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Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1.

ABSTRACT: Vitamin D deficiency (hypovitaminosis D) causes osteomalacia and poor long bone mineralization. In apparent contrast, hypovitaminosis D has been reported in patients with primary brain calcifications ("Fahr's disease"). We evaluated the expression of two phosphate transporters which we have found to be associated with primary brain calcification (SLC20A2, whose promoter has a predicted vitamin D receptor binding site, and XPR1), and one unassociated (SLC20A1), in an in vitro model of calcification. Expression of all three genes was significantly decreased in calcifying human bone osteosarcoma (SaOs-2) cells. Further, we confirmed that vitamin D (calcitriol) reduced calcification as measured by Alizarin Red staining. Cells incubated with calcitriol under calcifying conditions specifically maintained expression of the phosphate transporter SLC20A2 at higher levels relative to controls, by RT-qPCR. Neither SLC20A1 nor XPR1 were affected by calcitriol treatment and remained suppressed. Critically, knockdown of SLC20A2 gene and protein with CRISPR technology in SaOs2 cells significantly ablated vitamin D mediated inhibition of calcification. This study elucidates the mechanistic importance of SLC20A2 in suppressing the calcification process. It also suggests that vitamin D might be used to regulate SLC20A2 gene expression, as well as reduce brain calcification which occurs in Fahr's disease and normal aging.

SUBMITTER: Keasey MP

PROVIDER: S-EPMC4868979 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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Project description:Constitutively activated STAT1 and elevated IFN-? are both characteristic of T cell large granular lymphocytic leukemia (T-LGLL), a rare incurable leukemia with clonal expansion of cytotoxic T cells due to defective apoptosis. Interferon gamma (IFN-?) is an inflammatory cytokine that correlates with worse progression and symptomology in multiple autoimmune diseases and cancers. In canonical IFN-?-STAT1 signaling, IFN-? activates STAT1, a transcription factor, via phosphorylation of tyrosine residue 701 (p-STAT1). p-STAT1 then promotes transcription of IFN-?, creating a positive feedback loop. We previously found that calcitriol treatment of the TL-1 cell line, a model of T-LGLL, significantly decreased IFN-? secretion and p-STAT1 while increasing the vitamin D receptor (VDR) protein. Here we further explore these observations. Using TL-1 cells, IFN-? decreased starting at 4h following calcitriol treatment, with a reduction in the intracellular and secreted protein levels as well as the mRNA content. A similar reduction in IFN-? transcript levels was observed in primary T-LGLL patient peripheral blood mononuclear cells (PBMCs). p-STAT1 inhibition followed a similar temporal pattern and VDR upregulation inversely correlated with IFN-? levels. Using EB1089 and 25(OH)D3, which have high or low affinity for VDR, respectively, we found that the decrease in IFN-? correlated with the ability of EB1089, but not 25(OH)D3, to upregulate VDR. However, both compounds inhibited p-STAT1; thus the reduction of p-STAT1 is not solely responsible for IFN-? inhibition. Conversely, cells treated with VDR siRNA exhibited decreased basal IFN-? production upon VDR knockdown in a dose-dependent manner. Calcitriol treatment upregulated VDR and decreased IFN-? regardless of initial VDR knockdown efficiency, strengthening the connection between VDR upregulation and IFN-? reduction. Our findings suggest multiple opportunities to further explore the clinical relevance of the vitamin D pathway and the potential role for vitamin D supplementation in T-LGLL.

Project description:Rationale & Objective:Mineral and bone disorder in chronic kidney disease (CKD) is associated with progression of coronary artery calcification (CAC). Mineral and bone disorder often is treated with calcitriol and other vitamin D receptor activators, including paricalcitol, agents that may have differential effects on calcium, phosphate, and parathyroid hormone levels. Accordingly, we investigated whether these agents have differential effects on CAC progression in patients with CKD. Study Design:Randomized, double-concealed, 48-week clinical trial. Setting & Participants:CKD stage 3 or 4 with secondary hyperparathyroidism with CAC score > 0 and no prior treatment with activated vitamin D. Intervention:Calcitriol versus paricalcitol. Outcomes:The primary outcome was log-transformed CAC change. Secondary outcomes included percent change in CAC volume, valvular calcifications, and bone mineral metabolism markers. Results:Among 44 individuals randomly assigned, mean age was 65 years and mean estimated glomerular filtration rate was 27 mL/min/1.73 m2. Median CAC score was 140 (IQR, 55-277) Agatston units at baseline. There was no significant difference in CAC progression between treatment arms (P = 0.06). After adjustment for baseline CAC score (log), treatment group remains nonsignificant (P = 0.08). Further adjustment for creatinine level and/or CKD stage did not change the association. In secondary analyses adjusting for dose level of activated vitamin D, treatment group was significant (P = 0.01), and when dose level was also included in the model, the coefficient for individuals in the paricalcitol group was significantly associated with CAC progression (P = 0.02). An interaction term between dosing level and CKD stage was significant at the highest dosing level (P = 0.04). Limitations:Pilot single-center study. Conclusions:In patients with CKD with secondary hyperparathyroidism naive to activated vitamin D therapy, there was no difference in CAC or valvular progression in participants receiving calcitriol compared with paricalcitol during a 48-week period. Funding:Abbvie, Inc. Trial Registration:NCT00752102.

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Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1.

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