Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Immunology and Allergology IA, 12203 Berlin, Germany.
Institute of Allergology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, Hindenburgdamm 30, 12203 Berlin, Germany.
Cells. 2023 Dec 24;13(1):42. doi: 10.3390/cells13010042.
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.
皮肤肥大细胞（MCs）是急性过敏反应的关键效应细胞，它们参与荨麻疹和特应性皮炎和接触性皮炎等慢性皮肤病。KIT 代表细胞的关键受体酪氨酸激酶，它在整个生命周期中协调增殖、存活和功能程序。环磷酸腺苷反应元件结合蛋白（CREB）是一种进化上高度保守的转录因子（TF），调节多种细胞程序，但它在 MCs 中的功能知之甚少。我们最近报道 CREB 是 SCF（干细胞因子）/KIT 轴的效应物。在这里，我们询问 CREB 是否也可以作为 KIT 的上游作用物来协调其功能。从皮肤中分离出原代人 MCs 并在 SCF+IL-4（白细胞介素 4）中培养。使用药理学抑制（666-15）和 RNA 干扰来操纵 CREB 功能。我们使用流式细胞术和 RT-qPCR 研究 KIT 表达，使用免疫印迹研究 KIT 介导的信号转导，使用散点图和 caspase-3 活性研究细胞存活。BrdU 掺入后定量细胞增殖和周期阶段。瞬时 CREB 干扰导致 KIT 表达减少。相反，小眼畸形转录因子（MITF）对于 KIT 的维持不是必需的。由于 CREB 导致的 KIT 衰减与 KIT 功能输出严重受损有关，例如抗细胞凋亡和细胞周期进程。同样，KIT 诱导的 ERK1/2（细胞外信号调节激酶 1/2）、AKT 和 STAT5（信号转导和转录激活因子）磷酸化在 CREB 抑制后大大减少。令人惊讶的是，CREB 的长期干扰导致完全细胞消除，这种方式超过了 KIT 抑制。总的来说，我们揭示 CREB 在 MCs 中不可或缺，其缺失与皮肤 MCs 的存在不兼容。由于 SCF/KIT 调节 CREB 活性，反之亦然，CREB 是 KIT 功能所必需的，这些元素之间的正反馈环决定了皮肤 MCs 的命运。
