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.
Objectives Recent prospective studies have found the associations between type 1 diabetes (T1D) and vitamin D deficiency. We investigated the role of vitamin D in the regulation of 25OHD-1α-hydroxylase (CYP27B1) and VDR expression in different tissues of T1D rats. Design T1D was induced in male Wistar rats by streptozotocin (55 mg/k b.w.). After 2 weeks of T1D, the animals were treated orally with or without vitamin D3 (cholecalciferol; 100 IU/rat, 30 days). Methods Serum 25-hydroxyvitamin D (25OHD) was detected by ELISA. CYP27A1, CYP2R1, CYP27B1, and VDR were assayed by RT-qPCR and Western blotting or visualized by immunofluorescence staining. Results We demonstrated that T1D led to a decrease in blood 25OHD, which is probably due to the established downregulation of CYP27A1 and CYP2R1 expression. Vitamin D deficiency was accompanied by elevated synthesis of renal CYP27B1 and VDR. Conversely, CYP27B1 and VDR expression decreased in the liver, bone tissue, and bone marrow. Cholecalciferol administration countered the impairments of the vitamin D-endo/para/autocrine system in the kidneys and extrarenal tissues of diabetic rats. Conclusions T1D-induced vitamin D deficiency is associated with impairments of renal and extrarenal CYP27B1 and VDR expression. Cholecalciferol can be effective in the amelioration of diabetes-associated abnormalities in the vitamin D-endo/para/autocrine system.
Актуальність. Довготривале введення глюкокортикоїдів (ГК) супроводжується порушенням мінерального обміну і, як наслідок, призводить до розвитку вторинного остеопорозу. Важливу роль у регулюванні процесу ремоделювання кісткової тканини відіграє вітамін D, який може реалізовувати свої біологічні ефекти через рецептор вітаміну D — VDR-опосередкований вплив на цитокінові системи, зокрема RANKL-RANK-OPG (ліганд рецептора активатора ядерного фактора транскрипції NF-κB — рецептор RANKL — остеопротегерин). Мета. Дослідження ГК-індукованих змін експресії детермінантних для процесів формування та резорбції кісткової тканини регуляторних протеїнів (RANK, RANKL, OPG, остеокальцин, VDR) залежно від біодоступності вітаміну D. Матеріали та методи. Щури-самиці лінії Wistar отримували преднізолон (5 мг/кг маси тіла) разом з вітаміном D3 (100 МО) або без нього протягом 30 днів. Рівні VDR, остеокальцину, RANK, RANKL і OPG у кістковій тканині визначали за методом вестерн-блот-аналізу. Рівень гідроксивітаміну D (25OHD) у сироватці крові аналізували, використовуючи метод ELISA. Вміст мінеральних компонентів (Ca2+ та Pi), активність лужної фосфатази (ЛФ) та її ізоформ визначали спектрофотометрично. Результати. Преднізолон значно знижував рівень 25OHD у сироватці крові та вміст протеїну VDR у кістковій тканині. Це супроводжувалось підвищенням загальної активності сироваткової ЛФ і її кісткової ізоформи, гіпокальціємією, гіпофосфатемією, а також зниженням вмісту мінеральних компонентів у кістковій тканині. Спостерігалось значне зниження експресії остеокальцину, ключового маркера остеосинтезу. Зміни у D-вітамінному статусі тварин, викликані ГК, призводили до зниження рівня RANK та OPG у кістковій тканині, тоді як рівень RANKL залишався без змін. Уведення вітаміну D3, підвищуючи рівні 25OHD і VDR, сприяло частковій або повній нормалізації викликаних преднізолоном змін мінерального обміну й кісткового гомеостазу через підвищення рівнів RANK, OPG та остеокальцину. Висновки. Преднізолон-індуковане дерегулювання цитокінової системи RANKL — RANK — OPG та остеокальцин-опосередковане формування кісткової тканини можуть бути тісно пов’язані зі зниженням біодоступності вітаміну D і порушенням клітинного сигналювання через VDR. Відновлення біодоступності вітаміну D є необхідною умовою, що протидіє негативним ефектам глюкокортикоїдної терапії у кістковій тканині.
Vitamin D (VD) is a molecule that can be synthesized directly in the humans' body or enter the organism with food in the form of inactive precursors. To exert its biological action, VD undergoes two-stage hydroxylation (at the 25th and 1st position) catalyzed by cytochromes P450, the presence of which has already been shown in almost all tissues of the human body. The product of hydroxylation is hormone-active form of vitamin D-1,25(OH) 2 D. 1,25(OH) 2 D binds to specific vitamin D receptor (VDR) and regulates the expression of genes involved in bone remodeling (classical function) and genes that control immune response, hormone secretion, cell proliferation, and differentiation (nonclassical functions). VD deficiency is prevalent around the globe and may be one of the key factors for diabetes development. The direct association between vitamin D deficiency and type 1 (T1D) and type 2 (T2D) diabetes has been proven. Detection of VDR in pancreas and adipose tissue, skeletal muscles, and immune cells allowed implying the antidiabetic role of vitamin D by enhancing insulin synthesis and exocytosis, increasing the expression of the insulin receptor, and modulating immune cells' functions. This chapter summarizes data about relationship between VD insufficiency/deficiency and development of T1D and T2D, and their complications.
Osteoporosis is a devastating side effect of chronic glucocorticoid (GC) treatment. Despite the crucial role of vitamin D (VD) in bone homeostasis, the precise molecular mechanisms of its action on GC-induced disturbances of bone remodeling remain undefined. The study was performed to elucidate the relation of VD status to GC-induced changes of the angiogenesis/osteogenesis/bone resorption coupling in bone tissue. Female Wistar rats received prednisolone (5 mg/kg of b.w.) with or without VD3 (1000 IU/kg of b.w., for 30 days). Biomechanical parameters of rat femurs were assessed by the three-point bending test. The levels of calcium, inorganic phosphate, activity of total alkaline phosphatase (ALP), and its isoenzymes were determined spectrophotometrically. Vascular endothelial growth factor-A (VEGF-A) and caspase-3 protein levels were detected by western blotting. Vdr and Cyp27b1 mRNAs were measured by qRT-PCR. Receptor activator of nuclear factor κB (RANK) expression in bone sections was visualized immunohistochemically. Serum 25(OH)D was assayed by ELISA. GC administration led to a decrease in maximal load (by 1.2-fold) and stiffness and toughness (by 1.3-fold), which was accompanied by a 3-fold reduction of 25(OH)D level, an elevation of the ALP bone isoenzyme activity in serum, hypocalcaemia, and hypophosphatemia. Along with prednisolone-induced VD deficiency, an impaired synthesis of Vdr (−30%) and Cyp27b1 (+71%) mRNA was observed, reflecting deregulation of bone tissue VD-auto-/paracrine system. GC caused an increase in caspase-3 content, suppressed the synthesis of the osteoclastic marker RANK, and altered angiogenesis/osteogenesis coupling by significantly reducing the level of VEGF-A.VD3 treatment restored serum 25(OH)D content and the expression of key components of the VD-auto-/paracrine system. VD3 supplementation diminished cell apoptosis and strongly improved angiogenesis/osteogenesis coupling as well as mineral metabolism and biomechanical parameters of femurs in GC-administered rats. Thus, VD3 can have a beneficial effect on the correction of GC-induced pathological changes in bone remodeling.
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