Objectives Lung fibrosis leads as the cause of death in Systemic Sclerosis (SSc), with no cure currently available. Antifibrotic Endostatin (ES) production does not reach therapeutic levels in SSc patients, suggesting a deficit in its release from Collagen-XVIII by the main cleavage enzyme, Cathepsin L (CTSL). Thus, elucidating a potential deficit in CTSL expression and activity unravels an underlying molecular cause for SSc-driven lung fibrosis. Methods Fibrosis was induced experimentally using transforming growth factor-β (TGF-β) in vivo, in primary human lung fibroblasts (pLFs), and ex-vivo, in human lung tissues. ES and CTSL expression were quantified using ELISA, RT-qPCR, immunoblotting, or immunofluorescence. Recombinant NC1-FLAG peptide was used to assess CTSL cleavage activity. CTSL expression was also compared between SSc vs normal (NL)-derived pLFs and lung tissues. Results ES levels were significantly reduced in media conditioned by TGF-β-induced pLFs. TGF-β-stimulated pLFs significantly reduced expression and secretion of CTSL into the extracellular matrix (ECM). CTSL was also sequestered in its inactive form into extracellular vesicles (EVs) further reducing its availability in the ECM. Media conditioned by TGF-β-induced pLFs showed reduced cleavage of NC1-Flag and reduced release of the antifibrotic ES fragment. SSc-derived pLFs and lung tissues expressed significantly lower levels of CTSL compared with NL. Conclusions Our findings identify CTSL as a protein protective against lung fibrosis via its activation of antifibrotic ES, and whose expression in SSc pLFs and lung tissues is suppressed. Identifying strategies to boost CTSL endogenous levels in SSc patients could serve as a viable therapeutic strategy.

中文翻译：

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减少组织蛋白酶 L 表达和分泌到细胞外环境导致系统性硬化症肺纤维化

目标 肺纤维化是系统性硬化症 (SSc) 的主要死因，目前尚无治愈方法。抗纤维化内皮抑素 (ES) 的产生未达到 SSc 患者的治疗水平，表明主要裂解酶组织蛋白酶 L (CTSL) 从胶原蛋白 XVIII 中释放的缺陷。因此，阐明 CTSL 表达和活性的潜在缺陷揭示了 SSc 驱动的肺纤维化的潜在分子原因。方法 使用转化生长因子-β (TGF-β) 在体内、原代人肺成纤维细胞 (pLF) 和体外人肺组织中实验诱导纤维化。使用 ELISA、RT-qPCR、免疫印迹或免疫荧光定量 ES 和 CTSL 表达。重组 NC1-FLAG 肽用于评估 CTSL 切割活性。还比较了 SSc 与正常 (NL) 衍生的 pLF 和肺组织之间的 CTSL 表达。结果 在由 TGF-β 诱导的 pLF 条件化的培养基中，ES 水平显着降低。TGF-β 刺激的 pLF 显着降低了 CTSL 的表达和分泌到细胞外基质 (ECM) 中。CTSL 还以其非活性形式被隔离到细胞外囊泡 (EV) 中，进一步降低了其在 ECM 中的可用性。由 TGF-β 诱导的 pLF 调节的培养基显示 NC1-Flag 的切割减少和抗纤维化 ES 片段的释放减少。与 NL 相比，SSc 衍生的 pLF 和肺组织表达显着较低水平的 CTSL。结论 我们的研究结果将 CTSL 鉴定为一种通过激活抗纤维化 ES 来保护肺纤维化的蛋白质，并且其在 SSc pLF 和肺组织中的表达受到抑制。