Plasma membrane ion fluxes and NFAT-dependent gene transcription contribute to c-met-induced epithelial scattering

Abstract

SummaryHepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and causing them to migrate as solitary cells, a process that parallels epithelial-mesenchymal transition. HGF binds and activates the c-met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. We have applied a chemical biology approach to identify components of HGF signaling that are required for scattering in an in vitro model system. This approach yields a number of small molecules that block HGF-induced scattering, including a calcium channel blocker. We show that HGF stimulation results in sudden and transient increases in ion channel influxes at the plasma membrane. Although multiple channels occur in the membranes of our model system, we find that TrpC6 is specifically required for HGF-induced scattering. We further demonstrate that HGF-induced ion influxes through TrpC6 channels coincide with a transient increase in nuclear factor of activated T-cells (NFAT)-dependent gene transcription and that NFAT-dependent gene transcription is required for HGF-induced cell scattering.

Control of cell mechanics by RhoA and calcium fluxes during epithelial scattering

“…31 One obvious interpretation of this result is that microtubuledependent delivery of vesicles to the plasma membrane is required for the occurrence of high frequency calcium influxes seen after HGF stimulation. This interpretation is further supported by the observation that calcium influxes increase simultaneously for all the 3 distinct channel types detected at the plasma membrane by electrophysiological recording.…”

“…30 Electrophysiological recording of MDCK cells treated with hepatocyte growth factor reveals 2 bursts of high frequency calcium influxes at the plasma membrane. 31 In MDA-MB-468 cells, which undergo epithelial scattering in response to epidermal growth factor (EGF), cytosolic calcium levels quickly rise after EGF stimulation, then gradually level off in under 800 seconds. 32 This transient pulse results in a prolonged increase in vimentin expression, 32 indicating that transient calcium increases have long-lasting impacts on cell behavior.…”

“…36 TRPC6 inhibitors prevent HGFinduced proliferation, migration, and morphogenesis of HK2 cells. 37 Additionally, Langford et al 31 concluded that TRPC6 is specifically required for HGFinduced scattering in MDCK cells, though calcium fluxes through TRPV4 and other channels are all increased at the plasma membrane following HGF stimulation.…”

“…A key finding of Langford et al 31 is that HGF-induced calcium fluxes recorded at the plasma membrane require an intact microtubule cytoskeleton; pretreatment of cells with microtubule destabilizing agents prevents bursts of high frequency calcium influxes that normally follow HGF stimulation. 31 One obvious interpretation of this result is that microtubuledependent delivery of vesicles to the plasma membrane is required for the occurrence of high frequency calcium influxes seen after HGF stimulation.…”

“…This interpretation is further supported by the observation that calcium influxes increase simultaneously for all the 3 distinct channel types detected at the plasma membrane by electrophysiological recording. 31 This is most easily accounted for by different channel types being cargoes in the same vesicle populations, rather than by different channels sharing the same mechanism of post-translational control at the plasma membrane. Further supporting this argument, EGF has been shown to induce translocation of TRPC5 channels to the cell surface, an event that is mediated by PI-3 kinase.…”