Primary keratinocytes were isolated and cultured as previously described (17). In brief, 3-day postnatal mice were sacrificed by CO2 asphyxiation. The bodies were rinsed in 75% ethanol and dried with sterile gauze. The mouse was cut from the nose to the tail, and the skin was removed in one piece. The skin was rinsed several times in washing solution [PBS (pH 7.4), containing penicillin (100 U/ml), streptomycin (100 μg/ml), fungizone (0.25 μg/ml), and gentamicin (50 μg/ml)] and incubated in medium containing dispase [dispase II (5 U/ml; Sigma-Aldrich), streptomycin (100 μg/ml), fungizone (0.25 μg/ml), and gentamicin (50 μg/ml)] overnight at 4°C. The dermis was separated from the epidermis with forceps, minced, and digested in 0.25% trypsin for 10 min. Rat keratinocyte culture medium containing defined KM2 (PromoCell) without EGF was used. Keratinocytes were plated into 24- or 6-well plates at previously established cell densities. Media were changed every 2 days. The cells were trypsinized, and three independent batches of cells were counted.

For stimulation experiments, 1 μM S1P (Cayman Chemical) or EGF (100 ng/ml; PeproTech) was added into KM2 and cells were treated for 30 min. In some cases, cells were pretreated with inhibitors (10 μM) for 30 min, followed by S1P (1 μM) or EGF (100 ng/ml), and incubated for another 30 min. The inhibitors were as follows: gefitinib (EGFR inhibitor, Tocris Bioscience), AG1478 (EGFR inhibitor, Sigma-Aldrich), erlotinib (EGFR inhibitor, Sigma-Aldrich), and PD-153053 (EGFR inhibitor, Tocris Bioscience); W146 (S1PR1 inhibitor, Tocris Bioscience), JTE013 (S1PR2 inhibitor, Tocris Bioscience), and VPC23109 (S1PR3 inhibitor, Tocris Bioscience); and BB94 (nonspecific metalloprotease inhibitor, Tocris Bioscience), TAPI-2 (ADAM17 inhibitor, Enzo), GI254023X (ADAM10 inhibitor, Tocris Bioscience), and MMP-2/MMP-9 inhibitor I (MMP-2/MMP-9 inhibitor, Millipore).

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