Interference with the contractile machinery of the fibroblastic chondrocyte cytoskeleton induces re-expression of the cartilage phenotype through involvement of PI3K, PKC and MAPKs
Interference with the contractile machinery of the fibroblastic chondrocyte cytoskeleton induces re-expression of the cartilage phenotype through involvement of PI3K, PKC and MAPKs
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DOI：
10.1016/j.yexcr.2013.11.004
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发表时间：
2014-01-15
影响因子：
3.7
通讯作者：
Maniura-Weber, Katharina
中科院分区：
文献类型：
--
作者：
Rottmar, Markus;Mhanna, Rami;Maniura-Weber, Katharina
当软骨细胞在2D基质上生长时，其II型胶原和聚集蛋白聚糖的表型表达迅速丧失。通常已经观察到，具有强肌动蛋白肌球蛋白收缩性的成纤维细胞形态抑制软骨形成，而软骨形成可以通过应力纤维的解聚和/或肌动蛋白应力纤维与ECM之间的物理连接的破坏来促进，如在3D水凝胶中的情况。在这里，我们研究了肌动蛋白肌球蛋白细胞骨架和软骨形成标记物的表达之间的关系，通过培养成纤维细胞软骨细胞中存在的细胞松弛素D和星形孢菌素。这两种药物诱导II型胶原蛋白的重新表达，然而，重新糖胺聚糖的合成，只能观察到治疗后与星形孢菌素。当将一种肌球蛋白/肌动蛋白收缩性抑制剂blebbistatin加入到星形孢菌素刺激的培养物中时，星形孢菌素的软骨形成作用增强。此外，在3D藻酸盐培养物中，诱导软骨形成所需的星形孢菌素的量与2D培养物相比低得多（0.625 nM对2.5 nM）。使用选择的特异性信号传导途径抑制剂，发现PI 3 K-、PKC-和p38-MAPK途径正调节软骨形成，而发现ERK途径在星形孢菌素诱导的再分化中是负调节剂，而通过siRNA下调ILK表明ILK不是软骨细胞再分化的决定因素。此外，星形孢菌素类似物米多替林仅显示出有限的软骨形成作用，这表明一组特定的关键信号传导分子的激活/失活可以控制软骨形成表型的表达。这项研究证明了机械生物学因素在软骨形成中的至关重要性，表明肌动蛋白细胞骨架的结构及其收缩性控制关键信号分子，这些信号分子决定软骨细胞表型是否将沿着成纤维细胞或软骨形成路径被引导沿着。All rights reserved.
Chondrocytes rapidly lose their phenotypic expression of collagen II and aggrecan when grown on 2D substrates. It has generally been observed that a fibroblastic morphology with strong actin myosin contractility inhibits chondrogenesis, whereas chondrogenesis may be promoted by depolymerization of the stress fibers and/or disruption of the physical link between the actin stress fibers and the ECM, as is the case in 3D hydrogels. Here we studied the relationship between the actin myosin cytoskeleton and expression of chondrogenic markers by culturing fibroblastic chondrocytes in the presence of cytochalasin D and staurosporine. Both drugs induced collagen II re-expression; however, renewed glycosaminoglycan synthesis could only be observed upon treatment with staurosporine. The chondrogenic effect of staurosporine was augmented when blebbistatin, an inhibitor of myosin/actin contractility, was added to the staurosporine-stimulated cultures. Furthermore, in 3D alginate cultures, the amount of staurosporine required to induce chondrogenesis was much lower compared to 2D cultures (0.625 nM vs. 2.5 nM). Using a selection of specific signaling pathway inhibitors, it was found that PI3K-, PKC- and p38-MAPK pathways positively regulated chondrogenesis while the ERK-pathway was found to be a negative regulator in staurosporine-induced re-differentiation, whereas down-regulation of ILK by siRNA indicated that ILK is not determining for chondrocyte redifferentiation. Furthermore, staurosporine analog midostaurin displayed only a limited chondrogenic effect, suggesting that activation/deactivation of a specific set of key signaling molecules can control the expression of the chondrogenic phenotype. This study demonstrates the critical importance of mechanobiological factors in chondrogenesis suggesting that the architecture of the actin cytoskeleton and its contractility control key signaling molecules that determine whether the chondrocyte phenotype will be directed along a fibroblastic or chondrogenic path. (C) 2013 Elsevier Inc. All rights reserved.