M. R. Torrisi - Role of the keratinocyte growth factor receptor on the molecular and cellular alterations itivity to identify cycling cells, was much less pronounced in HaCaT cells expressing E5 compared to the control cells. These results indicate that E5 expression down-modulates KGFR transcription and the consequent KGF-induced proliferation and differentation of human keratinocytes. Since 16E5 is known to inhibit the EGFR endocytic degradative pathway and to enhance receptor recycling, we performed also a series of experiments to investigate if the E5 protein would be able to interfere with the two alternative KGFR endocytic pathways. In fact, we have demonstrated that FGF10/KGF2 induces receptor recycling, whereas KGF/FGF7 stimulates receptor degradation (Belleudi et al., 2007): since the recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared to KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport, our hypothesis was that 16E5 would play a similar role in targeting KGFR to the recycling route. To this aim, first we performed transient transfection of HaCaT cells with a pCIneo-HA E5 expression vector (kindly provided by Dr. Venuti) and, 48 hours post-transfection, we immunostained E5 protein with anti-HA antibodies. Immunfluorescence and confocal analysis showed that the E5 signal colocalized with the endoplasmic reticulum marker calreticulin, as expected, while only a minor colocalization was observed with the marker of the Golgi complex giantin and no colocalization was detected with the mitochondrial marker MitoTracker. Next, to analyze if E5 expression could affect KGFR endocytosis, we studied the intracellular endocytic traffic followed by KGFR during internalization induced by the two ligands KGF and FGF10 in HaCaT E5/KGFR cotrasfected cells. In particular, we focused our attention on the late steps of KGFR internalization to search for a possible differential sorting of receptors destined to recycling or degradation in the presence of E5. The KGFR endocytic intracellular transport was analyzed in detail at immunofluorescence and confocal microscopy and the intracellular endocytic structures and compartments involved in the KGFR inter- 26 nalization process induced by the ligands were identified using the following markers: EEA1 for early endosomes, LysoTracker for lysosomes and internalized Transferrin for the recycling compartment. HaCaT KGFR/E5 cotrasfected and HaCaT KGFR transfected cells were treated with the ligands and with the anti-KGFR antibody at 4°C and warmed for 1h at 37°C. Cells were also incubated in the presence of LysoTracker to identify lysosomes or in the presence of Tf-Tx to identify the juxtanuclear recycling compartment. The results showed that, upon KGF treatment of cells expressing E5, most of KGFRs did not reach the lysosomes, as demonstrated by the very low percentage of colocalization with LysoTracker. In contrast, in agreement with our previous results (Belleudi et al., 2007), the receptor extensively colocalized with LysoTracker in cells overexpressing KGFR alone. After FGF10 treatment, the KGFR signal colocalized with internalized Tf, but not with LysoTracker in both cotransfeted or singly transfected cells. suggesting that E5 expression does not inhibit receptor recycling to the plasma membrane. Thus, E5 appears to interfere with the KGF-induced KGFR transport to the degradative pathway, but not with the FGF10-induced receptor traffic through the recycling compartment (Belleudi et al. in preparation). Selected Publications Belleudi F, Leone L, Nobili V, Raffa S, Francescangeli F, Maggio M, Morrone S, Marchese C, Torrisi MR. Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways. Traffic 2007, 8:1854-72. Lotti LV, Rotolo S, Francescangeli F, Frati L, Torrisi MR, Marchese C. AKT and MAPK signaling in KGF-treated and UVB-exposed human epidermal cells. J Cell Physiol. 2007, 212:633-42. Cardinali G, Bolasco G, Aspite N, Lucania G, Lotti LV, Torrisi MR, Picardo M. Melanosome transfer promoted by keratinocyte growth factor in light and dark skin derived keratinocytes. J Invest Dermatol. 2008, 128:558-67.
AREA3 Molecular genetics of eukaryotes