BACKGROUND Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. METHODS Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. RESULTS cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. CONCLUSIONS Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.

中文翻译：

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低强度超声预处理间充质基质细胞：对软骨形成和定向SOX9信号通路的影响。

背景技术在不存在外源添加的转化生长因子-β（TGFβ）的情况下，连续低强度超声（cLIUS）促进人间充质基质细胞（MSC）的软骨分化，通过上调软骨形成的主要调控因子转录因子SOX9的表达来实现。本研究评估了在cLIUS下与影响SOX9基因和蛋白质表达的信号通路相关的分子事件。方法将人类骨髓来源的MSC在14 kPa（5 MHz，2.5 Vpp）的cLIUS刺激下暴露5分钟。评价了SOX9的基因和蛋白质表达。通过用选择性信号分子的小分子抑制剂处理MSC，然后进行cLIUS处理，可以确定cLIUS下SOX9上调的特异性。通过基因表达，免疫荧光染色和蛋白质印迹分析了在cLIUS下调节SOX9表达的信号事件。结果与非cLIUS刺激的对照组相比，cLIUS上调了SOX9的基因表达并增强了SOX9蛋白的核定位。注意到cLIUS增强了信号分子ERK1 / 2的磷酸化。PD98059对MEK / ERK1 / 2的抑制导致cLIUS诱导的SOX9表达的有效消除，表明cLIUS诱导的SOX9上调取决于ERK1 / 2的磷酸化。整联蛋白和TRPV4的抑制，ERK1 / 2的上游细胞表面效应器，不能抑制ERK1 / 2的磷酸化，因此不能消除cLIUS诱导的SOX9表达，从而提示其他机械感受器的参与。所以，评估了cLIUS对肌动蛋白细胞骨架（一种调节SOX9的机械敏感性受体）的作用。通过使用细胞骨架药物Y27632处理，在cLIUS下在MSC中观察到的弥散和破坏的肌动蛋白纤维与肌动蛋白破坏非常相似，已知它可以增加SOX9的基因表达。因此，在cLIUS下SOX9的上调与cLIUS诱导的肌动蛋白重组有关。还发现肌动蛋白重组诱导的SOX9上调依赖于ERK1 / 2的磷酸化。结论总的来说，cLIUS对MSC的预处理导致SOX9的核定位，ERK1 / 2的磷酸化和肌动蛋白丝的破坏，而SOX9的表达取决于cLIUS下ERK1 / 2的磷酸化。被评估。通过使用细胞骨架药物Y27632处理，在cLIUS下在MSC中观察到的弥散和破坏的肌动蛋白纤维与肌动蛋白破坏非常相似，已知它可以增加SOX9的基因表达。因此，在cLIUS下SOX9的上调与cLIUS诱导的肌动蛋白重组有关。还发现肌动蛋白重组诱导的SOX9上调依赖于ERK1 / 2的磷酸化。结论总的来说，cLIUS对MSC的预处理导致SOX9的核定位，ERK1 / 2的磷酸化和肌动蛋白丝的破坏，而SOX9的表达取决于cLIUS下ERK1 / 2的磷酸化。被评估。通过使用细胞骨架药物Y27632处理，在cLIUS下在MSC中观察到的弥散和破坏的肌动蛋白纤维与肌动蛋白破坏非常相似，已知它可以增加SOX9的基因表达。因此，在cLIUS下SOX9的上调与cLIUS诱导的肌动蛋白重组有关。还发现肌动蛋白重组诱导的SOX9上调依赖于ERK1 / 2的磷酸化。结论总的来说，cLIUS对MSC的预处理导致SOX9的核定位，ERK1 / 2的磷酸化和肌动蛋白丝的破坏，而SOX9的表达取决于cLIUS下ERK1 / 2的磷酸化。已知它可以增加SOX9的基因表达。因此，在cLIUS下SOX9的上调与cLIUS诱导的肌动蛋白重组有关。还发现肌动蛋白重组诱导的SOX9上调依赖于ERK1 / 2的磷酸化。结论总的来说，cLIUS对MSC的预处理导致SOX9的核定位，ERK1 / 2的磷酸化和肌动蛋白丝的破坏，而SOX9的表达取决于cLIUS下ERK1 / 2的磷酸化。已知它可以增加SOX9的基因表达。因此，在cLIUS下SOX9的上调与cLIUS诱导的肌动蛋白重组有关。还发现肌动蛋白重组诱导的SOX9上调依赖于ERK1 / 2的磷酸化。结论总的来说，cLIUS对MSC的预处理导致SOX9的核定位，ERK1 / 2的磷酸化和肌动蛋白丝的破坏，而SOX9的表达取决于cLIUS下ERK1 / 2的磷酸化。