Impact of 25-OH and 1,25-OH Vitamin D on Osteoblast Activity

Objectives: Introduction: Vitamin D is a fat-soluble molecule involved in bone homeostasis as a major player in the homeostatic balance between bone-building osteoblasts and bone-resorbing osteoclasts. There are two main types of Vitamin D found in the body 25-OHD (circulating VitD) and 1,25-OHD (active VitD). It is not known which of these forms are more potent in regulating the osteoblast function. Since osteoblast and osteoclast coupling is mediated by the OPG/RANKL interaction, we hypothesized that the VitD will regulate the OPG and RANKL in osteoblast function.

Aims: Our aim was to measure the impact of 25-OH and 1,25-OH VitD on osteoblast function and OPG/RANKL response.
Methods: Materials and Methods: Osteoblasts were harvested from the calvaria of 3-5 day old healthy mice and seeded into 12-well plates at 20,000 cells/well. They were then treated with 1,25-OHD and 25-OHD in different concentrations (1-100 nmol and 100-400 nmol, respectively). OPG and RANKL levels were measured using ELISA and RT-qPCR. Osteoblast function was measured by Alkaline Phosphatase (ALP) activity assay. All measurements were performed at 3,7 and 10 days of culture.
Results: Results: Both types of VitD resulted in a dose-dependent decrease in OPG levels especially at 7 days at their highest concentrations (1,25-OHD 100nmol p= 0.002 and 25-OHD 400nmol p=0.034). In parallel, RANKL levels were dose-dependently increased in response to both forms of VitD, notably at 3 days 1,25OHD 10nmol (p=0.044). ALP activity was not significantly changed between groups and over time.
Conclusions: Conclusion: Both 1,25-OHD and 25-OHD affect OPG and RANKL expression by osteoblasts indicating an active involvement of Vitamin D on bone formation.