M1 macrophage-derived oncostatin M induces osteogenic differentiation of ligamentum flavum cells through the JAK2/STAT3 pathway

IF 3.4 3区医学 Q1 ORTHOPEDICS JOR Spine Pub Date : 2023-10-18 DOI:10.1002/jsp2.1290

Jun Yang, Guanghui Chen, Tianqi Fan, Xiaochen Qu

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Abstract

Background

M1 macrophages (Mφs) are involved in osteogenic differentiation of ligamentum flavum (LF) cells and play an important role in heterotopic ossification. However, the mechanism by which M1 Mφs influence osteogenic differentiation of LF cells has not been studied.

Methods

The effect of conditioned medium including secretions of M1 Mφs (CM-M1) on LF cells was analyzed by GeneChip profiling and ingenuity pathway analysis (IPA). THP-1 cells were polarized into M1 Mφs and CM-M1 was used to induce LF cells. In addition, LF cells were induced by CM-M1 in the presence of cyclooxygenase 2 (COX-2) inhibitors or oncostatin M (OSM)-neutralizing antibodies. Based on the presence of OSM, knockout of OSMR or GP130 receptors, or addition of the Janus kinase 2 (JAK2) inhibitor AZD1480 or signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic were examined for effects on osteogenic differentiation of LF cells. OSM secretion was quantified by ELISA, while qPCR and western blot were used to evaluate expression of osteogenic genes and receptor and signaling pathway-related proteins, respectively.

Results

GeneChip and IPA results indicate that the OSM signaling pathway and its downstream signaling molecules JAK2 and STAT3 are significantly activated. ELISA results indicate that OSM is highly expressed in cells treated with CM-M1 and lowly expressed in cells treated with CM-M1 and a COX-2 inhibitor. Besides, CM-M1 induces osteogenic differentiation of LF cells, which is weakened when COX-2 inhibitors or OSM-neutralizing antibody are added to it. Recombinant OSM could induce osteogenic differentiation of LF cells and upregulate expression of OSMR, GP130, phosphorylated (P)-JAK2, and P-STAT3. Upon knockdown of OSMR or GP130, or the addition of AZD1480 or Stattic, P-JAK2 and P-STAT3 expression were decreased and osteogenic differentiation was reduced.

Conclusion

M1 Mφ-derived OSM induces osteogenic differentiation of LF cells and the JAK2/STAT3 signaling pathway plays an important role.

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M1 巨噬细胞衍生的 oncostatin M 通过 JAK2/STAT3 通路诱导黄韧带细胞成骨分化

背景 M1 巨噬细胞（Mφs）参与黄韧带（LF）细胞的成骨分化，并在异位骨化中发挥重要作用。然而，M1 Mφs 影响黄韧带细胞成骨分化的机制尚未得到研究。方法通过基因芯片分析和巧妙通路分析（IPA）分析包括 M1 Mφs 分泌物（CM-M1）在内的条件培养基对 LF 细胞的影响。将 THP-1 细胞极化为 M1 Mφs 并用 CM-M1 诱导 LF 细胞。此外，在环氧合酶2（COX-2）抑制剂或oncostatin M（OSM）中和抗体存在的情况下，用CM-M1诱导LF细胞。在OSM存在的基础上，敲除OSMR或GP130受体，或添加Janus激酶2（JAK2）抑制剂AZD1480或信号转导和激活转录3（STAT3）抑制剂Stattic，以检测其对LF细胞成骨分化的影响。用酶联免疫吸附法（ELISA）定量检测 OSM 的分泌，用 qPCR 和 western 印迹法分别评估成骨基因和受体及信号通路相关蛋白的表达。结果基因芯片和 IPA 结果表明，OSM 信号通路及其下游信号分子 JAK2 和 STAT3 被显著激活。ELISA 结果表明，OSM 在用 CM-M1 处理的细胞中高表达，而在用 CM-M1 和 COX-2 抑制剂处理的细胞中低表达。此外，CM-M1还能诱导LF细胞成骨分化，而加入COX-2抑制剂或OSM中和抗体后，这种作用会减弱。重组OSM可诱导LF细胞成骨分化，并上调OSMR、GP130、磷酸化（P）-JAK2和P-STAT3的表达。敲除 OSMR 或 GP130 或添加 AZD1480 或 Stattic 后，P-JAK2 和 P-STAT3 的表达降低，成骨分化减少。结论 M1 Mφ 衍生的 OSM 可诱导 LF 细胞成骨分化，JAK2/STAT3 信号通路在其中发挥了重要作用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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