Negatively charged synthetic hydrogels have been known to facilitate various cellular responses including cell adhesion, proliferation, and differentiation; however, the molecular mechanism of hydrogel-dependent control of cell behavior remains unclear. Recently, we reported that negatively charged poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel induces chondrogenic differentiation of ATDC5 cells via novel protein reservoir function. In this study, we identified the cell adhesion molecules binding to PAMPS gels that act as mechanoreceptors. First, we performed a pull-down assay by particle gels using cell membrane proteins of ATDC5, and found that multiple membrane proteins bound to the PAMPS gel, whereas the uncharged poly(N,N′-dimethylacrylamide) gel as control did not bind to any membrane proteins. Western blot analysis indicated differential binding of integrin (ITG) isoforms to the PAMPS gel, in which the α4 isoform, but not α5 and αv, efficiently bound to the PAMPS gel. ITG α4 knockdown decreased cell spreading of ATDC5 on PAMPS gels, whereas the enhanced expression increased the behavior. Furthermore, ITG α4 depletion suppressed PAMPS gel-induced expression of bone morphogenic protein (BMP) 4 contributing to chondrogenic differentiation, in concordance with the reduction of ERK activation. These results demonstrated that membrane protein binding to PAMPS gels occurred in a charge-dependent manner, and that ITG α4 plays a crucial role in cell spreading on PAMPS gels and acts as a mechanoreceptor triggering cellular signaling leads to chondrogenic differentiation.

已知带负电荷的合成水凝胶可促进各种细胞反应，包括细胞粘附，增殖和分化；然而，水凝胶依赖性控制细胞行为的分子机制仍不清楚。最近，我们报道了带负电荷的聚（2-丙烯酰胺基-2-甲基丙烷磺酸）（PAMPS）凝胶可通过以下途径诱导ATDC5细胞的软骨分化新的蛋白质储库功能。在这项研究中，我们确定了细胞粘附分子与充当机械感受器的PAMPS凝胶结合。首先，我们使用ATDC5的细胞膜蛋白通过颗粒凝胶进行了下拉分析，发现多个膜蛋白与PAMPS凝胶结合，而未充电的聚（N，N'-二甲基丙烯酰胺）凝胶不与PAMPS凝胶结合任何膜蛋白。Western印迹分析表明整联蛋白（ITG）同工型与PAMPS凝胶的差异结合，其中α4同工型（而非α5和αv）有效结合至PAMPS凝胶。ITGα4敲低可减少ATDC5在PAMPS凝胶上的细胞扩散，而增强的表达可提高行为。此外，ITGα4耗竭抑制了PAMPS凝胶诱导的骨形态发生蛋白（BMP）4表达与减少ERK活化相一致，有助于软骨形成分化。这些结果表明，膜蛋白与PAMPS凝胶的结合以电荷依赖的方式发生，并且ITGα4在PAMPS凝胶上的细胞扩散中起着至关重要的作用，并作为触发细胞信号转导软骨形成的机械受体起作用。