白介素-6/可溶性白介素-6受体诱导的牙龈成纤维细胞组织蛋白酶B和L的分泌受caveolin-1和ERK1/2通路的调控。

IF 1.8 Q3 DENTISTRY, ORAL SURGERY & MEDICINE
Frontiers in dental medicine Pub Date : 2025-03-11 eCollection Date: 2025-01-01 DOI:10.3389/fdmed.2025.1547222
Ayaka Goto, Kazuhiro Omori, Tomoko Yamaguchi-Tomikawa, Hiroya Kobayashi, Yuki Shinoda-Ito, Kimito Hirai, Atsushi Ikeda, Shogo Takashiba
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引用次数: 0

摘要

目的:组织蛋白酶是一种重要的溶酶体酶,通过降解细胞外底物来维持生物体的稳态。炎症细胞因子白介素-6 (IL-6)通过小窝蛋白-1 (Cav-1)和c-Jun n-末端激酶(JNK)信号通路增加组织蛋白酶的产生,这些信号通路与牙周组织的破坏有关。本研究探讨了IL-6/可溶性IL-6受体(sIL-6R)复合物对人牙龈成纤维细胞(HGFs)胞外分泌组织蛋白酶的影响,并在体外酸性培养条件下考察了细胞外分泌组织蛋白酶B和L的功能。方法:从健康志愿者供体中分离hgf。通过转染靶向Cav-1的小干扰RNA (siRNA)抑制Cav-1的表达。采用western blotting和酶联免疫吸附法检测细胞外IL-6/sIL-6R诱导组织蛋白酶B和L的表达水平。在IL-6/sIL-6R刺激后,使用甲基香豆胺底物进行荧光检测,评估细胞外组织蛋白酶活性。在抑制细胞外信号调节激酶(ERK) 1/2和/或JNK信号的条件下,定量分析IL-6/sIL-6R诱导hgf中组织蛋白酶B和L的表达,这两种信号通路都是由IL-6/sIL-6R激活的转导途径。这种定量也在抑制Cav-1表达的HGFs中使用western blotting进行。结果:HGFs以前体形式分泌组织蛋白酶B和L,在il -6/sIL-6R刺激后24、48和72 h,组织蛋白酶B和L的蛋白水平显著升高。在酸性培养条件下,组织蛋白酶B的活性在48和72 h时增加。无论IL-6/sIL-6R是否刺激,Cav-1的抑制均抑制组织蛋白酶B的分泌,而组织蛋白酶L的分泌仅在IL-6/sIL-6R刺激48 h后才减少。在IL-6/sIL-6R刺激48 h后,抑制ERK1/2和JNK通路可降低组织蛋白酶B的分泌,而在类似条件下,抑制JNK可降低组织蛋白酶L的分泌。结论:IL-6/sIL-6R刺激可增加hgf细胞外组织蛋白酶B和L前体的分泌,且这些前体在酸性条件下被激活。Cav-1和ERK1/2参与调节组织蛋白酶B前体的分泌。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Interleukin-6/soluble IL-6 receptor-induced secretion of cathepsin B and L from human gingival fibroblasts is regulated by caveolin-1 and ERK1/2 pathways.

Aims: Cathepsins are essential lysosomal enzymes that maintain organismal homeostasis by degrading extracellular substrates. The inflammatory cytokine interleukin-6 (IL-6) increases the production of cathepsins through the caveolin-1 (Cav-1) and c-Jun N-terminal kinase (JNK) signaling pathways, which have been implicated in the destruction of periodontal tissue. This study investigated the effect of the IL-6/soluble IL-6 receptor (sIL-6R) complex on the extracellular secretion of cathepsins in human gingival fibroblasts (HGFs) and examined the function of extracellularly secreted cathepsins B and L under acidic culture conditions in vitro.

Methods: HGFs were isolated from healthy volunteer donors. The expression of Cav-1 was suppressed via transfection with small interfering RNA (siRNA) targeting Cav-1. The expression levels of cathepsins B and L induced by extracellular IL-6/sIL-6R were measured using western blotting and enzyme-linked immunosorbent assay. Extracellular cathepsin activity following IL-6/sIL-6R stimulation was assessed using a methylcoumarylamide substrate in a fluorescence-based assay. IL-6/sIL-6R-induced expression of cathepsins B and L in HGFs was quantified under inhibitory conditions for extracellular signal-regulated kinase (ERK) 1/2 and/or JNK signaling, both of which are transduction pathways activated by IL-6/sIL-6R. This quantification was also performed in HGFs with suppressed Cav-1 expression using western blotting.

Results: Cathepsins B and L were secreted in their precursor forms from HGFs, with significantly elevated protein levels observed at 24, 48, and 72 h post-IL-6/sIL-6R stimulation. Under acidic culture conditions, cathepsin B activity increased at 48 and 72 h. Cav-1 suppression inhibited the secretion of cathepsin B regardless of IL-6/sIL-6R stimulation, whereas the secretion of cathepsin L was reduced only after 48 h of IL-6/sIL-6R stimulation. Inhibition of ERK1/2 and JNK pathways decreased the secretion of cathepsin B after 48 h of IL-6/sIL-6R stimulation, and JNK inhibition reduced the secretion of cathepsin L under similar conditions.

Conclusion: IL-6/sIL-6R stimulation increased the extracellular secretion of cathepsin B and L precursors in HGFs, and these precursors became activated under acidic conditions. Cav-1 and ERK1/2 are involved in regulating the secretion of cathepsin B precursors.

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