Altered Expression of Vitamin D Metabolism Genes and Circulating MicroRNAs in PBMCs of Patients with Type 1 Diabetes: Their Association with Vitamin D Status and Ongoing Islet Autoimmunity.

BACKGROUND: The immunomodulatory role of 1,25-Dihydroxy vitamin D3 (1,25(OH)2D3) is exerted through its interaction with the vitamin D receptor (VDR) present on pancreatic and immune cells. While a deficiency in vitamin D has been linked to Type 1 Diabetes Mellitus (T1DM), the exact molecular mechanism driving this down-regulation in T1DM is yet to be fully understood. This study aimed to decipher differences in the expression of genes associated with vitamin D metabolism in T1DM patients and to ascertain if there is a correlation between serum 1,25(OH)2D3 levels and the expression of these genes. We also sought to understand the influence of specific microRNAs (miRNAs) on the expression of vitamin D metabolism genes in peripheral blood mononuclear cells (PBMCs) of T1DM patients. Furthermore, the study delved into the potential implications of altered vitamin D metabolism genes and miRNAs on autoimmune processes. METHODS: Utilizing real-time PCR, we assessed the expression profiles of genes encoding for 1-hydroxylases (CYP27B1) and 24-hydroxylases (CYP24A1), as well as related miRNAs, in PBMCs from 30 T1DM patients and 23 healthy controls. ELISA tests facilitated the measurement of 1,25(OH)2D3, GAD65, and IA-2 levels. RESULTS: Our findings showcased downregulated CYP27B1 mRNA levels, while CYP24A1 expression remained stable compared to healthy subjects (CYP27B1, p = 0.0005; CYP24A1, p = 0.205, respectively). In T1DM patients, the levels of has-miR-216b-5p were found to be increased, while the levels of has-miR-21-5p were decreased in comparison to the control group. Notably, no correlation was identified between the expression of CYP27B1 in T1DM patients and the levels of has-miR-216b-5p, has-miR-21-5p, and 1,25(OH)2D3. A significant negative correlation was identified between CYP27B1 mRNA levels in PBMCs of T1DM and IA2, but not with GAD65. CONCLUSIONS: The study highlights there were reduced levels of both CYP27B1 mRNA and has-miR-21-5p, along with elevated levels of has-miR-216b-5p in the PBMCs of T1DM. However, the absence of a correlation between the expression of CYP27B1, levels of has-miR-216b-5p, and the status of 1,25(OH)2D3 suggests the possible existence of other regulatory mechanisms. Additionally, the inverse relationship between IA2 autoantibodies and CYP27B1 expression in T1DM patients indicates a potential connection between this gene and the autoimmune processes inherent in T1DM.

25-Hydroxycholecalciferol Inhibits Cell Growth and Induces Apoptosis in SiHa Cervical Cells via Autocrine Vitamin D Metabolism.

Preclinical studies show that the anticancer actions of vitamin D metabolites are mediated by apoptosis, inhibition of cell proliferation and induction of cell cycle arrest. Cervical cancer cells express an autocrine vitamin D metabolising system (VDMS) comprised of a vitamin D receptor, vitamin D catabolic enzyme (CYP24A1), and the activating enzyme of 25-hydroxycholecalciferol (25(OH)D3), CYP27B1. We assessed the anticancer effects of 25(OH)D3 at clinically relevant concentrations on a cervical squamous cell cancer cell line, SiHa. We evaluated cell health parameters (cell count, viability, and cell cycle), cell death modes (apoptosis, autophagic-dependent death, and necrosis by flow cytometry and transmission electron microscopy), and autocrine VDMS gene and protein expression by qPCR and Western blot, respectively. Our study demonstrates that physiological and supraphysiological doses of 25(OH)D3 inhibit cell growth and viability and induce biochemical and morphological apoptosis in SiHa cells. These growth effects are mediated by alteration in the VDMS gene and protein expression, with prominent negative feedback at supraphysiological treatment dose. These data identify promising therapeutic potential of 25(OH)D3 in cervical cancer, which warrants further clinical translational investigations.

Effect of Different Concentrations of Astragali Radix Containing Serum on CYP24A1，CYP27B1 mRNA and Protein in Osteogenic Differentiation of Aging Bone Marrow Mesenchymal Stem Cells / 中国实验方剂学杂志

Objective:To investigate the effects of different concentrations of Astragali Radix containing serum on the expression of 24-hydroxylase（CYP24A1），1α-OHase（CYP27B1） mRNA and protein in rat bone marrow mesenchymal stem cells （BMSCs）， and to explore the mechanism of primary osteoporosis （OP）. Method:The experiment was divided into 6 groups，like normal group， model group， low ，middle and high dose group of Astragali Radix containing serum（20%，40%，60%），Vitamin D group. Cell proliferation toxicity assay（CCK-8） was used to detect the effect of different concentrations of Astragali Radix containing serum on survival rate of aging BMSCs.Real-time quantitative PCR（Real-time PCR） and Western blot was used to detect the expression of CYP24A1 CYP27B1 mRNA and protein in senile BMSCs osteogenic differentiation cells by different concentrations of Astragali Radix containing serum. Result:Compared with normal group， the proliferation and survival rate of BMSCs osteoblasts induced by D-galactose in model group was significantly lower than that in normal group （P<0.01）. Compared with model group， medium and high dose groups and Vitamin D group could improve the proliferation and differentiation of aging BMSCs into osteoblasts in different degrees（P<0.01）. The relative expression of CYP27B1 mRNA and protein in model group was significantly lower than that in normal group， while the relative expression of CYP24A1 mRNA and protein in model group was significantly higher than that in normal group. Compared with model group， high dose Astragali Radix containing serum group could increase the relative expression of CYP27B1 mRNA and protein， and decrease the relative expression of CYP24A1 mRNA and protein in a dose-dependent manner（P<0.01）. Conclusion:The mechanism of different concentrations of Astragali Radix containing serum in the treatment of osteoporosis may be related to the regulation of CYP24A1， CYP27B1 mRNA and protein in the osteogenic differentiation of aging BMSCs.

Analysis of the binding sites of vitamin D 1α-hydroxylase (CYP27B1) and vitamin D 24-hydroxylase (CYP24A1) for the design of selective CYP24A1 inhibitors: Homology modelling, molecular dynamics simulations and identification of key binding requirements.

A homology model of human CYP27B1 was built using MOE and was further optimised by molecular dynamics simulations of the hCYP27B1 homology model and a hCYP27B1-SDZ-88357 complex. Docking results from the hCYP27B1-SDZ-88357 complex showed amino acids Arg107, Asn387 and Asp320 have an important role in binding interaction, with Asp320 part of the important acid-alcohol pair situated in the I-helix with the conserved sequence (A/G) GX (E/D) (T/S), which assumes an essential role in the binding of an oxygen molecule for catalysis. Additional docking experiments with selective hCYP27B1 or hCYP24A1 inhibitors using both the hCYP27B1 model and a triple mutant hCYP24A1 model provided further support for the importance of H-bonding interactions with the three identified active site amino acids. To confirm the role of Arg107, Asn387 and Asp320 in the active site of hCYP27B1 compounds were designed that would form H-bonding interactions, as determined from docking experiments with the hCYP27B1 model. Subsequent synthesis and CYP24A1 and CYP27B1 enzyme assays of the designed compounds 1a and 1b showed a∼5-fold selectivity for CYP27B1 confirming the importance of Asp320 in particular and also Asn387 and Arg107 as important amino acids for CYP27B1 inhibitory activity.

Eldecalcitol reduces osteoporotic fractures by unique mechanisms.

Eldecalcitol shows higher binding affinity for vitamin D-binding protein (DBP), tighter binding to vitamin D receptor (VDR), and resistance to metabolic degradation via 24-hydroxylation. In silico analysis of the mode of binding demonstrated that the 3-hydroxypropyloxy (3-HP) group of eldecalcitol offers additional hydrogen bond and CH-π interaction for the binding to DBP and VDR. However, the 3-HP group interferes with the binding of eldecalcitol to CYP24A1, causing poor metabolic clearance of eldecalcitol by this enzyme. These characteristics may contribute to the stronger effect of eldecalcitol than calcitriol. The present post-hoc analysis also demonstrate that the incidence of hypercalcemia and hypercalciuria is slightly higher in eldecalcitol than in alfacalcidol group especially in patients with CKD stage 3B, that both serum and urinary calcium return to the baseline levels shortly after cessation of the treatment in both treatment groups, that the incidence of urolithiasis is higher in patients with higher eGFR and is similar between alfacalcidol and eldecalcitol groups, and that eGFR is transiently reduced by both alfacalcidol and eldecalcitol treatment especially among patients with higher eGFR but recovers after the end of both treatment. Eldecalcitol can be used for the treatment of osteoporosis without Ca supplementation to reduce the incidence of hypercalcemia and hypercalciuria, and enough hydration is recommended in order to avoid hypercalcemia, urolithiasis and deterioration of renal function.

Effects of 25-hydroxyvitamin D3 on cathelicidin production and antibacterial function of human oral keratinocytes.

Vitamin D and its metabolites have been recognized as key determinants in innate immune modulation. In this study, we investigated the regulation of antibacterial functions of oral keratinocyte cells by 25-hydroxyvitamin D3 (25VD3). OKF6/TERT2 cells, an immortalized human oral keratinocyte cell line, were transfected with or without 24-hydroxylase small interfering RNA (siRNA) and incubated with different amounts of 25VD3. These epithelial cells expressed high levels of inactivating 24-hydroxylase (CYP24A1) and relatively low levels of activating 1α-hydroxylase (CYP27B1) in the presence of 25VD3. 25VD3 influenced the expression of vitamin D-driven genes and cathelicidin in a dose-related manner. SiRNA specific to 24-hydroxylase augmented the cathelicidin production and subseqently influenced the antibacterial activity on multispecies of oral pathogens. These observations suggest that 25VD3 is capable of stimulating cathelicidin production and modulating antibacterial function upon CYP24A1 knochdown in oral epithelial cells, and indicate novel mechanisms that 25VD3 may enhance antibacterial ability in oral keratinocytes.