Autophagy in human keratinocytes: role of the fibroblast growth factor receptor 2b and its signaling

Signaling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. In this work I investigated, in our cell model of HaCaT keratinocytes, if the two processes might be related and the possible involvement of PLCγ signaling in the autophagy triggered by FGFR2b activation. Using fluorescence and electron microscopy we demonstrated that the FGFR2b-induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signaling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor-triggered autophagy requires PLCγ signaling, which in turn activates JNK1 via PKCδ. Finally we found that, in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways depend on the level of receptor expression. These results suggest that FGFR2b signaling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation. Since the early oncoprotein E5 of the human papillomavirus type 16 (16E5) is able to down-regulate FGFR2b expression and since it has been recently proposed a possible role of the entire “early protein pool” of HPV16 in inhibiting the autophagic process in epithelial cells, I also proposed to investigate the possible impairment of FGFR2b-induced autophagy in keratinocytes expressing 16E5. The results showed that the presence of 16E5 strongly inhibited the autophagic process, while forced expression and activation of FGFR2b counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy. In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition. Finally, molecular approaches showed that the viral protein interferes with the transcriptional regulation of autophagy also through the impairment of p53 function, indicating that 16E5 uses parallel mechanisms for autophagy impairment. Overall our results further support the hypothesis that a transcriptional crosstalk among 16E5 and FGFR2b might be the crucial molecular driver of epithelial deregulation during early steps of HPV infection and transformation.