Medical Science Research Center, College of Medicine, Korea University, Seoul 02841, Republic of Korea.
Korean Medicine Clinical Trial Center, Kyung Hee University Korean Medicine Hospital, Seoul 02447, Republic of Korea.
Mediators Inflamm. 2024 Nov 16;2024:8892514. doi: 10.1155/mi/8892514. eCollection 2024.
Keratinocytes can be activated by , leading to the production of proinflammatory cytokines via toll-like receptors (TLRs) 2 and 4. Although several studies have investigated keratinocytes, the mechanism of calcium-mediated activation remains unclear. Herein, we investigated whether calcium influx via TLR2 and TLR4 stimulation was involved in cytokine secretion by keratinocytes in HaCaT cells. Although TLR2 stimulation by peptidoglycan (PGN) increased intracellular calcium influx, TLR4 stimulation by lipopolysaccharide (LPS) did not increase it, as analyzed using flow cytometry with the calcium indicator Fluo-3. However, activation by either TLR2 or TLR4 ligands upregulated the intracellular calcium influx in THP-1 monocytes. Additionally, the expression of major proinflammatory cytokines and chemokines, such as interleukin (IL)-6, IL-8, IL-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein-1 (MCP-1), was significantly increased by TLR2 in HaCaT cells. Moreover, treatment with the intracellular calcium chelator, BAPTA-AM, disrupted PGN-mediated induction of IL-6, IL-8, and MCP-1 production. Real-time quantitative polymerase chain reaction (PCR) and western blotting revealed that TLR2 stimulation induced expression of the epidermal differentiation marker keratin 1. In conclusion, TLR2-induced intracellular calcium influx plays a pivotal role in the secretion of proinflammatory cytokines, such as IL-6 and MCP-1, in keratinocytes. Moreover, the continuous influx of calcium via TLR2 activation leads to keratinization. In vitro studies using HaCaT cells provide basic research on the effect of TLR2-induced calcium on -mediated inflammation in keratinocytes. These studies are limited in their ability to clinically predict what happens in human keratinocytes. Clinical studies on patients with acne, including three-dimensional (3D) cultures of primary keratinocytes, are required to develop new diagnostic markers for determining the severity of acne vulgaris.
角质形成细胞可被激活，通过 Toll 样受体（TLR）2 和 4 产生促炎细胞因子。尽管已有几项研究调查了角质形成细胞，但钙介导的激活机制仍不清楚。在此，我们研究了 TLR2 和 TLR4 刺激引起的钙内流是否参与 HaCaT 细胞中角质形成细胞细胞因子的分泌。虽然肽聚糖（PGN）刺激 TLR2 增加了细胞内钙内流，但脂多糖（LPS）刺激 TLR4 并没有增加，这可以通过用钙指示剂 Fluo-3 通过流式细胞术进行分析。然而，TLR2 或 TLR4 配体的激活均上调了 THP-1 单核细胞中的细胞内钙内流。此外，主要促炎细胞因子和趋化因子（如白细胞介素（IL）-6、IL-8、IL-1、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和单核细胞趋化蛋白-1（MCP-1）的表达在 HaCaT 细胞中也被 TLR2 显著上调。此外，用细胞内钙螯合剂 BAPTA-AM 处理可破坏 PGN 介导的 IL-6、IL-8 和 MCP-1 产生的诱导作用。实时定量聚合酶链反应（PCR）和蛋白质印迹分析显示，TLR2 刺激诱导表皮分化标记物角蛋白 1 的表达。总之，TLR2 诱导的细胞内钙内流在角质形成细胞中促炎细胞因子（如 IL-6 和 MCP-1）的分泌中起关键作用。此外，TLR2 激活引起的钙持续内流导致角化。使用 HaCaT 细胞的体外研究为 TLR2 诱导的钙对角质形成细胞中炎症的影响提供了基础研究。这些研究在预测人类角质形成细胞中发生的情况方面存在局限性。需要对痤疮患者进行包括原代角质形成细胞的 3D 培养在内的临床研究，以开发用于确定寻常痤疮严重程度的新诊断标志物。
