FGF22 in Skin Development and Repair Figure 2. Normal skin and pelage hair in fgf22 knockout mice. A) Histological analysis of H&E stained back (A1 and A2) and tail (A3 and A4) skin sections from four-month-old fgf22 wild type and knockout mice. No differences were observed in skin thickness or morphology, when back skin or tail skin sections from control and fgf22 mice were compared. (EP epidermis, DE dermis, AD adipose tissue, PC panniculus carnosus, HF hair follicle, * sebaceous gland). Scale bar (200 mm). B) Comparison of pelage hair structure. All major hair shaft types (zigzag, guard and awl/auchene) were present in both wild type and knockout animals. The morphology of different hair types showed no difference between wild type and fgf22 knockout mice as shown by light microscopic analysis. Hairs were plucked from eight-week-old wild type mice (n = 100 hairs) and compared with hairs of fgf22 knockout mice (n = 100 hairs). Scale bar = 200 mm. C) Tail epidermis whole-mount preparations from 10 weeks old mice, stained with Mayer’s haemalum, revealed no difference between wild type and fgf22 knockout mice in terms of sebaceous gland number and morphology. Hair follicle is indicated by arrow and sebaceous gland by asterisk. doi:10.1371/journal.pone.0039436.g002 wound gap just as effectively and rapidly as those from wild type animals at all timepoints (Fig. 4A and B). Finally, we challenged the mice with a classical two-step skin carcinogenesis assay using DMBA and TPA. These experiments were performed on female mice, since they involved long-term observation and multiple mice could be kept together. When cohorts of fgf22 knockout female mice were subjected to two-step (DMBA/TPA) skin carcinogenesis treatment, they developed significantly less papillomas than wild type mice. Fgf22 wild type and knockout mice subjected to TPA treatment alone never developed papillomas (Fig. 5A). Over the duration of the experiment, only 50% of DMBA treated knockout mice developed papillomas, compared to 100% of treated wild type mice (Fig. 5A). The time when 50% of mice developed papillomas was delayed by 16 weeks in fgf22 knockout mice compared with wild type controls (Fig. 5A). Tumour multiplicity was difficult to judge since these studies were performed on a tumour refractory C57Bl6/J background, so few papillomas formed during the study. With the exception of one outlier knockout female that developed five papillomas, the remaining knockout mice never developed more than one papilloma per mouse. The maximum number of papillomas on a DMBA treated wild type female was two (Fig. 5B). Papillomas that did develop showed identical histology between genotypes, with an exophytic appearance and no areas of epithelial invasion (Fig. 5C). These findings raised the question of whether FGF22 might interfere with the tumour-suppressive effect of FGFR2b (Fig. 5D). We therefore investigated whether treatment with FGF22 altered the biochemical response to FGF treatment in HaCaT cells, an immortalised human keratinocyte cell line [27]. In order to ensure that our HaCaT cell line exhibited a standard response to FGF stimulation, serum starved cells were stimulated with FGFs 7, 10 and 22 (100 ng/ml) in the presence of Heparin (300 ng/ml) in serum free media for 15, 30 and 60 minutes. Western blotting using an antibody specific to phosphorylated ERK confirmed that stimulation with FGFs 7 and 10 triggered rapid ERK phosphor- ylation, and this response was blocked by pre-treatment with the FGFR inhibitor, PD173074 (1.7 mM; 30 minutes; Fig. 6A). In contrast, FGF22 elicited no such response, with P-ERK levels remaining the same as in unstimulated cells (Fig. 6A; NB: blot was exposed for longer time relative to FGF7 and FGF10 to allow visualisation of the P-ERK signal). Total ERK levels from the same blot stripped and re-probed are shown as a loading control. To investigate whether FGF22 might modulate the capacity of FGF7 and 10 to activate ERK signalling, HaCaT cells were treated with FGF7 and 10 either concomitantly with FGF22 or following a four hour pre-treatment with FGF22 (all at 100 ng/ml; Fig. 6B and C). As above, cells were lysed after 0, 15, 30 and 60 minutes and lysates analysed by Western blot. Neither pre- treatment (four hours at 100 ng/ml) or co-treatment with FGF22 showed any effect on the timecourse of ERK phosphorylation elicited by stimulation with FGF7 or FGF10 (Fig. 6B and C respectively), although basal levels of ERK phosphorylation were raised very slightly by pre-treatment with FGF22, as shown by ImageJ quantitation of five independent experiments. Further- more, we used an siRNA pool to knock down FGF22 expression in HaCaT cells (Sup Fig. S1) and confirmed that this also had no effect on response to stimulation with either FGF7 or 10 (Fig. 6D). Expression levels of the FGF7 target gene NRF2 were also unaltered in knockdown cells (Sup Fig. S1). Finally, performing Realtime RT-PCR on RNA isolated from back skin of eight-week- old control and knockout female mice, we confirmed that expression levels of FGF7 and FGF10 were not altered signifi- cantly between genotypes (Sup Fig. S2). Discussion Numerous studies on FGF7, FGF10 and their receptors have shown the importance of this subfamily of FGFs for normal embryonic development and adult tissue homeostasis. However, PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39436