Protein disulfide isomerase A3 might be involved in the regulation of 24-dehydrocholesterol reductase via vitamin D equilibrium in primary cortical neurons

Abstract

Vitamin D is a secosteroid hormone mediating its functions via vitamin D receptor (VDR) and an endoplasmic reticulum chaperone, protein disulfide isomerase A3 (PDIA3). From a physiological perspective, there is also a well-established association of cholesterol and vitamin D synthesis, since both share a common metabolic substrate, 7 dehydrocholesterol (7-DHC). Yet, the potential basic pathways, of the biological interplay of DHCR24 and vitamin D equilibrium, on neuronal level, are yet to be determined. In this study, we aimed to investigate the relation between vitamin D pathways and DHCR24 in primary cortical neuron cultures. The neocortex of Sprague-Dawley rat embryos (E16) was used for the preparation of primary cortical neuron cultures. DHCR24 mRNA and protein expression levels were determined by qRT-PCR, Western blotting, and immunofluorescent labeling in 1,25-dihydroxyvitamin D3-treated or VDR/PDIA3-silenced primary cortical neurons. The mRNA expression of DHCR24 was significantly decreased in the cortical neurons treated with 10^-8M 1,25-dihydroxyvitamin D₃ (p<0.001). In parallel with the mRNA results, DHCR24 protein expression in cortical neurons treated with 10^-8M 1,25-dihydroxyvitamin D₃ was also significantly lower than untreated neurons (p<0.05). These data were also confirmed with immunofluorescent labeling and fluorescence intensity measurements of DHCR24 (p<0.001). Finally, DHCR24 mRNA expression level was significantly increased in PDIA3 siRNA-treated neurons (p<0.05). Similar to the mRNA results, the DHCR24 protein expression of PDIA3 siRNA-treated neurons was also statistically higher than the other groups (p<0.05). Results of this mechanistic experimental basic study demonstrate that DHCR24 mRNA expression and protein concentrations attenuated in response to vitamin D treatment. Furthermore, we observed that PDIA3 might be involved in this modulatory effect. Our findings indicate a complex interaction of DHCR24 and vitamin D equilibrium, through the involvement of PDIA3 and vitamin D in the modulation of cholesterol metabolism in neuronal cells, requiring future studies on the field.

Keywords: 24-dehydrocholesterol reductase; Neurodegeneration; Primary neuronal culture; Protein disulfide isomerase A3; Vitamin D.