Miao Hui, Nickel Christian H, Cantley Lloyd G, Bruggeman Leslie A, Bennardo Laura N, Wang Bingcheng
Rammelkamp Center for Research, MetroHealth Campus, Cleveland, OH 44109, USA.
J Cell Biol. 2003 Sep 29;162(7):1281-92. doi: 10.1083/jcb.200304018.
Eph kinases and their ephrin ligands are widely expressed in epithelial cells in vitro and in vivo. Our results show that activation of endogenous EphA kinases in Madin-Darby canine kidney (MDCK) cells negatively regulates hepatocyte growth factor/scatter factor (HGF)-induced branching morphogenesis in collagen gel. Cotreatment with HGF and ephrin-A1 reduced sprouting of cell protrusions, an early step in branching morphogenesis. Moreover, addition of ephrin-A1 after HGF stimulation resulted in collapse and retraction of preexisting cell protrusions. In a newly developed assay that simulates the localized interactions between Ephs and ephrins in vivo, immobilized ephrin-A1 suppressed HGF-induced MDCK cell scattering. Ephrin-A1 inhibited basal ERK1/2 mitogen-activated protein kinase activity; however, the ephrin-A1 effect on cell protrusion was independent of the mitogen-activated protein kinase pathway. Ephrin-A1 suppressed HGF-induced activation of Rac1 and p21-activated kinase, whereas RhoA activation was retained, leading to the preservation of stress fibers. Moreover, dominant-negative RhoA or inhibitor of Rho-associated kinase (Y27632) substantially negated the inhibitory effects of ephrin-A1. These data suggest that interfering with c-Met signaling to Rho GTPases represents a major mechanism by which EphA kinase activation inhibits HGF-induced MDCK branching morphogenesis.
Eph激酶及其ephrin配体在体外和体内的上皮细胞中广泛表达。我们的结果表明，在Madin-Darby犬肾（MDCK）细胞中内源性EphA激酶的激活负向调节胶原凝胶中肝细胞生长因子/散射因子（HGF）诱导的分支形态发生。HGF与ephrin-A1共同处理可减少细胞突起的萌发，这是分支形态发生的早期步骤。此外，在HGF刺激后添加ephrin-A1会导致先前存在的细胞突起塌陷和回缩。在一项新开发的模拟体内Ephs与ephrins之间局部相互作用的实验中，固定化的ephrin-A1抑制了HGF诱导的MDCK细胞散射。Ephrin-A1抑制基础ERK1/2丝裂原活化蛋白激酶活性；然而，ephrin-A1对细胞突起的作用独立于丝裂原活化蛋白激酶途径。Ephrin-A1抑制HGF诱导的Rac1和p21活化激酶激活，而RhoA激活得以保留，导致应力纤维得以保留。此外，显性负性RhoA或Rho相关激酶抑制剂（Y27632）基本上消除了ephrin-A1的抑制作用。这些数据表明，干扰c-Met向Rho GTP酶的信号传导是EphA激酶激活抑制HGF诱导的MDCK分支形态发生的主要机制。
