Cholesterol sulfate is abundant in the human epidermis and is a putative natural ligand for retinoic acid receptor-related orphan receptor alpha (ROR alpha). Although direct binding of cholesterol sulfate is expected to activate ROR alpha, cholesterol sulfate can also induce ROR alpha expression and increase ROR alpha target gene expression. The purpose of this study was to determine whether cholesterol sulfate induces profilaggrin expression, a precursor of the barrier protein filaggrin in the epidermis, through activation of ROR alpha by directly binding to ROR alpha, or through increased ROR alpha expression. Immunohistochemical and polymerase chain reaction (PCR) analyses showed that ROR alpha was expressed in normal human epidermal keratinocytes (NHEKs) and that its expression increased during keratinocyte differentiation in parallel with that of profilaggrin and cholesterol sulfotransferase, which catalyzes the synthesis of cholesterol sulfate. Exogenous cholesterol sulfate significantly increased both ROR alpha and profilaggrin expression in NHEKs, whereas no effect on profilaggrin expression was observed in cells in which ROR alpha was knocked down with small interfering RNA (siRNA). Additionally, a luciferase reporter gene assay revealed that exogenous ROR alpha dose-dependently increased the activity of the profilaggrin gene promoter even in the absence of cholesterol sulfate, and that this response involves activator protein-1. In conclusion, the results of this study indicate that cholesterol sulfate induces filaggrin expression through increased ROR alpha expression. Further studies are required to fully elucidate the mechanisms involved. (C) 2012 Elsevier Inc. All rights reserved.