Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures
Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures
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DOI：
10.1016/j.cellsig.2013.12.001
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发表时间：
2014-03-01
影响因子：
4.8
通讯作者：
Zakany, Roza
中科院分区：
文献类型：
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作者：
Juhasz, Tumas;Matta, Csaba;Zakany, Roza
生物力学刺激在胎儿早期关节软骨的形成中起着重要作用，而最佳力学负荷是成人软骨细胞代谢和功能的重要调节因素。在这项研究中，我们分析了体外软骨形成过程中的机械转导途径。从4日龄鸡胚的肢体芽中分离软骨祖细胞作为高密度细胞培养物培养6天。机械刺激是通过自行设计的生物反应器进行的，通过培养基以静水压力和流体剪切的方式向分化细胞施加单轴间歇性循环载荷。加载方案（0.05 Hz, 600 Pa, 30 min）应用于培养第2和第3天，此时模型中软骨祖细胞发生最终承诺和分化。施加的机械负荷通过增强主要软骨生成转录因子Sox9的表达、磷酸化和核信号，显著增加软骨基质的生成和几种软骨基质成分mRNA的表达，包括II型胶原和聚集蛋白核心蛋白，以及生成基质的透明质酸合成酶。随着cAMP水平的升高，蛋白激酶a （PI(a)）活性也显著增强，MA信号传导的典型下游转录因子CREB在机械刺激下表现出磷酸化水平升高和更强的核信号。pka抑制剂H89可使上述作用减弱。HJC0197抑制与pica无关的camp -介质Epac1和Epac2导致软骨形成增强，这是机械刺激的附加作用，这意味着机械负荷促进软骨形成的作用是独立于Epac的。同时，机械负荷后PP2A活性降低，使用PP2A抑制剂冈田酸处理能够模拟干预的效果。我们的研究结果表明，适当的机械刺激通过促进软骨细胞的分化和基质的产生来增强体外软骨的形成，而PKA/CREB-Sox9和PP2A信号通路的相反调控在这一现象中至关重要。(C) 2014爱思唯尔公司版权所有。
Biomechanical stimuli play important roles in the formation of articular cartilage during early foetal life, and optimal mechanical load is a crucial regulatory factor of adult chondrocyte metabolism and function. In this study, we undertook to analyse mechanotransduction pathways during in vitro chondrogenesis. Chondroprogenitor cells isolated from limb buds of 4-day-old chicken embryos were cultivated as high density cell cultures for 6 days. Mechanical stimulation was carried out by a self-designed bioreactor that exerted uniaxial intermittent cyclic load transmitted by the culture medium as hydrostatic pressure and fluid shear to differentiating cells. The loading scheme (0.05 Hz, 600 Pa; for 30 min) was applied on culturing days 2 and 3, when final commitment and differentiation of chondroprogenitor cells occurred in this model. The applied mechanical load significantly augmented cartilage matrix production and elevated mRNA expression of several cartilage matrix constituents, including collagen type II and aggrecan core protein, as well as matrix-producing hyaluronan synthases through enhanced expression, phosphorylation and nuclear signals of the main chondrogenic transcription factor Sox9. Along with increased cAMP levels, a significantly enhanced protein kinase A (PI(A) activity was also detected and CREB, the archetypal downstream transcription factor of MA signalling, exhibited elevated phosphorylation levels and stronger nuclear signals in response to mechanical stimuli. All the above effects were diminished by the PKA-inhibitor H89. Inhibition of the PICA-independent cAMP-mediators Epac1 and Epac2 with HJC0197 resulted in enhanced cartilage formation, which was additive to that of the mechanical stimulation, implying that the chondrogenesis-promoting effect of mechanical load was independent of Epac. At the same time, PP2A activity was reduced following mechanical load and treatments with the PP2A-inhibitor okadaic acid were able to mimic the effects of the intervention. Our results indicate that proper mechanical stimuli augment in vitro cartilage formation via promoting both differentiation and matrix production of chondrogenic cells, and the opposing regulation of the PKA/CREB-Sox9 and the PP2A signalling pathways is crucial in this phenomenon. (C) 2014 Elsevier Inc. All rights reserved.