Hansen L K, Mooney D J, Vacanti J P, Ingber D E
Department of Surgery, Children's Hospital, Boston, Massachusetts 02115.
Mol Biol Cell. 1994 Sep;5(9):967-75. doi: 10.1091/mbc.5.9.967.
This study was undertaken to determine the importance of integrin binding and cell shape changes in the control of cell-cycle progression by extracellular matrix (ECM). Primary rat hepatocytes were cultured on ECM-coated dishes in serum-free medium with saturating amounts of growth factors (epidermal growth factor and insulin). Integrin binding and cell spreading were promoted in parallel by plating cells on dishes coated with fibronectin (FN). Integrin binding was separated from cell shape changes by culturing cells on dishes coated with a synthetic arg-gly-asp (RGD)-peptide that acts as an integrin ligand but does not support hepatocyte extension. Expression of early (junB) and late (ras) growth response genes and DNA synthesis were measured to determine whether these substrata induce G0-synchronized hepatocytes to reenter the growth cycle. Cells plated on FN exhibited transient increases in junB and ras gene expression (within 2 and 8 h after plating, respectively) and synchronous entry into S phase. Induction of junB and ras was observed over a similar time course in cells on RGD-coated dishes, however, these round cells did not enter S phase. The possibility that round cells on RGD were blocked in mid to late G1 was confirmed by the finding that when trypsinized and replated onto FN-coated dishes after 30 h of culture, they required a similar time (12-15 h) to reenter S phase as cells that had been spread and allowed to progress through G1 on FN. We have previously shown that hepatocytes remain viable and maintain high levels of liver-specific functions when cultured on these RGD-coated dishes. Thus, these results suggest that ECM acts at two different points in the cell cycle to regulate hepatocyte growth: first, by activating the G0/G1 transition via integrin binding and second, by promoting the G1/S phase transition and switching off the default differentiation program through mechanisms related to cell spreading.
本研究旨在确定整合素结合和细胞形状变化在细胞外基质（ECM）控制细胞周期进程中的重要性。原代大鼠肝细胞在含有饱和量生长因子（表皮生长因子和胰岛素）的无血清培养基中，培养于涂有ECM的培养皿上。通过将细胞接种在涂有纤连蛋白（FN）的培养皿上，可同时促进整合素结合和细胞铺展。通过在涂有合成精氨酸-甘氨酸-天冬氨酸（RGD）肽的培养皿上培养细胞，将整合素结合与细胞形状变化分开，该肽作为整合素配体，但不支持肝细胞伸展。检测早期（junB）和晚期（ras）生长反应基因的表达以及DNA合成，以确定这些基质是否诱导G0期同步化的肝细胞重新进入生长周期。接种在FN上的细胞在junB和ras基因表达上表现出短暂增加（分别在接种后2小时和8小时内），并同步进入S期。在涂有RGD的培养皿上的细胞中，在相似的时间进程中观察到junB和ras的诱导，然而，这些圆形细胞未进入S期。培养30小时后，将RGD上的圆形细胞胰蛋白酶消化并重新接种到涂有FN的培养皿上，发现它们重新进入S期所需的时间（12 - 15小时）与在FN上已铺展并通过G1期的细胞相似，这一发现证实了RGD上的圆形细胞在G1期中期至晚期被阻滞的可能性。我们之前已经表明，当在这些涂有RGD的培养皿上培养时，肝细胞保持存活并维持高水平的肝脏特异性功能。因此，这些结果表明，ECM在细胞周期的两个不同点发挥作用来调节肝细胞生长：第一，通过整合素结合激活G0/G1期转换；第二，通过促进G1/S期转换并通过与细胞铺展相关的机制关闭默认的分化程序。
