METHODS: Osteogenesis of MEFs was observed by alkaline phosphatase (ALP) staining, alizarin red staining, and Von Kossa staining assays. Activation of unfolded protein response and Shh signaling were examined using real-time quantitative PCR and western blot assays. IRE1α-deficient MEFs were used to explore the effect of IRE1α on N-Shh-driven osteogenesis.
RESULTS: N-Shh increased ALP activity, matrix mineralization, and the expression of Alp and Col-I in MEFs under osteogenic conditions; notably, this was reversed when combined with the ER stress activator Tm treatment. Interestingly, the administration of N-Shh decreased the expression of IRE1α. Abrogation of IRE1α increased the expression of Shh pathway factors in osteogenesis-induced MEFs, contributing to the osteogenic effect of N-Shh. Moreover, IRE1α-deficient MEFs exhibited elevated levels of osteogenic markers.
CONCLUSIONS: Our findings suggest that the IRE1α-mediated unfolded protein response may alleviate the ossification of MEFs by attenuating Shh signaling. Our research has identified a strategy to inhibit excessive ossification, which may have clinical significance in preventing temporomandibular joint bony ankylosis.
方法:通过碱性磷酸酶(ALP)染色观察MEFs的成骨作用,茜素红染色,和VonKossa染色测定。使用实时定量PCR和蛋白质印迹测定检查未折叠蛋白应答的激活和Shh信号传导。使用IRE1α缺陷的MEFs来探索IRE1α对N-Shh驱动的成骨作用。
结果:N-Shh增加了ALP活性,基质矿化,以及成骨条件下MEFs中Alp和Col-I的表达;当与ER应激激活剂Tm治疗结合使用时,这种情况可以逆转。有趣的是,N-Shh的给药降低了IRE1α的表达。IRE1α的消除增加了Shh途径因子在成骨诱导的MEFs中的表达,有助于N-Shh的成骨作用。此外,IRE1α缺陷型MEFs显示成骨标志物水平升高。
结论:我们的发现表明IRE1α介导的未折叠蛋白反应可能通过减弱Shh信号来减轻MEFs的骨化。我们的研究确定了一种抑制过度骨化的策略,这可能对预防颞下颌关节骨性强直具有临床意义。