Department of Anesthesiology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China.
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China.
Bone. 2020 Feb;131:115059. doi: 10.1016/j.bone.2019.115059. Epub 2019 Sep 12.
The osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is critical for bone homeostasis. Here, we investigated the regulation of Galectin-3 and tripartite motif protein 16 (TRIM16) on osteogenic differentiation of hBMSCs through autophagy.
Quantitative PCR (qPCR) and western blot were performed to determine the expression of osteogenic markers, autophagic markers, Galectin-3 and TRIM16. Short-hairpin RNAs (shRNAs) and overexpression plasmids were used to manipulate the expression of Galectin-3, TRIM16 and Unc-51 like autophagy activating kinase 1 (ULK1). Alkaline phosphatase (ALP) activity was measured by ALP staining assay. Calcium deposition in differentiated hBMSCs was assessed by Alizarin Red S staining. LC3 puncta formation was monitored by immunofluorescence staining. The interaction between indicated proteins was confirmed by co-immunoprecipitation (Co-IP) assay.
Either Galectin-3 or TRIM16 knockdown led to impaired ALP activity, reduced calcium deposition, down-regulation of pro-osteogenic markers as well as restrained autophagy in osteogenic-induced hBMSCs. However, overexpression of Galectin-3 or TRIM16 promoted osteogenic differentiation of hBMSCs, which was then compromised by autophagy inhibition. Co-IP experiment demonstrated that TRIM16 associated with Galectin-3 through ULK1. Meanwhile, osteogenic induction enhanced the association between TRIM16 and ULK1 or coiled-coil myosin-like BCL2-interacting protein (Beclin1), and TRIM16 increased the stability of ULK1 and Beclin1. Moreover, either TRIM16 or ULK1 knockdown dampened the pro-osteogenic effect of Galectin-3, which elucidated that Galectin-3 mediated osteogenic differentiation was at least partly dependent on TRIM16 and ULK1.
In summary, the present study revealed Galectin-3 and TRIM16 co-regulated osteogenic differentiation of hBMSCs at least partly via enhancing autophagy, which might provide a promising approach for osteoporosis treatment in future.
人骨髓间充质干细胞（hBMSCs）的成骨分化对于骨稳态至关重要。在这里，我们通过自噬研究了半乳糖凝集素-3（Galectin-3）和三联基序蛋白 16（TRIM16）对 hBMSCs 成骨分化的调节作用。
采用实时定量 PCR（qPCR）和 Western blot 检测成骨标志物、自噬标志物、Galectin-3 和 TRIM16 的表达。使用短发夹 RNA（shRNA）和过表达质粒来操纵 Galectin-3、TRIM16 和自噬激活激酶 1（Unc-51 样）（ULK1）的表达。通过碱性磷酸酶（ALP）染色测定 ALP 活性。通过茜素红 S 染色评估分化的 hBMSCs 中的钙沉积。通过免疫荧光染色监测 LC3 斑点形成。通过免疫共沉淀（Co-IP）测定证实指示蛋白之间的相互作用。
Galectin-3 或 TRIM16 敲低均导致成骨诱导的 hBMSCs 中 ALP 活性降低、钙沉积减少、成骨前标志物下调以及自噬受到抑制。然而，Galectin-3 或 TRIM16 的过表达促进了 hBMSCs 的成骨分化，随后自噬抑制会损害其成骨分化。Co-IP 实验表明，TRIM16 通过 ULK1 与 Galectin-3 结合。同时，成骨诱导增强了 TRIM16 与 ULK1 或卷曲螺旋肌球蛋白样 BCL2 相互作用蛋白（Beclin1）之间的结合，TRIM16 增加了 ULK1 和 Beclin1 的稳定性。此外，TRIM16 或 ULK1 敲低减弱了 Galectin-3 的成骨作用，这表明 Galectin-3 介导的成骨分化至少部分依赖于 TRIM16 和 ULK1。
综上所述，本研究揭示了 Galectin-3 和 TRIM16 通过增强自噬至少部分协同调节 hBMSCs 的成骨分化，这为未来骨质疏松症的治疗提供了一种有前途的方法。
