Kwok Carsten Tsun-Ka, Wong Chun-Chak, Li Jing-Jing, Kwan Yiu-Wa, Leung George Pak-Heng, Tsoi Bun, Chow Franklin Wang-Ngai, Seto Sai-Wang
Department of Food Science and Nutrition, The Hong Kong Polytechnic University, Hong Kong, SAR, China.
Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, SAR, China.
Inflamm Res. 2025 Jan 25;74(1):27. doi: 10.1007/s00011-024-01987-1.
Sclerostin (SOST) is traditionally regarded as an osteocyte-derived secreted glycoprotein that regulates bone mineralization. Recent studies reported that SOST is also released from non-skeletal sources, especially during inflammation. However, the cellular source and regulatory mechanisms governing SOST generation in inflammation remain unclear. This study investigated whether macrophages produce SOST in response to inflammatory stimuli and determined associated regulatory pathways.
The effect of lipopolysaccharide (LPS)-induced inflammation in SOST generation and its underlying regulatory mechanism was examined on mouse macrophage RAW 264.7 by western blot and immunofluorescent staining. Transfection with miR-92a-3p mimic and inhibitor were used to validate its role in SOST production. The role of NF-κB and TLR4 were studied using pharmacological inhibitors BAY 11-7085 and TAK242, respectively. The involvement of NF-κB and TLR4 in LPS-induced SOST production was further validated through nuclear NF-κB p65 immunoprecipitation and TLR4 small interfering RNA (siRNA) experiments, respectively.GW4869 and manumycin A (extracellular vesicles (EV) biogenesis inhibitors) were used to examine the associated of SOST and EV. Finally, SOST expression and characteristics of the isolated EV were assessed by Western blot and nanoparticle tracking analysis (NTA).
LPS significantly induced SOST protein expression and secretion in RAW 264.7. MiR-92a-3p was upregulated by LPS stimulation in macrophages. Transfection of miR-92a-3p mimic increased SOST generation in RAW 264.7. Inhibition of TLR4 and NF-κB signalling pathways using pharmacological inhibitors significantly suppressed LPS-induced SOST in RAW 264.7. Similarly, TLR4 siRNA effectively suppressed LPS-induced SOST level. However, the LPS-induced upregulation of miR-92a-3p was only regulated by TLR4, but not by NF-κB. NF-κB was found to directly bind to the mouse sost promoter, thereby activating sost transcription. Additionally, SOST secretion was found predominantly associated with EV from LPS-stimulated cells, and inhibition of EV biogenesis suppressed SOST production in RAW 264.7 cells.
In conclusion, our study showed, for the first time, that LPS induced SOST generation and secretion via TLR4/miR-92a-3p/PTEN/NF-κB singling pathway in murine macrophage RAW 264.7 cells. Moreover, we showed that SOST is secreted from the RAW 264.7 cells in the form of extracellular vesicle. This study identified macrophage as a novel source of SOST, highlighting its potential role in inflammatory diseases.
硬化蛋白（SOST）传统上被认为是一种由骨细胞分泌的调节骨矿化的糖蛋白。最近的研究报道，SOST也可从非骨骼来源释放，尤其是在炎症期间。然而，炎症中SOST产生的细胞来源和调控机制仍不清楚。本研究调查了巨噬细胞是否会对炎症刺激产生SOST，并确定了相关的调控途径。
通过蛋白质免疫印迹法和免疫荧光染色，检测脂多糖（LPS）诱导的炎症对小鼠巨噬细胞RAW 264.7中SOST产生的影响及其潜在调控机制。转染miR-92a-3p模拟物和抑制剂以验证其在SOST产生中的作用。分别使用药理学抑制剂BAY 11-7085和TAK242研究核因子κB（NF-κB）和Toll样受体4（TLR4）的作用。分别通过核NF-κB p65免疫沉淀和TLR4小干扰RNA（siRNA）实验进一步验证NF-κB和TLR4在LPS诱导的SOST产生中的作用。使用GW4869和曼诺霉素A（细胞外囊泡（EV）生物发生抑制剂）检测SOST与EV的相关性。最后，通过蛋白质免疫印迹法和纳米颗粒跟踪分析（NTA）评估分离的EV的SOST表达和特征。
LPS显著诱导RAW 264.7细胞中SOST蛋白的表达和分泌。LPS刺激巨噬细胞后，miR-92a-3p上调。转染miR-92a-3p模拟物可增加RAW 264.7细胞中SOST的产生。使用药理学抑制剂抑制TLR4和NF-κB信号通路可显著抑制LPS诱导的RAW 264.7细胞中SOST的产生。同样，TLR4 siRNA有效抑制LPS诱导的SOST水平。然而，LPS诱导的miR-92a-3p上调仅受TLR4调节，不受NF-κB调节。发现NF-κB直接与小鼠sost启动子结合，从而激活sost转录。此外，发现SOST分泌主要与LPS刺激细胞产生的EV相关，抑制EV生物发生可抑制RAW 264.7细胞中SOST的产生。
总之，我们的研究首次表明，LPS通过TLR4/miR-92a-3p/磷酸酶和张力蛋白同源物（PTEN）/NF-κB信号通路诱导小鼠巨噬细胞RAW 264.7细胞中SOST的产生和分泌。此外，我们表明SOST以细胞外囊泡的形式从RAW 264.7细胞中分泌。本研究确定巨噬细胞是SOST的新来源，突出了其在炎症性疾病中的潜在作用。
