FGFR2b is the FGFR2 epithelial splicing variant whose signaling triggers different outcomes in epithelial cells, including differentiation. Starting from the evidence that several PKC isoforms participate in the regulation of cell differentiation, in this project we propose to assess if the well-known role of FGFR2b in early steps of keratinocyte differentiation could involve PKCdelta pathway. At the same time we also propose to clarify if this receptor can also control intermediate differentiation always via PKCdelta and/or late differentiation through PKCalpha. Using 2D cultures or 3D skin equivalents prepared with keratinocytes stably overexpressing or not FGFR2b, we propose to analyze the expression and the distribution throughout the epidermal layers of early, intermediate and late differentiation markers by molecular, immunofluorescence and biochemical approaches, while the ultrastructural aspects of skin equivalent differentiation will be checked in detail by transmission electron microscopy. Then, the possible differential contribution of PKCdelta and PKCalpha in FGFR2b-induced differentiation will be investigated by the use of isoform specific inhibitors of PKC activity and definitely confirmed using silencing approaches. Because it is well known that, in keratinocytes, PKCs isoforms control early/intermediate and late differentiation through a functional crosstalk with KLF4 and DLX3 respectively, the possible ability of FGFR2b to modulate these two transcription factors in both 2D cultures and 3D rafts of the different clones will be also investigated by molecular approaches.
The expected results will make it possible to advance in the knowledge of the complex signaling network through which FGFRs might control keratinocyte differentiation. These networks involving PKCs/transcription factors axis underlies the physiological progression along the differentiation program, and could be unbalanced in several differentiation disorders, including SCCs.
On the light of the widely accepted role of the epithelial isoform of the FGFR2 (FGFR2b) in the epidermal early differentiation as well as in the skin homeostasis (Petiot et al., 2003; Grose et al., 2007; Yang et al., 2010; Belleudi et al. 2011; Purpura et al., 2013) in this project we propose to investigate the possible role of this receptor in the control of the entire program of keratinocyte differentiation, analyzing its possible involvement also in the intermediate as well as the late steps of the process. To this aim, we will take advantage of the use of keratinocytes transiently or stably overexpressing FGFR2b WT (HaCaT FGFR2b WT) or the empty vector as control (HaCaT pBp) (Ranieri et al., 2016). In particular stable clones will be used also for 3D organotypic culture preparation that will be used to analyze all the aspects of keratinocyte differentiation, in term of cell phenotype, stratification ability, differentiation marker expression and distribution along the skin equivalent layers. These approaches will permit to verify if FGFR2b could trigger an enhancement and/or a precocious onset not only of the early differentiation program, as expected, but also of the following steps of the process. These kind of evidences will allow to strength the assumption of a more general function of FGFR2b in the control of epidermal differentiation, which is consistent with the tumor suppressive role widely proposed for this receptor (Feng et al., 1997; Zhang et al., 2001; Turner and Grose, 2010; Haugsten et al., 2010).
This project will also allow to progress in the knowledge of molecular mechanisms and the signaling pathways activated by FGFRs involved in keratinocyte differentiation. Starting from the evidence that PI3K/Akt is one of, but not the exclusive, signaling pathway involved in FGFR2b-mediated keratinocyte differentiation (Belleudi et al., 2011), here we will focus on the PKCs, which are identified as key substrates of RTKs and in particular of FGFRs controlling the differentiation program in several tissues including epidermis (Kim et al., 2003; Breitkreutz et al., 2007; Miraoui et al., 2009). In particular, PKCdelta, which is expressed in basal keratinocytes, seems to be involved in the triggering of the early and intermediate steps, while PKCalpha, whose expression starts from the suprabasal layers, appears to regulate the terminal step of the process (Breitkreutz et al., 2007). Taking advantage of the use of PKC isoform inhibitors or PKCs specific siRNA interference approaches, we expect to identify the specific, differential role of these PKCs in the different steps of keratinocyte differentiation, which will result controlled by FGFR2b.
Indeed, several evidences have recently highlighted that epidermal differentiation is closely controlled by multiple functional axis involving PKCs and transcription factors, whose unbalance leads to skin disorders associated with altered differentiation, including psoriasis and squamous cell carcinomas (SCCs) (Hwang et al., 2011; Palazzo et al., 2016). Since DLX3 is the effector of PKCalpha (Palazzo et al., 2017), while KLF4 appears to act downstream PKCdelta (Chew et al., 2011; Chew et al., 2013), in this project we will also investigate the possible ability of FGFR2b to functionally modulate these transcription factors. If so, our finding could focus on the receptor as the key upstream player able to regulate the various PKC/transcription factor axis and consequently the expression of a PKC-dependent gene signature characterized by epidermal differentiation.
It is well known that a correct epidermal differentiation and stratification is also consequent to the molecular down-regulation of the transcription factor p63 (mainly in its isoform DeltaNp63) (Candi et al., 2008) and a double loop between DLX3 and DeltaNp63 has been proposed, in which DLX3 is responsible for DeltaNp63 proteasomal degradation (Di Costanzo et al., 2009).
Since we previously found that p63 is also regulated by a fine negative loop with FGFR2b whose unbalance may be responsible for the impairment of keratinocyte differentiation (Purpura et al., 2013), to be able to demonstrate an ability of FGFR2b to modulate DLX3 expression will mean to have identified an additional molecular mechanism through which this receptor could control p63 protein level and consequently keratinocyte differentiation.
Overall the evidences that we propose to obtain in this project will permit to further progress in the identification of the complex signaling network starting downstream FGFRs and involving several FGFR substrates and transcription factors, whose differential activation underlies the physiological progression along the differentiation program, and whose unbalance could be responsible for the onset of several differentiation disorders, including SCCs.