Abstract
Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked β-N-acetylglucosamine transferase (OGT), which adds an O-linked β-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid–induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression.
Highlights
Glucocorticoids are effective in inflammatory diseases and blood cancers. [1, 2]
Despite evidence of cellular target engagement of O-linked β-N-acetylglucosaminease (OGA) by thiamet-G, there was no evidence of a potentiating effect on glucocorticoid receptor (GR)-mediated transrepression of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-controlled genes or pro-apoptotic effects in glucocorticoid resistant cell lines treated with dexamethasone
Target engagement by the OGA inhibitor was observed in HTS-43 cells using flow cytometry as levels of O-GlcNAcylated protein
Summary
Glucocorticoids are effective in inflammatory diseases and blood cancers. [1, 2]. steroid insensitivity among asthmatics represents a large unmet medical need [3,4,5]. It was concluded that O-GlcNAcylation is required to form the preinitiation complex on the DNA promoter, but that removal of O-GlcNAc by OGA is required to allow phosphorylation and initiation of transcription This would suggest that blocking either enzyme would impair transcription. By association, that the increase in glucocorticoid efficiency following OGT overexpression, as seen by Li’s group, should be observed by the direct inhibition of OGA This would result in the inhibition of O-GlcNAc removal from pol II, impairing transcription. Despite evidence of cellular target engagement of OGA by thiamet-G, there was no evidence of a potentiating effect on GR-mediated transrepression of NF-κB-controlled genes or pro-apoptotic effects in glucocorticoid resistant cell lines treated with dexamethasone (dex) These studies suggest pharmacological inhibition of OGA does not increase sensitivity to glucocorticoid-mediated transrepression
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