Phosphate promotes osteogenic differentiation through non-canonical Wnt signaling pathway in human mesenchymal stem cells
Phosphate promotes osteogenic differentiation through non-canonical Wnt signaling pathway in human mesenchymal stem cells
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磷酸盐通过非经典Wnt信号通路促进人间充质干细胞的成骨分化
DOI：
10.1016/j.bone.2022.116525
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发表时间：
2022
期刊：
影响因子：
4.1
通讯作者：
Ozono Keiichi
中科院分区：
文献类型：
--
作者：
Rui Shumin;Kubota Takuo;Ohata Yasuhisa;Yamamoto Kenichi;Fujiwara Makoto;Takeyari Shinji;Ozono Keiichi
背景磷酸盐在骨形成和矿化过程中是不可缺少的。然而，磷酸盐促进成骨分化的机制尚未完全了解。在这项研究中，我们研究了磷酸盐对成骨分化的影响，以及磷酸盐诱导的信号通路在这个过程中，诱导人骨髓间充质干细胞分化为成骨细胞的培养基中含有β-甘油磷酸盐（GP），1 mM无机磷酸盐，或3 mM无机磷酸盐（Pi）的变化。通过成骨细胞分化标志物的表达和钙沉积来验证成骨细胞的分化。进行RNA测序，以评估转录组在早期阶段的成骨differentiation.ResultsOsteogenic分化和矿化促进在3 mM Pi组相比，在GP和1 mM Pi组在第7天的文化。RNA测序结果显示，在第7天，与GP中的那些相比，在3 mM Pi组中参与成骨的基因表达和Wnt信号通路中的组分增加。qPCR和Western blot分析表明，在第7天，3 mM Pi组的非经典Wnt信号通路中的组分上调，包括WNT 5 b和磷酸化-c-Jun。在第7天，2个诱导组中WNT 11 mRNA表达增加。siRNA抑制WNT 5 b后，WNT 5 b、WNT 11、ROR 2 mRNA表达和磷酸化c-Jun蛋白表达均明显减弱。结论Pi通过上调WNT 5 b、WNT 11、p-c-Jun/c-Jun等非经典Wnt信号通路促进成骨细胞分化，促进成骨细胞的分化。这些发现提供了一个新的角度到协会的Pi和非经典的Wnt信号通路在成骨分化。
BackgroundPhosphate is indispensable in osteogenesis and mineralization. However, mechanisms by which phosphate enhances osteogenic differentiation are not fully understood. In this study, we studied the effect of phosphate on osteogenic differentiation as well as signaling pathways induced by phosphate in the process.MethodInduced human bone marrow-derived mesenchymal stem cells differentiation into osteoblasts by the change of media containing β-glycerophosphate (GP), 1 mM inorganic phosphate, or 3 mM inorganic phosphate (Pi). The differentiation of osteoblasts was verified by the expression of osteoblast differentiation markers and calcium deposition. RNA sequencing was performed to assess transcriptome in the early stage of osteogenic differentiation.ResultsOsteogenic differentiation and mineralization were promoted in the 3 mM Pi group compared to those in the GP and 1 mM Pi groups on day 7 of culture. RNA sequencing revealed that the gene expressions involved in osteogenesis and the components in the Wnt signaling pathway was increased in 3 mM Pi group compared with those in the GP on day 7. Analysis with qPCR and Western blot suggested upregulation of components in the non-canonical Wnt signaling pathway, including WNT5b and phosphorylated-c-Jun in the 3 mM Pi group on day 7.WNT11mRNA expression was increased in the 2 induction groups on day 7. Inhibition of WNT5b by siRNA experiment attenuated the components in non-canonical Wnt signaling expression, includingWNT5b,WNT11andROR2mRNA expression and phosphorylated-c-Jun protein expression. In addition, osteogenic differentiation and mineralization were partly decreased in 3 mM Pi group on day 7 by the inhibition of WNT5b.ConclusionPi promoted osteogenic differentiation through the up-regulation of the non-canonical Wnt signaling pathway, including WNT5b, WNT11, p-c-Jun/c-Jun, in the early stage of differentiation. These findings provide a new perspective into the association of Pi and the non-canonical Wnt signaling pathway during osteogenic differentiation.