BACKGROUND Dysfunction in survival and differentiation of osteoblasts commonly occurs in patients with osteoporosis. Cannabinoid receptor type 2 (CNR2) is a major receptor of endocannabinoid system that is crucial for bone mass homeostasis. Our group prior demonstrated that activation of CNR2 signaling promoted osteogenic differentiation of bone marrow derived mesenchymal stem cells in vitro. Autophagy is reported to participate in osteoblastic differentiation. Whether autophagy is regulated by CNR2-mediated cannabinoid signaling is unknown, and how the autophagy-CNR2 interaction affects osteoblastic differentiation requires further elucidation. METHODS hFOB 1.19 osteoblasts were treated with CNR2 agonists HU308 (5, 10, 25, 50 or 100 nM) and JWH133 (1, 2, 5, 10 or 20 μM) in presence or absence of autophagy inhibitor 3-Methyladenine (3-MA). The differentiation of hFOB 1.19 cells was determined via evaluating their alkaline phosphatase (ALP) activity and mineralization ability (Alizarin red staining). Alterations in autophagy-related molecules and osteogenic markers were analyzed via real-time PCR and/or immunoblotting assays. RESULTS hFOB 1.19 cells spontaneously differentiated towards mature osteoblasts under 39 °C, during which CNR2 expression increased, and autophagy was activated. The strongest autophagy flux was observed at 192 h post differentiation─LC3I to LC3II conversion was enhanced and Beclin 1 expression was upregulated considerably, while p62 expression was downregulated. Treatment of HU308 and JWH133 promoted autophagy in a dose-dependent manner, and suppressed mTOR signaling pathway in hFOB 1.19 cells. In CNR2-silenced cells, HU308's and JWH133's effects on autophagy were weakened. HU308 and JWH133 enhanced the ALP activity and mineralization, and upregulated the expression of osteogenic markers, osteopontin and osteocalcin, in hFOB 1.19 cells. Intriguingly, such pro-osteogenic effects induced by CNR2 activation were markedly mitigated by 3-MA. In addition to provoking autophagy, CNR2 agonists also reduced nuclear Nrf2 accumulation and increased Keap1 expression. Further, re-expression of p62 inhibited CNR2 agonists-induced Nrf2 degradation. CONCLUSIONS Osteogenic differentiation induced by CNR2 signaling activation involves autophagy induction and p62-mediated Nrf2 deactivation.

中文翻译：

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大麻素2型受体诱导的成骨分化的激活涉及自噬诱导和p62介导的Nrf2失活。

背景技术成骨细胞存活和分化的功能障碍通常发生在骨质疏松症患者中。大麻素2型受体（CNR2）是内源性大麻素系统的主要受体，对于骨质稳态是至关重要的。我们的研究小组先前证明，CNR2信号的激活在体外促进了骨髓来源的间充质干细胞的成骨分化。据报道自噬参与成骨细胞分化。自噬是否受CNR2介导的大麻素信号传导调控尚不清楚，并且自噬-CNR2相互作用如何影响成骨细胞分化还需要进一步阐明。方法在存在或不存在自噬抑制剂3-甲基腺嘌呤（3-MA）的情况下，用CNR2激动剂HU308（5、10、25、50或100 nM）和JWH133（1、2、5、10或20μM）处理hFOB 1.19成骨细胞）。hFOB 1.19细胞的分化通过评估其碱性磷酸酶（ALP）活性和矿化能力（茜素红染色）来确定。通过实时PCR和/或免疫印迹测定法分析自噬相关分子和成骨标记的变化。结果在39°C下，hFOB 1.19细胞自发分化为成熟的成骨细胞，在此期间CNR2表达增加，并且自噬被激活。分化后192 h观察到最强的自噬通量-LC3I向LC3II的转化增强，Beclin 1的表达明显上调，而p62的表达下调。HU308和JWH133的治疗以剂量依赖性方式促进自噬，并抑制hFOB 1.19细胞中的mTOR信号传导途径。在CNR2沉默的细胞中，HU308和JWH133' 对自噬的作用减弱。HU308和JWH133增强了hFOB 1.19细胞的ALP活性和矿化作用，并上调了成骨标记物骨桥蛋白和骨钙素的表达。有趣的是，3-MA明显减轻了由CNR2激活诱导的促成骨作用。除了引起自噬外，CNR2激动剂还减少了核Nrf2积累并增加了Keap1表达。此外，p62的重新表达抑制了CNR2激动剂诱导的Nrf2降解。结论CNR2信号激活诱导的成骨分化涉及自噬诱导和p62介导的Nrf2失活。3-MA明显减轻了由CNR2激活引起的这种促成骨作用。除了引起自噬外，CNR2激动剂还减少了核Nrf2积累并增加了Keap1表达。此外，p62的重新表达抑制了CNR2激动剂诱导的Nrf2降解。结论CNR2信号激活诱导的成骨分化涉及自噬诱导和p62介导的Nrf2失活。3-MA明显减轻了由CNR2激活引起的这种促成骨作用。除了引起自噬外，CNR2激动剂还减少了核Nrf2积累并增加了Keap1表达。此外，p62的重新表达抑制了CNR2激动剂诱导的Nrf2降解。结论CNR2信号激活诱导的成骨分化涉及自噬诱导和p62介导的Nrf2失活。