O-GlcNAc transferase regulates H2O2 production via p38 MAPK

Jones, L. I.; Vang, S.; McIntire-Ray, H. J.; Petersen, H. A.; Morales, A. N.; Acevedo Rua, V. E.; Anderson, J. C.; Gonzalez Coba, A. J.; Krick, S.; Barnes, J. W.

doi:10.64898/2026.05.27.728188

bioRxiv · 10.64898/2026.05.27.728188

O-GlcNAc transferase regulates H2O2 production via p38 MAPK

Jones, L. I.·Vang, S.·McIntire-Ray, H. J.·Petersen, H. A.·Morales, A. N.·Acevedo Rua, V. E.·Anderson, J. C.·Gonzalez Coba, A. J.·Krick, S.·Barnes, J. W.

View original source ↗Export metadata ↓

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

Explore related subjects

molecular biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Jones, L. I., Vang, S., McIntire-Ray, H. J., Petersen, H. A., Morales, A. N., Acevedo Rua, V. E., Anderson, J. C., Gonzalez Coba, A. J., Krick, S., Barnes, J. W.. 2026-05-31. O-GlcNAc transferase regulates H2O2 production via p38 MAPK. https://doi.org/10.64898/2026.05.27.728188

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A feed-forward UHRF1 read-write mechanism supports H3 multi- mono-ubiquitination and DNA methylation maintenance at CpG-sparse regions

The epigenetic inheritance of mammalian DNA methylation requires DNMT1 and its E3 ligase cofactor UHRF1. At newly replicated chromatin, UHRF1 recognition of hemi-methylated DNA and histone H3 N-terminal tails directs catalysis of H3K14, H3K18, and/or H3K23 mono-ubiquitination to recruit DNMT1. While it is appreciated that UHRF1 can deposit multiple mono-ubiquitin marks on a single H3 tail and that DNMT1 recognizes this state through tandem ubiquitin interacting motifs, the mechanism that promotes successive ubiquitination and the biological function of multi-mono-ubiquitination are unknown. Here, we show that UHRF1 directly binds its mono-ubiquitinated H3 products through a previously uncharacterized LGDDSL loop in Tudor 2 of its tandem Tudor domain (TTD) to promote further ubiquitin deposition. Disruption of this ubiquitin reading activity impairs H3 multi-mono-ubiquitination and accelerates DNA methylation loss within late-replicating, CpG-sparse genomic regions that are characteristic of partially methylated domains (PMDs) in cancer and aging cells. These methylation defects overlap those observed by disruption of UHRF1 ubiquitin ligase activity, providing convergent evidence that both writing and reading of H3 ubiquitination support CpG-sparse DNA methylation maintenance. Together, these findings establish a feed-forward ubiquitin read-write mechanism that generates multi-mono-ubiquitinated H3 and safeguards DNMT1-dependent DNA methylation maintenance at vulnerable genomic regions of the mammalian methylome.

molecular biology↗

bioRxiv2026

Calcium dysregulation amplifies fibrotic responses to TGFβ in human Friedreich's ataxia fibroblasts

Friedreich's ataxia (FA) is an inherited disease caused by loss of frataxin (FXN) and characterized by neurodegeneration and fatal cardiomyopathy. Cardiac fibrosis contributes to cardiomyopathy by stiffening the heart wall, yet the underlying mechanisms remain unknown. Here, we investigated pro-fibrotic predisposition in FA patient-derived fibroblasts, focusing on the role of cytosolic calcium (Ca) in TGF{beta}-driven fibroblast-to-myofibroblast transition (FMT). We found pro-fibrotic transcriptional priming in FA fibroblasts, alongside elevated expression of genes controlled by the Ca-responsive transcription factor NFAT. Upon FMT, FA myofibroblasts showed amplified induction of pro-fibrotic (CCN2, NOX4) and suppression of anti-fibrotic (CCN3) genes, which were inversely correlated with residual FXN. Mechanistically, FA fibroblasts exhibited elevated cytosolic Ca and strongly downregulated expression of the Na-Ca exchanger NCX1, which directly correlated with FXN. Furthermore, NCX1 inhibition in control fibroblasts recapitulated FA Ca phenotypes, whereas NCX1 transduction in FA fibroblasts normalized Ca dynamics and blunted CCN2 induction in FMT. These findings highlight NCX1 as a modulator of fibrotic reprogramming in FA and identify Ca dyshomeostasis as an intrinsic mechanism of fibrosis that could be targeted therapeutically.

molecular biology↗

bioRxiv2026

Stromal CTHRC1 protects the valvular interstitium from macrophage-associated inflammatory remodeling and calcification

Background: Calcific aortic valve disease (CAVD) is characterized by progressive inflammatory and fibrocalcific remodeling. Although valvular interstitial cells (VICs) are generally considered to drive fibrosis and osteogenic remodeling, whether injury-activated VICs mount endogenous protective responses that preserve the valvular interstitial microenvironment and restrain calcification remains unknown. Methods: We performed spatial transcriptomic profiling of aortic valves in a mouse model of endothelial injury-induced CAVD to define early injury-responsive programs within the valvular interstitium. The cellular origin and spatial distribution of candidate protective factors were examined by immunohistochemistry and lineage tracing, and their relevance to human disease was assessed using stenotic aortic valves. The functional role of CTHRC1 was investigated using genetic Cthrc1 deficiency combined with longitudinal hemodynamic assessment, histological analysis, and spatial transcriptomic profiling. Results: Spatial transcriptomics identified Cthrc1 as a prominent component of an early injury-induced stromal response in the expanding valvular interstitium. CTHRC1 was strongly expressed in activated VICs within thickened murine valve leaflets and human stenotic aortic valves. Lineage tracing demonstrated that the expanded VIC population arose predominantly from PDGFR{beta}+ resident interstitial cells, with minimal endothelial contribution. Despite comparable early hemodynamic responses to endothelial injury, Cthrc1 deficiency exacerbated chronic valvular calcification. Spatial profiling of Cthrc1-deficient valves revealed pronounced interstitial accumulation of galectin-3+ foamy macrophages, accompanied by mitochondrial respiratory-chain signature loss and cell death-associated pathway activation. These findings indicate that transient CTHRC1 induction after endothelial injury defines an endogenous stromal protective response that preserves the valvular interstitial microenvironment and limits macrophage-associated tissue injury and subsequent dystrophic calcification. Conclusions: Injury-activated VICs are not merely effectors of pathological remodeling, but can engage an endogenous tissue-protective response through CTHRC1. These findings identify a previously unrecognized stromal defense mechanism linking endothelial injury to macrophage-associated inflammatory remodeling and dystrophic calcification and suggest CTHRC1-dependent stromal protection as a potential therapeutic axis for limiting CAVD progression.

molecular biology↗