Raf plus TGFbeta-dependent EMT is initiated by endocytosis and lysosomal degradation of E-cadherin.

Oncogenic Ras interferes with adhesive functions of epithelial cells, but requires tumor growth factor beta (TGFbeta) signaling to cause epithelial-mesenchymal transition (EMT) and tumor progression in model systems. To investigate the mechanisms by which Ras and TGFbeta pathways cooperate in EMT induction, we introduced a tamoxifen-inducible version of Raf-1 (RafER) into fully polarized, mammary epithelial cells (EpH4). EMT characterized by loss of E-cadherin expression and upregulation of invasiveness-promoting genes was induced by TGFbeta plus 4-hydroxytamoxifen (4HT) activation of RafER. Downregulation of E-cadherin by RafER plus TGFbeta was detectable in total cell lysates after 48 h and much earlier in detergent-insoluble fractions of E-cadherin. Both pathways cooperated to strongly enhance endocytosis of E-cadherin, mainly via the clathrin-dependent route. Pulse-chase experiments showed decreased E-cadherin protein stability in cells stimulated with TGFbeta and 4HT and increased E-cadherin half-life in the presence of monensin. Monensin and chloroquine prevented E-cadherin degradation to different extent, but only monensin effectively blocked the loss of E-cadherin from the junctional complexes. Both lysosome inhibitors caused accumulation of E-cadherin vesicles, some of which were positive for Cathepsin D and lysosome-associated membrane protein 1 (LAMP-1). In addition, TGFbeta and mitogen-activated protein kinase hyperactivation synergistically induced E-cadherin ubiquitination, suggesting that the cooperation of Raf and TGFbeta favors lysosomal degradation of E-cadherin instead of its recycling. Our data indicate that early stages of EMT involve cooperative, post-translational downregulation of E-cadherin, whereas loss of E-cadherin via transcriptional repression is a late event in EMT.

Raf induces TGFbeta production while blocking its apoptotic but not invasive responses: a mechanism leading to increased malignancy in epithelial cells.

c-Raf-1 is a major effector of Ras proteins, responsible for activation of the ERK MAP kinase pathway and a critical regulator of both normal growth and oncogenic transformation. Using an inducible form of Raf in MDCK cells, we have shown that sustained activation of Raf alone is able to induce the transition from an epithelial to a mesenchymal phenotype. Raf promoted invasive growth in collagen gels, a characteristic of malignant cells; this was dependent on the operation of an autocrine loop involving TGFbeta, whose secretion was induced by Raf. TGFbeta induced growth inhibition and apoptosis in normal MDCK cells: Activation of Raf led to inhibition of the ability of TGFbeta to induce apoptosis but not growth retardation. ERK has been reported previously to inhibit TGFbeta signaling via phosphorylation of the linker region of Smads, which prevents their translocation to the nucleus. However, we found no evidence in this system that ERK can significantly influence the function of Smad2, Smad3, and Smad4 at the level of nuclear translocation, DNA binding, or transcriptional activation. Instead, strong activation of Raf caused a broad protection of these cells from various apoptotic stimuli, allowing them to respond to TGFbeta with increased invasiveness while avoiding cell death. The Raf-MAP kinase pathway thus synergizes with TGFbeta in promoting malignancy but does not directly impair TGFbeta-induced Smad signaling.

Immediate and long-term galvanotactic responses of Amoeba proteus to dc electric fields.

The long-term and immediate galvanotactic responses of Amoeba proteus to the direct current electric fields (dcEFs) were studied with the methods of computer-aided image analysis. It was found that in contrast to earlier reports, amoebae continued locomotion towards cathode (the negative pole) for hours and the increase in the field strength in the range 300-600 mV/mm caused the straightening of cell trajectories accompanied by the decreased frequency of the lateral pseudopods formation and lesser change in the speed of cell movement. In the cell regions pointing to the anode, the formation of new pseudopodia was prevented and the higher cEFs strength the more extended were the regions in which formation of new pseudopods was inhibited. Replacement of calcium with magnesium in the extracellular medium reduced the galvanotactic cell responses. Research on the localisation and kinetics of the primary cell responses to the dcEF or to change in its direction revealed that the primary cell responses occurred at the anode oriented cell regions. The cell response to the field reversal appeared to be localised and to take place in less than 1 sec. First the retraction and withdrawal of the anode-directed pseudopodium was observed whereas the uroid (cell tail) moved for 10-40 sec in the original direction before it begun to react to the field reversal. The exposure of amoebae to the dcEFs sensitised them to the reversion in the field direction and induced an acceleration of cell responses. The results presented are difficult to reconcile with the attempt to explain the cell galvanotaxis as a consequence of the membrane protein lateral electrophoresis or electroosmosis. It is suggested that the lateral electrophoresis of ions and the modification of ionic conditions at the vicinity of ion channels may be involved in the induction of fast responses of cells to external dcEFs.