Figure 4. In vitro wound healing. Scratch wound assay was performed on primary keratinocytes isolated from wild type and fgf22 knockout male back skin. A) Photographs show the migration of wild type and knockout primary keratinocytes to wounded regions of the monolayer culture at 0, 12, 18 and 32 h post wounding. Scale bar (200 mm). B) Wound closure was monitored, photographed and measured at all timepoints after wounding. Error bars represent SEM. doi:10.1371/journal.pone.0039436.g004 with skin specific FGFR1/2 double knockout mice having a defective skin barrier and developing epidermal hyperthickening with age, as a result of chronic inflammation [25]. Given that fgf22 knockout mice do not phenocopy those changes, FGF22 clearly is not essential for normal skin physiology. Therefore, we analysed the capacity of FGF22 mouse skin to respond to a challenging stimulus; either to wounding (Fig. 3) or FGF22 in Skin Development and Repair to classical two-step skin carcinogenesis (Fig. 5), since both of these interventions cause an increase in keratinocyte proliferation; either transiently in the case of wound repair or more permanently in the case of carcinogenesis. These studies allowed us to question whether FGF22 was capable of driving keratinocyte differentiation and/or inhibiting proliferation. During wound healing, in contrast to FGF7 and FGF10, which appear to drive the healing process, FGF22 expression is up regulated towards the end of repair [12]. Thus, we hypothesised that it may act as a negative regulator of FGFR2 signalling, either decreasing proliferation or increasing differentiation, to safeguard against over-proliferation of keratinocytes. However, fgf22 knock- out mice did not display any defects in wound healing (Fig. 3). It is possible that, as repair progresses, the decrease in expression of pro-mitogenic ligands is sufficient to dampen the epithelial healing process. This apparent redundancy in the FGF-FGFR axis during repair is not without precedent, as mice lacking fgf7 or fgfr1b showed no wound healing phenotype, despite expression of these molecules being reported during the repair process [9,12]. Support for a redundant role of FGF22 in repair comes from the lack of difference in in vitro wound closure by primary keratinocytes isolated from wild type and knockout mice (Fig. 4). Our skin carcinogenesis data suggest that FGF22 may play a role in skin carcinogenesis, with the decreased sensitivity to chemically induced carcinogenesis in fgf22 knockout mice implying that basal expression of FGF22 confers sensitivity to skin carcinogenesis. In contrast, mice lacking FGFR2b in the skin develop spontaneous papillomas and show great sensitivity to chemically induced carcinogenesis [19]. Thus the ligand and its receptor appear to have antagonizing effects, with a tumour suppressive receptor, FGFR2b, appearing to be activated by a putative oncogenic ligand, FGF22. One model that might explain this is that FGF22 might act to antagonise the tumour suppressive role of FGFR2b, by competing with the protective ligands FGF7 and FGF10 (Fig. 5D). In this case, lack of FGF22 would cause increased signalling via FGF7 and FGF10. However, our cell-based investigations in the spontaneously immortalized HaCaT kerati- nocyte cell line showed FGF22 to have no effect on FGF7- or FGF10-dependent ERK activation (Fig. 6 and Sup Fig. S2). FGF7 is known to regulate genes that encode for mediators of cytoprotection, including the antioxidant Peroxiredoxin VI and the transcription factor Nrf2 [30,31,32,33]. In fgfr2b knockout mice, this cytoprotective mechanism is impaired due to lack of signalling receptor, and this most likely contributes to the dramatic incidence of papillomas in response to chemically induced carcinogenesis. Therefore we hypothesised that in fgf22 knockout mice, where FGF7 and/or FGF10 do not have to compete with FGF22 for receptor binding, it may allow stronger FGF-mediated cytoprotective signalling and, consequently, reduced papilloma formation (Fig. 5D). However, knock down of endogenous FGF22 mRNA in HaCaT cells did not result in a change in expression of NRF-2, suggesting that this potential mechanism needs further investigation. Due to the inability to detect FGF22 protein by western blot, we cannot absolutely exclude that FGF22 protein levels remain unaffected despite clear mRNA knock down. FGF7, in particular, is known to be cytoprotective for keratinocytes, with a recombinant form of FGF7 used clinically in the treatment of oral mucositis in patients undergoing bone marrow transplantation [34]. Future studies generating mice over-expressing FGF22 in the skin will allow us to address this mechanism. Taken together, our data provide evidence that FGF22 is not essential for skin development or repair but may play an important role in skin tumorigenesis, though the mechanism remains to be determined. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39436