The Cell Collection and Research Center, National Institutes for Food and Drug Control, No. 2 Tiantan Xili, Dongcheng District, Beijing, 100050, China.
BMC Cancer. 2020 Nov 4;20(1):1064. doi: 10.1186/s12885-020-07542-5.
Immunomodulatory activities of human mesenchymal stromal /stem cells (hMSCs) has been widely recognized as the most critical function of hMSCs for exerting its therapeutic effects. However, the detailed mechanisms responsible for regulating the immunomodulation of hMSCs still remain largely unknown. Previous studies revealed that the Notch1 protein exerted a pro-immunomodulatory function probably through interacting with the protein(s) subjective to proteasome-mediated protein degradation. The DLC-1 protein represents a well characterized tumor suppressor subjective to proteasome-mediated degradation. However, the detailed signaling pathway of Notch1 and the involvement of DLC-1 in regulating the immunomodulation of hMSCs have not been studied before.
The transfection with cDNA or siRNA into hMSCs assisted by co-culture of hMSCs with peripheral blood mononuclear cells and small molecule inhibitors of signaling proteins, followed by immunoprecipitation, Western blotting, RT-PCR, and flowcytometry, were employed to characterize the Notch1 signaling, to identify DLC-1 as a candidate proteasome-targeted protein, and to characterize DLC-1 signaling pathway and its interaction with the Notch1 signaling, in the regulation of immunomodulation of hMSCs, specifically, the inhibition of pro-inflammatory CD4-Th1 lymphocytes, and the release of immunomodulatory molecule IDO1.
One-way ANOVA was utilized as a statistical tool to analyze the data presented as means ± SEM of at least three separate experiments.
The present study revealed that the Notch1-Hey1 axis, but not the Notch1-Hes1 axis, was likely responsible for mediating the pro-immunomodulatory function of the Notch1 signaling. The DLC-1 protein was found subjective to proteasome-mediated protein degradation mediated by the DDB1 and FBXW5 E3 ligases and served as an inhibitor of the immunomodulation of hMSCs through inhibiting Rock1, but not Rock2, downstream the DLC-1 signaling. The Notch1 signaling in the Notch1-Hey1 pathway and the DLC-1 signaling in the DLC-1-Rock1-FBXW5 pathway exhibited a mutual exclusion interaction in the regulation of immunomodulation of hMSCs.
The present study uncovers a novel function of DLC-1 tumor suppressor in regulating the immunomodulation of hMSCs. It also proposes a novel mutual exclusion mechanism between the DLC-1 signaling and the Notch1 signaling that is possibly responsible for fine-tuning the immunomodulation of hMSCs with different clinical implications in hMSCs therapy.
人们普遍认为，人类间充质基质/干细胞（hMSCs）的免疫调节活性是其发挥治疗作用的最重要功能。然而，负责调节 hMSC 免疫调节的确切机制在很大程度上仍然未知。先前的研究表明，Notch1 蛋白通过与受蛋白酶体介导的蛋白质降解影响的蛋白质（s）相互作用发挥促免疫调节功能。DLC-1 蛋白是一种经过充分表征的肿瘤抑制因子，受蛋白酶体介导的降解影响。然而，Notch1 信号通路的详细信号通路以及 DLC-1 在调节 hMSC 免疫调节中的作用以前尚未研究过。
通过共培养 hMSC 和外周血单核细胞并使用信号蛋白的小分子抑制剂转染 hMSC 的 cDNA 或 siRNA，随后进行免疫沉淀、Western 印迹、RT-PCR 和流式细胞术，以表征 Notch1 信号通路，鉴定 DLC-1 作为候选蛋白酶体靶向蛋白，并表征 DLC-1 信号通路及其与 Notch1 信号通路的相互作用，以调节 hMSC 的免疫调节，具体而言，抑制促炎 CD4-Th1 淋巴细胞，并释放免疫调节分子 IDO1。
采用单向方差分析作为统计工具，分析至少三个独立实验的平均值±SEM 表示的数据。
本研究表明，Notch1-Hey1 轴，而不是 Notch1-Hes1 轴，可能负责介导 Notch1 信号的促免疫调节功能。发现 DLC-1 蛋白受 DDB1 和 FBXW5 E3 连接酶介导的蛋白酶体介导的蛋白质降解影响，并通过抑制 Rock1（而非 Rock2）作为 hMSC 免疫调节的抑制剂，发挥作用。Notch1 信号通路在 Notch1-Hey1 通路中的作用和 DLC-1 信号通路在 DLC-1-Rock1-FBXW5 通路中的作用在调节 hMSC 的免疫调节中表现出相互排斥的相互作用。
本研究揭示了 DLC-1 肿瘤抑制因子在调节 hMSC 免疫调节中的新功能。它还提出了 DLC-1 信号通路和 Notch1 信号通路之间的一种新的互斥机制，该机制可能负责微调 hMSC 治疗中具有不同临床意义的 hMSC 免疫调节。
