O-GlcNAc Transferase Knockdown Research Articles

O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis. We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-β) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation. We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-β1 induced collagen expression/deposition. OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

407 The OGT/O-GlcNAc Axis Regulates Fibrosis Resolution in Idiopathic Pulmonary Fibrosis

OBJECTIVES/GOALS: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by dysregulated collagen accumulation in the lung parenchyma. Our goal is to investigate the role of O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in pulmonary fibrosis to ultimately discover novel therapies for fibrosis resolution. METHODS/STUDY POPULATION: Lung tissue from IPF and non-IPF donors was subjected to immunohistochemistry (IHC) to assess O-GlcNAc levels. Primary human lung fibroblasts were treated with OGT or O-GlcNAcase (OGA) inhibitors followed by transforming growth factor-beta 1 (TGF-β1) stimulation to assess O-GlcNAc regulation of fibroblast-to-myofibroblast transition (FMT) markers [alpha smooth muscle actin (α-SMA) and type 1 and type 3 collagen (COL1α1, COL3α1)] In Drosophila melanogaster, OGT knockdown (KD)/overexpression (OE) was conditionally induced to assess pericardin, a type IV collagen-like protein, regulation by immunofluorescence. Lastly, a mouse model of bleomycin-induced pulmonary fibrosis was examined following OGT KD and assessed for fibrosis resolution via histology, hydroxyproline assay, and western blotting. RESULTS/ANTICIPATED RESULTS: O-GlcNAc staining was increased in IPF lung tissue compared to non-IPF control lungs. In primary human lung fibroblasts, TGF-α1 administration resulted in increased FMT markers (α-SMA, COL1α1, and COL3α1), which were reduced or increased by OGT or OGA inhibition, respectively. Genetic manipulation in the Drosophila models showed decreased pericardin expression with OGT KD compared to the wild-type, whereas OGT OE increased pericardin compared to control. Additionally, OGT KD in bleomycin treated aged mice resulted in reduced collagen levels at the transcript and protein level and concurrent fibrosis resolution as assessed by Masson’s trichrome staining and total hydroxyproline analysis. Collectively, showing OGT/O-GlcNAc regulating collagen in fibrosis resolution. DISCUSSION/SIGNIFICANCE: These data suggest that the OGT/O-GlcNAc axis regulates collagen deposition in pulmonary fibrosis, and we show that O-GlcNAc is implicated in the pathogenesis of IPF. We identified OGT as a therapeutic target to overcome current drug limitations, opening new horizons for biomedical treatment.

O-GlcNAc regulates the mitochondrial integrated stress response by regulating ATF4.

Accumulation of mitochondrial dysfunctional is a hallmark of age-related neurodegeneration including Alzheimer's disease (AD). Impairment of mitochondrial quality control mechanisms leading to the accumulation of damaged mitochondria and increasing neuronal stress. Therefore, investigating the basic mechanisms of how mitochondrial homeostasis is regulated is essential. Herein, we investigate the role of O-GlcNAcylation, a single sugar post-translational modification, in controlling mitochondrial stress-induced transcription factor Activating Transcription Factor 4 (ATF4). Mitochondrial dysfunction triggers the integrated stress response (ISRmt), in which the phosphorylation of eukaryotic translation initiation factor 2α results in the translation of ATF4. We used patient-derived induced pluripotent stem cells, a transgenic mouse model of AD, SH-SY5Y neuroblastoma and HeLa cell-lines to examine the effect of sustained O-GlcNAcase inhibition by Thiamet-G (TMG) on ISRmt using biochemical analyses. We show that TMG elevates ATF4 protein levels upon mitochondrial stress in SH-SY5Y neuroblastoma and HeLa cell-lines. An indirect downstream target of ATF4 mitochondrial chaperone glucose-regulated protein 75 (GRP75) is significantly elevated. Interestingly, knock-down of O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc, in SH-SY5Y increases ATF4 protein and mRNA expression. Additionally, ATF4 target gene Activating Transcription Factor 5 (ATF5) is significantly elevated at both the protein and mRNA level. Brains isolated from TMG treated mice show elevated levels of ATF4 and GRP75. Importantly, ATF4 occupancy increases at the ATF5 promoter site in brains isolated from TMG treated mice suggesting that O-GlcNAc is regulating ATF4 targeted gene expression. Interestingly, ATF4 and GRP75 are not induced in TMG treated familial Alzheimer's Disease mice model. The same results are seen in a human in vitro model of AD. Together, these results indicate that in healthy conditions, O-GlcNAc regulates the ISRmt through regulating ATF4, while manipulating O-GlcNAc in AD has no effect on ISRmt.

O-GlcNAcylation of TLR4 inhibits osteogenic differentiation of periodontal ligament stem cells.

Toll-like receptor 4 (TLR4)-mediated inflammatory responses are associated with diabetes and periodontitis, which are dysregulated by O-GlcNAcylation. This study aimed to investigate the effects of O-GlcNAc transferase (OGT)-mediated TLR4 O-GlcNAcylation on the osteogenesis of periodontal ligament stem cells (PDLCs). PDLCs were treated with high glucose (HG) to establish a cell model. Osteogenic differentiation was evaluated using western blotting, an alkaline phosphatase activity assay, and an alizarin red S staining assay. The regulation of OGT on the O-GlcNAcylation of TLR4 was analyzed using co-immunoprecipitation, immunoprecipitation, western blotting, and immunofluorescence staining. The results showed that HG inhibited osteogenic differentiation and promoted inflammatory response. Knockdown of OGT promoted osteogenic differentiation of HG-treated PDLCs. OGT interacted with TLR4 and increased the O-GlcNAcylation and protein levels of TLR4 in the cytomembrane of PDLCs. Moreover, silenced TLR4 reversed the effects on osteogenic differentiation induced by OGT in HG-treated PDLCs. O-GlcNAcylation of TLR4 induced by OGT suppresses osteogenic differentiation of PDLCs after HG stimulation. The findings suggest a promising strategy for treating DP.

O-GlcNAc transferase promotes glioblastoma by modulating genes responsible for cell survival, invasion, and inflammation

Metabolic reprogramming has emerged as one of the key hallmarks of cancer cells. Various metabolic pathways are dysregulated in cancers, including the hexosamine biosynthesis pathway (HBP). Protein O-GlcNAcylation is catalyzed by the enzyme O-GlcNAc transferase (OGT), an effector of HBP that is found to be upregulated in most cancers. Post-translational O-GlcNAcylation of various signaling and transcriptional regulators could promote cancer cell maintenance and progression by regulating gene expression, as gene-specific transcription factors and chromatin regulators are among the most highly O-GlcNAcylated proteins. Here, we investigated the role of OGT in glioblastoma. We demonstrate OGT knockdown and chemical inhibition led to reduced glioblastoma cell proliferation and downregulation of many genes known to play key roles in glioblastoma cell proliferation, migration, and invasion. We show genes downregulated due to OGT reduction are also known to be transcriptionally regulated by transcriptional initiation/elongation co-factor BRD4. We found BRD4 to be O-GlcNAcylated in glioblastoma cells, however OGT knockdown/inhibition neither changed its expression nor its chromatin association on promoters. Intriguingly, we observed OGT knockdown led to reduced Pol II-Ser2P chromatin association on target genes independent of BRD4 and without affecting other transcription initiation/elongation factors. Finally, we found chemical inhibition of BRD4 potentiated the effects of OGT inhibition in reducing glioblastoma cell proliferation, invasion, and migration. We propose BRD4 and OGT act independently in the transcriptional regulation of a common set of genes and that combined inhibition of OGT and BRD4 could be utilized therapeutically for more efficient glioblastoma cell targeting than targeting of either protein alone.

O-GlcNAcylation of NLRP3 Contributes to Lipopolysaccharide-Induced Pyroptosis of Human Gingival Fibroblasts.

Periodontitis is a leading chronic oral disorder and poses a serious burden on public health. O-GlcNAc glycosylation (O-GlcNAcylation) is regulated only by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) and participates in the regulation of human gingival fibroblasts (HGFs) function. Hence, the purpose of this study is to investigate whether HGFs cell function and periodontitis pathogenesis are regulated by O-GlcNAcylation. Herein, we first established cell model of periodontitis induced by lipopolysaccharide (LPS). The cell viability was measured with CCK-8 assay. Pyroptosis was measured by flow cytometry and western blot. The inflammatory factors levels were detected with ELISA kits. Afterward, our findings indicated that LPS elevated the O-GlcNAcylation level of HGFs and inhibition of O-GlcNAcylation improved LPS-induced pyroptosis of HGFs. Mechanistically, LPS heightened the expression of OGT to induce the O-GlcNAcylation of NLRP3. Subsequently, we certified that Thr542 was the O-GlcNAcylation site of NLRP3. More importantly, upregulation of NLRP3 reversed the effects of OGT knockdown on LPS-induced pyroptosis. In general, the current research demonstrated that LPS contributed to the pyroptosis of HGFs by enhancing the OGT expression to promote O-GlcNAcylation of NLRP3, which suggested that O-GlcNAcylation of NLRP3 was a driving factor for periodontitis and offered a novel insight into the treatment of this disease.

O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer's disease.

Aberrant activation of Extracellular Signal-Regulated Kinase (ERK) signaling is associated with Alzheimer's disease (AD) pathogenesis. For example, enhanced ERK signal activation mediated by Apolipoprotein E4 (APOE4), which is a critical genetic risk factor for AD, increases the transcription of amyloid precursor protein (APP). We hypothesize that O-linked N-acetylglucosamine (O-GlcNAc) regulates the phosphorylation and activation of ERK. O-GlcNAc is a single sugar post-translational modification that dynamically cycles on and off proteins in response to nutrient changes by the action of the enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, O-GlcNAc quickly returns to a baseline level after stimulus removal (called O-GlcNAc homeostasis). We did a serum reactivation time-course followed by western blot in SH-SY5Y neuroblastoma cells after long-term O-GlcNAcase (OGA) inhibition by Thiamet-G (TMG) treatment, O-GlcNAc transferase (OGT) knock-down (KD) and OGA KD. Brain tissues of C57BL6/J mice and 5XFAD Alzheimer's disease mice intra-peritoneally injected with TMG for 1 month and C57BL6/J mice intra-peritoneally injected with TMG for 6 months were also used for western blot. We found that ERK1/2 phosphorylation at Thr 202/Tyr204 and Thr183/Tyr185 (p-ERK) are amplified and hence ERK1/2 are activated after long-term OGA inhibition in SH-SY5Y cells. In addition to pharmacological treatment, genetic disruption of O-GlcNAc by OGT KD and OGA KD also increased p-ERK in SH-SY5Y cells suggesting O-GlcNAc homeostasis controls ERK signaling. To determine how O-GlcNAc regulates p-ERK, we probed the expression of phosphorylated mitogen-activated protein kinase-kinase (p-MEK) which phosphorylates and activates ERK and Dual specificity phosphatase-4 (DUSP4) which dephosphorylates and inactivates ERK in SH-SY5Y cells. p-MEK increases in TMG treated and OGT KD cells whereas total DUSP4 decreases in OGT KD and OGA KD cells with serum reactivation time course. Next, we probed the role of OGA inhibition in regulating ERK activation using mice brain-tissue samples. Interestingly, 6-month intra-peritoneal TMG injection in C57BL/6J mice showed an increase in amplitude of p-ERK and APP protein levels, indicating long-term OGA inhibition potentially contributes to AD progression. Furthermore, 1-month TMG injection was sufficient to increase the amplitude of p-ERK in 5XFAD AD mice brains suggesting AD phenotype contributes to the acceleration of ERK activation mediated by OGA inhibition. Together, these results indicate that disruptions to O-GlcNAc homeostasis amplify ERK signal activation in AD.

O‑GlcNAcylation mediates endometrial cancer progression by regulating the Hippo‑YAP pathway.

The incidence of endometrial cancer (EC) is rapidly increasing worldwide. The majority of endometrial cancers are diagnosed at an early stage and are associated with a good prognosis; however, patients with advanced‑stage EC have a poor prognosis and present with invasive metastasis. The mechanisms responsible for the invasion and metastasis of endometrial cancer remain unknown. Here, the present study aimed to examine the effects of O‑GlcNAcylation on the malignancy of EC and its association with Yes‑associated protein (YAP). It was found that the expression of O‑GlcNAc transferase (OGT) and O‑GlcNAcylation were increased in EC tissues; the decrease in O‑GlcNAcylation levels was found to lead to the decreased proliferation, migration and invasion of EC cells. Mass spectrometric analysis revealed that OGT knockdown reduced the O‑GlcNAcylation of YAP. Furthermore, it was found that the reduction in the O‑GlcNAcylation of YAP promoted its phosphorylation, which in turn inhibited the access of YAP to the nucleus and downstream target gene activation, demonstrating that the level of O‑GlcNAcylation affects the development of EC. On the whole, the findings of the present study indicate that YAP is a key molecule linking the O‑GlcNAcylation and Hippo pathways, which together regulate the progression of EC.

O-GlcNAcylation of RAB10 promotes hepatocellular carcinoma progression.

Ras-related protein Rab-10 (RAB10) is involved in tumorigenesis and progression of hepatocellular carcinoma (HCC). Here, we found RAB10, O-GlcNAc transferase (OGT), and O-GlcNAcylation were upregulated in HCC. In addition, RAB10 protein level was prominently positively correlated with the expression of OGT. O-GlcNAcylation modification of RAB10 was then investigated. Here we showed that RAB10 interacts directly with OGT in HCC cell lines, Meanwhile, O-GlcNAcylation enhanced RAB10 protein stability. Furthermore, knockdown of OGT suppressed aggressive behaviors of HCC in vitro and in vivo, while elevated RAB10 reversed these. Taken together, these results indicated that OGT mediated O-GlcNAcylation stabilized RAB10, thus accelerating HCC progression.

Glutamate shall not pass: a mechanistic role for astrocytic O-GlcNAc transferase in stress and depression.

Major depressive disorder, characterized by aberrant glutamatergic signaling in the prefrontal cortex (PFC), is a leading cause of disability worldwide. Depression is highly comorbid with metabolic disorders, but a mechanistic link is elusive. In this issue of the JCI, Fan and coauthors report that elevated posttranslational modification with the glucose metabolite N-acetylglucosamine (GlcNAc) by O-GlcNAc transferase (OGT) contributed to stress-induced establishment of depression-like behaviors in mice. This effect was specific to medial PFC (mPFC) astrocytes, with glutamate transporter-1 (GLT-1) identified as an OGT target. Specifically, O-GlcNAcylation of GLT-1 resulted in diminished glutamate clearance from excitatory synapses. Further, astrocytic OGT knockdown restored stress-induced deficits in glutamatergic signaling, promoting resilience. These findings provide a mechanistic link between metabolism and depression and have relevance for antidepressant targets.

Targeted Inhibition of O-Linked β-N-Acetylglucosamine Transferase as a Promising Therapeutic Strategy to Restore Chemosensitivity and Attenuate Aggressive Tumor Traits in Chemoresistant Urothelial Carcinoma of the Bladder.

Acquisition of acquired chemoresistance during treatment cycles in urothelial carcinoma of the bladder (UCB) is the major cause of death through enhancing the risk of cancer progression and metastasis. Elevated glucose flux through the abnormal upregulation of O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) controls key signaling and metabolic pathways regulating diverse cancer cell phenotypes. This study showed that OGT expression levels in two human UCB cell models with acquired resistance to gemcitabine and paclitaxel were significantly upregulated compared with those in parental cells. Reducing hyper-O-GlcNAcylation by OGT knockdown (KD) markedly facilitated chemosensitivity to the corresponding chemotherapeutics in both cells, and combination treatment with OGT-KD showed more severe growth defects in chemoresistant sublines. We subsequently verified the suppressive effects of OGT-KD monotherapy on cell migration/invasion in vitro and xenograft tumor growth in vivo in chemoresistant UCB cells. Transcriptome analysis of these cells revealed 97 upregulated genes, which were enriched in multiple oncogenic pathways. Our final choice of suspected OGT glycosylation substrate was VCAN, S1PR3, PDGFRB, and PRKCG, the knockdown of which induced cell growth defects. These findings demonstrate the vital role of dysregulated OGT activity and hyper-O-GlcNAcylation in modulating treatment failure and tumor aggression in chemoresistant UCB.

The beneficial effect of global O-GlcNAcylation on odontogenic differentiation of human dental pulp cells via mTORC1 pathway

ObjectiveTo investigate whether and how global O-linked N-Acetylglucosamine modification (O-GlcNAcylation), a prevalent nutrient-sensitive post-translation modification, regulates odontogenic differentiation and mineralization in human dental pulp cells (hDPCs). DesignFirst, immunostaining assays on sections of dental pulp tissue were performed to detect the distributions of O-GlcNAcylation and its exclusive enzyme set O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Then global O-GlcNAcylation was determined by anti O-linked N-Acetylglucosamine (RL2) Western blot during odontogenesis of hDPCs. Further, inhibition or knockdown of OGT and OGA were achieved by specific inhibitors or siRNA in vitro, respectively. The odonto-induction effect of O-GlcNAcylation ex vivo was investigated by a subcutaneous transplantation experiment. Moreover, the O-GlcNAc modification of RAPTOR was confirmed by immunoprecipitation. Odontogenic differentiation assays also investigated the indispensable role of RAPTOR during enhanced global O-GlcNAcylation. ResultsThe signals of O-GlcNAc became more enriched in the odontoblasts compared to pulp fibroblasts. During odontogenesis of hDPCs, global O-GlcNAcylation was significantly increased. An increase or decrease of O-GlcNAcylation significantly boosted or blunted odontogenic differentiation, respectively. The fluctuation of O-GlcNAcylation continuously impacted the downstream targets of mTORC1. Consistently, RAPTOR was modified by O-GlcNAcylation, which was necessary for inducing odontogenesis. ConclusionsGlobal O-GlcNAcylation participated in and affected the odontogenic differentiation of hDPCs, which was mediated by the mTORC1 pathway. Thus, targeting O-GlcNAcylation might be a potential therapeutic intervention for pulp repair and regeneration.

Thymidylate synthase O-GlcNAcylation: a molecular mechanism of 5-FU sensitization in colorectal cancer.

Alteration of O-GlcNAcylation, a dynamic posttranslational modification, is associated with tumorigenesis and tumor progression. Its role in chemotherapy response is poorly investigated. Standard treatment for colorectal cancer (CRC), 5-fluorouracil (5-FU), mainly targets Thymidylate Synthase (TS). TS O-GlcNAcylation was reported but not investigated yet. We hypothesize that O-GlcNAcylation interferes with 5-FU CRC sensitivity by regulating TS. In vivo, we observed that combined 5-FU with Thiamet-G (O-GlcNAcase (OGA) inhibitor) treatment had a synergistic inhibitory effect on grade and tumor progression. 5-FU decreased O-GlcNAcylation and, reciprocally, elevation of O-GlcNAcylation was associated with TS increase. In vitro in non-cancerous and cancerous colon cells, we showed that 5-FU impacts O-GlcNAcylation by decreasing O-GlcNAc Transferase (OGT) expression both at mRNA and protein levels. Reciprocally, OGT knockdown decreased 5-FU-induced cancer cell apoptosis by reducing TS protein level and activity. Mass spectrometry, mutagenesis and structural studies mapped O-GlcNAcylated sites on T251 and T306 residues and deciphered their role in TS proteasomal degradation. We reveal a crosstalk between O-GlcNAcylation and 5-FU metabolism in vitro and in vivo that converges to 5-FU CRC sensitization by stabilizing TS. Overall, our data propose that combining 5-FU-based chemotherapy with Thiamet-G could be a new way to enhance CRC response to 5-FU.

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.

O‐GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics‐oriented study

This study aimed to reveal the possible mechanisms by which O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulates osteoblast differentiation using a series of bioinformatics-oriented experiments. To examine the influence of O-GlcNAcylation levels on osteoblast differentiation, osteoblastic MC3T3-E1 cells were treated with O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) inhibitors. Correlations between the levels of O-GlcNAcylation and the expression of osteogenic markers as well as OGT were evaluated by qPCR and western blotting. The O-GlcNAcylated proteins assumed to correlate with Runx2 expression were retrieved from several public databases and used for further bioinformatics analysis. Following the findings of the bioinformatics analysis, intracellular calcium ([Ca2+ ]i ) was monitored in the cells treated with OGT and OGA inhibitors using a confocal laser-scanning microscope (CLS). The interaction effect between O-GlcNAcylation and [Ca2+ ]i on osteogenic marker expression was determined using stable OGT knockdown MC3T3-E1 cells. O-GlcNAcylation was positively associated with osteoblast differentiation. The time-course profile of global O-GlcNAcylated proteins showed a distinctive pattern with different molecular weights during osteoblast differentiation. The expression pattern of several O-GlcNAcylated proteins was significantly similar to that of Runx2 expression. Bioinformatic analysis of the retrieved Runx2-related-O-GlcNAcylated-proteins revealed the importance of [Ca2+ ]i . CLS showed that alteration of O-GlcNAcylation rapidly changed [Ca2+ ]i in MC3T3-E1 cells. O-GlcNAcylation and [Ca2+ ]i showed an interaction effect on the expression of osteogenic markers. OGT knockdown disrupted the [Ca2+ ]i -induced expression changes of osteogenic markers. O-GlcNAcylation interacts with [Ca2+ ]i and elicits osteoblast differentiation by regulating the expression of osteogenic markers.

Sustained O‐GlcNAcylation causes ERK Signal and APP Amplification

Activation of ERK (Extra-cellular Regulated Kinase) signal plays a major role in metabolic diseases such as cancer and Alzheimer's. Ingenuity Pathway Analysis (IPA) of RNA-Seq data from long-term O-GlcNAcase (OGA) enzyme inhibitor- Thiamet-G (TMG) treated SH-SY5Y (neuroblastoma) cells, which elevates O-GlcNAc levels, indicated ERK Signaling as a top upregulated pathway. O-GlcNAcylation consists of the addition of a single N-acetyl-glucosamine residue (GlcNAc) to specific serine/threonine residues of proteins by the enzyme OGT (O-GlcNAc Transferase) and the removal of O-GlcNAc is catalyzed by the enzyme OGA. O-GlcNAc is highly abundant and is known to modulate kinase activity. Thus, to further investigate the mechanism by which O-GlcNAcylation activates ERK, we did a serum reactivation time-course and found that there is an amplification in ERK phosphorylation after long-term TMG treatment in HeLa and SH-SY5Y cells. Next, TGF-β stimulation that specifically activates ERK signaling also indicated an increase in ERK phosphorylation with long-term TMG treatment, confirming a robust ERK activation with TMG treatment. ERK phosphorylation oscillates with time after mitogen activation; therefore, we wanted to see if TMG treatment influenced ERK oscillation for a period of 24 hours. Serum reactivation revealed an oscillation in phosphorylated-ERK expression within a 24-hour period, where it reaches a maximum at 4 hours and slowly decreases at 8,12 and 24 hours upon long-term TMG treatment. Next, we wanted to further evaluate the mechanism by which TMG treatment increases ERK phosphorylation. Of note, we did not measure any O-GlcNAcylation on ERK; hence, we probed the expression of activated mitogen activated protein kinase-kinase (phosphorylated MEK) which phosphorylates ERK and Dual specificity phosphatases (DUSPs), which dephosphorylate ERK. Interestingly, serum reactivation time course showed an increase in both phosphorylated-MEK and total DUSP4 with TMG treatment. In addition to the use of pharmacological OGA inhibitor, we measured the effect of OGT knock-down (KD) and OGA KD on ERK signaling in SH-SY5Y and HeLa cells. There was amplification in ERK phosphorylation even after OGT knock-down (OGT KD) which lowers the level of O-GlcNAcylation, in contrast to long-term TMG treatment which increases O-GlcNAcylation. These data suggest that the cycling of the modification on and off substrates is key to ERK pathway regulation. Recent research also shows APOE4 stimulates the transcription of amyloid precursor protein (APP) via a non-canonical ERK signaling pathway, leading to increased amyloid-beta secretion in Alzheimer's disease pathogenesis. Therefore, we wanted to see if there is an increase in the expression of APP in the long-term TMG treated SH-SY5Y cells after serum reactivation, and we saw an increase in APP expression correlating with amplification of ERK signaling. Further evaluation of these molecular mechanisms to elucidate how O-GlcNAcylation amplifies ERK signaling and understand if O-GlcNAcylation and ERK activation work together to increase the transcription of APP are necessary.

Changes in O‐GlcNAcylation Alter Mitochondrial Function

Mitochondrial dysfunction is a common characteristic of many diseases, such as Alzheimer's disease, diabetes, and cancer. Normal mitochondrial and cellular function is regulated by post-translational modifications (PTM) of proteins, which alter protein function to adapt to changes in the cellular environment. One PTM that plays a key role in protein function is the attachment of a single N-acetylglucosamine (O-GlcNAc) sugar to nuclear, mitochondrial, or cytoplasmic proteins at their serine or threonine residues. This process, known as O-GlcNAcylation, is catalyzed by only two enzymes: O-GlcNAc transferase (OGT), which attaches the sugar, and O-GlcNAcase (OGA), which removes the sugar. O-GlcNAcylation is sensitive to changes in various metabolic pathways, such as the metabolism of glucose, fatty acids, and amino acids. Thus, this modification is necessary for maintaining cellular homeostasis. Our previous data showed that changes in O-GlcNAcylation, such as loss of OGT or inhibition of OGA via thiamet-G (TMG) treatment in the liver, showed disruption of the electron transport chain (ETC); however, we still do not know which complexes are most affected by these changes. One potential explanation lies in the relationship between O-GlcNAcylation and acetylation, as acetylation is a highly abundant mitochondrial PTM responsible for the regulation of many components of the ETC. A variety of cell line knockouts and animal models were studied to more fully understand the role of O-GlcNAcylation in regulating mitochondrial function and acetylation. We generated a stable cell line in SH-SY5Y human neuroblastoma cells with shRNA mediated knockdown of OGT. These cells demonstrated an increase in acetylation in the mitochondria as a result of the loss of OGT. Further analysis also showed changes in the expressions of two proteins that are critical for mitochondrial acetylation: acetyl-coenzyme A synthetase 2-like (ACSS1) and NAD-dependent deacetylase sirtuin-3 (SIRT3). Taken together, these findings suggest that reduced O-GlcNAcylation alters the mitochondrial proteome including proteins that are regulated via acetylation. We also observed that oxidative phosphorylation rates were altered in the OGT knockdown cell lines, demonstrating alterations in mitochondrial function due to the reduction of OGT. To further elucidate the role of O-GlcNAcylation in the electron transport chain, ETC complex assays were run to assess changes in complex function due to different levels of O-GlcNAcylation. SH-SY5Y cells treated with and without TMG expressed different levels of complex activity, providing key insights about the importance of O-GlcNAcylation in the ETC. Based on these findings, more studies are needed to understand how O-GlcNAcylation and acetylation work in tandem to maintain electron transport chain function.

OGT knockdown counteracts high phosphate-induced vascular calcification in chronic kidney disease through autophagy activation by downregulating YAP

AimsPathological vascular calcification (VC), a major risk factor for cardiovascular mortality, is a highly prevalent finding in patients with chronic kidney disease (CKD). We previously analyzed several pathways protecting against high phosphate-induced VC through induction of autophagy. Here, we explored how O-GlcNAc transferase (OGT) affected high phosphate-induced VC of CKD though mediation of autophagy. Main methodsIn the rats with CKD induced by 5/6 nephrectomy, the VC process was accelerated by a high phosphate diet. The calcification of vascular smooth muscle cells (VSMCs) was induced by high phosphate treatment. We then experimentally tested the effect of OGT on high phosphate-induced VC by conducting loss-of-function experiments. Co-immunoprecipitation and GST pull-down assays were performed to evaluate interaction between OGT and Yes-associated protein (YAP). In mechanistic studies of this pathway, we measured autophagy protein expression and autophagosome formation, as well as calcium deposition and calcium content in VSMCs and in vivo in response to altered expression of OGT and/or YAP. Key findingsOGT was up-regulated in high phosphate-induced VC models in vitro and in vivo. High phosphate-induced calcification in the rat aorta and VSMCs were suppressed by OGT silencing. OGT promoted the glycosylation of YAP to enhance its stability. Importantly, over-expressing YAP reduced autophagy and OGT expedited high phosphate-induced VC by inhibiting autophagy through upregulation of YAP. SignificanceOGT silencing downregulated YAP to induce autophagy activation, thus suppressing high phosphate-induced VC, which highlighted a promising preventive target against high phosphate-induced VC in CKD.

Regulation of pancreatic cancer TRAIL resistance by protein O-GlcNAcylation

AbstractTRAIL-activating therapy is promising in treating various cancers, including pancreatic cancer, a highly malignant neoplasm with poor prognosis. However, many pancreatic cancer cells are resistant to TRAIL-induced apoptosis despite their expression of intact death receptors (DRs). Protein O-GlcNAcylation is a versatile posttranslational modification that regulates various biological processes. Elevated protein O-GlcNAcylation has been recently linked to cancer cell growth and survival. In this study, we evaluated the role of protein O-GlcNAcylation in pancreatic cancer TRAIL resistance, and identified higher levels of O-GlcNAcylation in TRAIL-resistant pancreatic cancer cells. With gain- and loss-of-function of the O-GlcNAc-adding enzyme, O-GlcNActransferase (OGT), we determined that increasing O-GlcNAcylation rendered TRAIL-sensitive cells more resistant to TRA-8-induced apoptosis, while inhibiting O-GlcNAcylation promoted TRA-8-induced apoptosis in TRAIL-resistance cells. Furthermore, we demonstrated that OGT knockdown sensitized TRAIL-resistant cells to TRA-8 therapy in a mouse model in vivo. Mechanistic studies revealed direct O-GlcNAc modifications of DR5, which regulated TRA-8-induced DR5 oligomerization. We further defined that DR5 O-GlcNAcylation was independent of FADD, the adapter protein for the downstream death-inducing signaling. These studies have demonstrated an important role of protein O-GlcNAcylation in regulating TRAIL resistance of pancreatic cancer cells; and uncovered the contribution of O-GlcNAcylation to DR5 oligomerization and thus mediating DR-inducing signaling.

Epigenetic activation of O-linked β-N-acetylglucosamine transferase overrides the differentiation blockage in acute leukemia.

BackgroundOverriding the differentiation blockage in acute myeloid leukemia (AML) is the most successful mode-of-action in leukemia therapy – now curing the vast majority of patients with acute promyelocytic leukemia (APL) using all-trans retinoic acid (ATRA)-based regimens. Similar approaches in other leukemia subtypes, such as IDH1/2-mutated AML, are under active investigation. We herein present successful release of the differentiation blockage upon treatment with the natural (−)-Δ9-Tetrahydrocannabinol isomer dronabinol in vitro and in vivo.MethodsCellular maturation and differentiation were followed in two patients employing whole genome methylation profiling, proteome analyses, NGS deep sequencing and multispectral imaging flow cytometry. For functional studies lentiviral OGT knock-down in vitro and ex vivo cell models were created to evaluate proliferative, apoptotic and differentiating effects of OGT in acute leukemia.FindingsIn here, we provide molecular evidence that dronbinol is capable to override the differentiation blockage of acute leukemia blasts at the state of the leukemia-initiating clone. We further identify the O-linked β-N-acetyl glucosamine (O-GlcNAc) transferase (OGT) to be crucial in this process. OGT is a master regulator enzyme adding O-GlcNAc to serine or threonine residues in a multitude of target proteins. Aberrant O-GlcNAc modification is implicated in pathologies of metabolic, neurodegenerative and autoimme diseases as well as cancers. We provide evidence that dronabinol induces transcription of OGT via epigenetic hypomethylation of the transcription start site (TSS). A lentiviral OGT-knock out approach proves the central role of OGT exerting antileukemic efficacy via a dual-mechanism of action: High concentrations of dronabinol result in induction of apoptosis, whereas lower concentrations drive cellular maturation. Most intriguingly, overriding of the differentiation blockage of acute leukemia blasts is validated in vivo following two patients treated with dronabinol.InterpretationIn conclusion, we provide evidence for overcoming the differentiation blockage in acute leukemia in subentities beyond promyelocytic and IDH1/2-mutated leukemia and thereby identify O-GlcNAcylation as a novel (drugable) field for future leukemia research.FundingUnrestricted grant support by the IZKF Program of the Medical Faculty Tübingen (MMS) and Brigitte Schlieben-Lange Program as well as the Margarete von Wrangell Program of the Ministry of Science, Research and the Arts, Baden-Württemberg, Germany (KKS) and Athene Program of the excellence initiative University of Tübingen (KKS).