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First published online 7 November 2006
doi: 10.1242/jcs.03259

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Keratinocyte growth factor protects epidermis and hair follicles from cell death induced by UV irradiation, chemotherapeutic or cytotoxic agents

¹ Institute of Cell Biology, Department of Biology, ETH Zurich, Honggerberg, 8093 Zurich, Switzerland
² Department of Dermatology, University Hospital Schleswig-Holstein, University of Lübeck, 23538 Lübeck, Germany

View larger version (50K): [in this window] [in a new window]   Fig. 1. KGF is cytoprotective for keratinocytes in vitro and for hair follicle keratinocytes in organ culture. (A,B) Quiescent keratinocytes were pre-treated with KGF or vehicle for 16 hours and 1 hour before the addition of menadione. Results of representative experiments of MTT assays with HaCaT keratinocytes (A) or primary human foreskin keratinocytes (B) are shown. All measurements were performed in quadruplicate and the means ± s.d. are shown. The values obtained with the non-treated cells and the KGF-treated cells without application of the cytotoxic agent were arbitrarily set as 1. This was necessary because KGF affects the survival of starved cells, i.e. although the cell number is the same in the non-treated and the KGF-treated population, the mitochondrial activity is higher in the KGF-treated cell population. (C-F) Human anagen VI follicles were isolated from scalp skin, pre-treated with 20 ng/ml KGF for 12 hours and for one additional hour with fresh KGF, and subsequently incubated with 50 µM menadione. Frozen sections from the treated hair follicles [(C) vehicle treatment; (D) 50 µM menadione; (E) 20 ng/ml KGF; (F) 20 ng/ml KGF and 50 µM menadione], were analyzed by TUNEL staining for the presence of apoptotic cells. DP, dermal papilla; MK, matrix keratinocytes; PM, precortical matrix. (G) TUNEL-positive cells and all cells (excluding the cells in the dermal papilla) were counted and the ratio between both numbers was determined (ctrl, n=10; menadione, n=9; KGF, n=11; KGF+menadione, n=12; n, number of hair follicles). Means ± s.e.m. are shown. Statistical analysis was performed using the Mann-Whitney U test. *P<0.05; **P<0.01.

View larger version (28K): [in this window] [in a new window]   Fig. 2. The protective effect of KGF is dependent on de novo protein synthesis. (A) In cytoprotection assays (see legend to Fig. 1A) quiescent HaCaT cells were pre-treated with KGF for different periods of time (1 hour, overnight, or overnight plus 1 hour) or left untreated before the addition of menadione. (B) Results of cytoprotection assays in the absence or presence of the protein biosynthesis inhibitor cycloheximide (CHX) are shown: 2-hour treatment with DMSO before KGF addition (left panel), 2-hour treatment with 10 µg/ml CHX in DMSO before KGF addition (right panel). Results are mean ± s.d. *P<0.05 using the Mann-Whitney U test.

View larger version (28K): [in this window] [in a new window]   Fig. 3. The protective effect of KGF is not dependent on a single signaling pathway. (A) HaCaT keratinocytes were rendered quiescent by serum starvation (lane 1), treated with either the MEK1/2 inhibitor U0126 (lanes 3,7,11), the PI3K inhibitor LY294002 (lanes 4,8,12), a combination of both inhibitors (lanes 5,9,13) or solvent control (lanes 2,6,10), and subsequently incubated with 10 ng/ml KGF for the time periods indicated. Whole cell lysates were analyzed for the activation of Erk1/2, Akt1/2/3, MARCKS, JNK and p38 using phospho-specific antibodies. Levels of total Erk1/2 and total Akt1/2 were determined as loading controls. As positive controls (pos., lane 14), cells were treated with EGF (20 ng/ml for 5 minutes) for activation of Erk1/2 and Akt1/2/3, UVB irradiated (40 mJ/cm2 and incubated for 45 minutes after irradiation) for activation of JNK and p38, or treated with phorbol-12-myristate-13-acetate (200 nM, 30-minute incubation) for activation of MARCKS. The results were reproduced in three independent experiments. (B) Cytoprotection assays were carried out in the absence (upper left panel) or presence of inhibitors of different signaling pathways: MEK1/2 inhibitor U0126 (10 µM, upper right panel), PI3K inhibitor LY294002 (10 µM, lower left panel), U0126 and LY294002 (10 µM each, lower right panel). Results are mean ± s.d. **P<0.01 using the Mann-Whitney U test.

View larger version (73K): [in this window] [in a new window]   Fig. 4. KGF protects keratinocytes from UV-induced cell damage in vitro and in vivo. (A,B) Quiescent HaCaT cells were treated with KGF or vehicle 16 hours and 1 hour before irradiation with different doses of UVA (A) and UVB (B) as indicated. UV-irradiated cells were subjected to MTT assay 24 hours after irradiation. (C) The shaved back skin of transgenic mice expressing a dominant-negative FGFR2IIIb (tg) and their wild-type littermates (wt) was irradiated with 100 mJ/cm2 UVB. One group of mice of each genotype received a subcutaneous injection of 10 µg KGF 24 hours before irradiation (n=8 for each genotype), whereas the control group (n=7 for each genotype) was only injected with the solvent (0.5% BSA in PBS). Representative histological pictures of each treatment group and genotype are depicted. The arrows indicate sunburn cells. E, epidermis; D, dermis; HF, hair follicle. (D) Apoptotic cells (arrows) were detected by TUNEL staining (green). Propidium iodide (PI) staining (red) was used to visualize the nuclei. The basement membrane is indicated with a dashed line. Four animals were used of each genotype for vehicle control and five animals of each genotype for KGF injections. Sunburn cells (E) or TUNEL-positive cells (F) were counted and their number per mm basement membrane was calculated. For quantitative analyses in E and F one section from each animal was analyzed; ten pictures were taken from each section. Results are mean ± s.d. For statistical analysis the Mann-Whitney U test was used. *P<0.05; ***P<0.001.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Proposed mechanism of the cytoprotective effect of KGF. The cell-damaging effects of UV irradiation and of xenobiotics with electrophilic and oxidative properties are alleviated by KGF. Upon binding of KGF to its receptor signaling pathways are activated, which induces the expression of cytoprotective genes. A higher activity of cytoprotective proteins renders a cell more resistant against the cellular damage caused by UV irradiation, menadione, tBHQ and other substances.