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The adaptor 3BP2 is required for KIT receptor expression and human mast cell survival.

ABSTRACT: SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein that acts as a positive regulator in mast cell Fc?RI-dependent signaling. The KIT receptor whose ligand is the stem cell factor is necessary for mast cell development, proliferation, and survival as well as for optimal IgE-dependent signal. Activating mutations in KIT have been associated with several diseases including mastocytosis. In the present work, we found that 3BP2 silencing impairs KIT signaling pathways, thus affecting phosphoinositide 3-kinase and MAPK pathways in human mast cells (huMCs) from HMC-1, LAD2 (huMC lines), and CD34(+)-derived mast cells. Unexpectedly, silencing of 3BP2 reduces KIT expression in normal huMCs as well as in HMC-1 cells where KIT is mutated, thus increasing cellular apoptosis and caspase-3/7 activity. 3BP2 silencing reduces KIT transcription expression levels. Interestingly, 3BP2 silencing decreased microphthalmia-associated transcription factor (MITF) expression, a transcription factor involved in KIT expression. Reconstitution of 3BP2 in knockdown cells leads to reversal of KIT expression as well as survival phenotype. Accordingly MITF reconstitution enhances KIT expression levels in 3BP2-silenced cells. Moreover, downregulation of KIT expression by miRNA-221 overexpression or the proteasome inhibitor bortezomib also reduced 3BP2 and MITF expression. Furthermore, KIT tyrosine activity inhibition reduced 3BP2 and MITF expression, demonstrating again a tight and reciprocal relationship between these molecules. Taken together, our results show that 3BP2 regulates huMC survival and participates in KIT-mediated signal transduction by directly controlling KIT receptor expression, suggesting its potential as a therapeutic target in mast cell-mediated inflammatory diseases and deregulated KIT disorders.

SUBMITTER: Ainsua-Enrich E

PROVIDER: S-EPMC4402263 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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The adaptor 3BP2 is required for KIT receptor expression and human mast cell survival.

Ainsua-Enrich Erola E Serrano-Candelas Eva E Álvarez-Errico Damiana D Picado César C Sayós Joan J Rivera Juan J Martín Margarita M

Journal of immunology (Baltimore, Md. : 1950) 20150325 9

SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein that acts as a positive regulator in mast cell FcεRI-dependent signaling. The KIT receptor whose ligand is the stem cell factor is necessary for mast cell development, proliferation, and survival as well as for optimal IgE-dependent signal. Activating mutations in KIT have been associated with several diseases including mastocytosis. In the present work, we found that 3BP2 silencing impairs KIT signaling pathways, thus affecting phosp ...[more]

PMID: 25810396

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Project description:Skin mast cells (MCs) are critical effector cells in acute allergic reactions, and they contribute to chronic dermatoses like urticaria and atopic and contact dermatitis. KIT represents the cells' crucial receptor tyrosine kinase, which orchestrates proliferation, survival, and functional programs throughout the lifespan. cAMP response element binding protein (CREB), an evolutionarily well-conserved transcription factor (TF), regulates multiple cellular programs, but its function in MCs is poorly understood. We recently reported that CREB is an effector of the SCF (Stem Cell Factor)/KIT axis. Here, we ask whether CREB may also act upstream of KIT to orchestrate its functioning. Primary human MCs were isolated from skin and cultured in SCF+IL-4 (Interleukin-4). Pharmacological inhibition (666-15) and RNA interference served to manipulate CREB function. We studied KIT expression using flow cytometry and RT-qPCR, KIT-mediated signaling using immunoblotting, and cell survival using scatterplot and caspase-3 activity. The proliferation and cycle phases were quantified following BrdU incorporation. Transient CREB perturbation resulted in reduced KIT expression. Conversely, microphthalmia transcription factor (MITF) was unnecessary for KIT maintenance. KIT attenuation secondary to CREB was associated with heavily impaired KIT functional outputs, like anti-apoptosis and cell cycle progression. Likewise, KIT-elicited phosphorylation of ERK1/2 (Extracellular Signal-Regulated Kinase 1/2), AKT, and STAT5 (Signal Transducer and Activator of Transcription) was substantially diminished upon CREB inhibition. Surprisingly, the longer-term interference of CREB led to complete cell elimination, in a way surpassing KIT inhibition. Collectively, we reveal CREB as non-redundant in MCs, with its absence being incompatible with skin MCs' existence. Since SCF/KIT regulates CREB activity and, vice versa, CREB is required for KIT function, a positive feedforward loop between these elements dictates skin MCs' fate.

Dataset Information

The adaptor 3BP2 is required for KIT receptor expression and human mast cell survival.

Publications

The adaptor 3BP2 is required for KIT receptor expression and human mast cell survival.

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