Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, No. 1, University Road, Tainan City 701, Taiwan, ROC.
National Institute of Cancer Research, National Health Research Institutes, No. 367, Shengli Rd., Tainan City 704, Taiwan, ROC.
Biochim Biophys Acta Mol Cell Res. 2019 Dec;1866(12):118517. doi: 10.1016/j.bbamcr.2019.07.012. Epub 2019 Jul 21.
Cathepsin S (CTSS), a lysosomal cysteine protease, has been reported to be associated with extracellular matrix (ECM) degradation, thus promoting cell migration and invasion, but whether CTSS regulates other intracellular mechanisms during metastasis remains unknown. The expression of CTSS was knocked down using siRNA transfection, and enzymatic activity was inhibited by the highly-selective CTSS inhibitor RJW-58. The results of in vitro functional assays, western blot analysis, and an in vivo colonization model demonstrated that CTSS was positively related to cellular adhesive ability. Moreover, both CTSS knockdown and inhibition significantly decreased Ca influx via store-operated Ca entry (SOCE) without changing STIM1 and Orai1 expression levels, while RJW-58 dose-dependently reduced the activation of the Ca-dependent downstream effectors, NFAT1 and Rac1. The results of immunoprecipitation assays demonstrated that CTSS could bind to STIM1, which was reversed by CTSS inhibition. In addition, confocal microscopy and super-resolution imaging showed that CTSS inhibition led to STIM1 puncta accumulation in the endoplasmic reticulum and reduced the interaction between active STIM1 and EB1. In conclusion, we have demonstrated for the first time that the lysosomal cysteine protease, CTSS, plays an important role in mediating Ca homeostasis by regulating STIM1 trafficking, which leads to the suppression of cell migration and invasion.
组织蛋白酶 S（CTSS）是一种溶酶体半胱氨酸蛋白酶，已被报道与细胞外基质（ECM）降解有关，从而促进细胞迁移和侵袭，但 CTSS 是否在转移过程中调节其他细胞内机制尚不清楚。使用 siRNA 转染下调 CTSS 的表达，并使用高选择性 CTSS 抑制剂 RJW-58 抑制酶活性。体外功能测定、Western blot 分析和体内定植模型的结果表明，CTSS 与细胞黏附能力呈正相关。此外，CTSS 敲低和抑制均显著降低通过钙库操纵钙内流（SOCE）的钙内流，而不改变 STIM1 和 Orai1 的表达水平，而 RJW-58 呈剂量依赖性降低钙依赖性下游效应物 NFAT1 和 Rac1 的激活。免疫沉淀测定的结果表明 CTSS 可以与 STIM1 结合，而 CTSS 抑制可逆转这种结合。此外，共聚焦显微镜和超分辨率成像显示 CTSS 抑制导致内质网中 STIM1 斑点积累，并减少活性 STIM1 和 EB1 之间的相互作用。总之，我们首次证明溶酶体半胱氨酸蛋白酶 CTSS 通过调节 STIM1 运输在调节钙稳态中发挥重要作用，从而抑制细胞迁移和侵袭。
