Biology subjects

Kauffmann, M.

Publications and source records attributed to Kauffmann, M..

3 recordsLinked to original sources

bioRxiv2025

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

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.

immunology↗

bioRxiv2023

SR-BI regulates the synergistic mast cell response by modulating the plasma membrane-associated cholesterol pool

The high-affinity IgE receptor Fc{varepsilon}RI is the fundamental mast cell (MC) receptor responsible for the involvement of MCs in IgE-associated allergic disorders. Activation of the Fc{varepsilon}RI is achieved via crosslinking by multivalent antigen (Ag) recognized by IgE, which results in degranulation and pro-inflammatory cytokine production. In comparison to the T and B cell receptor complexes, for which several co-receptors that orchestrate the initial signaling have been described, information is scarce about Fc{varepsilon}RI-associated proteins. Additionally, it is not completely clear how Fc{varepsilon}RI signaling synergizes with input from other receptors and how potential regulators affect this synergistic response. We aimed at identifying new regulators of Fc{varepsilon}RI and found that the HDL receptor SR-BI (gene name: Scarb1/SCARB1) is expressed in MCs, functionally associates with Fc{varepsilon}RI and regulates the local plasma membrane cholesterol content in cholesterol-rich plasma membrane nanodomains as shown by using the cholesterol-sensitive probe GFP-D4. This impacted on the activation of murine MCs upon co-stimulation of the Fc{varepsilon}RI with different receptors known to synergize with Fc{varepsilon}RI-signaling pathways. Amongst them we investigated the co-activation of the Fc{varepsilon}RI with the receptor tyrosine kinase KIT, the IL-33 receptor and GPCRs activated by adenosine or PGE2. Scarb1-deficient bone marrow-derived MCs (BMMCs) showed reduced cytokine secretion in response to these co-stimulation conditions suggesting a role for plasma membrane-associated cholesterol regulating MC-driven inflammation. Mimicking Scarb1 deficiency by membrane cholesterol depletion employing M{beta}CD, we identified PKB and PLC{gamma}1 as cholesterol-sensitive signaling molecules activated downstream of Fc{varepsilon}RI in BMMCs. Specifically, when murine MCs were stimulated with SCF and Ag in combination, PLC{gamma}1 activation appeared to be drastically boosted and this could be mitigated by cholesterol depletion. Inhibiting SR-BI in BMMCs phenocopied this effect. Similarly, SR-BI inhibition also attenuated the synergistic response to PGE2 and anti-IgE in the human ROSAKIT WT mast cell line suggesting that SR-BI is a crucial regulator of synergistic MC activation by regulating the local plasma membrane cholesterol concentration.

immunology↗

bioRxiv2023

Transformation of primary murine peritoneal mast cells by constitutive KIT activation as a result of lost Cdkn2a/Arf expression

Mast cells (MCs) are immune cells of the myeloid lineage distributed in tissues throughout the body. Phenotypically, they are a heterogeneous group characterized by different protease repertoires stored in secretory granules and differential presence of receptors. To adequately address aspects of MC biology either primary MCs isolated from human or mouse tissue or different human MC lines, like HMC-1.1 and -1.2, or rodent MC lines like L138.8A or RBL-2H3 are frequently used. Nevertheless, cellular systems to study MC functions are very limited. We have generated a murine connective tissue-like MC line, termed PMC-306, derived from primary peritoneal MCs (PMCs), which spontaneously transformed. We analyzed PMC-306 cells regarding MC surface receptor expression, effector functions and respective signaling pathways, and found that the cells reacted very similar to primary wildtype (WT) PMCs. In this regard, stimulation with MAS-related G-protein-coupled receptor member B2 (MRGPRB2) ligands induced respective signaling and effector functions. Furthermore, PMC-306 cells revealed significantly accelerated cell cycle progression, which however was still dependent on IL-3 and stem cell factor (SCF). Phenotypically, PMC-306 cells adopted an immature connective tissue-like MCs appearance. The reason for immortalization most likely is the loss of the two critical cell cycle regulators Cdkn2a/INK4A and Arf/p19, respectively. The loss of Cdkn2a and Arf expression could be mimicked in primary bone marrow-derived mast cells (BMMCs) by SCF supplementation strongly arguing for an involvement of KIT activation in the transformation process. Hence, this new cell line might be a useful tool to study further aspects of PMC function and to address tumorigenic processes associated with MC leukemia.

immunology↗