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bioRxiv · 10.1101/2025.07.27.666827

SMAD2 sets divergent thresholds for TGF-β-induced SMAD1/5 signaling and IgE-mediated pro-inflammatory activation of mast cells

Bronneberg, G.·Meurer, S. K.·Kauffmann, M.·Kuo, C.-C.·Liedtke, C.·Weiskirchen, R.·Huber, M.
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Abstract

ObjectiveTGF-{beta}/SMAD signaling controls mast cell (MC) development and exerts anti-inflammatory functions, while antigen (Ag)-triggered Fc{varepsilon}RI/MAPK activation commands pro-inflammatory processes. SMAD2 is a signaling hub integrating both, TGF-{beta}/ALK5-mediated SMAD2 C-terminal-and ERK-mediated SMAD2 linker-phosphorylation. Here, we analyzed the role of SMAD2 in TGF-{beta}-and Ag-stimulated signaling, and their cross-talk which regulate MC responses. MethodsThe CRISPR-Cas method was used to disable SMAD2 expression in the PMC-306 cell line. The transcriptome of the corresponding cells was analyzed by NGS in homeostatic and stimulated conditions. Activation of signaling intermediates, target gene responses and effector function were analyzed by qPCR, Western blot and ELISAs. The causal association of SMAD2 with the found effects was proven by re-introduction of a tagged SMAD2 in knock out cells. ResultsThe absence of SMAD2 led to increased proliferation and survival, and decreased transcription of target genes like Smad7 and Jun in steady state and upon TGF-{beta} treatment. Intriguingly, SMAD2 was found to regulate TGF-{beta}-mediated SMAD1/5 activation, resulting in augmented expression of Id3 in SMAD2-deficient MCs. Unexpectedly, we found that SMAD2 is indispensable for Ag-triggered production of pro-inflammatory cytokines, such as IL-6 and TNF. Re-introducing SMAD2 restores these events with varying sensitivity depending on the receptor system triggered. ConclusionsOur findings highlight a previously unrecognized role of SMAD2 as a decisive hub in TGF-{beta}-ALK5-SMAD1/5 and Ag-Fc{varepsilon}RI-MAPK signaling, suggesting that pharmacological modulation of SMAD2 activity could be leveraged as regulator of both suppressive and inflammatory MC functions.

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Bronneberg, G., Meurer, S. K., Kauffmann, M., Kuo, C.-C., Liedtke, C., Weiskirchen, R., Huber, M.. 2025-07-31. SMAD2 sets divergent thresholds for TGF-β-induced SMAD1/5 signaling and IgE-mediated pro-inflammatory activation of mast cells. https://doi.org/10.1101/2025.07.27.666827

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