pathways involved in HSC activation. Promoter sequence analysis of the differentially expressed genes predicted serum response factor (SRF) as a key transcription factor regulated by OGT. Luciferase reporter assay confirmed that OGT repressed activity of SRF to induce α-SMA transcription. Mutations of specific O-GlcNAcylation sites on SRF increased its transcriptional activity, validating negative regulation of SRF by OGT-mediated O-GlcNAcylation. Our results suggest that OGT functions as a negative regulator of HSC activation by promoting SRF O-GlcNAcylation to protect against liver fibrosis.

MicroRNAs MiR-15a and MiR-26a cooperatively regulate O-GlcNAc-transferase to control proliferation in clear cell renal cell carcinoma.

MicroRNAs (miRNAs), a group of non-coding post-transcriptional regulators of gene expression, are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in carcinogenesis. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors associated with progression to metastatic disease. To investigate the impact of two of these dysregulated miRNA, miR-15a-5p and -26a-5p, in an effort to elucidate the mechanisms underpinning aggressive forms of stage I ccRCC. The ccRCC cell line 786-O was transfected with pre-miRs-15a-5p and -26a-5p to rescue expression. Cell proliferation was measured via MT Cell Viability Assay. O-GlcNAc-transferase (OGT), a known protein in ccRCC proliferation, was identified by bioinformatics analysis as a target of both miRNA and validated via luciferase reporter assay to confirm binding of each miR to the 3' untranslated region (UTR). OGT protein expression was evaluated via western blotting. Luciferase assay confirmed specificity of miR-15a-5p and -26a-5p for the OGT UTR. Western blot analysis for OGT showed reduced expression following co-transfection of both miRNAs compared to negative control or individual transfection. Co-transfection of these miRNAs greatly reduced proliferation when compared to negative control or the individual transfections. Our results indicate that the dysregulation of miR-15a-5p and -26a-5p contribute cooperatively to the proliferation of ccRCC through their regulation of OGT. These results give insight into the pathogenesis of aggressive early stage ccRCC and suggest potential therapeutic targets for future research.

Inhibition of mechanistic target of rapamycin signaling decreases levels of O-GlcNAc transferase and increases serotonin release in the human placenta.

Changes in placental function, in particular down-regulation of placental O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) in response to maternal stress and increased placental secretion of serotonin into the fetal circulation following maternal infection, have been mechanistically linked to adverse neurodevelopment in mice. We hypothesized that mechanistic target of rapamycin (mTOR) signaling is a key regulator of trophoblast serotonin synthesis and OGT protein expression and that serotonin is secreted by the human placenta into the fetal circulation. Placental homogenates (n=46) from elective terminations at 8-22 weeks of gestation and from healthy-term women were sexed and the protein levels of OGT and enzymes involved in serotonin synthesis was determined. Primary human trophoblast (PHT) cells were isolated from normal term placenta (n=27), cultured and transfected (n=8) with siRNA targeting a scramble sequence (control), raptor (inhibits mTOR Complex 1 (mTORC1)), or rictor (inhibits mTOR Complex 2 (mTORC2)). Subsequently, conditioned media and PHT cell lysates were collected. Free serotonin concentration was measured using ELISA in cell culture media and in platelet-depleted normal term umbilical vein and artery plasma (n=38). Both mTORC1 and mTORC2 inhibition down-regulated OGT levels in PHT cells. The level of serotonin synthesis enzyme tryptophan hydroxylase (TPH-1) was higher in early gestation female placentas and at term serotonin concentration was three-fold higher in the umbilical vein than in the umbilical artery. Inhibition of mTORC2, but not mTORC1, increased cultured PHT cell serotonin secretion. Our data are consistent with the model that mTOR signaling is a key regulator of trophoblast serotonin synthesis and OGT protein expression.

Non-canonical Interaction Between O-Linked N-Acetylglucosamine Transferase and miR-146a-5p Aggravates High Glucose-Induced Endothelial Inflammation.

Background and Aims: Increased O-GlcNAc transferase (OGT)–induced O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification is linked with diabetic complications. MicroRNA-146a-5p (miR-146a-5p) is a negative inflammatory regulator and is downregulated in diabetes. Here, we investigated the interaction between miR-146a-5p and OGT.Methods: Human aortic endothelial cells (HAECs) were stimulated with high glucose (25 mM) and glucosamine (25 mM) for 24 h. Western blot, real time PCR, bioinformatics analysis, luciferase reporter assay, miR-146a-5p mimic/inhibitor transfection, siRNA OGT transfection, miR-200a/200b mimic transfection, and OGT pharmacological inhibition (ST045849) were performed. The aorta from miR-146a-5p mimic-treated db/db mice were examined by immunohistochemistry staining.Results: HG and glucosamine upregulated OGT mRNA and protein expression, protein O-GlcNAcylation, and IL-6 mRNA and protein expression. Real time PCR analysis found that miR-146a-5p was decreased in HG- and glucosamine-stimulated HAECs. This suggested that OGT-induced protein O-GlcNAcylation as a mechanism to downregulate miR-146a-5p. Bioinformatic miR target analysis excluded miR-146a-5p as a post-transcriptional regulator of OGT. However, a luciferase reporter assay confirmed that miR-146a-5p mimic bound to 3′-UTR of human OGT mRNA, indicating that OGT is a non-canonical target of miR-146a-5p. Transfection with miR-146a-5p mimic and inhibitor confirmed that miR-146a-5p regulated OGT/protein O-GlcNAcylation/IL-6 expression levels. Furthermore, OGT siRNA transfection, miR-200a/miR-200b mimic transfection, and ST045849 increased HG-induced miR-146a-5p expression levels, indicating that HG-induced miR-146a-5p downregulation is partially mediated through OGT-mediated protein O-GlcNAcylation. In vivo, intravenous injections of miR-146a mimic decreased endothelial OGT and IL6 expression in db/db mice.Conclusion: A non-canonical positive feedback interaction between miR-146a-5p and OGT is involved in a vicious cycle to aggravate HG-induced vascular complications.

O-GlcNAc levels are regulated in a tissue and time specific manner during post-natal development, independently of dietary intake

O-GlcNAcylation is a well-preserved, ubiquitous, post-translational modification involved in response to stress, considered as a nutrient sensor. GlcNAc moiety is added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Metabolic sources vary from carbohydrate in utero, to fatty acid after birth then a mix once adult. Study the evolution of O-GlcNAc levels during the first period of life and decipher the impact of O-GlcNAc on cardiac proteins. Heart and brain were harvested from Wistar rats before and after birth (D-1 and D0), in suckling animals (D12), after weaning (D28) with a standard or a carbohydrate free diet, and when adults (D84). O-GlcNAc levels, GFAT, OGT and OGA protein expression were evaluated by western blot. Mass spectrometry approaches were performed to (i) quantify known regulator of O-GlcNAc and (ii) identify cardiac O-GlcNAcylated proteins at D0 and D28. Cardiac protein O-GlcNAc levels decrease progressively from D-1 to D84 (13 fold, P < 0.05), whereas protein O-GlcNAc levels tend to increase in the brain. Both cardiac and brain protein O-GlcNAc levels were similar between the two weaning diets. Moreover, levels of UDP-GlcNAc and glutamine are not correlated with changes in O-GlcNAc levels. Mass spectrometry analysis allowed us to identify proteins of interest involved in cardiac stress response (including those implicated in protein and RNA biosynthesis, metabolism). Interestingly, several proteins are only O-GlcNAcylated at D28 despite a decrease in O-GlcNAc levels between D0 and D28. We demonstrate for the first time that O-GlcNAc levels are not linked to the dietary sources. Our results suggest that glutamine and UDP-GlcNAc are not the main regulatory element of O-GlcNAc levels. Proteins identified by untargeted mass spectrometry will need to be specifically studied to better understand the impact of O-GlcNAcylation during development, growth or stress response.

OR14-07 Association Between Placental Glucose Uptake and Protein O-Glcnacylation and Birth Outcomes in Obese Non-Diabetic Mothers

Increased transport of nutrients such as glucose, across the placenta, has been linked to abnormal fetal growth and pregnancy complications in obese non-diabetic mothers (1); however, the underlying mechanisms are poorly understood. We hypothesized that in placenta, the metabolic and nutrient sensor O-GlcNAc transferase (OGT), highly sensitive to glucose flux through the hexosamine biosynthetic pathway (HBP), responds to maternal obesogenic environment by increasing O-GlcNAc post-translational modification of nucleocytoplasmic proteins in the placenta altering fetal growth trajectories. Tissue biopsies were isolated from placentas collected at term from 26 non-diabetic mothers alongside routine biochemistry and anthropometric data (maternal fasting glucose, glycated hemoglobin (HbA1c), early pregnancy body weight (BMI) and birth weight). OGT and glucose transporter 1 (GLUT1) protein expression as well as tissue levels of O-GlcNAcylation were determined by immunoblotting using specific antibodies. The BeWo choriocarcinoma cell line was also used as an in vitro model of trophoblast to test the effect of high glucose and GLUT1 silencing on the OGT activity by immunoblotting. Maternal BMI was positively correlated to birth weight centile (BWC) (p=0.0130, R2=0.231), maternal fasting glucose (p=0.0177, R2=0.221) and HbA1c levels (p= 0.0156, R2=0.229) as well as placental OGT protein expression (p=0.0294, R2=0.183). The latter was positively associated to the levels of protein O-GlcNAcylation (p=0.0023, R2=0.326). Interestingly, GLUT1 protein levels were positively correlated to BWC (p=0.0056, R2=0.279) and strongly correlated to protein O-GlcNAcylation (p<0.0001, R2=0.507), suggesting an increase in the placental flux of glucose. In agreement with findings in placenta biopsies, in BeWo cells total protein O-GlcNAcylation levels were altered by cell exposure to different glucose levels (5 mM vs 15 mM, p<0.01). This was prevented by downregulating OGT or GLUT1 expression (p<0.001) using gene silencing. In addition, OGT protein levels were negatively associated to AMP-activated protein kinase (AMPK) activation (p=0.0005, R2=0.402) in placenta biopsies identifying a novel cross-talk between two placental nutrient sensors, OGT and AMPK, previously shown in other tissues (2). Accordingly, the silencing of OGT promoted the activation of AMPK (p<0.01) and its downstream target acetyl-CoA carboxylase (ACC) (p<0.01) in BeWo cells, as demonstrated by increased phosphorylation of residues Thr172 and Ser79 for AMPK and ACC respectively. Such obesity-associated cross talk between metabolic and nutrient sensors might disrupt multiple cellular pathways involved in fetal development and growth.

Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts.

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a decoy 5′ splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since antisense targeting of decoy 5′ splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR and suggest that dynamic regulation of decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.

Cardiac O-GlcNAc levels variation with age: Impact of suckling to weaning transition

O-GlcNAcylation is a well-preserved, ubiquitous, post-translational modification, involved in the reprogramming of cardiac metabolism to enhance stress adaptation and acute cardiac O-GlcNAc stimulation is associated with beneficial effects. Two enzymes regulate O-GlcNAc levels: (i) O-GlcNAc Transferase (OGT) and (ii) O-GlcNAcAse (OGA) which adds and removes the GlcNAc residue respectively. GFAT, the rate-limiting enzyme of the hexosamines biosynthesis pathway (HBP), modulates O-GlcNAc levels through cellular metabolism. Cardiac metabolism varies during the first days of life with an important transition after birth, from carbohydrate in utero, to fatty acid in adults. Interestingly, hearts are less sensitive to ischemic stress during the first day of life. Study the variations in O-GlcNAc level and regulator during aging. Hearts were harvested from Wistar rats before and after birth (D-1 and D0), before and after weaning (D12 and D28) with a carbohydrate free diet (CHO-F) (D28F) to avoid metabolic transition and adult (D84). O-GlcNAc levels, GFAT, OGT and OGA protein and gene expression were evaluated. A Click-iT technique was used to identify O-GlcNAcylated proteins at D0 and/or D28. Cardiac O-GlcNAc levels decrease from D-1 to D84 (13 fold), along with GFAT1 (50 fold) and GFAT2 (10 fold) and OGT (6 fold) while OGA is nearly undetectable at D-1 and increases over development. Interestingly, metabolism had no impact on cardiac O-GlcNAc levels however CHO-F diet reduces OGA expression (5 fold). The mass spectrometry analysis allows to identify potential protein of interest as they can play an important role in stress response, transcription and metabolism. O-GlcNAcylation levels decrease throughout aging and are independent of metabolism. Proteins identified in mass spectrometry need to be explored more specifically now to evaluate the potential involvement of O-GlcNAc levels in stress response in young.

MicroRNA-200a/200b Modulate High Glucose-Induced Endothelial Inflammation by Targeting O-linked N-Acetylglucosamine Transferase Expression.

Background and Aims: Increased O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins by O-GlcNAc transferase (OGT) is associated with diabetic complications. Furthermore, oxidative stress promotes endothelial inflammation during diabetes. A previous study reported that microRNA-200 (miR-200) family members are sensitive to oxidative stress. In this study, we examined whether miR-200a and miR-200b regulate high-glucose (HG)-induced OGT expression in human aortic endothelial cells (HAECs) and whether miRNA-200a/200b downregulate OGT expression to control HG-induced endothelial inflammation.Methods: HAECs were stimulated with high glucose (25 mM) for 12 and 24 h. Real-time polymerase chain reaction (PCR), western blotting, THP-1 adhesion assay, bioinformatics predication, transfection of miR-200a/200b mimic or inhibitor, luciferase reporter assay, and transfection of siRNA OGT were performed. The aortic endothelium of db/db diabetic mice was evaluated by immunohistochemistry staining.Results: HG upregulated OGT mRNA and protein expression and protein O-GlcNAcylation levels (RL2 antibody) in HAECs, and showed increased intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Bioinformatics analysis revealed homologous sequences between members of the miR-200 family and the 3′-untranslated region (3′-UTR) of OGT mRNA, and real-time PCR analysis confirmed that members of miR-200 family were significantly decreased in HG-stimulated HAECs. This suggests the presence of an impaired feedback restraint on HG-induced endothelial protein O-GlcNAcylation levels because of OGT upregulation. A luciferase reporter assay demonstrated that miR-200a/200b mimics bind to the 3′-UTR of OGT mRNA. Transfection with miR-200a/200b mimics significantly inhibited HG-induced OGT mRNA expression, OGT protein expression; protein O-GlcNAcylation levels; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Additionally, siRNA-mediated OGT depletion reduced HG-induced protein O-GlcNAcylation; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion, confirming that HG-induced endothelial inflammation is partially mediated via OGT-induced protein O-GlcNAcylation. These results were validated in vivo: tail-vein injection of miR-200a/200b mimics downregulated endothelial OGT and ICAM-1 expression in db/db mice.Conclusion: miR-200a/200b are involved in modulating HG-induced endothelial inflammation by regulating OGT-mediated protein O-GlcNAcylation, suggesting the therapeutic role of miR-200a/200b on vascular complications in diabetes.

O‐GlcNAc Transferase Regulates Phenotypic Transformation of Vascular Smooth Muscle Cells in Response to Hyperglycemia

Risks of vascular complications are enhanced two‐to‐four fold in diabetes, accounting for increased morbidity and mortality in these patients. Hyperglycemia is an important risk factor for macrovascular complications associated with diabetes. Diabetic patients exhibit an increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a hallmark of VSMC phenotypic switching from the quiescent contractile state to a synthetic proliferative phenotype. Hyperglycemia increases glucose flux through the hexosamine biosynthetic pathway triggering an enhanced signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), the key enzyme catalyzing addition of O‐GlcNAc moieties to proteins. Protein O‐GlcNAcylation is a unique post‐translational protein modification implicated in diabetes and related cardiovascular complications. Previous studies have suggested both cardio‐protective and cardio‐detrimental effects of protein O‐GlcNAcylation in diabetes. However, the precise role of OGT in the etiology of diabetic atherogenesis remains incompletely understood. The goal of the current study was to investigate whether OGT modulates VSMC transition to a de‐differentiated phenotype under hyperglycemic conditions. Immunoblotting experiments revealed that in primary cultures of human aortic smooth muscle cells (HASMC) in vitro, benzyl 2‐deoxy α‐D‐galactopyranoside (BG), a pharmacological inhibitor of OGT, decreased high glucose‐induced OGT and O‐GlcNAc protein expression, and this was accompanied with attenuated PCNA (proliferation marker), reduced vimentin (SM synthetic marker) and increased SM‐MHC (SM contractile marker) expression. Consistent with these findings, deletion of OGT using siRNA gene silencing significantly lowered protein O‐GlcNAc and PCNA expression in glucose‐stimulated HASMC compared with glucose‐treated cells transfected with control siRNA. Notably, under glucose‐stimulated conditions, OGT knockdown enhanced calponin (SM contractile marker) expression in HASMC transfected with OGT siRNA vs cells transfected with control siRNA. Finally, electrophoretic mobility shift assay (EMSA) revealed that specific OGT inhibitors (BG and ST045849) attenuated high glucose‐induced DNA‐binding activity of YY1 and Elk1 (transcriptional repressors of SM contractile phenotype) in HASMC compared to cells treated with glucose alone. Taken together, these data clearly demonstrate that loss of OGT inhibits VSMC phenotypic de‐differentiation in response to hyperglycemia, suggesting OGT as a potential therapeutic target in diabetic atherosclerosis.Support or Funding InformationAmerican Heart Association ‐ Grant‐in‐Aid 16GRNT31200034This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 176: O-GlcNAc Transferase Promotes Vascular Smooth Muscle Cell De-differentiation

Vascular smooth muscle cells (VSMCs) in mature vessels have a differentiated, contractile phenotype but retain the ability to reversibly de-differentiate in response to environmental cues. Plasticity is necessary for development and wound healing, but also contributes to pathologies such as atherosclerosis and intimal hyperplasia. O- linked N- acetylglucosamine ( O -GlcNAc) transferase (OGT) mediates nutrient-sensitive post-translational modification of serine and threonine residues and has been implicated in multiple diseases, but its role in VSMC phenotypic modulation is not known. In human coronary SMCs, we found that OGT knockdown significantly increased contractile marker expression, including MYH11 and ACTA2, at the mRNA and protein levels. OGT knockdown also decreased proliferation rates after serum treatment in VSMCs. In addition, rapamycin, a differentiating agent, decreased OGT protein expression and O- GlcNAc levels. Conversely, de-differentiation with PDGF treatment increased OGT expression and global O- GlcNAc. Neither treatment affected OGT mRNA levels. OGT has been shown to increase the protein stability of EZH2, the enzyme that generates the inhibitory histone epigenetic mark H3K27me3. We found that OGT knockdown did not affect EZH2 mRNA levels but significantly decreased EZH2 protein expression as well as global H3K27me3 in VSMCs. Similar to OGT, EZH2 expression was decreased upon rapamycin treatment and increased with PDGF. A decrease in ubiquitination of EZH2 was observed after PDGF treatment, suggesting an increase in protein stability. Importantly, EZH2 knockdown increased protein expression of SMC contractile markers while EZH2 overexpression repressed these markers. Wire injury of mouse femoral arteries increased H3K27me3 levels after 21 days. OGT expression was elevated in the innermost layer of the neointima at the same time point. These data demonstrate that OGT inhibits VSMC differentiation and that OGT and its target EZH2 are both dynamically regulated during phenotypic modulation. Future work will illuminate if OGT promotes VSMC de-differentiation through EZH2-mediated histone modification at contractile gene promoters.

MTOR/MYC Axis Regulates O-GlcNAc Transferase Expression and O-GlcNAcylation in Breast Cancer.

Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-β-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K-mTOR-MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade. Evidence indicates OGT as a therapeutic target in c-MYC-amplified cancers.

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.

Glutathione depletion and acute exercise increase O-GlcNAc protein modification in rat skeletal muscle.

Post-translational modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) profoundly affects protein structure, function, and metabolism. Although many skeletal muscle proteins are O-GlcNAcylated, the modification has not been extensively studied in this tissue, especially in the context of exercise. This study investigated the effects of glutathione depletion and acute exercise on O-GlcNAc protein modification in rat skeletal muscle. Diethyl maleate (DEM) was used to deplete intracellular glutathione and rats were subjected to a treadmill run. White gastrocnemius and soleus muscles were analyzed for glutathione status, O-GlcNAc and O-GlcNAc transferase (OGT) protein levels, and mRNA expression of OGT, O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase. DEM and exercise both reduced intracellular glutathione and increased O-GlcNAc. DEM upregulated OGT protein expression. The effects of the interventions were significant 4 h after exercise (P < 0.05). The changes in the mRNA levels of O-GlcNAc enzymes were different in the two muscles, potentially resulting from different rates of oxidative stress and metabolic demands between the muscle types. These findings indicate that oxidative environment promotes O-GlcNAcylation in skeletal muscle and suggest an interrelationship between cellular redox state and O-GlcNAc protein modification. This could represent one mechanism underlying cellular adaptation to oxidative stress and health benefits of exercise.

Study of the O-linked-N-acetyl-glucosaminylation of proteins induced by high doses of glucosamine and its correlation with osteoarthritis progression

Study of the O-linked-N-acetyl-glucosaminylation of proteins induced by high doses of glucosamine and its correlation with osteoarthritis progression

O-GlcNAc Protein Modification in Cancer Cells Increases in Response to Glucose Deprivation through Glycogen Degradation

When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.

AMP-activated Protein Kinase and p38 MAPK Activate O-GlcNAcylation of Neuronal Proteins during Glucose Deprivation

We have demonstrated previously that a wide array of stress signals induces O-GlcNAc transferase (OGT) expression and increases O-GlcNAcylation of many intracellular proteins, a response that is critical for cell survival. Here, we describe a mechanism by which glucose deprivation induces OGT expression and activity in Neuro-2a neuroblastoma cells. Glucose deprivation increases OGT mRNA and protein expression in an AMP-activated protein kinase-dependent manner, whereas OGT enzymatic activity is regulated in a p38 MAPK-dependent manner. OGT is not phosphorylated by p38, but rather it interacts directly with p38 through its C terminus; this interaction increases with p38 activation during glucose deprivation. Surprisingly, the catalytic activity of OGT, as measured toward peptide substrates, is not altered by glucose deprivation. Instead, p38 regulates OGT activity within the cell by recruiting it to specific targets, including neurofilament H. Neurofilament H is O-GlcNAcylated during glucose deprivation in a p38-dependent manner. Interestingly, neurofilament H solubility is increased by glucose deprivation in an O-GlcNAc-dependent manner, suggesting that O-GlcNAcylation of neurofilament H regulates its disassembly from filaments. Not only do these data help to reveal how OGT is regulated by stress, but these findings also describe a possible mechanism by which defective brain glucose metabolism, as found in aging and ischemia, may directly affect axonal structure.