Fig. 1. Role of ceramide in UVA radiation-induced ICAM-1 expression in long-term cultured, normal human keratinocytes. (A) Time-dependent activation of transcription factor AP-2 after stimulation of cells with 10 µM C2-ceramide. Nuclear extracts were analysed by electrophoretic mobility shift assay using a radiolabelled AP-2 consensus site derived from the human ICAM-1 promoter. (B) Time-dependent ICAM-1 mRNA expression after stimulation of cells with 10 µM C2-ceramide (grey bar). ICAM-1 mRNA expression was assessed by differential RT–PCR based on the housekeeping gene β-actin. Unstimulated control (white bar) is set as 1. (C) C2-ceramide-induced reporter gene activity in transiently transfected human keratinocytes detected as relative specific luciferase activity (RLU/µg protein) (grey bars). Different deletion constructs based on the human ICAM-1 promoter linked to luciferase were assessed in triplicate. Unstimulated but transfected cells were set as 1 (white bar). (D) UVA-induced ceramide release was detected in human keratinocytes by sequential HPTLC in triplicate. Cells were sham irradiated (white bar) or irradiated with 30 J/cm² UVA (black bars) and harvested at the indicated time points. Data were obtained as c.p.m./500 µg of protein (mean of three experiments) and are given as fold increase (control was set as 1). (E) Effect of l-cycloserine on UVA-induced ICAM-1 mRNA expression. UVA radiation-induced ICAM-1 mRNA expression was assessed by differential RT–PCR in cells that had been left untreated (black bar) or treated with 1 mM l-cycloserine (striped bar) for 3 days prior to, during and after UVA irradiation, or were additionally stimulated with 10 µM C2-ceramide (grey bar).