Galectin-3 and TRIM16 coregulate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells at least partly via enhancing autophagy
Galectin-3 and TRIM16 coregulate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells at least partly via enhancing autophagy
复制标题
DOI：
10.1016/j.bone.2019.115059
复制
发表时间：
2020-02-01
期刊：
影响因子：
4.1
通讯作者：
Wang, Bo-Wei
中科院分区：
文献类型：
--
作者：
Chen, Wen-Ting;Zhang, Fan;Wang, Bo-Wei
背景：人骨髓间充质干细胞(HBMSCs)的成骨分化是维持骨内环境稳定的关键。在此，我们通过自噬研究了Galectin-3和TRIM16对hBMSCs成骨分化的调节作用。方法：采用定量聚合酶链式反应(QPCR)和免疫印迹法检测成骨标志物、自噬标志物、Galectin-3和TRIM16的表达。利用短发夹状RNA(ShRNAs)和高表达载体调控Galectin-3、TRIM16和UNC-51类自噬激活蛋白1(ULK1)的表达。碱性磷酸酶(ALP)染色检测ALP活性。茜素红S染色检测分化的hBMSCs的钙沉积情况。免疫荧光染色观察LC_3斑点的形成。结果：Galectin-3和TRIM16基因敲除均导致成骨诱导的hBMSCs碱性磷酸酶活性降低，钙沉积减少，促成骨标志物表达下调，自噬抑制。然而，Galectin-3或TRIM16的过表达促进了hBMSCs的成骨分化，这一作用随后被自噬抑制所抑制。共IP实验证明TRIM16通过ULK1与Galectin-3结合。同时，成骨诱导增强了TRIM16与ULK1或卷曲盘状肌球蛋白样BCL2相互作用蛋白(Beclin1)的结合，TRIM16增加了ULK1和Beclin1的稳定性。此外，TRIM16和ULK1基因敲除都抑制了Galectin-3的成骨作用，说明Galectin-3介导的成骨分化至少部分依赖于TRIM16和ULK1。结论：本研究揭示Galectin-3和TRIM16至少部分通过促进自噬来共同调节hBMSCs的成骨分化，为今后骨质疏松的治疗提供了一条新的途径。
Background: 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.Methods: 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.Results: 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.Conclusion: 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.