O-GlcNAc transferase regulates H2O2 production via p38 MAPK

Abstract

i.Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by augmented transforming growth factor-{beta} (TGF-{beta}) signaling leading to excessive extracellular matrix (ECM) deposition. The fibroblast-to-myofibroblast-transition (FMT) and metabolic reprogramming of lung fibroblasts (HLFs) are essential to IPF pathogenesis, yet the connection between nutrient metabolism and fibrogenesis remains poorly defined. The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a nutrient-sensitive enzyme that adds O-GlcNAc moieties to substrates. We previously showed that loss of OGT reverses bleomycin-induced pulmonary fibrosis in mice. Here, using unbiased kinomics, we show that pharmacologic inhibition of OGT suppressed non-canonical TGF-{beta}-induced mitogen-activated protein kinase (MAPK) signaling. Molecular confirmation revealed that TGF-{beta}-induced phosphorylation of p38, but not ERK or JNK, was reduced by OGT blockade. Furthermore, p38 itself was O-GlcNAc-modified, which enhanced its phosphorylation and promoted downstream phosphorylation of the NADPH oxidase subunit, p47phox. Inhibition of OGT, p38, or p47phox reduced reactive oxygen species (ROS) in HLFs, revealing a previously unknown role of OGT-p38-p47phox signaling in ROS production. Collectively, this work establishes that O-GlcNAc-modified p38 enhances p47phox-dependent H2O2 production. HighlightsO_LIUsing PamChip STK arrays, we show that OGT inhibition causes broad kinomic remodeling, including suppression of non-canonical TGF-{beta} MAPKs and multiple CDKs. C_LIO_LIOGT blockade selectively attenuates p38 phosphorylation, despite TGF-{beta}-induced substrate redundancy with ERK and JNK. C_LIO_LIWe provide evidence that p38 MAPK undergoes O-GlcNAcylation in human lung fibroblasts, a modification not previously reported. C_LIO_LIThe study identifies a new signaling axis where O-GlcNAc modification of p38 modulates the phosphorylation of p47phox, therefore regulating NOX-dependent H2O2 production. C_LIO_LIBlocking OGT or inhibiting p38/p47phox dramatically reduces TGF-{beta}-driven H2O2 production in human lung fibroblasts. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/728188v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@114210eorg.highwire.dtl.DTLVardef@8647b8org.highwire.dtl.DTLVardef@1ce814corg.highwire.dtl.DTLVardef@8addec_HPS_FORMAT_FIGEXP M_FIG C_FIG