A Cross-Sectional Exploratory Study of Cardiovascular Risk Biomarkers in Non-Obese Women with and without Polycystic Ovary Syndrome: Association with Vitamin D.

Vitamin D is proposed to have a protective effect against cardiovascular disease, though the mechanism is unclear. Vitamin D deficiency is common in polycystic ovary syndrome (PCOS), where it is strongly related to obesity, insulin resistance (IR) and risk of cardiovascular disease. To determine if the inherent pathophysiology of PCOS or vitamin D levels are linked to dysregulation of cardiovascular risk proteins (CVRPs), a study in non-obese women with PCOS and without IR was undertaken. Our hypothesis was that the levels of vitamin D3 and its active metabolite would be associated with CVRPs comparably in women with and without PCOS. In women with PCOS (n = 29) and controls (n = 29), 54 CVRPs were determined by Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement and correlated to 25-hydroxyvitamin D3 (25(OH)D3) and the active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) measured by gold standard isotope-dilution liquid chromatography tandem mass spectrometry. Women with PCOS had comparable IR and systemic inflammation (normal C-reactive protein) to control women, though had higher free androgen index and anti-Mullerian hormone levels. 25(OH)D3 and 1,25(OH)2D3 levels did not differ between groups. Nine CVRPs were higher in PCOS (p < 0.05) (Galectin-9, Brother of CDO, C-motif chemokine 3, Interleukin-18 receptor-1, Thrombopoietin, Interleukin-1 receptor antagonist protein, Programmed cell death 1 ligand-2, Low-affinity immunoglobulin gamma Fc-region receptor II-b and human growth hormone), whilst 45 CVRPs did not differ. 25(OH)D3 correlated with five CVRPs in PCOS and one in controls (p < 0.05). Despite the women with PCOS not exhibiting overt systemic inflammation, 9 of 54 CVRPs were elevated, all relating to inflammation, and 5 of these correlated with 25(OH)D3, suggesting an ongoing underlying inflammatory process in PCOS even in the absence of obesity/IR.

1,25-Dihydroxyvitamin D3 Suppresses UV-Induced Poly(ADP-Ribose) Levels in Primary Human Keratinocytes, as Detected by a Novel Whole-Cell ELISA.

Photoprotective properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to reduce UV-induced DNA damage have been established in several studies. UV-induced DNA damage in skin such as single or double strand breaks is known to initiate several cellular mechanisms including activation of poly(ADP-ribose) (pADPr) polymerase-1 (PARP-1). DNA damage from UV also increases extracellular signal-related kinase (ERK) phosphorylation, which further increases PARP activity. PARP-1 functions by using cellular nicotinamide adenine dinucleotide (NAD+) to synthesise pADPr moieties and attach these to target proteins involved in DNA repair. Excessive PARP-1 activation following cellular stress such as UV irradiation may result in excessive levels of cellular pADPr. This can also have deleterious effects on cellular energy levels due to depletion of NAD+ to suboptimal levels. Since our previous work indicated that 1,25(OH)2D3 reduced UV-induced DNA damage in part through increased repair via increased energy availability, the current study investigated the effect of 1,25(OH)2D3 on UV-induced PARP-1 activity using a novel whole-cell enzyme- linked immunosorbent assay (ELISA) which quantified levels of the enzymatic product of PARP-1, pADPr. This whole cell assay used around 5000 cells per replicate measurement, which represents a 200-400-fold decrease in cell requirement compared to current commercial assays that measure in vitro pADPr levels. Using our assay, we observed that UV exposure significantly increased pADPr levels in human keratinocytes, while 1,25(OH)2D3 significantly reduced levels of UV-induced pADPr in primary human keratinocytes to a similar extent as a known PARP-1 inhibitor, 3-aminobenzamide (3AB). Further, both 1,25(OH)2D3 and 3AB as well as a peptide inhibitor of ERK-phosphorylation significantly reduced DNA damage in UV-exposed keratinocytes. The current findings support the proposal that reduction in pADPr levels may be critical for the function of 1,25(OH)2D3 in skin to reduce UV-induced DNA damage.

Calcifediol: a review of its pharmacological characteristics and clinical use in correcting vitamin D deficiency.

BACKGROUND: In addition to the role of vitamin D in bone mineralization, calcium and phosphate homeostasis, and skeletal health, evidence suggests an association between vitamin D deficiency and a wide range of chronic conditions. This is of clinical concern given the substantial global prevalence of vitamin D deficiency. Vitamin D deficiency has traditionally been treated with vitamin D3 (cholecalciferol) or vitamin D2 (ergocalciferol). Calcifediol (25-hydroxyvitamin D3) has recently become available more widely. METHODS: By means of targeted literature searches of PubMed, this narrative review overviews the physiological functions and metabolic pathways of vitamin D, examines the differences between calcifediol and vitamin D3, and highlights clinical trials conducted with calcifediol in patients with bone disease or other conditions. RESULTS: For supplemental use in the healthy population, calcifediol can be used at doses of up to 10 µg per day for children ≥ 11 years and adults and up to 5 µg/day in children 3-10 years. For therapeutic use of calcifediol under medical supervision, the dose, frequency and duration of treatment is determined according to serum 25(OH)D concentrations, condition, type of patient and comorbidities. Calcifediol differs pharmacokinetically from vitamin D3 in several ways. It is independent of hepatic 25-hydroxylation and thus is one step closer in the metabolic pathway to active vitamin D. At comparable doses to vitamin D3, calcifediol achieves target serum 25(OH)D concentrations more rapidly and in contrast to vitamin D3, it has a predictable and linear dose-response curve irrespective of baseline serum 25(OH)D concentrations. The intestinal absorption of calcifediol is relatively preserved in patients with fat malabsorption and it is more hydrophilic than vitamin D3 and thus is less prone to sequestration in adipose tissue. CONCLUSION: Calcifediol is suitable for use in all patients with vitamin D deficiency and may be preferable to vitamin D3 for patients with obesity, liver disease, malabsorption and those who require a rapid increase in 25(OH)D concentrations.

Vitamin D and bone metabolism in Graves' disease: a prospective study.

PURPOSE: Vitamin D and osteoporosis in Graves' disease (GD) have been examined in cross-sectional studies with divergent results. Here, we prospectively studied vitamin D metabolism and bone health in patients with newly diagnosed GD. METHODS: Thirty consecutive patients with de novo overt thyrotoxicosis diagnosed with GD were included. At diagnosis, none of the patients were treated with vitamin D or anti-osteoporotic drugs. All patients were initially treated with antithyroid drugs. Blood samplings were taken at baseline and at 6 weeks, 3, 6, 12 and 24 months after treatment start. Serum levels of 25OHD3, 1,25OH2D3, calcium, parathyroid hormone (PTH), and C-terminal telopeptides of Type I collagen (CTX-I) were analysed. Bone mineral density (BMD) was measured at baseline, and 1 and 2 years after treatment initiation. RESULTS: At diagnosis, patients with GD did not have vitamin D deficiency. There were no significant correlations between levels of 25OHD3 and thyrotoxicosis. Upon treatment of the thyrotoxicosis, serum calcium fell transiently, and PTH and 1,25OH2D3 increased. 25OHD3 fell within the normal range and stabilised at 6 months. CTX-I fell over 12 months, BMD increased significantly up to 2 years, p = 0.002, < 0.001 and 0.005 in the spine, left total hip and left femoral neck, respectively. CONCLUSIONS: The present data underline that thyrotoxicosis has a negative impact on bone health and demonstrate fine-tuned dynamics in bone and vitamin D metabolism. Upon treatment, bone health improved over a follow-up period of 24 months despite rising PTH. Increased conversion of 25OHD3 to 1,25OH2D3 occurs during treatment of GD.

Brk/PTK6 and Involucrin Expression May Predict Breast Cancer Cell Responses to Vitamin D3.

The process of human embryonic mammary development gives rise to the structures in which mammary cells share a developmental lineage with skin epithelial cells such as keratinocytes. As some breast carcinomas have previously been shown to express high levels of involucrin, a marker of keratinocyte differentiation, we hypothesised that some breast tumours may de-differentiate to a keratinocyte-derived 'evolutionary history'. To confirm our hypothesis, we investigated the frequency of involucrin expression along with that of Brk, a tyrosine kinase expressed in up to 86% of breast carcinomas whose normal expression patterns are restricted to differentiating epithelial cells, most notably those in the skin (keratinocytes) and the gastrointestinal tract. We found that involucrin, a keratinocyte differentiation marker, was expressed in a high proportion (78%) of breast carcinoma samples and cell lines. Interestingly, tumour samples found to express high levels of involucrin were also shown to express Brk. 1,25-dihydroxyvitamin D3, a known differentiation agent and potential anti-cancer agent, decreased proliferation in the breast cancer cell lines that expressed both involucrin and Brk, whereas the Brk/involucrin negative cell lines tested were less susceptible. In addition, responses to 1,25-dihydroxyvitamin D3 were not correlated with vitamin D receptor expression. These data contribute to the growing body of evidence suggesting that cellular responses to 1,25-dihydroxyvitamin D3 are potentially independent of vitamin D receptor status and provide an insight into potential markers, such as Brk and/or involucrin that could predict therapeutic responses to 1,25-dihydroxyvitamin D3.

Vitamin D3 and Salinomycin synergy in MCF-7 cells cause cell death via endoplasmic reticulum stress in monolayer and 3D cell culture.

1α, 25, dihydroxyvitamin D3 (1,25D), the active form of vitamin D3, has antitumor properties in several cancer cell lines in vitro. Salinomycin (Sal) has anticancer activity against cancer cell lines. This study aims to examine the cytotoxic and antiproliferative effect of Sal associated with 1,25D on MCF-7 breast carcinoma cell line cultured in monolayer (2D) and three-dimensional models (mammospheres). We also aim to evaluate the molecular mechanism of Sal and 1,25D-mediated effects. We report that Sal and 1,25D act synergistically in MCF-7 mammospheres and monolayer causing G1 cell cycle arrest, reduction of mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) overproduction with a long-lasting cytotoxic response represented by clonogenic and mammosphere assay. We observed the induction of cell death by apoptosis with upregulation in mRNA levels of apoptosis-related genes (CASP7, CASP9, and BBC3). Extensive cytoplasmic vacuolization, a morphological characteristic found in paraptosis, was also seen and could be triggered by endoplasmic reticulum stress (ER) as we found transcriptional upregulation of genes related to ER stress (ATF6, GADD153, GADD45G, EIF2AK3, and HSPA5). Overall, Sal and 1,25D act synergistically, inhibiting cell proliferation by activating simultaneously multiple death pathways and may be a novel and promising luminal A breast cancer therapy strategy.

1,25-dihydroxyvitamin D3 ameliorates lupus nephritis through inhibiting the NF-κB and MAPK signalling pathways in MRL/lpr mice.

BACKGROUND: Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus (SLE). However, the aetiology and pathogenesis of LN remain unknown. 1,25-dihydroxyvitamin D3 [1,25-(OH)2-VitD3] is the active form of vitamin D, and it has been shown to perform important functions in inflammatory and immune-related diseases. In this study, we investigated the time-dependent effects of 1,25-dihydroxyvitamin D3 and explored the underlying mechanism in MRL/lpr mice, a well-studied animal model of LN. METHODS: Beginning at 8 weeks of age, 24-h urine samples were collected weekly to measure the levels of protein in the urine. We treated female MRL/lpr mice with 1,25-dihydroxyvitamin D3 (4 µg/kg) or 1% DMSO by intraperitoneal injection twice weekly for 3 weeks beginning at the age of 11 weeks. The mice were separately sacrificed, and serum and kidney samples were collected at the ages of 14, 16, 18, and 20 weeks to measure creatinine (Cr) levels, blood urea nitrogen (BUN) levels, histological damage, immunological marker (A-ds DNA, C1q, C3, IgG, IgM) levels, and inflammatory factor (TNF-α, IL-17, MCP-1) levels. Furthermore, the nuclear factor kappa B (NF-κB) and the mitogen-activated protein kinase (MAPK) signalling pathways were also assessed to elucidate the underlying mechanism. RESULTS: We found that MRL/lpr mice treated with 1,25-dihydroxyvitamin D3 displayed significantly attenuated LN. VitD3-treated mice exhibited significantly improved renal pathological damage and reduced proteinuria, BUN, SCr, A-ds DNA antibody and immune complex deposition levels (P < 0.05) compared with untreated MRL/lpr mice. Moreover, 1,25-dihydroxyvitamin D3 inhibited the complement cascade, inhibited the release of proinflammatory cytokines, such as TNF-α, IL-17, and MCP-1, and inhibited NF-κB and MAPK activation (P < 0.05). CONCLUSION: 1,25-dihydroxyvitamin D3 exerts a protective effect against LN by inhibiting the NF-κB and MAPK signalling pathways, providing a potential treatment strategy for LN. Interestingly, the NF-κB and MAPK signalling pathways are time-dependent mediators of LN and may be associated with lupus activity.

Protective Effects of 1,25 Dihydroxyvitamin D3 against High-Glucose-Induced Damage in Human Umbilical Vein Endothelial Cells Involve Activation of Nrf2 Antioxidant Signaling.

AIM: To explore the protective effects and related mech-anisms of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) on en-dothelial dysfunction under hyperglycemic conditions. METHODS: Cultured human umbilical vein endothelial cells (HUVECs) were treated with normal glucose (glucose concentration of 5.5 mmol/L), high glucose (glucose concentration of 33 mmol/L), and high glucose plus 1,25(OH)2D3, respectively. Cell viability and apoptosis, intracellular reactive oxygen species (ROS) and nitric oxide (NO) contents, antioxidant enzyme activities, proinflammatory cytokine mRNA levels, and expression levels of proteins involved were measured. RESULTS: High glucose decreased HUVEC viability, promoted ROS production and apoptosis, and reduced NO generation, which was associated with decreased activities of antioxidant enzymes and increased levels of proinflam-matory cytokines. 1,25(OH)2D3 treatment enhanced HUVEC viability, attenuated ROS generation and apoptosis, and -increased NO production, which was accompanied by -enhanced antioxidant enzyme activities and reduced -proinflammatory factors. Mechanically, 1,25(OH)2D3 promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in a vitamin D receptor (VDR)-dependent manner, and Nrf2 siRNA abolished the antioxidative and -anti-inflammatory effects of 1,25(OH)2D3. CONCLUSIONS: 1,25(OH)2D3 attenuates high-glucose-induced endothelial oxidative injury through upregulation of the Nrf2 antioxidant pathway in a VDR-dependent manner.

Impacts of deficiency in vitamin D derivatives on disease severity in adult bronchial asthma patients.

Vitamin D affects innate and adaptive immunity processes that impact treatment, severity, and morbidity of acute asthma episodes. Several vitamin D forms may help modulate immunity, including vitamin D2 (D2), vitamin D3 (D3), 25-hydroxyvitamin D3 (25(OH)D3), and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study assessed serum levels of vitamin D derivatives in bronchial asthma patients and their correlation with disease markers. One hundred thirteen subjects, divided into two groups, were enrolled. The first group included 73 asthmatic patients (57 males and 16 females), and the second included 40 healthy adults (31 males and 9 females) as a control group. All subjects were evaluated with a careful history and clinical examination, a chest X-ray with a posteroanterior view, routine laboratory examination, spirometry, and asthma control tests (ACT). Vitamin D serum levels were assessed using ultra-performance liquid chromatography (UPLC) with tandem mass spectrometry. Disease markers were assessed and correlated with serum levels of vitamin D forms. Markers included forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), FEV1/FVC%, peak expiratory flow (PEF), forced expiratory flow25-75% (FEF25-75%), eosinophilic blood count, and total immunoglobulin E (IgE). Asthmatic patients had significantly lower serum levels of vitamin D than healthy controls (p ≤ 0.001). Further, serum vitamin D levels decreased significantly in uncontrolled asthmatic patients than partially controlled and controlled patients. Correlations for 25(OH)D3 and 1,25-(OH) 2D3 were stronger than for D2 and D3. There were negative correlations for eosinophilic blood count, total IgE, and ACT. Serum levels of all vitamin D forms were reduced in asthmatic patients with moderate to strong correlations with disease severity. Vitamin D deficiency or even insufficiency may thus play a role in disease severity.

1,25-dihydroxyvitamin D3 regulates t helper and b lymphocyte responses substantially in drug-naive primary Sjögren's syndrome patients' mononuclear cells

Background/aim: A correlation between vitamin D deficiency and primary Sjögren's syndrome (pSS) has already been described. The limited data has been reported regarding the pathological relevance of vitamin D in primary Sjögren's syndrome. In this study, the peripheral blood mononuclear cells were cocultured with 1,25-dihydroxyvitamin D3 to determine the modulatory effect of vitamin D3 on T and B lymphocyte phenotypes in pSS. Materials and methods: Venous blood samples were collected from 11 patients in the treatment phase and 9 drug-naive pSS patients. Peripheral blood mononuclear cells (PBMC) were isolated and separately cultured in the presence and absence of 1,25-dihydroxyvitamin D3 (10 mM) for 5 days of culture period. Lymphocyte proliferation was analyzed for CFSE signaling via flow cytometry. CD3+CD4+ cells were analyzed for intracellular IFN- and IL-17 expressions. CD19+IgD cells were analyzed for CD38 and CD27 expressions to evaluate naive and total memory B cell subsets. Culture supernatants were analyzed for the IFN-, IL-17, and IL-10 cytokine secretions via flow cytometry. Results: 1,25-dihydroxyvitamin D3 significantly decreased Th lymphocyte proliferative responses in drug-naive (p < 0.005) and treated pSS patients (p < 0.05), and B lymphocyte proliferation in drug-naive pSS PBMC cultures (p < 0.01) compared to mononuclear cell cultures alone. 1,25-dihydroxyvitamin D3 significantly decreased IFN- and IL-17 secreting Th cells in both drug naive (p < 0.005 and p < 0.01, respectively) and treated subjects (p < 0.05 and p < 0.05, respectively) by increasing FoxP3 expressing CD4+CD25+ Treg cell frequency. Plasma B lymphocytes significantly reduced in the presence of 1,25-dihydroxyvitamin D3 in drug naive pSS (p < 0.001) and treated patients (p < 0.05) mononuclear cell cultures compared to PBMC cultures alone. Total memory B cell subsets significantly increased with 1,25-dihydroxyvitamin D3 in drug naive pSS when compared with PBMC cultures alone (p < 0.005). IFN- and IL-17 cytokine levels in culture supernatants significantly reduced (p < 0.05 and p < 0.01, respectively) in drug naive pSS patients' PBMC cultures with 1,25-dihydroxyvitamin D3, and IL-10 levels significantly enhanced in both drug-naive (p < 0.01) and treated pSS patients' PBMC cultures (p < 0.01) in the presence of 1,25-dihydroxyvitamin D3. Conclusion: In conclusion, 1,25-dihydroxyvitamin D3 regulated immune responses in both treated and drug-naive pSS patients, but have a more pronounced modulatory effect on mononuclear cell responses in drug-naive pSS patients.

Targeted genomic deletions identify diverse enhancer functions and generate a kidney-specific, endocrine-deficient Cyp27b1 pseudo-null mouse.

Vitamin D3 is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) via cytochrome P450 family 27 subfamily B member 1 (CYP27B1), whose gene is regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase-like 1 (Mettl1) and Mettl21b that mediate basal and PTH-induced expression of Cyp27b1 and FGF23- and 1,25(OH)2D3-mediated repression. Gross deletion of these segments in mice has severe effects on Cyp27b1 regulation and skeletal phenotype but does not affect Cyp27b1 expression in nonrenal target cells (NRTCs). Here, we report a bimodal activity in the Mettl1 intronic enhancer with components responsible for PTH-mediated Cyp27b1 induction and 1,25(OH)2D3-mediated repression and additional activities, including FGF23 repression, within the Mettl21b enhancers. Deletion of both submodules eliminated basal Cyp27b1 expression and regulation in the kidney, leading to systemic and skeletal phenotypes similar to those of Cyp27b1-null mice. However, basal expression and lipopolysaccharide-induced regulation of Cyp27b1 in NRTCs was unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescued the skeletal phenotype of this mutant mouse, creating an ideal in vivo model to study nonrenal 1,25(OH)2D3 production in health and disease. Finally, we confirmed a conserved chromatin landscape in human kidney that is similar to that in mouse. These findings define a finely balanced homeostatic mechanism involving PTH and FGF23 together with protection from 1,25(OH)2D3 toxicity that is responsible for both adaptive vitamin D metabolism and mineral regulation.

1α,25-Dihydroxyvitamin D3 prevents renal oxidative damage via the PARP1/SIRT1/NOX4 pathway in Zucker diabetic fatty rats.

Diabetic nephropathy (DN) is one of the most important renal complications associated with diabetes, and the mechanisms are yet to be fully understood. To date, few studies have shown the antioxidant effects of 1α,25-dihydroxyvitamin-D3 [1,25(OH)2D3] on hyperglycemia-induced renal injury. The aim of the present study was to explore the potential mechanism by which 1,25(OH)2D3 reduced oxidative stress in diabetic rat kidneys. In this study, we established a vitamin D-deficient spontaneous diabetes model: 5-6 wk of age Zucker diabetic fatty (ZDF) rats were treated with or without 1,25(OH)2D3 for 7 wk, age-matched Zucker lean rats served as control. Results showed that ZDF rats treated with 1,25(OH)2D3 had decreased body mass, food intake, water intake, and urine volume. 1,25(OH)2D3 ameliorated urine glucose, blood glucose and abnormal glucose tolerance. Additionally, 1,25(OH)2D3 significantly lowered microalbuminuria, decreased the glomerular basement membrane thickness, and in some degree inhibited glomerular hypertrophy, mesangial expansion, and tubular dilatation. Furthermore, 1,25(OH)2D3 attenuated renal oxidative damage, as reflected by the levels of malondialdehyde, reduced glutathione, 4-hydroxynonenal, 8-hydroxy-2'-deoxyguanosine, and reactive oxygen species production, and notably inhibited poly(ADP-ribose) polymerase-1 (PARP1), activated sirtuin 1 (SIRT1), and decreased the expression of NADPH oxidase 4 (NOX4). Of interest, the abovementioned proteins could be involved in the antioxidant mechanism of 1,25(OH)2D3 in diabetic rat kidneys. Our study showed that oxidative stress might be a major contributor to DN pathogenesis and uncovered the antioxidant role of 1,25(OH)2D3 in diabetic nephropathy that was associated with the PARP1/SIRT1/ NOX4 pathway.

Vitamin D and Bone.

Vitamin D is a principal factor required for mineral and skeletal homeostasis. Vitamin D deficiency during development causes rickets and in adults can result in osteomalacia and increased risk of fracture. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, is responsible for the biological actions of vitamin D which are mediated by the vitamin D receptor (VDR). Mutations in the VDR result in early-onset rickets and low calcium and phosphate, indicating the essential role of 1,25(OH)2D3/VDR signaling in the regulation of mineral homeostasis and skeletal health. This chapter summarizes our current understanding of the production of the vitamin D endocrine hormone, 1,25(OH)2D3, and the actions of 1,25(OH)2D3 which result in the maintenance of skeletal homeostasis. The primary role of 1,25(OH)2D3 is to increase calcium absorption from the intestine and thus to increase the availability of calcium for bone mineralization. Specific actions of 1,25(OH)2D3 on the intestine, kidney, and bone needed to maintain calcium homeostasis are summarized, and the impact of vitamin D status on bone health is discussed.

Association of vitamin D genetic pathway with asthma susceptibility in the Kurdish population.

BACKGROUND: Vitamin D (Vit D) function in asthma progression has been studied well. The effects of genetic variations in Vit D pathway molecules have been also studied, although the results are contradicted. In the present study, for the first time we examined the Vit D pathway molecules included serum Vit D and vitamin D-binding protein (VDBP) and also genetic variations in the vitamin D receptor (VDR) and VDBP in a Kurdish population with asthma. METHODS: An enzyme-linked immunosorbent assay (ELISA) method was used to measure the serum Vit D and VDBP. VDR rs1544410 and rs2228570 and VDBP rs7041 were assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The serum level of Vit D significantly decreased in asthmatic patients versus controls (16.26 ± 6.76 vs 23.05 ± 10.57 ng/mL, P value = .001). We observed an indirect correlation between Vit D and clinical findings. We also found an increased level of serum VDBP in patients as compared to the controls (1044.6 ± 310.82 vs 545.95 ± 121.73 µg/mL, P value < .0001). Besides, the risk of asthma progression was increased in patients with the VDR rs2228570 CC and VDBP rs7041 GG genotypes (OR = 3.56, P = .0382 and OR = 2.58, P = .01, respectively). CONCLUSION: In summary, our results explain the influence of the genetic variations in VDR and VDBP in addition to Vit D and VDBP serum concentrations on asthma susceptibility in the Kurdish population.

Histone demethylase KDM6B regulates 1,25-dihydroxyvitamin D3-induced senescence in glioma cells.

Vitamin D is a fat-soluble vitamin and plays an important role in calcium absorption and bone development, whose lack can cause a variety of diseases, including cancer. Human epidemiological studies suggested that vitamin D3 deficiency might increase glioma incidence, but molecular mechanism is less understood. In this study, we show that 1,25-dihydroxyvitamin D3 (the active form of vitamin D3) induces senescence of glioma cells and increases the expression of senescence markers, INK4A and cyclin-dependent kinase inhibitor 1A (CDKN1A). 1,25-Dihydroxyvitamin D3 also upregulates the expression of histone demethylase, KDM6B. Knockdown of KDM6B attenuates 1,25-dihydroxyvitamin D3-induced senescence and upregulation of INK4A and CDKN1A. KDM6B promotes the transcription of INK4A by eliminating the trimethylation of repressive marker H3K27me3 near its promoter. This study reveals a new regulatory mechanism involved in vitamin D3 inhibition on gliomas, which is beneficial to prevention and adjuvant therapy of glioma.

Mechanisms and regulation of epithelial phosphate transport in ruminants: approaches in comparative physiology.

Ruminants have a unique utilization of phosphate (Pi) based on the so-called endogenous Pi recycling to guarantee adequate Pi supply for ruminal microbial growth and for buffering short-chain fatty acids. Large amounts of Pi enter the gastrointestinal tract by salivary secretion. The high saliva Pi concentrations are generated by active secretion of Pi from blood into primary saliva via basolateral sodium (Na+)-dependent Pi transporter type II. The following subsequent intestinal absorption of Pi is mainly carried out in the jejunum by the apical located secondary active Na+-dependent Pi transporters NaPi IIb (SLC34A2) and PiT1 (SLC20A1). A reduction in dietary Pi intake stimulates the intestinal Pi absorption by increasing the expression of NaPi IIb despite unchanged plasma 1,25-dihydroxyvitamin D3 concentrations, which modulate Pi homeostasis in monogastric species. Reabsorption of glomerular filtrated plasma Pi is mainly mediated by the Pi transporters NaPi IIa (SLC34A1) and NaPi IIc (SLC34A3) in proximal tubule apical cells. The expression of NaPi IIa and the corresponding renal Na+-dependent Pi capacity were modulated by high dietary phosphorus (P) intake in a parathyroid-dependent manner. In response to reduced dietary Pi intake, the expression of NaPi IIa was not adapted indicating that renal Pi reabsorption in ruminants runs at a high level allowing no further increase when P intake is diminished. In bones and in the mammary glands, Na+-dependent Pi transporters are able to contribute to maintaining Pi homeostasis. Overall, the regulation of Pi transporter activity and expression by hormonal modulators confirms substantial differences between ruminant and non-ruminant species.

1α,25(OH)2 D3 alleviates high glucose-induced lipid accumulation in rat renal tubular epithelial cells by inhibiting SREBPs.

Lipid accumulation is a vital event in the progression of diabetic nephropathy. 1,25-Dihydroxyvitamin D3 (1α,25(OH)2 D3 ) is considered to have a protective effect on diabetic nephropathy. However, it remains unclear whether 1α,25(OH)2 D3 can inhibit lipid accumulation, and the potential mechanisms responsible for lipid metabolism are incompletely understood. In this study, we evaluated the effects of 1α,25(OH)2 D3 on lipid metabolism in high glucose-exposed rat renal tubular epithelial NRK-52E cells. Results indicated that high glucose-enhanced lipid accumulation in NRK-52E cells and 1α,25(OH)2 D3 can remarkably decrease high glucose-induced lipid accumulation. Western blot showed that 1α,25(OH)2 D3 alleviated high glucose-induced upregulation of sterol regulatory element-binding protein-1c (SREBP-1c) and SREBP2, along with their established target genes fatty acid synthase (FASN) and hydroxymethylglutaryl CoA reductases (HMGCR). Overall, these findings suggest that 1α,25(OH)2 D3 downregulated the expressions of SREBPs to inhibit high glucose-induced lipid accumulation, which provides new sights into the protective effects of 1α,25(OH)2 D3 on diabetic nephropathy.

1,25-Dihydroxyvitamin D3 induces human myeloid cell differentiation via the mTOR signaling pathway.

1,25-Dihydroxyvitamin D3 or 1,25(OH)2D3 is known to play an important role in the differentiation of human myeloid cells. However, the molecular mechanism underlying the 1,25(OH)2D3-mediated differentiation of human myeloid cells is incompletely understood. Here, we report that 1,25(OH)2D3 induces differentiation of human myeloid cell lines such as U937 and THP-1 cells via the mammalian target of rapamycin (mTOR) signaling pathway. Both the expression of the differentiation marker CD14 and activation of the mTOR signaling pathway were induced by 1,25(OH)2D3 in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 and THP-1 cells. The 1,25(OH)2D3-induced expression of CD14 in PMA-differentiated U937 and THP-1 cells was prevented by mTOR inhibitors, PP242 and Torin1. The 1,25(OH)2D3-induced morphological changes as characteristics of differentiated myeloid cells were also reversed after PP242 and Torin1 treatment. Silencing of either regulatory-associated protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) in PMA-differentiated THP-1 cells with small-interfering RNA resulted in the inhibition of CD14 expression and morphological changes induced by 1,25(OH)2D3, indicating that both mTORC1 and mTORC2 were important for the differentiation of myeloid THP-1 cells. Previous studies have shown that phosphatidic acid (PA) maintains the stability of the mTOR complex. Here we found that the attenuation of PA production with 1-butanol or a PLD inhibitor prevented the 1,25(OH)2D3-induced upregulation of CD14. Taken together, our results show that 1,25(OH)2D3 enhances the differentiation of human myeloid cells through the mTOR signaling pathway.

1.25 Dihydroxyvitamin D3 Attenuates Hypertrophy Markers in Cardiomyoblast H9c2 Cells: Evaluation of Sirtuin3 mRNA and Protein Level.

Background: The cellular and molecular mechanisms of cardioprotective effects of Vitamin D are poorly understood. Given the essential role of sirtuin-3 (SIRT3) as an endogenous negative regulator of cardiac hypertrophy, this study was designed to investigate the effect of 1, 25-dihydroxyvitamin D3 (calcitriol) on hypertrophy markers and SIRT3 mRNA and protein levels following angiotensin II induced - hypertrophy in cardiomyoblast H9c2 cells. Methods: Rat cardiomyoblast H9c2 cells were treated for 48 hr with angiotensin II alone (Ang group) or in combination with 1, 10 and 100 nM of calcitriol (C + Ang groups). Intact cells served as control (Ctl). The cell area was measured using methylene blue staining. Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and SIRT3 transcription levels were measured by real time RT-PCR. SIRT3 protein expression was evaluated using western blot technique. Results: The results showed that in Ang group cell size was increase by 128.4 ± 15% (P < 0.001 vs. Ctl) whereas in C100 + Ang group it was increased by 21.3 ± 6% (P < 0.001 vs. Ang group). Calcitriol pretreatment decreased ANP mRNA level significantly (P < 0.05) in comparison with Ang group (Ang: 75.5 ± 15%, C100 + Ang: 19.2 ± 9%). There were no significant differences between Ang group and cells pretreated with 1 and 10 nM of calcitriol. SIRT3 at mRNA and protein levels did not change significantly among the experimental groups. Conclusions: In conclusion, pretreatment with calcitriol (100 nM) prevents Ang II-induced hypertrophy in cardiomyoblast H9c2 cells. This probably occurs through other pathways except SIRT3 upregulation.

Investigating the Role of VDR and Megalin in Semi-Selectivity of Side-Chain Modified 19-nor Analogs of Vitamin D.

1,25-dihydroxyvitamin D3 (1,25D3) is implicated in many cellular functions, including cell proliferation and differentiation, thus exerting potential antitumor effects. A major limitation for therapeutic use of 1,25D3 are potent calcemic activities. Therefore, synthetic analogs of 1,25D3 for use in anticancer therapy should retain cell differentiating potential, with calcemic activity being reduced. To obtain this goal, the analogs should effectively activate transcription of genes responsible for cell differentiation, leaving the genes responsible for calcium homeostasis less active. In order to better understand this phenomenon, we selected a series of structurally related 19-nor analogs of 1,25D (PRI-5100, PRI-5101, PRI-5105, and PRI-5106) and tested their activities in blood cells and in cells connected to calcium homeostasis. Affinities of analogs to recombinant vitamin D receptor (VDR) protein were not correlated to their pro-differentiating activities. Moreover, the pattern of transcriptional activities of the analogs was different in cell lines originating from various vitamin D-responsive tissues. We thus hypothesized that receptors which participate in transport of the analogs to the cells might contribute to the observed differences. In order to study this hypothesis, we produced renal cells with knock-out of the megalin gene. Our results indicate that megalin has a minor effect on semi-selective activities of vitamin D analogs.