Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA.
J Cell Mol Med. 2021 Mar;25(5):2390-2403. doi: 10.1111/jcmm.16071. Epub 2021 Jan 28.
Osteoclasts (OCs) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodelling in both homeostasis and pathology. Various immune and non-immune cells help initiating activation of myeloid cells for differentiation, whereas hyper-activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, we show the efficacy of dental pulp-derived stem cells (DPSCs) in limiting RAW 264.7 cell differentiation and underlying molecular mechanism, which has the potential for future therapeutic application in bone-related disorders. We found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co-cultured in a contact-free system. DPSCs reduced expression of key OC markers, such as NFATc1, cathepsin K, TRAP, RANK and MMP-9 assessed by quantitative RT-PCR, Western blotting and immunofluorescence detection methods. Furthermore, quantitative RT-PCR analysis revealed that DPSCs mediated M2 polarization of RAW 264.7 cells. To define molecular mechanisms, we found that osteoprotegerin (OPG), an OC inhibitory factor, was up-regulated in RAW 264.7 cells in the presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, the addition of recombinant OPG inhibited OC differentiation in a dose-dependent manner by reducing the expression of OC differentiation markers, NFATc1, cathepsin K, TRAP, RANK and MMP9 in RAW 264.7 cells. RNAKL and M-CSF phosphorylate AKT and activate PI3K-AKT signalling pathway during osteoclast differentiation. We further confirmed that OPG-mediated inhibition of the downstream activation of PI3K-AKT signalling pathway was similar to the DPSC co-culture-mediated inhibition of OC differentiation. This study provides novel evidence of DPSC-mediated inhibition of osteoclastogenesis mechanisms.
破骨细胞（OCs）来源于单核/巨噬细胞谱系，在稳态和病理条件下对骨吸收和重塑起关键调节作用。各种免疫和非免疫细胞有助于启动髓样细胞的分化激活，而过度激活则导致发病机制，其机制尚未完全理解。在此，我们展示了牙髓干细胞（DPSCs）在限制 RAW 264.7 细胞分化及其潜在分子机制中的功效，这为骨相关疾病的未来治疗应用提供了潜力。我们发现，当在无接触系统中进行共培养时，DPSCs 可抑制 RAW 264.7 细胞诱导的 OC 分化。通过定量 RT-PCR、Western blot 和免疫荧光检测方法，DPSCs 降低了 NFATc1、组织蛋白酶 K、TRAP、RANK 和 MMP-9 等关键 OC 标志物的表达。此外，定量 RT-PCR 分析显示，DPSCs 介导了 RAW 264.7 细胞的 M2 极化。为了确定分子机制，我们发现，在存在 DPSCs 的情况下，OC 抑制因子护骨素（OPG）在 RAW 264.7 细胞中上调。此外，DPSCs 还持续分泌 OPG，有助于限制 OC 分化。最后，添加重组 OPG 通过降低 RAW 264.7 细胞中 OC 分化标志物 NFATc1、组织蛋白酶 K、TRAP、RANK 和 MMP9 的表达，以剂量依赖的方式抑制 OC 分化。RNAKL 和 M-CSF 磷酸化 AKT 并激活 PI3K-AKT 信号通路，在破骨细胞分化过程中。我们进一步证实，OPG 介导的对 PI3K-AKT 信号通路下游激活的抑制与 DPSC 共培养介导的 OC 分化抑制相似。本研究为 DPSCs 介导的抑制破骨细胞生成机制提供了新的证据。
