1 Role of FGFR2b in the crosstalk between autophagy and differentiation: 1 involvement of JNK signaling. 2

The FGFR2b is a receptor tyrosine kinase expressed exclusively in epithelial cells. We previously 28 demonstrated that FGFR2b induces autophagy and that this process is required for the triggering of 29 FGFR2b-mediated keratinocytes early differentiation. However, the molecular mechanisms 30 regulating this interplay remain to be elucidated. Since we have also recently shown that JNK1 31 signaling is involved in FGFR2b-induced autophagy and a possible role of JNK pathway in 32 epidermal differentiation has been suggested but it is still debated, here we investigated the 33 crosstalk between FGFR2b-mediated autophagy and differentiation focusing on the downstream 34 JNK signaling. Biochemical, molecular and immunofluorescence approaches in 2D keratinocyte 35 cultures and 3D organotypic skin equivalents confirmed that FGFR2b overexpression increased 36 both autophagy and early differentiation. The use of FGFR2b substrate inhibitors and the silencing 37 of JNK1 highlighted that this signaling is required not only for autophagy but also for the triggering 38 of early differentiation. In contrast, ERK1/2 pathway did not appear to be involved in the two 39 processes and AKT signaling, whose activation contributes to the FGFR2b-mediated onset of 40 keratinocyte differentiation, was not required for the triggering of autophagy. Overall, our results 41 point to JNK1 as a signaling hub that regulates the interplay between FGFR2b-induced autophagy 42 and differentiation. 43 44 45 46 47 48 50 51 ATG5 and BECN1 mRNA transcripts induced by FGF7 stimulation is significantly counteracted by JNK1 depletion. Results are expressed as mean values ± SE. The quantitative analysis and Student’s t test were performed as above: *p < 0.05 vs the corresponding FGF7- unstimulated cells; **p < 0.05 vs the corresponding control siRNA cells. (C) HaCaT pBp-FGFR2b cells were cotransfected with JNK1 siRNA or control siRNA and mCherry-EGFP-LC3. Cells were 646 then left untreated or stimulated with FGF7 as above. Quantitative fluorescence analysis shows that JNK1 depletion significantly inhibits the increase of both yellow and red dots per cell induced by