Endocrine Abstracts

Background: Calcifediol (25(OH)D₃), the major circulating form and the direct precursor of the biologically active form of vitamin D, has been identified as an agonist ligand for vitamin D receptor (VDR) with anti-proliferative effects and gene regulatory function despite having a lower receptor affinity respect than the biologically active form of vitamin D₃. In fact, recent studies have suggested that 25(OH)D₃ can regulate gene expression by direct binding to VDR. So, considering this, our purpose has been to investigate whether 25(OH)D₃ could be able to activate rapid non-genomic pathways, similarly to what was observed with the biologically active form of vitamin D. In this light, we analyzed in vitro the Ca²⁺ intracellular following exposure to 10^-5 M 25(OH)D₃ in mesenchymal stem cells (MSCs) derived from human adipose tissue (hADMSCs), which had been previously described as excellent cell model systems for studying the hormonal effects of 1α,25(OH)₂D₃.

Methods: hADMSC lines were established from small subcutaneous adipose tissue biopsies. Changes in intracellular Ca²⁺ concentration on hADMSCs exposed to 10^-5 M of 25(OH)D₃ were determined in Fluo-4-AM loaded cells and evaluated by Laser Scanning Confocal Microscopy (LSCM). As a positive control, cells were treated with 10^-5 M calcium ionophore A23187. Statistical analysis was carried out by ANOVA followed by Bonferroni’s test with predefined experimentwise (αT) = 0.05.

Results: We showed that 4 cells, one for each cell line tested, exposed to 10^-5 M calcium ionophore A23187 as well as 17 cells treated with 10^-5 M 25(OH)D₃ responded positively with a significant increase of intracellular Ca²⁺ levels. In particular, three of them reached a maximum comparable to what was achieved in cells treated with 10^-5 M calcium ionophore A23187, whereas the other cells showed a weaker response. The average ratio of the maximum fluorescence intensity changes of the cells treated with 25(OH)D₃ and calcium ionophore compared to the baseline fluorescence was 356% and 473%, respectively.

Conclusions and future perspective: Our data demonstrated for the first time the ability of 25(OH)D₃ to trigger rapid non-genomic effects like other steroid hormones, such as the increase in intracellular Ca²⁺ levels in hADMSCs. Nowadays, we are profiling experiments on the same cell model to study whether calcifediol could be able also to stimulate rapid cAMP signaling pathway. These findings could lead to a greater vitamin D endocrine system understanding, paving the way for the identification of novel therapeutical targets.