Claudin-7 in keratinocytes is downregulated by the inhibition of HMG-CoA reductase and is highly expressed in the stratum granulosum of the psoriatic epidermis

Background: Cholesterol is de novo synthesized in the upper epidermis and plays an important role in maintaining the normality of skin. Studying the impact of the inhibition of cholesterol de novo synthesis in the epidermis may help understand how skin homeostasis is regulated. Objective: In this study, we created a gene expression profile to investigate the effect of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors on epidermal homeostasis. Methods: A microarray analysis was performed using normal keratinocytes with or without HMG-CoA reductase inhibitor (pitavastatin) treatment. Real-time PCR confirmed the reproducibility of genes with altered expression in keratinocytes treated with HMG-CoA reductase inhibitors. Among these genes, we focused on reduced expression of claudin 7 histologically confirmed by immunohistochemical staining, in situ hybridization, and immunoelectron microscopy. Results: Claudin-7 was highly expressed in the stratum granulosum of psoriatic lesions but was not expressed in the normal epidermis. Immunoelectron microscopy revealed that claudin-7 was localized in the keratohyalin granules of psoriatic lesions. Conclusion: These results indicate that claudin-7 expression was regulated by HMG-CoA reductase in the epidermis and might play a pathogenic role in the keratohyalin granules found in the epidermal granular layer of psoriasis. Adult human epidermal keratinocytes were used. According to the preliminary studies, the administration concentration was set to 0.1 M, at which level pitavastatin does not affect cell proliferation abilities. The cells (1.0 106) were seeded in a 10 cm dish and cultured in 10 mL of EpiLife medium (Thermo Fisher Scientific, USA) for 3 days. Pitavastatin solution was then added to the culture medium to reach a final concentration of 0.1 M, and the solution was cultured for 6 hours. The same amount of DMSO alone was added as a control and cultured (culture conditions: 5% CO2, 37 C). The cells treated with pitavastatin and the cells treated with the vehicle had the same passage number (each n = 2). After culturing, the cells were collected, and total RNA was extracted using the SV Total RNA Isolation System (Promega, Tokyo, JAPAN). The resulting RNA was labeled with Cyanine-3 and was used for the microarray analysis. Three normalization procedures were performed on the samples. First, genes with signal intensities of 0.01 or less were corrected to 0.01. The normalized value of the corresponding pitavastatin-treated sample was then divided by the normalized value of the measured vehicle-treated sample on each chip. Genes with low expression levels with a signal intensity of less than 100 were filtered out in both the vehicle-treated samples and the pitavastatin-treated samples. A comparative analysis of the expression variability in genes that passed filtering was performed.