The FGFRs are receptor tyrosine kinases expressed by tissue-specific alternative splicing in epithelial IIIb or mesenchymal IIIc isoforms. Deregulation of FGF/FGFR signaling unbalances the epithelial-stromal homeostasis and may lead to cancer development. In the epithelial-context, while FGFR2b/KGFR acts as tumor suppressor, FGFR2c appears to play an oncogenic role. Based on our recent observation that the switching of FGFR2b versus FGFR2c induces EMT, here we investigated the biological outcome of the ectopic expression of FGFR2c in normal human keratinocytes. Morphological analysis showed that, differently from FGFR2b overexpression, the forced expression and activation of FGFR2c drive the epithelial cells to acquire a mesenchymal-like shape and actin reorganization. Moreover, the appearance of invasiveness and anchorage-independent growth ability in FGFR2c transfected keratinocytes was consistent with the potential tumorigenic role proposed for this receptor variant. Biochemical and molecular approaches revealed that the observed phenotypic changes were accompanied by modulation of EMT biomarkers and indicated the involvement of EMT transcription factors and miRs. Finally, the analysis of the expression pattern of discriminating markers strongly suggested that activation of FGFR2c triggers a process corresponding to the initiation of the pathological type III EMT, but not to the more physiological type II EMT occurring during FGFR2b-mediated wound healing.

The altered isoform switching of the fibroblast growth factor receptor 2 (FGFR2) and aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells is involved in cancer progression. We have recently described that the ectopic expression of FGFR2c in normal human keratinocytes induces epithelial‐mesenchymal transition and leads to invasiveness and anchorage‐independent growth. Here, we extended our analysis to the effects of this FGFR2c forced expression on human keratinocyte differentiation and stratification. Our findings demonstrated that, differently from cells overexpressing the epithelial splicing variant FGFR2b, keratinocytes ectopically expressing FGFR2c are not able to form a monolayer and display decreased expression of early differentiation markers. This impaired ability to enter the differentiation program is related to the up‐modulation of the transcription factor ΔNp63. In addition, FGFR2c‐expressing keratinocytes undergo defective stratification and invasion of the collagen matrix in 3D organotypic cultures, further suggesting their tumorigenic potential. Taken together, our results support the hypothesis that the receptor switching and the consequent appearance of the mesenchymal FGFR2c variant in the epithelial context would drive early steps of carcinogenesis, unbalancing the p63/FGFR interplay, and altering the paracrine response to the microenvironment.

The microgravitational environment is known to affect the cellular behaviour inducing modulation of gene expression and enzymatic activities, epigenetic modifications and alterations of the structural organization. Simulated microgravity, obtained in the laboratory setting through the use of a Random Positioning Machine (RPM), represents a well recognized and useful tool for the experimental studies of the cellular adaptations and molecular changes in response to weightlessness. Short exposure of cultured human keratinocytes to the RPM microgravity influences the cellular circadian clock oscillation. Therefore, here we searched for changes on the regenerative ability and response to tissue damage of human epidermal cells through the analysis of the effects of the simulated microgravity on the re-epithelialization phase of the repair and wound healing process. Combining morphological, biochemical and molecular approaches, we found that the simulated microgravity exposure of human keratinocytes promotes a migratory behavior and triggers the epithelial-mesenchymal transition (EMT) through expression of the typical EMT transcription factors and markers, such as Snail1, Snail2 and ZEB2, metalloproteases, mesenchymal adhesion molecules and cytoskeletal components.

Fibroblast growth factor receptor 2b (FGFR2b) is a receptor tyrosine kinase expressed exclusively in epithelial cells. We previously demonstrated that FGFR2b induces autophagy and that this process is required for the triggering of FGFR2b-mediated early differentiation of keratinocytes. However, the molecular mechanisms regulating this interplay remain to be elucidated. Since we have also recently shown that Jun N-terminal protein kinase 1 (JNK1) signaling is involved in FGFR2b-induced autophagy and a possible role of the JNK pathway in epidermal differentiation has been suggested (though it is still debated), we investigated here the cross talk between FGFR2b-mediated autophagy and differentiation, focusing on the downstream JNK signaling. Biochemical, molecular, and immunofluorescence approaches in 2-dimensional (2-D) keratinocyte cultures and three-dimensional (3-D) organotypic skin equivalents confirmed that FGFR2b overexpression increased both autophagy and early differentiation. The use of FGFR2b substrate inhibitors and the silencing of JNK1 highlighted that this signaling is required not only for autophagy but also for the triggering of early differentiation. In contrast, the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway did not appear to be involved in the two processes, and AKT signaling, whose activation contributes to the FGFR2b-mediated onset of keratinocyte differentiation, was not required for the triggering of autophagy. Overall, our results point to JNK1 as a signaling hub that regulates the interplay between FGFR2b-induced autophagy and differentiation.