Oxysterol-induced oxidative disruption of skin junction integrity

The cutaneous tissue is persistently exposed to environmental stressors, including a wide range of airborne pollutants. This chronic exposure often leads to a condition of oxidative stress, with the outermost layer of epidermis, the stratum corneum (SC), being especially vulnerable due to its high lipid content. Notably, approximately 40 % of SC lipids consist of cholesterol, present in both esterified and unesterified forms. The oxidative imbalance induced by environmental stressors and constantly associated with inflammatory skin diseases promotes the formation and accumulation of cholesterol oxidation products, belonging to the oxysterols' family, which are known for their potent pro-oxidant and pro-inflammatory properties. In addition, harmful oxysterols of dietary origin could reach the epidermis via the vascularized dermis, thus adding another route of exposure. 7β-Hydroxycholesterol (7βOHC) and 7-ketocholesterol (7 KC), two highly toxic oxysterols of non-enzymatic origin, have been shown to significantly downregulate proteins involved in adherens and tight junctions in the intestinal epithelium. Given the structural similarity of extracellular junction proteins across tissues, it is reasonable to expect that oxysterols may similarly disrupt the integrity of the epidermal barrier. To investigate this, supraphysiologic concentrations of 7 KC and 7βOHC were added to the medium of human keratinocytes. Immunofluorescence analysis revealed a consistent and significant reduction in the levels of Claudin-1, Zonulin-1 (ZO1), and E-cadherin, key proteins of tight and adherens junctions, respectively, in oxysterol-treated cells compared to controls. Notably, oxysterol exposure also led to a reduction of mitochondrial membrane potential and an increased mitochondrial reactive oxygen species (ROS) production. Both mitochondrial damage and the disruption of skin junctions were efficiently prevented by mitoTEMPO, a selective mitochondrial superoxide scavenger, suggesting the pro-oxidant activity of oxysterols mediates these effects in keratinocytes. Finally, experiments conducted using a 3D skin model corroborated findings observed in keratinocyte cultures, reinforcing the role of oxysterols in compromising the skin barrier integrity.