Characterization of serotonin-5- HTR1E signaling pathways and its role in cell survival

Raw data files for the manuscript "Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival" published in The FASEB Journal, DOI: 10.1096/fj.202300128R.

5-Hydroxytryptamine receptor 1E (5-HTR1E) is reported to activate cyclic AMP (cAMP) and extracellular-signal related kinases (ERK) pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin-induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin-induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of
serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gái-linked cascade. We also observed that GBg and Gq were not associated with 5-HTR1E activation, while blocking protein kinase A (PKA) inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, B-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockdown studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E
knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 (RAMP1), Nuclear receptor 1 (NR4A1) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.

本数据集对应发表于《美国实验生物学会联合会会志（The FASEB Journal）》（DOI: 10.1096/fj.202300128R）的论文《5-羟色胺5-HTR1E信号通路特征及其在细胞存活中的作用》的原始数据文件。

据报道，5-羟色胺受体1E（5-HTR1E）可通过其配体与结合伴侣激活环腺苷酸（cAMP）及细胞外信号调节激酶（ERK）通路，但血清素诱导的5-HTR1E信号转导的具体分子机制仍未阐明。本研究中，我们在过表达5-HTR1E的HEK293细胞中，探究了血清素诱导5-HTR1E激活后，ERK与cAMP信号通路的细胞调控因子。实验发现，百日咳毒素（PTX）处理可完全逆转血清素-5-HTR1E介导的信号对cAMP与ERK通路的影响，证实了Gαi偶联级联反应的参与。我们还观察到，Gβγ与Gq并未参与5-HTR1E的激活过程；而蛋白激酶A（PKA）抑制剂仅能抑制ERK信号通路，对cAMP通路无影响。此外，在β-抑制蛋白缺陷型HEK293细胞中，血清素刺激诱导的ERK1/2磷酸化水平与对照组相似，且该过程完全依赖于G蛋白信号转导。在SH-SY5Y细胞中开展的小干扰RNA（siRNA）介导的基因敲低实验显示，抑制5-HTR1E的表达可降低c-Myc、细胞周期蛋白D1、细胞周期蛋白E及B细胞淋巴瘤因子2（BCL2）的转录水平，这些基因均与细胞周期调控及细胞存活相关。MTT实验结果表明，在SH-SY5Y与U118细胞中敲低5-HTR1E可显著抑制细胞存活能力。除信号转导机制外，我们还对过表达5-HTR1E的HEK293细胞开展了RNA测序（RNA-seq）分析，发现5-HTR1E可调控受体活性修饰蛋白1（RAMP1）、核受体1（NR4A1）及其他细胞周期蛋白基因的表达。上述研究结果表明，血清素与5-HTR1E受体结合后，可同时在HEK293细胞中激活cAMP与ERK通路，且该受体的表达对细胞存活至关重要。