Abstract
Many intracellular proteins are reversibly modified by O-linked GlcNAc (O-GlcNAc), a post-translational modification that dynamically regulates fundamental cellular processes in response to diverse environmental cues. Accumulating evidence indicates that both excess and deficiency of protein O-GlcNAcylation can have deleterious effects on the cell, suggesting that maintenance of O-GlcNAc homeostasis is essential for proper cellular function. However, the mechanisms through which O-GlcNAc homeostasis is maintained in the physiologic state and altered in the disease state have not yet been investigated. Here, we demonstrate the existence of a homeostatic mechanism involving mutual regulation of the O-GlcNAc-cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) at the transcriptional level. Specifically, we found that OGA promotes Ogt transcription through cooperation with the histone acetyltransferase p300 and transcription factor CCAAT/enhancer-binding protein β (C/EBPβ). To examine the role of mutual regulation of OGT and OGA in the disease state, we analyzed gene expression data from human cancer data sets, which revealed that OGT and OGA expression levels are highly correlated in numerous human cancers, particularly in pancreatic adenocarcinoma. Using a KrasG12D -driven primary mouse pancreatic ductal adenocarcinoma (PDAC) cell line, we found that inhibition of extracellular signal-regulated kinase (ERK) signaling decreases OGA glycosidase activity and reduces OGT mRNA and protein levels, suggesting that ERK signaling may alter O-GlcNAc homeostasis in PDAC by modulating OGA-mediated Ogt transcription. Our study elucidates a transcriptional mechanism that regulates cellular O-GlcNAc homeostasis, which may lay a foundation for exploring O-GlcNAc signaling as a therapeutic target for human disease.
Highlights
Many intracellular proteins are reversibly modified by Olinked GlcNAc (O-GlcNAc), a post-translational modification that dynamically regulates fundamental cellular processes in response to diverse environmental cues
Aberrant protein O-GlcNAcylation is being identified as a key pathological feature of an increasing number of human disorders; elucidating the mechanisms through which cellular O-GlcNAc homeostasis is maintained in the physiologic state and altered in the disease state is of significant biomedical interest
We previously hypothesized that cellular O-GlcNAcylation levels are maintained within an optimal zone by mutual regulation of the O-GlcNAc– cycling enzymes O-GlcNAc transferase (OGT) and OGA [2]
Summary
O-GlcNAc, O-linked GlcNAc; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; C/EBP, CCAAT/enhancer-binding protein; PDAC, pancreatic ductal adenocarcinoma; ERK, extracellular signal– regulated kinase; HEK, human embryonic kidney; TMG, Thiamet G; ChIPseq, ChIP sequencing; LAP, liver-enriched activator protein; LIP, liver-enriched inhibitor protein; TCGA, The Cancer Genome Atlas; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; FBS, fetal bovine serum; 4MU, 4-methylumbelliferyl; GalNAc, N-acetyl-D-galactosamine; qPCR, quantitative PCR. E, primary hepatocytes isolated from Ogt-floxed mice were transduced with adenoviral vectors expressing GFP or CRE (n ϭ 6), and Ogt and Oga mRNA levels were measured by RT– qPCR. We show that OGT and OGA mRNA levels are highly correlated across a wide range of human cancers, in pancreatic adenocarcinoma. We demonstrate that the OGA–C/EBP axis regulates Ogt expression in primary mouse pancreatic ductal adenocarcinoma (PDAC) cells. We show that loss of extracellular signal–regulated kinase (ERK) signaling in these cells decreases OGA glycosidase activity and reduces OGT mRNA and protein levels, raising the possibility that oncogenic ERK signaling may impinge upon OGA-mediated Ogt transcription to increase OGT expression in pancreatic cancer
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