1,25(OH)2-vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte

The steroid hormone 1,25(OH)₂-vitamin D₃ [1,25D] has been shown to affect the growth and proliferation of primary cultures of ventricular myocytes isolated from neonatal rat hearts. The research presented here shows that the vitamin D receptor [VDR] is present in murine cardiac myocytes (HL-1 cells), and that 1,25D affects the growth, proliferation and morphology of these cells. In addition we show that 1,25D effects expression of ANP, myotrophin, and c-myc. Furthermore, 1,25D effects expression and localization of the VDR within the cell. Murine HL-1 cardiac myocytes were grown and treated with 1,25D in culture, and growth and morphology were assessed with microscopic analysis. Cells were counted and protein levels were evaluated through Western blot analysis. Subcellular localization of the VDR was determined using immunofluorescence and confocal microscopy. 1,25D was found to decrease proliferation and alter cellular morphology of the HL-1 cells. Treatment with 1,25D increased expression of myotrophin while decreasing expression of atrial natriuretic peptide [ANP] and c-myc. 1,25D treatment also increased expression and nuclear localization of the VDR in these cardiac myocytes. Thus 1,25D is an important hormone involved in modulating and maintaining heart cell structure and function.     

Renal 25-hydroxyvitamin D-3 1α-hydroxylase in guinea-pig: Activity variations during development and pregnancy

The renal 25-hydroxyvitamin D-3-1α-hydroxylase (1α-hydroxylase) activity and circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)₂D) were measured in pregnant guinea-pigs and their offspring. Serum levels of 1,25(OH)₂D were significantly elevated in pregnant guinea-pigs but the renal enzyme activity was not different from non-pregnant animals. The fetal renal 1α-hydroxylase activity was about 6-fold higher than the maternal level, whereas circulating 1,25(OH)₂D was low. Treatment with pharmacological doses of 1,25(OH)₂D3 increased circulating 1,25(OH)₂D and depressed the renal 1α-hydroxylases both in the mother and the fetus. In newborn guinea-pigs the enzyme activity was up to 10-times that seen in adults. It declined over the first 3 weeks, showing no difference between the sexes. In sexually mature animals the males had a significantly higher 1α-hydroxylase activity than the female. However, this higher enzyme activity was not correlated to serum testosterone. Around the time the animals reached sexual maturity serum 1,25(OH)₂D increased in both sexes. In the males this rise was correlated to an increase in serum testosterone. It is concluded that the maternal renal 1α-hydroxylase activity is unchanged in late pregnancy, compared to non-pregnant females. The data indicate that the fetus produces 1,25(OH)₂D, and may contribute to the maternal circulating 1,25(OH)₂D. The sex difference in 1α-hydroxylase activity previously demonstrated is manifest at about the time of puberty.     

Vitamin D substrate–product relationship in idiopathic hypercalciuria

Absorptive hypercalciuria (AH) is associated with elevated levels of 1,25-dihydroxyvitamin D (1,25(OH)₂D). While no increase of 1,25(OH)₂D after oral administration of 25-hydroxyvitamin D (25OHD) at high doses has been claimed in normal subjects, a substrate–product relationship has been reported in normal children, young people after UV irradiation, older persons, postmenopausal women, primary hyperparathyroidism, renal failure, osteomalacia, and sarcoidosis. No data of this relationship in AH is available. To investigate 25OHD-1,25(OH)₂D substrate–product relationship in AH, 161 AH patients (mean age 60.9 ± 11.7 years) and 110 age- and sex-matched controls (mean age 61.5 ± 12.4 years) were studied. In 57 controls and 52 AH subjects 25OHD-1,25(OH)₂D relationship in basal conditions and after 2-week oral 25OHD (25 μg/day) administration were evaluated. In basal conditions 25OHD and 1,25(OH)₂D were correlated in both, controls and AH; 25OHD treatment was followed by an increase in serum 25OHD and 1,25(OH)₂D in both groups. However, delta responses of 25OHD and 1,25(OH)₂D to 25OHD were higher in AH suggesting an enhanced activity of 1α-hydroxylase. In conclusion, the higher response of 1,25(OH)₂D after oral 25OHD in AH patients suggests a differential capacity between both groups in handling the increases in 1,25(OH)₂D.     

Serum Concentrations of Vitamin D Metabolites in Cayo Santiago Rhesus Macaques

Serum 25-hydroxyvitamin D (25[OH]D), an index of vitamin D nutrition, and the calcium-regulating hormone 1,25-dihydroxyvitamin D (1,25[OH]₂D) were measured in rhesus macaques of various ages. Both metabolites were much higher in monkeys than in man. For 25(OH)D there was no sex difference, and this metabolite increased with age (P > 0.05). The 1,25(OH)₂D fell with age for males (P < 0.02), but not for females. Pregnant or lactating females had significantly elevated 1,25(OH)₂D levels (P < 0.025).     

Mineral regulation of vitamin D metabolism

The pediatrician's interest in vitamin D metabolism stems from the once-endemic rachitic deformities induced by vitamin D deficiency; later, clinical research of inherited forms of rickets established further principles of vitamin D metabolism and action. Constantine Anast, as both clinician and researcher, maintained an enthusiastic interest in vitamin D metabolism. His investigative esprit fostered my interest, as a fellow in his laboratory, in the synthetic pathway of active vitamin D.The best known active metabolite of vitamin D, 1,25(OH)₂D, is formed by 1βhydroxylation of 25(OH)D, the most abundant circulating form of the vitamin. This well-characterized biochemical conversion is the rate-limiting reaction in the synthesis of 1,25(OH)₂D [1]. The classical homeostatic role of 1,25(OH)₂D is predominantly that of a calcemie agent, an action largely resulting from the metabolite's stimulation of intestinal transport of calcium [2]. Intestinal phosphorus transport, to a lesser extent than calcium transport can be stimulated by 1,25(OH)₂D [3]. Furthermore, skeletal [4] and perhaps renal activity [5] of 1,25(OH)₂D can increase circulating concentrations of calcium. These in vivo effects of 1,25(OH)₂D on mineral homeostasis raise the question of whether feedback control, via mineral regulation of 1,25(OH)₂D production, exists, and the significant mechanisms involved. Here, I will briefly review evidence from earlier studies supporting the notion of calcium and phosphorus regulation of 1α-hydroxylase activity, and present data generated in collaboration with Dr Anast examining vitamin D metabolism in magnesium deficiency.     

Activation of rapid signaling pathways does not contribute to 1α,25‐dihydroxyvitamin D3‐induced growth inhibition of mouse prostate epithelial progenitor cells

The active form of vitamin D, 1α,25‐dihydroxyvitamin D₃ (1,25(OH)₂D) inhibits the growth of prostate epithelial cells, however the underlying mechanisms have not been clearly delineated. In the current study, the impact of 1,25(OH)₂D on the rapid activation of extracellular‐regulated kinase (ERK) 1/2 and protein kinase C α (PKCα), and the role of these pathways in growth inhibition was examined in immortalized mouse prostate epithelial cells, MPEC3, that exhibit stem/progenitor cell characteristics. 1,25(OH)₂D treatment suppressed the growth of MPEC3 in a dose and time dependent manner (e.g., 21% reduction at three days with 100 nM 1,25(OH)₂D treatment). However, ERK1/2 activity was not altered by 100 nM 1,25(OH)₂D treatment for time points from 1 min to 1 h in either serum‐containing or serum‐free medium. Similarly, PKCα activation (translocation onto the plasma membrane) was not regulated by short‐term treatment of 100 nM 1,25(OH)₂D. In conclusion, 1,25(OH)₂D did not mediate rapid activation of ERK1/2 or PKCα in MPEC3 and therefore the growth inhibitory effect of 1,25(OH)₂D is independent of rapid activation of these signaling pathways in this cell type. J. Cell. Biochem. 107: 1031–1036, 2009. © 2009 Wiley‐Liss, Inc. This article was published online 2 June 2009. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 15 June 2009.     

Altered Calcium and Vitamin D Homeostasis in First-Time Calcium Kidney Stone-Formers

Background

Elevated serum 1,25-dihydroxyvitamin D (1,25(OH)₂D) concentrations have been reported among cohorts of recurrent calcium (Ca) kidney stone-formers and implicated in the pathogenesis of hypercalciuria. Variations in Ca and vitamin D metabolism, and excretion of urinary solutes among first-time male and female Ca stone-formers in the community, however, have not been defined.

Methods

In a 4-year community-based study we measured serum Ca, phosphorus (P), 25-hydroxyvitamin D (25(OH)D), 1,25(OH)₂D, 24,25-dihydroxyvitamin D (24,25(OH)₂D), parathyroid hormone (PTH), and fibroblast growth factor-23 (FGF-23) concentrations in first-time Ca stone-formers and age- and gender frequency-matched controls.

Results

Serum Ca and 1,25(OH)₂D were increased in Ca stone-formers compared to controls (P = 0.01 and P = 0.001). Stone-formers had a lower serum 24,25(OH)₂D/25(OH)D ratio compared to controls (P = 0.008). Serum PTH and FGF-23 concentrations were similar in the groups. Urine Ca excretion was similar in the two groups (P = 0.82). In controls, positive associations between serum 25(OH)D and 24,25(OH)₂D, FGF-23 and fractional phosphate excretion, and negative associations between serum Ca and PTH, and FGF-23 and 1,25(OH)₂D were observed. In SF associations between FGF-23 and fractional phosphate excretion, and FGF-23 and 1,25(OH)₂D, were not observed. 1,25(OH)₂D concentrations associated more weakly with FGF-23 in SF compared with C (P <0.05).

Conclusions

Quantitative differences in serum Ca and 1,25(OH)₂D and reductions in 24-hydroxylation of vitamin D metabolites are present in first-time SF and might contribute to first-time stone risk.     

Vitamin D Status among Thai School Children and the Association with 1,25-Dihydroxyvitamin D and Parathyroid Hormone Levels

In several low latitude countries, vitamin D deficiency is emerging as a public health issue. Adequate vitamin D is essential for bone health in rapidly growing children. In the Thai population, little is known about serum 25-hydroxyvitamin D [25(OH)D] status of infants and children. Moreover, the association between 25(OH)D and the biological active form of 1,25-dihydroxyvitamin D [1,25(OH)]₂D is not clear. The specific aims of this study were to characterize circulating serum 25(OH)D, 1,25(OH)₂D and their determinants including parathyroid hormone (PTH), age, sex, height and body mass index (BMI) in 529 school-aged Thai children aged 6–14 y. Adjusted linear regression analysis was performed to examine the impact of age and BMI, and its interaction with sex, on serum 25(OH)D concentrations and 1,25(OH)₂D concentrations. Serum 25(OH)D, 1,25(OH)₂D and PTH concentrations (geometric mean ± geometric SD) were 72.7±1.2 nmol/L, 199.1±1.3 pmol/L and 35.0±1.5 ng/L, respectively. Only 4% (21 of 529) participants had a serum 25(OH)D level below 50 nmol/L. There was statistically significant evidence for an interaction between sex and age with regard to 25(OH)D concentrations. Specifically, 25(OH)D concentrations were 19% higher in males. Moreover, females experienced a statistically significant 4% decline in serum 25(OH)D levels for each increasing year of age (P = 0.001); no decline was seen in male participants with increasing age (P = 0.93). When BMI, age, sex, height and serum 25(OH)D were individually regressed on 1,25(OH)₂D, height and sex were associated with 1,25(OH)₂D with females exhibiting statistically significantly higher serum 1,25(OH)₂D levels compared with males (P<0.001). Serum 1,25(OH)₂D among our sample of children exhibiting fairly sufficient vitamin D status were higher than previous reports suggesting an adaptive mechanism to maximize calcium absorption.     

1α,25(OH)2-vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival

1α,25(OH)₂-vitamin D₃ (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca²⁺ channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca²⁺ concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca²⁺-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca²⁺-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.     

Identification of novel mediators of Vitamin D signaling and 1,25(OH)2D3 resistance in mammary cells

Since the discovery of the Vitamin D receptor (VDR) in mammary cells, the role of the Vitamin D signaling pathway in normal glandular function and in breast cancer has been extensively explored. In vitro studies have demonstrated that the VDR ligand, 1,25(OH)₂D₃, modulates key proteins involved in signaling proliferation, differentiation and survival of normal mammary epithelial cells. Anti-proliferative and pro-differentiating effects of 1,25(OH)₂D₃ have also been observed in VDR positive breast cancer cells, indicating that transformation per se does not abolish Vitamin D signaling. However, many breast cancer cell lines are less sensitive to 1,25(OH)₂D₃ than normal mammary epithelial cells. Reduced sensitivity to 1,25(OH)₂D₃ has been linked to alterations in Vitamin D metabolizing enzymes as well as down regulation of VDR expression or function. In this report, we describe results from a proteomics screening approach used to search for proteins involved in dictating sensitivity or resistance to Vitamin D mediated apoptosis in breast cancer cells. Several proteins not previously linked to 1,25(OH)₂D₃ signaling were identified with this approach, and a distinct subset of proteins was linked to 1,25(OH)₂D₃ resistance. Follow-up studies to determine the relevance of these proteins to Vitamin D signaling in general are in progress.     

Independent Association of Circulating Vitamin D Metabolites with Anemia Risk in Patients Scheduled for Cardiac Surgery

Background

Preoperative anemia is considered an independent risk factor of poor clinical outcome in cardiac surgical patients. Low vitamin D status may increase anemia risk.

Methods

We investigated 3,615 consecutive patients scheduled for cardiac surgery to determine the association between preoperative anemia (hemoglobin [Hb] <12.5 g/dL) and circulating levels of the vitamin D metabolites 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25[OH]₂D).

Results

Of the study cohort, 27.8 % met the criteria for anemia. In patients with deficient 25OHD levels (<30 nmol/l) mean Hb concentrations were 0.5 g/dL lower than in patients with adequate 25OHD levels (50.0–125 nmol/l; P<0.001). Regarding 1,25(OH)₂D, mean Hb concentrations were 1.2 g/dL lower in the lowest 1,25(OH)₂D category (<40 pmol/l) than in the highest 1,25(OH)₂D category (>70 pmol/l; P<0.001). In multivariable–adjusted logistic regression analyses, the odds ratios for anemia of the lowest categories of 25OHD and 1,25(OH)₂D were 1.48 (95%CI:1.19-1.83) and 2.35 (95%CI:1.86-2.97), compared with patients who had adequate 25OHD levels and 1,25(OH)₂D values in the highest category, respectively. Anemia risk was greatest in patients with dual deficiency of 25OHD and 1,25(OH)₂D (multivariable-adjusted OR = 3.60 (95%CI:2.40-5.40). Prevalence of deficient 25OHD levels was highest in anemia of nutrient deficiency, whereas low 1,25(OH)₂D levels were most frequent in anemia of chronic kidney disease.

Conclusion

This cross-sectional study demonstrates an independent inverse association between vitamin D status and anemia risk. If confirmed in clinical trials, preoperative administration of vitamin D or activated vitamin D (in case of chronic kidney disease) would be a promising strategy to prevent anemia in patients scheduled for cardiac surgery.     

Seasonal variation of 1,25-dihydroxyvitamin D and its association with body mass index and age

Under most normal conditions the serum level of 1,25-dihydroxyvitamin D is constant throughout the year, due to tight biochemical regulation. In contrast to this, the level of 25-hydroxyvitamin D is variable through the year, being largest in late summer, due to photosynthesis in the skin. The vitamin D status is usually assessed by measuring the level of the latter vitamin D derivative, rather than that of the presumably most active derivative 1,25(OH)₂ vitamin D.We here show that for persons with a high body mass index (BMI) there is a significant seasonal variation, not only of 25(OH) vitamin D, but also of 1,25(OH)₂ vitamin D. The variation seems to be largest for those with the poorest vitamin D status. Furthermore, there seems to be a correlation between the levels of the two vitamin D metabolites, indicating that the regulation of 1,25(OH)₂ vitamin D is not always tight, notably in persons with high BMI.     

Regulation of TNF-α release from bone marrow–derived macrophages by vitamin D

The calcium-regulating hormone 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] is recognized as an immuno-modulator affecting the activities of macrophages and lymphocytes. We have shown that macrophages harvested from vitamin D–deficient mice (–D MPs) exhibit impaired phagocytic and tumoricidal activities as compared with control cells (+D MPs), and that bone marrow–derived macrophage (BMDM) differentiation is modulated by 1,25(OH)₂D₃. The release of tumor necrosis factor–α (TNF-α) by macrophages is considered a major mechanism by which these cells exert their tumoricidal function. This cytokine was also implicated in modulation of bone resorption. In the present study we examine the role of 1,25(OH)₂D₃ in TNF-α synthesis and release. BMDMs were harvested from +D and ?D mice, cultured in vitro, and their conditioned media were analyzed for the presence of TNF-α. BMDMs did not release measurable amounts of TNF-α without stimulation. Addition of endotoxin (LPS) to the cultures resulted in a marked stimulation of TNF-α release. 1,25(OH)₂D₃ increased the stimulatory action of LPS, but failed to elicit a stimulatory effect in the absence of LPS. The use of another macrophage activator, interferon-γ (IFN-γ), yielded essentially similar results. +D and ?D mice were injected with LPS and TNF-α levels in the serum were measured. A marked reduction (～ fourfold) in the TNF-α levels was observed in the serum of ?D mice as compared with +D mice. Western blot and immunoprecipitation analyses suggested that the main effect of 1,25(OH)₂D₃ is on TNF-α synthesis. Our findings suggest that 1,25(OH)₂D₃ plays a role in the regulation of TNF-α secretion by mononuclear phagocytes. © 1994 Wiley-Liss, Inc.     

Vitamin D inhibition of pro-fibrotic effects of transforming growth factor β1 in lung fibroblasts and epithelial cells

The mechanisms that control fibroproliferation and matrix deposition in lung fibrosis remain unclear. We speculate that vitamin D deficiency may contribute to pulmonary fibrosis since vitamin D deficiency has been implicated in several diseases. First, we confirmed the presence of vitamin D receptors (VDRs) in cultured NIH/3T3 and lung fibroblasts. Fibroblasts transfected with a vitamin D response element–reporter construct and exposed to the active vitamin D metabolite, 1,25(OH)₂D₃, showed increased promoter activity indicating VDR functionality in these cells. Testing the effects of 1,25(OH)₂D₃ on fibroblasts treated with transforming growth factor β1 (TGFβ1), considered a driver of many fibrotic disorders, we found that 1,25(OH)₂D₃ inhibited TGFβ1-induced fibroblast proliferation in a dose-dependent fashion. 1,25(OH)₂D₃ also inhibited TGFβ1 stimulation of α-smooth muscle actin expression and polymerization and prevented the upregulation of fibronectin and collagen in TGFβ1-treated fibroblasts. Finally, we examined how 1,25(OH)₂D₃ affects epithelial–mesenchymal transformation of lung epithelial cells upon exposure to TGFβ1. We showed that the TGFβ1-induced upregulation of mesenchymal cell markers and abnormal expression of epithelial cell markers were blunted by 1,25(OH)₂D₃. These observations suggest that under TGFβ1 stimulation, 1,25(OH)₂D₃ inhibits the pro-fibrotic phenotype of lung fibroblasts and epithelial cells.     

Expressions of polypeptide: N-acetylgalactosaminyltransferase in leukemia cell lines during 1,25-dihydroxyvitamin D3 induced differentiation

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)₂D3] on two leukemia cell lines, K562 and SHI-1, and its relation to the expression of different subtypes of polypeptide: N-acetylgalactosaminyltransferase (pp-GalNAc-T) was studied. With morphological and cell flow-cytometric method, it was found that 1,25(OH)₂D3 induced the differentiation of both leukemia cell lines toward monocytic lineage, but not affected the cell growth and apoptosis. The expressions of different subtypes of pp-GalNAc-T, the initial glycosyltransferase in O-glycan synthesis, were studied with RT-PCR before and after the treatment of different concentrations of 1,25(OH)₂D3. Among fourteen subtypes of pp-GalNAc-T (T1 ∼ T14), K562 cells obviously expressed pp-GalNAc-T2, T4, T5, T7 (T2 was the highest) and SHI-1 cells apparently expressed pp-GalNAcT1, T2, T3 and T4 (T4 was the highest) only. After K562 cells were treated 1, 25(OH)₂D3 for 72 h, pp-GalNAc-T2, T4, T5, T7 were increased in a dose dependent manner. In contrast, pp-GalNAc-T1 and T2, especially T1, were up-regulated in SHI-1 cells by 1,25(OH)₂D3, but T3 was unchanged and T4 was down-regulated. The different alterations of pp-GalNAc-Ts in these two cell lines were probably related to the different structural changes of O-glycans during 1,25(OH)₂D3 induced differentiation.     

Inhibition of proliferation and induction of differentiation of osteoblastic cells by a novel 1,25-dihydroxyvitamin D3 analog with an extensively modified side chain (CB1093)

1,25-Dihydroxyvitamin D₃ (1,25D) is involved in the regulation of proliferation and differentiation of a variety of cell types including cancer cells. In recent years, numerous new vitamin D₃ analogs have been developed in order to obtain favorable therapeutic properties. The effects of a new 20-epi analog, CB1093 (20-epi-22-ethoxy-23-yne-24a,26a,27a-trihomo-1α,25(OH)₂D₃), on the proliferation and differentiation of human MG-63 osteosarcoma cell line were compared here with those of the parent compound 1,25D. Proliferation of the MG-63 cells was inhibited similarly by 22%, 50% and 59% after treatment with 0.1 μM 1,25D or CB1093 for 48 h, 96 h, and 144 h, respectively. In transfection experiments, the compounds were equipotent in stimulating reporter gene activity under the control of human osteocalcin gene promoter. In cell culture experiments, however, CB1093 was more potent than 1,25D at low concentrations and more effective for a longer period of time in activating the osteocalcin gene expression at mRNA and protein levels. Also, a 6-h pretreatment and subsequent culture for up to 120 h without 1,25D or CB1093 yielded higher osteocalcin mRNA and protein levels with analog-treated cells than with 1,25D-treated cells. The electrophoretic mobility shift assay (EMSA) revealed stronger VDR-VDRE binding with analog-treated MG-63 cells than with 1,25D-treated cells. The differences in the DNA binding of 1,25D-bound vs. analog-bound VDR, however, largely disappeared when the binding reactions were performed with recombinant hVDR and hRXRβ proteins. These results demonstrate that the new analog CB1093 was equally or even more effective than 1,25D in regulating all human osteosarcoma cell functions ranging from growth inhibition to marker gene expression and that the differences in effectivity most probably resulted from interactions of the hVDR:hRXRβ-complex with additional nuclear proteins. J. Cell. Biochem. 70:414–424, 1998. © 1998 Wiley-Liss, Inc.