Cholesterol sulfate induces expression of the skin barrier protein filaggrin in normal human epidermal keratinocytes through induction of RORα

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Abstract

Cholesterol sulfate is abundant in the human epidermis and is a putative natural ligand for retinoic acid receptor-related orphan receptor alpha (RORα). Although direct binding of cholesterol sulfate is expected to activate RORα, cholesterol sulfate can also induce RORα expression and increase RORα 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 RORα by directly binding to RORα, or through increased RORα expression. Immunohistochemical and polymerase chain reaction (PCR) analyses showed that RORα 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 RORα and profilaggrin expression in NHEKs, whereas no effect on profilaggrin expression was observed in cells in which RORα was knocked down with small interfering RNA (siRNA). Additionally, a luciferase reporter gene assay revealed that exogenous RORα 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 RORα expression. Further studies are required to fully elucidate the mechanisms involved.

Highlights

► RORα is expressed in normal human epidermal keratinocytes. ► Cholesterol sulfate increases both RORα and profilaggrin expression in keratinocytes. ► Cholesterol sulfate has no effect on profilaggrin expression in RORα-knockdown keratinocytes. ► RORα activates the filaggrin gene promoter even in the absence of cholesterol sulfate. ► Cholesterol sulfate induces filaggrin expression through increased RORα expression.

Introduction

The skin is an effective physical barrier that protects the body from exogenous pathogens. The outer component of the skin, the epidermis, forms a protective covering, its predominant cell type being the keratinocyte. It consists of four layers: basal, spinous, granular, and cornified layers (in that order from the basement membrane outwards) [1]. Each layer represents a distinct stage of differentiation. In the basal layer, keratinocytes are supplied by their cell division and gradually move towards the skin’s surface through a series of differentiation events [2]. When keratinocytes reach the granular layer, their keratin synthesis ceases and they instead begin producing various barrier proteins, including filaggrin [1], which is initially synthesized as the large precursor protein profilaggrin [3]. Profilaggrin is extensively phosphorylated and packaged into keratohyalin granules in cells of the granular layer. While phosphorylated profilaggrin is protected from proteolysis, dephosphorylated profilaggrin is not and its cleavage yields filaggrin monomers [3]. In the cornified layer, filaggrin becomes associated with keratin intermediate filaments, resulting in aggregation of the latter [4]. In the final step of the cell migration process, epidermal keratinocytes and their products are released from the cornified layer in a process known as desquamation [2].

Previous studies have suggested that the expression of profilaggrin in human epidermal keratinocytes is closely linked to cholesterol sulfate and RORα, a nuclear receptor expressed in various tissues [5], [6]. Although expression of RORα has not been demonstrated in human skin [5], [7], cholesterol sulfate dose-dependently increases the formation of filaggrin in primary mouse keratinocytes [8]. Additionally, expression of SULT2B1b, the enzyme responsible for cholesterol sulfate synthesis, has been detected in the granular layer of the human epidermis where cholesterol sulfate accumulates [9], [10], [11]. Because cholesterol sulfate binds to the ligand-binding domain of RORα with high affinity, and increases its transcriptional activity more strongly than cholesterol or any other cholesterol derivative [12], [13], cholesterol sulfate likely increases profilaggrin expression through activation of RORα. However, recent research showed that cholesterol sulfate can induce RORα expression, and that increased RORα expression itself can enhance its target gene expression regardless of the presence of cholesterol sulfate [14].

The purpose of the present study was to determine whether cholesterol sulfate induces filaggrin expression through activation of RORα by binding to RORα, or through increased RORα expression.

Section snippets

Immunohistochemistry

Expression of RORα, SULT2B1b, and filaggrin in normal human skin was examined by immunohistochemistry, as described previously [11]. A scalp biopsy was obtained from a healthy volunteer. Tissue sections were treated with anti-RORα (Santa Cruz Biotechnology, Santa Cruz, CA), anti-SULT2B1b, and anti-filaggrin (Biomedical Technologies, Stoughton, MA). Cell nuclei (SULT2B1b and filaggrin staining) were counterstained with Mayer’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan).

Culture of NHEKs

NHEKs were obtained

RORα expression in human skin

RORα, SULT2B1b, and filaggrin were detected in the outer parts of the suprabasal region of the normal human epidermis in immunohistochemical analyses performed using specific antibodies. Both RORα and SULT2B1b were weakly detected in the spinous layer and stained more intensely in the granular layer (Fig. 1). Filaggrin was expressed both in the granular layer and the lower part of the cornified layer. Moreover, while RORα localized exclusively to cell nuclei, SULT2B1b and filaggrin were

Discussion

In the present study, we indicated that cholesterol sulfate induces filaggrin expression through increased RORα expression. We found that exogenous cholesterol sulfate significantly increased RORα mRNA expression in NHEKs (Fig. 3A). Moreover, exogenous cholesterol sulfate significantly increased profilaggrin mRNA expression in NHEKs, whereas no effect on profilaggrin was observed in cells in which RORα was knocked down with siRNA (Fig. 3B). In addition, a luciferase reporter gene assay revealed

References (24)

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