Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene

Abstract

UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation-and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and͞or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response.

Ceramide and raft signaling are linked with each other in UVA radiation-induced gene expression

“…UVA irradiation results in a decrease of sphingomyelin and Chol in plasma membranes and rafts of keratinocytes To analyse the effects of UVA radiation on the lipid composition of rafts, normal human epidermal keratinocytes (NHEKs) were exposed to a dose of 30 J/cm 2 UVA, which previously was shown to be optimal for induction of the UVA signaling response in this cell type (Grether-Beck et al, 1996, 2005. After 30 min, plasma membranes or lipids rafts were isolated and their sphingomyelin and Chol content was quantified using high-performance thin-layer chromatography (TLC).…”

“…A dose of 5 mM bMCD decreased Chol content by about 50%, without altering cell viability even when Chol was added for 24 h, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Grether-Beck et al, 2003;data not shown). This dose was, therefore, used in subsequent experiments in which UVA-stress response was analysed by measuring UVA radiation-induced mRNA expression of intercellular adhesion molecule-1 (ICAM-1) as a model gene (Grether-Beck et al, 1996. Chol decrease increased sensitivity of NHEKs toward UVA radiation-induced ICAM-1 expression.…”

“…Inhibition of the UVA response does not involve Chol oxidation and can also be achieved by phytosterols We have shown previously that UVA radiation-induced signaling is initiated at the level of the cell membrane via Ceramide and raft signaling linkage in UVA radiation-induced gene expression S Grether-Beck et al generation of singlet oxygen (Grether-Beck et al, 1996). Chol can quench singlet oxygen (Korytowski and Girotti, 1999) or free radicals (Girotti, 1998), indicating the possibility that the UVA-inhibitory effect of Chol might be due to its antioxidant properties.…”

“…Cell culture Long-term cultured, normal human keratinocytes prepared from neonatal foreskin were cultured under serum free conditions for up to five passages (Grether-Beck et al, 1996, 2003, 2005. Depletion of Chol from cytoplasmic membranes was achieved through a 2-h preincubation of cells with 5 mM bMCD as previously described (Ridgway et al, 1998).…”

“…Human epidermal keratinocytes represent the primary cellular target for UVA radiation. In previous studies, we have shown that the UVA radiation-induced stress response is initiated in this cell type by a photo-oxidative process, which causes the generation of singlet oxygen and that UVA radiation/ singlet oxygen-induced gene expression critically depend on activation of transcription factor activator protein-2 (AP-2) (Grether-Beck et al, 1996). The steps that occur downstream of UVA radiation-induced singlet oxygen generation and upstream of AP-2 activation involve generation of the second messenger ceramide from cell membrane sphingomyelin indicating that UVA radiation-induced alterations at the level of cell membrane lipids are critically involved in this signaling cascade (Grether-Beck et al, 2000).…”