Epithelial to mesenchymal transition (EMT) is a process whereby epithelial cells, which act collectively through robust cell–cell interactions, take on mesenchymal characteristics, breaking cell–cell junctions to become solitary, invasive and motile. Our previous results show that a transient increase in calcium influxes through TRP channels at the plasma membrane is required for hepatocyte growth factor (HGF)– stimulated EMT. Since this transient increase requires an intact microtubule cytoskeleton, we propose that HGF stimulation results in the mobilization of calcium channels to the plasma membrane from an intracellular compartment via microtubule–dependent vesicle trafficking. Through immunofluorescence, we show that prior to HGF treatment, TRPV4 localizes to a perinuclear compartment that stains for rab11. After HGF stimulation, this colocalization is reduced and TRPV4 localizes more precisely to fibrous structures. Similarly, rab8 staining is seen throughout the cytoplasm prior to HGF treatment, but localizes primarily to tubular structures after HGF stimulation. This is indicative of endocytic recycling of TRPV4 via rab8. MDCK cells null for rab8 activator, rabin8, were developed using the CRISPR system and then analyzed for changes in epithelial scattering and trafficking of ion channels to the plasma membrane following HGF stimulation. Rabin8 KO cells had a decrease in TRPV4 vesicle trafficking. While rabin8 KO cells did undergo HGF-induced spreading and some disassembly of cell-cell junctions, they lost all motility. Also, HGF-treated rabin8 KO cells had similar calcium levels to untreated WT cells, which had fewer calcium spikes than HGF-treated WT cells. ERK1/2, a known downstream effector of HGF stimulation, has been shown to activate rabin8, and so we tested the effect of an ERK1/2 inhibitor on HGF-induced WT cells as well. These cells had decreased TRPV4 vesicle trafficking and loss of motility, similar to rabin8 KO cells, indicating that ERK1/2 may act upstream of rabin8 and rab8 in this pathway. Our results indicate that TRPV4 undergoes endocytic recycling via rab8 to the cell surface to allow a necessary calcium influx within one hour of HGF stimulation in MDCK cells, leading to EMT.
| Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-7224 |
| Date | 01 March 2016 |
| Creators | Haws, Hillary Jean |
| Publisher | BYU ScholarsArchive |
| Source Sets | Brigham Young University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | http://lib.byu.edu/about/copyright/ |
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