The fibroblast growth factor receptors (FGFRs) trigger divergent responses, such as proliferation and differentiation, and the cell type as well as the context-dependent signaling are crucial for the functional outcome. The FGFR2b/KGFR is expressed exclusively on epithelial cells and plays a key role in skin homeostasis. In the first part of this work I analyzed in vitro the role of KGFR in the early differentiation of keratinocytes modulating its expression by KGFR cDNA transient transfection or KGFR siRNA microinjection and inducing a synchronous wave of differentiation in pre-confluent cells. Immunofluorescence, biochemical and molecular approaches demonstrated that KGFR overexpression increased the early differentiation marker keratin 1 at both transcriptional and translational levels, while receptor depletion reduced it. Ligand-dependent receptor activation and signaling were required for this differentiative effect. Overexpression of kinase negative KGFR mutant or Tyr769 KGFR signaling mutant, which is not able to recruit and activate PLCγ showed that the receptor kinase activity, but not its PLCγ-mediated signaling, is required for differentiation. Reduction of K1 expression, obtained by AKT inhibition, demonstrated that the PI3K/Akt signaling pathway is involved in the control of KGFR-mediated keratinocyte differentiation. This in vitro experimental model indicates that KGFR expression represents a key event regulating keratinocyte early differentiation during the switch from undifferentiated to differentiating cells. Since membrane and actin cytoskeleton dynamics during phagocytosis can be triggered and amplified by the signal transduction of receptor tyrosine kinases and the epidermal keratinocytes appear to use the phagocytic mechanism of uptake to ingest melanosomes released by the melanocytes playing a pivotal role in the transfer process, in the second part of this work I investigate the contribution of KGFR expression, activation and signaling in regulating the phagocytic process. We have previously demonstrated that the keratinocyte growth factor KGF/FGF7 promotes the melanosome uptake through activation of its receptor tyrosine kinase KGFR. Phagocytosis was analyzed in vitro using fluorescent latex beads on human keratinocytes induced to differentiate. KGFR depletion by siRNA microinjection and overexpression by transfection of KGFR wild type or defective mutants previously described were performed to demonstrate the direct effect of the receptor on phagocytosis. Colocalization of the phagocytosed beads with the internalized receptors in phagolysosomes was analyzed by optical sectioning and 3D reconstruction. KGFR ligands triggered phagocytosis in differentiated keratinocytes and receptor kinase activity and signaling were required for these effects, suggesting that KGFR expression/activity and PLCγ signaling pathway play crucial roles in phagocytosis.
|Titolo:||Role of keratinocyte growth factor receptor (KGFR/FGFR2b) expression and signaling in the control of human keratinocyte differentiation|
|Data di pubblicazione:||26-gen-2012|
|Appartiene alla tipologia:||07b Tesi di Dottorato (EX-Padis)|