Vitamin D3 Modulates Impaired Crosstalk Between RANK and Glucocorticoid Receptor Signaling in Bone Marrow Cells After Chronic Prednisolone Administration

Ihor Shymanskyi, Olha Lisakovska, Anna Mazanova, Dmytro Labudzynskyi, Mykola Veliky

The effectiveness of vitamin D3 (cholecalciferol) in counteracting the side effects of glucocorticoid (GC) therapy has been demonstrated previously. Abnormalities in systemic hormonal and local (cytokine) regulation of bone marrow (BM) cells may underlie GC-induced imbalance between osteosynthesis and bone resorption. The cytokine system receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) is considered as an integrating link in the NF-κB-mediated interaction of various cells involved in maintaining osteoblastic-osteoclastic balance, which makes it a pharmacological target for regulation and correction of the bone remodeling process. We studied GC-induced impairments of the RANKL/RANK/OPG axis in BM cells depending on vitamin D bioavailability and whether these changes were mediated by glucocorticoid (GR) and/or vitamin D (VDR) receptors. Female Wistar rats administered with prednisolone (5 mg/kg b.w., 30 days) showed a decrease in the GR protein level and the number of GR-positive BM cells. GC caused a marked elevation of RANKL and RANK levels in BM, while OPG decreased. Flow cytometry data indicated GC-elicited increase in the number of circulating RANK-positive osteoclast precursors (OCPs) in BM, peripheral blood, and spleen. In full accordance with the data that the interaction of RANKL-RANK leads to transcriptional activation of NF-κB and subsequent differentiation of osteoclasts, we found an increase in the level of phosphorylated p65 subunit of NF-κB with a simultaneous decrease in the NF-κB inhibitor (IκB) level. These changes were accompanied by vitamin D insufficiency and downregulated expression of CYP27B1 and VDR, which are responsible for synthesis and hormonal signaling of 1,25(OH)2D. Notably, we observed VDR and RANK co-localization in OCPs. Cholecalciferol co-administration (1,000 IU/kg b.w., 30 days) with prednisolone resulted in elevated GR synthesis in BM. Cholecalciferol prevented prednisolone-elicited disturbances of the RANKL/RANK/OPG, which correlated with improved bioavailability and vitamin D signaling through VDR. This caused the lowering of phosphoNF-κB p65 level and inhibiting NF-κB translocation to the nucleus that could reduce the circulating OCPs pool in BM, peripheral blood, and spleen. Our findings suggest that prednisolone-induced abnormalities in GR and RANKL/RANK/OPG signaling pathways are associated with the impairments of vitamin D auto/paracrine system in BM cells and can be ameliorated by cholecalciferol supplementation.