P2X7R-primed keratinocytes are susceptible to apoptosis via GPCR-Gβγ-pERK signal pathways.

Tomoki Nishiguchi, Haruna Kimura, Yuki Saito, Takeaki Ozawa, Riichiro Abe, Akito Hasegawa
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Abstract

Background: Cell death constitutes a pivotal biological phenomenon essential for the preservation of homeostasis within living organisms. In the context of maintaining a functional skin barrier, keratinocytes exert positively and negatively control cell death signals. However, in patients with severe drug eruptions, anomalous overexpression of the formyl peptide receptor 1 (FPR1) in keratinocytes elicits a distinctive mode of cell death known as necroptosis, thereby suffering a loss of the skin barrier. The precise molecular mechanisms connecting FPR1 activation to this cell death remain unclear.

Objective: We have investigated the intracellular signal transduction cascade governing FPR1-mediated cell death in cultured keratinocytes.

Methods: We used HaCaT cells as a model keratinocyte. The expression of FPR1 was detected with qPCR. The presence of cell death events was monitored through live-cell fluorescent staining and LDH release assays. Furthermore, the phosphorylation of ERK was assessed via Western blot analysis. Intracellular signal pathways were investigated using specific inhibitors.

Results: Ligand stimulation of an endogenous ion channel, purinergic receptor P2X7 (P2X7R), increased the FPR1 expression level. This upregulated FPR1 demonstrated functional competence in the phosphorylation of downstream MAP kinase and the initiation of cell death. Notably, this cell death was ameliorated upon the administration of inhibitors targeting Gβγ, ERK, and caspases.

Conclusion: The induction and stimulation of FPR1 initiated apoptosis in keratinocytes via the Gβγ-pERK signaling pathway. Our findings postulate that the downstream components of FPR1 represent an alternative therapeutic target for preventing unintended keratinocyte cell death.

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P2X7R刺激的角质形成细胞易通过GPCR-Gβγ-pERK信号通路发生凋亡。
背景:细胞死亡是维持生物体内平衡的关键生物现象。在维持功能性皮肤屏障的过程中,角质形成细胞对细胞死亡信号进行正向和负向控制。然而,在严重药物疹患者中,角质形成细胞中甲酰肽受体 1(FPR1)的异常过度表达会引起一种称为坏死的独特细胞死亡模式,从而导致皮肤屏障的丧失。FPR1 激活与这种细胞死亡之间的确切分子机制仍不清楚:我们研究了细胞内调控 FPR1 介导的角质形成细胞死亡的信号转导级联:方法:我们使用 HaCaT 细胞作为模型角质形成细胞。方法:我们使用 HaCaT 细胞作为模型角质形成细胞,用 qPCR 检测 FPR1 的表达。通过活细胞荧光染色和 LDH 释放试验监测细胞死亡事件的存在。此外,还通过 Western 印迹分析评估了 ERK 的磷酸化情况。使用特异性抑制剂对细胞内信号通路进行了研究:结果:配体刺激内源性离子通道--嘌呤能受体 P2X7(P2X7R)可提高 FPR1 的表达水平。这种上调的 FPR1 在下游 MAP 激酶磷酸化和引发细胞死亡方面表现出功能性能力。值得注意的是,在使用针对 Gβγ、ERK 和 caspases 的抑制剂后,细胞死亡的情况得到了改善:结论:FPR1 的诱导和刺激可通过 Gβγ-pERK 信号通路引发角质形成细胞凋亡。我们的研究结果推测,FPR1 的下游成分是防止角质形成细胞意外死亡的另一个治疗靶点。
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