OGT Expression Research Articles

The correlation between cellular O-GlcNAcylation and sensitivity to O-GlcNAc inhibitor in colorectal cancer cells.

The upregulation of O-GlcNAc signaling has long been implicated in the development and progression of numerous human malignancies, including colorectal cancer. In this study, we characterized eight colorectal cancer cell lines and one non-cancerous cell line for O-GlcNAc-related profiles such as the expression of OGT, OGA, and total protein O-GlcNAcylation, along with their sensitivity toward OSMI-1 (Os), an OGT inhibitor (OGTi). Indeed, Os dose-dependently suppressed the viability of all colorectal cancer cell lines tested. Among the three O-GlcNAc profiles, our results revealed that Os IC50 exhibited the strongest correlation with total protein O-GlcNAcylation (Pearson Correlation Coefficient r = -0.73), suggesting that total O-GlcNAcylation likely serves as a better predictive marker for OGTi sensitivity than OGT expression levels. Furthermore, we demonstrated that Os exhibited a synergistic relationship with regorafenib (Re). We believed that this synergism could be explained, at least in part, by the observed Re-mediated increase of cellular O-GlcNAcylation, which was counteracted by Os. Finally, we showed that the Os:Re combination suppressed the growth of NCI-H508 tumor spheroids. Overall, our findings highlighted OGTi as a potential anticancer agent that could be used in combination with other molecules to enhance the efficacy while minimizing adverse effects, and identified total cellular O-GlcNAcylation as a potential predictive marker for OGTi sensitivity.

The circZEB1/miR-337–3p/OGT axis mediates angiogenesis and metastasis via O-GlcNAcylation and up-regulating YBX1 in breast cancer

BackgroundA growing corpus of research has revealed that circular RNAs (circRNAs) have become increasingly important for the growth of malignancies in recent years. CircRNAs as ideal candidates for breast cancer (BC) therapeutic targets is still absent. MethodsIn our study, the dysregulated circRNAs in BC progression were explored, we analysed the BC's circRNA expression profiles using publicly available datasets (GSE101124 and GSE101122). The expression of circZEB1 in BC and cell lines was investigated by qPCR. RNase and actinomycin D were used to examine the features of circZEB1. The function of circZEB1 was subsequently investigated through the utilisation of colony formation, tube formation, transwell assays, and xenograft animal models.RNA immunoprecipitation (RIP), luciferase reporter assays, immunoprecipitation (co-IP) test in conjunction with LC-MS, and ChIP-seq assay to investigate the molecular mechanism underlying the biological activity of circZEB1 in BC. ResultsAmong the circRNAs, we were particularly interested in hsa_circ_0000228, which is spliced from the oncogene ZEB1. In BC cell lines, CircZEB1 expression was upregulated. CircZEB1 knockdown prevented BC cells from migrating and invading, as well as HUVECs from forming tubes and developing. By sponging miR-337–3p, functional testing revealed that circZEB1 promoted O-GlcNAcylation, increased YBX1, and OGT expression. Moreover, circZEB1 overexpression is reversible, in contrast to YBX1 knockdown, which mostly results in the downregulation of multiple oncogenes. ConclusionOur study indicate that circZEB1 had oncogenic function in BC by focusing on circZEB1/miR-337–3p/OGT and YBX1. It might be inferred that circZEB1 could be a promising new target for BC treatment.

6-Gingerol alleviates ectopic lipid deposition in skeletal muscle by regulating CD36 translocation and mitochondrial function

Ectopic lipid deposition (ELD) and mitochondrial dysfunction are common causes of metabolic disorders in humans. Consuming too much fructose can result in mitochondrial dysfunction and metabolic disorders. 6-Gingerol, the main component of ginger (Zingiber officinale Roscoe), has been proven to alleviate metabolic disorders. This study seeks to examine the effects of 6-gingerol on metabolic disorders caused by fructose and uncover the underlying molecular mechanisms. In this study, the results showed that 6-Gingerol ameliorated high-fructose-induced metabolic disorders. Moreover, it inhibited CD36 membrane translocation, increased CD36 expression in the mitochondria, and decreased the O-GlcNAc modification of CD36 and OGT expression in vitro and vivo. In addition, 6-Gingerol enhanced the performance of mitochondria in the skeletal muscle and boosted the respiratory capability of L6 myotubes. This study provides a theoretical basis and new insights for the development of lipid-lowering drugs in clinical practice.

Effect of Abiotic Signals on the Accumulation of Saponarin in Barley Leaves in Hydroponics Under Artificial Lights.

Functional flavonoid production is a new agenda in the agricultural industry, and young barley leaves (YBL) are one of the highlighted crops due to their health-beneficial flavonoid, saponarin. For the year-round cultivation of a high saponarin content of YBL, abiotic signal effects on the biosynthesis and metabolism in YBL need to be understood clearly. In this research, the effects of reactive oxygen species (ROS)-related abiotic signals, such as light, potassium, and sodium, were investigated on the biosynthetic metabolism in YBL cultivation under artificial lights. A higher quantity of blue-rich white light (6500 K of light temperature) irradiation enhanced ROS levels and the related enzyme activities (APX and CAT), as well as photosynthesis and saponarin amount, while red-rich white light (3000 K of light temperature) increased the photosynthesis only. In addition, 1.0 g L-1 K+ treatment in water slightly reduced ROS levels and increased saponarin accumulation in YBL. These blue-rich light and K+ supplemental conditions relatively increased OGT expression and reduced 4-coumaric acid and isovitexin as saponarin precursors. Furthermore, the relative ratio of lutonarin as an oxidized product of saponarin increased in increments of light quantity. Finally, the abiotic conditions for saponarin production were optimized with the mixture solution treatment of 1.0 g L-1 Na+ and 1.0 g L-1 K+ under 500 PPFD of 6500 K light, and the saponarin amount per leaf was 219.5 μg plant-1; it was comparable amount with that under sunlight condition.

The oncogenic role and regulatory mechanism of PGK1 in human non-small cell lung cancer

BackgroundPhosphoglycerate kinase 1 (PGK1) is a metabolic enzyme that participates in various biological and pathological processes. Dysregulated PGK1 has been observed in numerous malignancies. However, whether and how PGK1 affects non-small cell lung cancer (NSCLC) is not yet fully elucidated.MethodsHerein, the non-metabolic function of PGK1 in NSCLC was explored by integrating bioinformatics analyses, cellular experiments, and nude mouse xenograft models. The upstream regulators and downstream targets of PGK1 were examined using multiple techniques such as RNA sequencing, a dual-luciferase reporter assay, Co-immunoprecipitation, and Western blotting.ResultsWe confirmed that PGK1 was upregulated in NSCLC and this upregulation was associated with poor prognosis. Further in vitro and in vivo experiments demonstrated the promoting effects of PGK1 on NSCLC cell growth and metastasis. Additionally, we discovered that PGK1 interacted with and could be O-GlcNAcylated by OGT. The inhibition of PGK1 O-GlcNAcylation through OGT silencing or mutation at the T255 O-GlcNAcylation site could weaken PGK1-mediated NSCLC cell proliferation, colony formation, migration, and invasion. We also found that a low miR-24-3p level led to an increase in OGT expression. Additionally, PGK1 exerted its oncogenic properties by augmenting ERK phosphorylation and MCM4 expression.ConclusionsPGK1 acted as a crucial mediator in controlling NSCLC progression. The miR-24-3p/OGT axis was responsible for PGK1 O-GlcNAcylation, and ERK/MCM4 were the downstream effectors of PGK1. It appears that PGK1 might be an attractive therapeutic target for the treatment of NSCLC.

Abstract 14841: Perm1 is a Novel Regulator of O-GlcNAcylation in the Heart

Background. Perm1 is a striated-muscle specific protein that is predominantly expressed in cardiac and skeletal muscle. We recently demonstrated that Perm1 is downregulated in the human and mouse failing hearts. However, whether and how Perm1 regulates cardiac function remains unknown. Beta-O-linkage of N-acetylglucosamine (O-GlcNAc) post-translational modification plays a role in stress response, and the excessive O-GlcNAc in the heart leads to the development of heart failure. We hypothesize that Perm1 regulates cardiac function through O-GlcNAcylation. Methods and Results. Our newly generated Perm1-knockout (KO) mice exhibited reduced cardiac function as compared to their wild-type (WT) littermates (50.1% vs. 34.7% in ejection fraction, p<0.05, n=15/group), which was associated with a significant increase in the total levels of O-GlcNAcylated proteins (139% from WT, p<0.05, n=6 in WT, n=8 in KO). To determine whether Perm1 downregulation increases the vulnerability to pressure overload, WT and Perm1 heterozygous (Perm1 +/- ) mice were subjected to transverse aortic constriction (TAC) surgery for 1 week. The total O-GlcNAcylated protein levels were markedly increased in Perm1 +/- -TAC mice as compared to WT-TAC mice (549% vs. 173% from non-TAC WT, p<0.05, Fig.1A, B), concurrent with lower ejection fraction than WT-TAC mice (50.0% vs. 41.5%, p=0.06, n=5 in WT-TAC, n=3 in Perm1 +/- -TAC). The excessive O-GlcNAc in Perm1 +/- mice was associated with upregulation of O-GlcNAc transferase (OGT, the enzymes that adds O-GlcNAc to proteins, p<0.05, Fig1A, C), while there was no significant difference in the expression of O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc from proteins) in WT vs. Perm1 +/- mice in post-TAC (Fig.1A, D). Conclusions. These data suggest that Perm1 suppresses excessive O-GlcNAcylation occurring in a stressed heart through regulating OGT expression. Hence, the maintenance of Perm1 expression may be protective in the failing heart.

Blocking ATM Attenuates SKOV3 Cell Proliferation and Migration by Disturbing OGT/OGA Expression via hsa-miR-542-5p.

Blocking ataxia telangiectasia mutated (ATM), a crucial player in DNA repair responses, has been proposed as a promising strategy in anti-cancer therapy. Most previous studies have focused on DNA damage response-related pathways after administration of ATM inhibitors. However, ATM inhibition could potentially influence a wide range of changes in gene expression, which remain poorly defined. Here, we report that administration of the ATM inhibitor KU60019 led to impaired migration and enhanced apoptosis in the ovarian cancer cell line SKOV3, accompanied by abnormally elevated O-GlcNAc transferase and O-GlcNAcase expression levels. In addition, KU60019 treatment significantly suppressed expression of hsa-miR-542-5p in SKOV3 cells. Up-regulation of hsa-miR-542-5p expression inhibited increases in OGT and OGA level, and reversed the effects of ATM inhibition on apoptosis and migration in SKOV3 cells. Finally, we found aberrant expression of OGT and OGA to be associated with ovarian cancer patient survival. Taken together, our results suggest that ATM inhibition may promote SKOV3 cell apoptosis via suppressing hsa-miR-542-5p and elevating OGT and OGA expression, providing new insights into the application of ATM inhibitors in cancer immunotherapy.

Ogt Demonstrated Conspicuous Clinical Significance in Cancers, from Pan-Cancer to Small-Cell Lung Cancer.

The clinical progression of small-cell lung cancer (SCLC) remains pessimistic. The aim of the present study was to promote the understanding of the clinical significance and mechanism of O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) in SCLC. Wilcoxon tests, standardized mean difference (SMD), and Kruskal–Wallis tests were utilized to compare OGT level differences among the experimental and control groups. The univariate Cox regression analysis, Kaplan–Meier curves, and receiver operating characteristic curves were applied to determine OGT's clinical relevance in cancers. The Spearman correlation analysis and enrichment analysis were utilized to explore the underlying mechanisms of OGT in cancers. For the first time in the field, we provide an overview of OGT in 32 cancers using a large number of samples (n = 21,196), determining distinct OGT expression in 25 cancers and its prognosis effects in 12 cancers. Furthermore, using 950 samples from multiple sources, upregulated OGT was found in both mRNA and protein levels in SCLC (SMD = 0.93, 95% CI [0.24, 1.63]). Higher OGT levels represented a more unfavorable disease-free interval for SCLC patients (p < 0.001). The research also identified OGT expression as a potential marker for SCLC prediction (sensitivity = 0.79, specificity = 0.86, and AUC = 0.88). The high expression of OGT in SCLC may result from the positive regulation of two transcription factors—DEK and XRN2. We primarily investigated the underlying mechanisms of OGT in SCLC. Herein, based on the analyses from pan-cancer to SCLC, OGT demonstrated conspicuous clinical significance. OGT may be an underlying biomarker for the treatment and identification of some cancers, including SCLC.

Sustained acylated ghrelin treatment lowers visceral adipose tissue insulin signalling in association with higher ogt expression in uremic rats

Rationale: Recent evidence shows that in chronic kidney disease (CKD), as in starvation, visceral adipose tissue (VAT) insulin sensitivity is reduced, in association with lower lipogenesis and increased lipolysis, contributing to promote energy availability. O-linked N-acetylglucosamine transferase (OGT) is a novel negative modulator of VAT insulin sensitivity which also promotes lipolysis and fat mass loss. Acylated ghrelin (AG) is a gastric orexigenic hormone and a relevant tissue-specific modulator of intermediate metabolism. It increases insulin signalling in skeletal muscle, while in it limits fat accumulation and reduces insulin signalling in the liver. The potential role of AG and OGT in VAT metabolism as well as their interaction in CKD is unknown.

Oncogenic microRNA-181d binding to OGT contributes to resistance of ovarian cancer cells to cisplatin

Ovarian cancer (OC), a common gynecological cancer, is characterized by a high malignant potential. MicroRNAs (miRNAs or miRs) have been associated with the chemo- or radiotherapeutic resistance of human malignancies. Herein, the current study set out to explore the regulatory mechanism of miR-181d involved in the cisplatin (DDP) resistance of OC cells. Firstly, in-situ hybridization method was performed to identify miR-181d expression in ovarian tissues of DDP-resistant or DDP-sensitive patients. In addition, miR-181d expression in A2780 cells and A2780/DDP cell lines was determined by RT-qPCR. Gain- and loss-of-function experiments were then performed to characterize the effect of miR-181d on OC cell behaviors. We probed the miR-181d affinity to OGT, as well as the downstream glycosylation of KEAP1 and ubiquitination of NRF2. Further, in vivo experiments were performed to define the role of miR-181d in tumor resistance to DDP. miR-181d was highly expressed in the ovarian tissues of DDP-resistant patients and the A2780/DDP cell line. Ectopic expression of miR-181d augmented DDP resistance in OC cells. In addition, miR-181d was found to target the 3′UTR of OGT mRNA, and negatively regulate the OGT expression. Mechanistic results indicated that OGT repressed NRF2 expression through glycosylation of KEAP1, thereby inhibiting the DDP resistance of OC cells. Furthermore, miR-181d negatively orchestrated the OGT/KEAP1/NRF2 axis to enhance the OC resistance to DDP in vivo. Overall, these findings suggest that miR-181d-mediated OGT inhibition restricts the glycosylation of KEAP1, and then reduces the ubiquitination and degradation of NRF2, leading to DDP resistance of OC. This study provides new insights for prevention and control of OC.

P53 suppresses the progression of hepatocellular carcinoma via miR-15a by decreasing OGT expression and EZH2 stabilization.

Existing literature has highlighted the tumour suppressive capacity of microRNA‐15a (miR‐15a); however, its role in hepatocellular carcinoma (HCC) remains relatively unknown. This study aimed to investigate the role of miR‐15a in HCC and the associated underlying mechanism. Initially, RT‐qPCR was performed to detect the expression of miR‐15a in HCC tissues and cells. Bioinformatics analysis, Pearson correlation coefficient, dual‐luciferase reporter assay, and molecular approaches were all conducted to ascertain the interaction between miR‐15a and O‐linked N‐acetylglucosamine (GlcNAc) transferase (OGT). PUGNAc treatment and cycloheximide (CHX) assay were performed to evaluate O‐GlcNAc and the stabilization of the enhancer of zeste homolog 2 (EZH2). Finally, gain‐ and loss‐of‐function studies were employed to elucidate the role of P53 and the miR‐15a/OGT/EZH2 axis in the progression of HCC, followed by in vivo experiments based on tumour‐bearing nude mice. Our results demonstrated that the miR‐15a expression was decreased in the HCC tissues and cells. P53 upregulated miR‐15a expression, which inhibited the proliferation, migration and invasion of HCC cells, while inducing apoptosis and triggering a G0/G1 cell cycle phase arrest. OGT stabilized EZH2 via catalysing O‐GlcNAc, which reversed the effect of P53 and miR‐15a. The results of our in vivo study provided evidence demonstrating that P53 could suppress the development of HCC via the miR‐15a/OGT/EZH2 axis. P53 was found to inhibit the OGT expression by promoting the expression of miR‐15a, which destabilized EZH2 and suppressed the development of HCC. The key findings of our study highlight a promising novel therapeutic strategy for the treatment of HCC.

OGT regulated O-GlcNacylation promotes migration and invasion by activating IL-6/STAT3 signaling in NSCLC cells

BackgroundO-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) is a key enzyme that regulates O-GlcNAc modification, which is significantly up-regulated and participates in the regulation of tumorigenesis. Although previous research indicated that OGT promotes epithelial-mesenchymal transition (EMT) of lung cancer, the underlying molecular mechanisms, especially within the tumor inflammatory microenvironment, require further elucidation. MethodsThe role of the inflammatory signaling Interleukin 6/Signal Transducer and activator of transcription 3 (IL-6/STAT3) in Non-small cell lung cancer (NSCLC) cells A549 were confirmed by Transwell assay, Scratch wound healing assay, Western blot, Immunofluorescence staining, and Nuclear and cytoplasmic extraction experiment. Western blot detected OGT expression and whole protein O-GlcNacylation after IL-6 stimulation in NSCLCs cells. The biological effects and related mechanism of OGT in NSCLC cells were investigated by Western blot, Transwell assay, Immunofluorescence staining and Immunoprecipitation. The up-stream mechanism of OGT expression was explored by employing the specific chemical inhibitors, and the expression and distribution of OGT and phosphorylated STAT3 in NSCLC samples were confirmed by immunohistochemical analysis. ResultsIL-6/STAT3 promoted the migration and invasion of NSCLC cells. IL-6 stimulation elevated OGT expression and the total protein O-GlcNacylation in A549 cells. Silencing OGT by shRNA significantly inhibited the IL-6 induced EMT marker (N-cadherin and Slug) expression, migration and invasion in A549 cells. OGT interacted with and mediated O-GlcNacylation of STAT3, which promoted STAT3 Y705 phosphorylation in IL-6 treated NSCLC cells. OGT expression was positively regulated by NF-κB p65 signaling pathway after IL-6 stimulation, instead of STAT3 signaling. OGT and phosphorylated STAT3 had an obviously higher expression in human NSCLC tissues, and phosphorylated STAT3 was mainly expressed in the nucleus. ConclusionThe above results showed that OGT regulated O-GlcNacylation promoted migration and invasion by activating IL-6/STAT3 signaling in lung cancer.

Hypertonic stress modulates eNOS function through O-GlcNAc modification at Thr-866

O-GlcNAcylation, an energy-sensitive posttranslational modification, can regulate the activity of endothelial nitric oxide synthase (eNOS). Previous studies found that Thr866 is the key site for low-glucose-mediated regulation of eNOS O-GlcNAc. However, it is not known whether this activity functions through the Thr866 site concomitant with other physical and chemical factors. Therefore, we first explored the effects of physical and chemical factors on eNOS O-GlcNAc and its Thr866 site. In this study, hypertonic stress, hyperthermia and hydrogen peroxide all increased the expression levels of eNOS O-GlcNAc, whereas hypoxia and high levels of alcohol had no effect. on the expression levels of eNOS O-GlcNAc; by contrast, low pH led to a decrease in eNOS O-GlcNAc levels. Notably, eNOS O-GlcNAc protein levels were unchanged after Thr866 site mutation only under hypertonic conditions, suggesting that hypertonic stress may act through the Thr866 site. Upon exploring the mechanism of hypertonic stress on eNOS O-GlcNAc activity and function, we found that hypertonic stress can upregulate the expression of O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), which is dependent on AMPK. When AMPK was knocked out, the upregulation of OGT expression and increased O-GlcNAc modifications induced by hypertonic stress were reversed.

Inhibition of O-GlcNAc Transferase Renders Prostate Cancer Cells Dependent on CDK9.

O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed a specific and potent inhibitor targeting this enzyme, and here, we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II to promote transcription elongation. We show that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from patients with metastatic prostate cancer, but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells, but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer-specific vulnerabilities. IMPLICATIONS: The primary contribution of OGT to cell proliferation is unknown, and in this study, we used a compound screen to indicate that OGT and CDK9 collaborate to sustain a cancer cell-specific pro-proliferative program. A better understanding of how OGT and CDK9 cross-talk will refine our understanding of this novel synthetic lethal interaction.

Intracellular O-linked glycosylation directly regulates cardiomyocyte L-type Ca2+ channel activity and excitation-contraction coupling.

Cardiomyocyte L-type Ca2+ channels (Cavs) are targets of signaling pathways that modulate channel activity in response to physiologic stimuli. Cav regulation is typically transient and beneficial but chronic stimulation can become pathologic; therefore, gaining a more complete understanding of Cav regulation is of critical importance. Intracellular O-linked glycosylation (O-GlcNAcylation), which is the result of two enzymes that dynamically add and remove single N-acetylglucosamines to and from intracellular serine/threonine residues (OGT and OGA respectively), has proven to be an increasingly important post-translational modification that contributes to the regulation of many physiologic processes. However, there is currently no known role for O-GlcNAcylation in the direct regulation of Cav activity nor is its contribution to cardiac electrical signaling and EC coupling well understood. Here we aimed to delineate the role of O-GlcNAcylation in regulating cardiomyocyte L-type Cav activity and its subsequent effect on EC coupling by utilizing a mouse strain possessing an inducible cardiomyocyte-specific OGT-null-transgene. Ablation of the OGT-gene in adult cardiomyocytes (OGTKO) reduced OGT expression and O-GlcNAcylation by > 90%. Voltage clamp recordings indicated an ~ 40% reduction in OGTKO Cav current (ICa), but with increased efficacy of adrenergic stimulation, and Cav steady-state gating and window current were significantly depolarized. Consistently, OGTKO cardiomyocyte intracellular Ca2+ release and contractility were diminished and demonstrated greater beat-to-beat variability. Additionally, we show that the Cav α1 and β2 subunits are O-GlcNAcylated while α2δ1 is not. Echocardiographic analyses indicated that the reductions in OGTKO cardiomyocyte Ca2+ handling and contractility were conserved at the whole-heart level as evidenced by significantly reduced left-ventricular contractility in the absence of hypertrophy. The data indicate, for the first time, that O-GlcNAc signaling is a critical and direct regulator of cardiomyocyte ICa achieved through altered Cav expression, gating, and response to adrenergic stimulation; these mechanisms have significant implications for understanding how EC coupling is regulated in health and disease.

Expression of Stress-Mediating Genes is Increased in Term Placentas of Women with Chronic Self-Perceived Anxiety and Depression.

Anxiety, chronical stress, and depression during pregnancy are considered to affect the offspring, presumably through placental dysregulation. We have studied the term placentae of pregnancies clinically monitored with the Beck’s Anxiety Inventory (BAI) and Edinburgh Postnatal Depression Scale (EPDS). A cutoff threshold for BAI/EPDS of 10 classed patients into an Index group (>10, n = 23) and a Control group (<10, n = 23). Cortisol concentrations in hair (HCC) were periodically monitored throughout pregnancy and delivery. Expression differences of main glucocorticoid pathway genes, i.e., corticotropin-releasing hormone (CRH), 11β-hydroxysteroid dehydrogenase (HSD11B2), glucocorticoid receptor (NR3C1), as well as other key stress biomarkers (Arginine Vasopressin, AVP and O-GlcNAc transferase, OGT) were explored in medial placentae using real-time qPCR and Western blotting. Moreover, gene expression changes were considered for their association with HCC, offspring, gender, and birthweight. A significant dysregulation of gene expression for CRH, AVP, and HSD11B2 genes was seen in the Index group, compared to controls, while OGT and NR3C1 expression remained similar between groups. Placental gene expression of the stress-modulating enzyme 11β-hydroxysteroid dehydrogenase (HSD11B2) was related to both hair cortisol levels (Rho = 0.54; p < 0.01) and the sex of the newborn in pregnancies perceived as stressful (Index, p < 0.05). Gene expression of CRH correlated with both AVP (Rho = 0.79; p < 0.001) and HSD11B2 (Rho = 0.45; p < 0.03), and also between AVP with both HSD11B2 (Rho = 0.6; p < 0.005) and NR3C1 (Rho = 0.56; p < 0.03) in the Control group but not in the Index group; suggesting a possible loss of interaction in the mechanisms of action of these genes under stress circumstances during pregnancy.

Is The Hexosamine Biosynthesis Pathway Affected By Inflammation In Endothelial Cells?

Excessive addition of β-linked N-acetylglucosamine (O-GlcNAc) via the hexosamine biosynthesis pathway (HBP) occurs with diabetes and decreases in muscles with exercise. HBP flux increases inflammation and may be specifically important in the activation phase of the inflammatory response. Inflammation plays a key role in insulin resistance and hyperglycemia (HG), with the process contributing to the development of diabetes and associated complications. β-cell development and differentiation are affected by the HBP, and O-GlcNAc has been indicated in insulin secretion from the pancreas (1). PURPOSE: We explored whether in the absence of HG, an inflammatory insult, can affect key factors in this pathway and total O-GlcNAcylation within the endothelium. Because African Americans are overrepresented in conditions involving chronic inflammation, potential racial differences were also examined. METHODS: Human umbilical vein endothelial cells (HUVECs), n=6, were stimulated with 10ng/mL of tumor necrosis factor alpha (TNFα) for 4 hours, or treated as a control. Western blot procedures were used to measure expression of the HBP rate-limiting enzyme (GFAT) and the transferase that adds the end-product onto proteins (OGT). Total protein O-GlcNAcylation (O-GlcNAc) was also measured. RESULTS: With TNFα stimulation, there was no difference in GFAT (0.63 + .09 vs 0.62 + .08, p=.89), OGT (0.49 + .09 vs 0.41 + .08, p=.56), nor total O-GlcNAc (1.56 + .38 vs 1.77 + .42, p=.71) expression compared to controls. There were no racial differences found among the various conditions (p>.05) with either of the proteins examined. CONCLUSIONS: The HBP is not altered with acute inflammation in endothelial cells (ECs), with no racial differences additionally found. Low-grade inflammation vitally contributes to diabetes, atherosclerosis, and their complications, with the HBP playing a role in each. We conclude that HG may be necessary to work in conjunction with inflammation to increase protein O-GlcNAcylation in ECs. Although increased flux though the HBP has been shown to upregulate inflammation, the reverse relationship cannot be confirmed. Future research is needed to examine links between this pathway, various conditions, and the ability of exercise to alter it within the endothelium.

SIRT1 regulates O-GlcNAcylation of tau through OGT.

Tau is modified with O-GlcNAcylation extensively in human brain. The O-GlcNAcylation levels of tau are decreased in Alzheimer’s disease (AD) brain. Sirtuin type 1 (SIRT1) is an enzyme that deacetylates proteins including transcriptional factors and associates with neurodegenerative diseases, such as AD. Aberrant SIRT1 expression levels in AD brain is in parallel with the accumulation of tau. cAMP response element binding protein (CREB), a cellular transcription factor, plays a critical role in learning and memory. In this present study, we found SIRT1 deacetylates CREB and inhibits phosphorylation of CREB at Ser133. The inactivated CREB suppresses OGT expression and therefore decreases the O-GlcNAcylation of tau and thus increases the phosphorylation of tau at specific sites. These findings suggest that SIRT1 may be a potential therapeutic target for treating tauopathies.

Regulation of Erythroblast Protein Expression By an RNA Splicing Mechanism, Decoy Exon-Mediated Intron Retention

The erythroid transcriptome is extensively remodeled during terminal erythropoiesis by dynamic changes in RNA splicing of cassette exons and retained introns. Mechanistic studies of these RNA processing networks will provide new insight into pathways that impact structure and function of the erythroid proteome during erythroblast differentiation. We previously showed that up-regulation of EPB41 exon 16 splicing imparts new functionality to the encoded protein, enhancing protein-protein interactions that mechanically strengthen the red cell membrane. More recently, RNA-seq analysis revealed that numerous erythroid transcripts exhibit up-regulation of intron retention (IR) events, some of which are controlled by a decoy exon-mediated mechanism that can reduce the output of translated mRNA so as to limit protein expression. Here we demonstrate that modulation of decoy-mediated IR quantitatively affects protein expression in primary human erythroid cells. We first studied the OGT gene (O-GlcNAc transferase), a key regulator of O-GlcNAC homeostasis. OGT expression responds to pharmacological inhibitors by regulating intron 4 retention, by a mechanism requiring a intronic splicing silencer (1) that functions as a decoy exon (2) to nonproductively engage annotated splice sites at the ends of the intron, thereby blocking excision and enforcing its retention. In erythroid CD34+ progenitors at day 7 of culture, we found that treatment with an OGT inhibitor (OSMI-1) reduced OGT IR and increased spliced OGT RNA and OGT protein. Conversely, an inhibitor of the antagonistic OGA enzyme (thiamet-G) induced greater OGT IR and reduced OGT protein expression. These results are similar to what was reported previously in established cell lines (1), and suggest that modulation of IR can vary mRNA and protein expression in primary cells &amp;gt;5-fold. To further explore the model, we independently blocked OGT IR in erythroid cultures by electroporation of an antisense morpholino directed against the OGT decoy exon 5' splice site. RT-qPCR and western blot analysis confirmed substantial reduction in IR, coupled with an increase in spliced RNA and elevated OGT protein expression, compared to control cells or cells treated with an irrelevant morpholino. The OGT-specific MO also substantially blocked IR induced by thiamet-G. These results show that pharmacological- or antisense-mediated alteration in IR can significantly change protein expression in primary erythroblast cultures. We propose that the abundance of IR transcripts in late erythropoiesis represents a widespread modulation of protein output by post-transcriptional pathways operating at the level of intron retention. Park SK, et al. (2017) Cell Rep. 20: 1088-99.Parra M et al. (2018) RNA 24: 1255-65. Disclosures No relevant conflicts of interest to declare.

64 - O-GlcNAcylation is decreased in aorta and mesenteric artery in normotensive and hypertensive pregnant rats

O-glycosylation by N-acetyl-glucosamine (O-GlcNAc) decreases nitric oxide synthase phosphorylation leading to a lower nitric oxide (NO) bioavailability. We performed the analysis of O-GlcNAc, OGT and OGA expression by Western Blot and OGA activity by biochemical analysis in aortic and mesenteric bed homogenates from non-pregnant (NP) and pregnant (P) Wistar and spontaneously hypertensive rats (SHR) with 12 weeks old (n= 6). Aorta rings or mesenteric artery rings (n= 6), with and without endothelium, or with endothelium in the presence of L-NAME (100 µM) were incubated in the absence or presence of PugNAc (100 μM) and stimulated with cumulative concentrations of PE. The results were compared among groups (Student`s t test or ANOVA, p