Cholesterol-rich plasma membrane domains (lipid rafts) in keratinocytes: importance in the baseline and UVA-induced generation of reactive oxygen species.
The biologic effects of ultraviolet radiation such as DNA damage, mutagenesis, cellular aging, and carcinogenesis are in part mediated by reactive oxygen species. In unirradiated cells the major known sources of reactive oxygen species are the mitochondrial respiratory chain and the membrane oxidases functionally coupled to several membrane growth factor receptors. There is evidence that mitochondria also play a role in oxidative stress after ultraviolet irradiation; however, it is unknown whether the biochemical processes at the level of the plasma membrane contribute to the regulation of reactive oxygen species synthesis. In order to elucidate this issue we examined here the importance of the microdomain plasma membrane organization in the regulation of oxidative stress in unirradiated and ultraviolet A (340-400 nm) irradiated HaCaT keratinocytes. Labeling of confluent HaCaT cultures with fluorescently tagged cholera toxin B subunit (FITC-CTx) revealed the presence of GM1 ganglioside and cholesterol-rich microdomains (lipid rafts) that formed junction-like structures in the membranes of adjacent cells and patchy microdomains elsewhere. There was a marked heterogeneity in the level of FITC-CTx labeling: there were groups of cells demonstrating prominent labeling (FITC-CTx(high)) whereas other cells were only weakly labeled (FITC-CTx(low)). When reactive oxygen species synthesis was measured with the fluorescent probe carboxy-2',7'-dichlorodihydrofluorescein diacetate, we found that (i) the baseline and ultraviolet-A-induced reactive oxygen species synthesis correlated with the magnitude of FITC-CTx labeling and was highest in the FITC-CTx(high) cells; (ii) reactive oxygen species synthesis was diminished in cells in which the integrity of membrane domains was disrupted by cholesterol sequestration with methyl-beta-cyclodextrin and filipin, or after treatment with GM1 ganglioside; (iii) reactive oxygen species synthesis in cholesterol-depleted cells was fully restored after cholesterol repletion. We conclude that the plasma membrane takes part in the regulation of oxidative stress in keratinocytes and disruption of its microdomain structure reduces reactive oxygen species synthesis both at the baseline and after ultraviolet A irradiation. We hypothesize that lipid-raft-associated protein(s) may be involved in the generation of reactive oxygen species and that pharmacologic modulation of membrane structure may provide a novel therapeutic approach relevant for photoprotection and cutaneous carcinogenesis.