TIMP1 promotes multi-walled carbon nanotube-induced lung fibrosis by stimulating fibroblast activation and proliferation

Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) may cause fibrosing lesions in animal lungs, raising health concerns about such exposure in humans. The mechanisms underlying fibrosis development remain unclear, but they are believed to involve the dysfunction of fibroblasts and myofibroblasts. Using a mouse model of MWCNT exposure, we found that the tissue inhibitor of metalloproteinase 1 (Timp1) gene was rapidly and highly induced in the lungs by MWCNTs in a time- and dose-dependent manner. Concomitantly, a pronounced elevation of secreted TIMP1 was observed in the bronchoalveolar lavage (BAL) fluid and serum. Knockout (KO) of Timp1 in mice caused a significant reduction in fibrotic focus formation, collagen fiber deposition, recruitment of fibroblasts and differentiation of fibroblasts into myofibroblasts in the lungs, indicating that TIMP1 plays a critical role in the pulmonary fibrotic response to MWCNTs. At the molecular level, MWCNT exposure significantly increased the expression of the cell proliferation markers Ki-67 and PCNA and a panel of cell cycle-controlling genes in the lungs in a TIMP1-dependent manner. MWCNT-stimulated cell proliferation was most prominent in fibroblasts but not myofibroblasts. Furthermore, MWCNTs elicited a significant induction of CD63 and integrin β1 in lung fibroblasts, leading to the formation of a TIMP1/CD63/integrin β1 complex on the surface of fibroblasts <i>in vivo</i> and <i>in vitro</i>, which triggered the phosphorylation and activation of Erk1/2. Our study uncovers a new pathway through which induced TIMP1 critically modulates the pulmonary fibrotic response to MWCNTs by promoting fibroblast activation and proliferation via the TIMP1/CD63/integrin β1 axis and ERK signaling.

肺部暴露于多壁碳纳米管（multi-walled carbon nanotubes, MWCNTs）可在动物肺部引发纤维化病变，由此引发了人类暴露于这类物质的健康安全担忧。纤维化发生的具体机制目前仍不明确，但普遍认为其与成纤维细胞及肌成纤维细胞的功能异常密切相关。本研究采用MWCNTs暴露小鼠模型，发现金属蛋白酶组织抑制剂1（tissue inhibitor of metalloproteinase 1, Timp1）基因在小鼠肺部被MWCNTs快速且高水平诱导，且该诱导过程呈现时间和剂量依赖性。与此同时，支气管肺泡灌洗液（bronchoalveolar lavage, BAL）与血清中分泌型TIMP1的水平也显著升高。小鼠Timp1基因敲除（knockout, KO）后，肺部纤维化病灶形成、胶原纤维沉积、成纤维细胞募集以及成纤维细胞向肌成纤维细胞的分化均显著降低，这表明TIMP1在MWCNTs诱导的肺部纤维化应答中发挥关键调控作用。在分子层面，MWCNTs暴露可通过TIMP1依赖的方式，显著上调肺部细胞增殖标志物Ki-67、PCNA以及一系列细胞周期调控基因的表达。MWCNTs刺激的细胞增殖效应在成纤维细胞中最为显著，而在肌成纤维细胞中则未观察到此现象。进一步研究发现，MWCNTs可诱导肺成纤维细胞中CD63与整合素β1的表达上调，进而在成纤维细胞表面于体内和体外环境下形成TIMP1/CD63/整合素β1复合物，该复合物可触发Erk1/2的磷酸化与激活。本研究揭示了一条全新的信号通路：诱导产生的TIMP1可通过TIMP1/CD63/整合素β1轴与ERK信号通路促进成纤维细胞活化与增殖，从而关键调控MWCNTs诱导的肺部纤维化应答。