Santos O F, Moura L A, Rosen E M, Nigam S K
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Dev Biol. 1993 Oct;159(2):535-48. doi: 10.1006/dbio.1993.1262.
MDCK cells cultured in Type I collagen gels can be induced to develop branching tubular structures with demonstrable lumens in the presence of hepatocyte growth factor (HGF). As we have now shown by immunofluorescent localization of specific marker proteins, these tubules retain apical-basolateral polarity. However, the secondary signaling events which lead to these characteristic morphogenetic changes induced by HGF remain largely unelucidated. In order to examine these signaling events, particularly the role of protein phosphorylation in the formation of branching tubular structures, Madin-Darby canine kidney (MDCK) cells in collagen gels were treated with HGF plus well-characterized agents that affect protein phosphorylation. We quantified the formation of branching processes, an early step in the development of tubular structures in this model. Protein kinase C (PKC) inhibition resulted in more complex branching processes in the presence of HGF, when compared with HGF alone. In contrast, treatment with activators of protein kinase A (PKA), as well as calmodulin antagonists, caused a marked decline in process formation. Consistent with an important role for protein phosphorylation in HGF-induced morphogenesis, protein phosphatase inhibition by okadaic acid or calyculin A was found to markedly inhibit process formation. Tyrosine kinase (TK) inhibition also decreased the percentage of processes. This is consistent with data indicating that one of the HGF receptors is identical to the c-met protooncogene product, which is known to possess TK activity. Our results suggest that the HGF-mediated induction of branching processes in MDCK cells, an early step in the development of branching tubular structures, can be modulated by multiple phosphorylation mechanisms including those mediated by PKC, PKA, and Ca2+/calmodulin-dependent kinase(s). We discuss how these phosphorylation events may play crucial roles in determining the degree of tubule formation and their length, as well as the extent of their arborization during the early development of epithelial tissues.
在I型胶原蛋白凝胶中培养的MDCK细胞，在肝细胞生长因子（HGF）存在的情况下，可被诱导形成具有可证实管腔的分支管状结构。正如我们现在通过特定标记蛋白的免疫荧光定位所显示的，这些小管保持顶端-基底外侧极性。然而，导致由HGF诱导的这些特征性形态发生变化的次级信号事件在很大程度上仍未阐明。为了研究这些信号事件，特别是蛋白磷酸化在分支管状结构形成中的作用，用HGF加上已充分表征的影响蛋白磷酸化的试剂处理胶原蛋白凝胶中的Madin-Darby犬肾（MDCK）细胞。我们对分支过程的形成进行了定量，这是该模型中管状结构发育的早期步骤。与单独使用HGF相比，蛋白激酶C（PKC）抑制在HGF存在时导致更复杂的分支过程。相反，用蛋白激酶A（PKA）激活剂以及钙调蛋白拮抗剂处理，导致过程形成显著下降。与蛋白磷酸化在HGF诱导的形态发生中起重要作用一致，发现用冈田酸或花萼海绵诱癌素A抑制蛋白磷酸酶可显著抑制过程形成。酪氨酸激酶（TK）抑制也降低了过程的百分比。这与数据一致，表明HGF受体之一与已知具有TK活性的c-met原癌基因产物相同。我们的结果表明，HGF介导的MDCK细胞中分支过程的诱导，这是分支管状结构发育的早期步骤，可通过多种磷酸化机制调节，包括由PKC、PKA和Ca2+/钙调蛋白依赖性激酶介导的机制。我们讨论了这些磷酸化事件如何在确定小管形成的程度及其长度以及上皮组织早期发育过程中其分支程度方面发挥关键作用。
