Potempa, Sandra Astrid; (1999) Characterisation of the signal transduction pathways mediating the hepatocyte growth factor/scatter response in MDCK cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

MDCK epithelial cells initially spread in response to hepatocyte growth factor/scatter factor (HGF/SF) followed by the disruption of cell-cell junctions and cell scattering. Their motility involves actin rearrangements mediated by Ras, but how Ras and its downstream effectors also regulate intercellular adhesions has not been established yet. Activation of HGF/SF was shown to induce sustained p42/p44 MAP kinase (MARK) activation, and this was required for spreading, disruption of adherens junctions and subsequent cell scattering as these responses were blocked by the p42/p44 MARK inhibitor PD98059. In addition, activation of phosphoinositide 3-kinase (PI3K) was also required for these responses as they were blocked by its inhibitor LY294002. Disassembly of adherens junctions required both activation of p42/p44 MAPK and PI3K and was a prerequisite for the disruption of desmosomes and tight junctions and subsequent cell scattering In polarized MDCK monolayers, HGF/SF induced a decrease in transepithelial resistance (TER) and loss of intercellular junctions monitored by transmission electron microscopy (TEM) which was PI3K- and p42/p44 MAPK- dependent. To analyse the roles of Rho, Cdc42 and Rac in HGF/SF-induced responses and to allow their expression in over 70% of cells, mutant forms of these GTPases were cloned into adenovirus vectors. Expression of N17RhoA, N17Cdc42, N17Racl and V12Rac1 affected tight junctions as they decreased the TER of MDCK cell monolayers although N17Rac1 transiently increased the TER. N19RhoA and N17Cdc42 dispersed adherens junctions and N17Cdc42 affected the localization of the tight junction protein ZO-1. N17Racl inhibited HGF/SF-induced scattering and disruption of adherens junctions whereas V12Racl enhanced scattering. In conclusion, these results show that HGF/SF- induced scattering and loss of junctions is PI3K- and p42/p44 MAPK-dependent and that these changes are mediated by Ras, Rho and Rac.

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