Epithelia are an essential organising feature of multicellular organisms and the selective permeability of these barriers is regulated by the junctional repertoire of tight junction proteins. The regulation of epithelial permeability is essential for the physiological function of various organs and is often pathologically deregulated, for example in inflammatory disease and cancer. Tight junctions are dynamically regulated in response to diverse stimuli through multiple signalling pathways. The RAF/MEK/ERK pathway has been reported to mediate junctional remodelling in response to various growth factors and hormones, although the unique contribution of this pathway and the mechanisms of reorganisation remain unclear. To address this, specific activation of the RAF/MEK/ERK pathway was achieved through the expression of BRAFWT or oncogenic BRAFV600E in Madin-Darby Canine Kidney (MDCK) II cells, a model epithelial cell line used to study tight junctions.Specific activation of the RAF/MEK/ERK pathway generated a transient increase in transepithelial resistance, which occurred concurrently with the differential regulation of tight junction protein expression levels and subcellular distribution. Claudin-2 protein levels were decreased, while junctional levels of claudin-4 were increased. Total levels of claudin-1, occludin and ZO-1 were unchanged and were retained at areas of cell contact although showing varying degrees of cytoplasmic accumulation.Conditionally active CRAF:ER fusion proteins were expressed to provide increased control of RAF/MEK/ERK signal duration and to study the rates of TJ protein synthesis, degradation and localisation. RAF-mediated downregulation of CLDN2 mRNA caused subsequent claudin-2 protein depletion without influencing rates of internalisation or degradation. In contrast, RAF activation caused the redistribution of claudin-1 and -4 from the lateral membrane to the apical junction. This junctional accumulation could not be attributed to changes in claudin protein levels, stability or endocytic trafficking. Taken together, these data reveal surprising diversity in RAF/MEK/ERK-mediated TJ control, where distinct combinations of claudin-specific regulatory mechanisms act in concert to regulate epithelial permeability.
|Date of Award||6 Jun 2015|
|Sponsors||Biotechnology and Biological Sciences Research Council|
|Supervisor||Christopher Caunt (Supervisor), Andrew Chalmers (Supervisor) & Paul Whitley (Supervisor)|