I. Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by augmented transforming growth factor-β (TGF-β) 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-β-induced mitogen-activated protein kinase (MAPK) signaling. Molecular confirmation revealed that TGF-β-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, p47 ^phox . Inhibition of OGT, p38, or p47 ^phox reduced reactive oxygen species (ROS) in HLFs, revealing a previously unknown role of OGT-p38-p47 ^phox signaling in ROS production. Collectively, this work establishes that O-GlcNAc-modified p38 enhances p47 ^phox -dependent H ₂ O ₂ production. Highlights: Using PamChip STK arrays, we show that OGT inhibition causes broad kinomic remodeling, including suppression of non-canonical TGF-β MAPKs and multiple CDKs. OGT blockade selectively attenuates p38 phosphorylation, despite TGF-β-induced substrate redundancy with ERK and JNK. We provide evidence that p38 MAPK undergoes O-GlcNAcylation in human lung fibroblasts, a modification not previously reported. The study identifies a new signaling axis where O-GlcNAc modification of p38 modulates the phosphorylation of p47 ^phox , therefore regulating NOX-dependent H ₂ O ₂ production. Blocking OGT or inhibiting p38/p47 ^phox dramatically reduces TGF-β-driven H ₂ O ₂ production in human lung fibroblasts. Graphical

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