1α, 25 Dihydroxyvitamin D (1,25(OH)2D) inhibits the T cell suppressive function of myeloid derived suppressor cells (MDSC).

Myeloid derived suppressor cells (MDSC) suppress the ability of cytotoxic T cells to attack and clear tumor cells from the body. The active form of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)2D), regulates myeloid cell biology and previous research showed that in mouse models 1,25(OH)2D reduced the tumor level of CD34+ cells, an MDSC precursor, and reduced metastasis. We tested whether MDSC are vitamin D target cells by examining granulocytic- (G-MDSC) and monocytic (M-MDSC) MDSC from tumors, spleen, and bone marrow. Vitamin D receptor (VDR) mRNA levels are low in MDSC from bone marrow and spleen but are 20-fold higher in tumor MDSC. At all sites, M-MDSC have 4-fold higher VDR mRNA expression than G-MDSC. Bone marrow MDSC were induced to differentiate in vitro into tumor MDSC-like cells by treating with IFN-γ, IL-13, and GM-CSF for 48 h. This treatment significantly elevated Arg1 and Nos2 levels, activated the T cell-suppressive function of MDSC, increased VDR expression 50-fold, and made the MDSC responsive to 1,25(OH)2D treatment. Importantly, 1,25(OH)2D treatment reduced the T cell suppressive capacity of cytokine-induced total MDSC and M-MDSC by ≥70 % and tumor-derived M-MDSC by 30-50 %. Consistent with this finding, VDR deletion (KO) increased T cell suppressive function of in vitro M-MDSC by 30 % and of tumor-derived M-MDSC by 50 % and G-MDSC by 400 %. VDR KO did not alter Nos2 mRNA levels but significantly increased Arg1 mRNA levels in tumor M-MDSC by 100 %. In contrast, 1,25(OH)2D treatment reduced nitric oxide production in both in vitro derived M- and G- MDSC. The major finding of this study is that 1,25(OH)2D signaling through the VDR decreases the immunosuppressive capability of MDSC. Collectively, our data suggest that activation of vitamin D signaling could be used to suppress MDSC function and release a constraint on T-cell mediated clearance of tumor cells.

Targeting 1,25(OH)2D-mediated calcium absorption machinery in proximal colon with calcitriol glycosides and glucuronides.

High intestinal calcium (Ca) absorption efficiency is associated with high peak bone mass in adolescents and reduced bone loss in adulthood. Transepithelial intestinal Ca absorption is mediated by 1,25-dihydroxyvitamin D (1,25(OH)2D, calcitriol) through the vitamin D receptor (VDR). Most research on Ca absorption focuses on the proximal small intestine but evidence shows that large intestine plays a crucial role in whole body Ca homeostasis. We directly assessed and compared Ca absorption capacity at the proximal colon and duodenum using in situ ligated loops (2 mM Ca, 10 min). In C57BL/6 J mice, the proximal colon (26.2 ±â€¯3.7 %) had comparable ability to absorb Ca as the duodenum (30.0 ±â€¯6.7 %). In VDR knockout (KO) mice, Ca absorption efficiency was reduced by 67 % in duodenum and 48 % in proximal colon. These data suggest that large intestine could be targeted to improve Ca absorption and protect bone in at risk-groups (e.g. bariatric patients). Glycoside forms of calcitriol found in Solanum Glaucophyllum (Sg) leaf are biologically inert but can be activated in the colon upon bacterial cleavage of the glycosides. We conducted a study to test whether Sg leaf, as well as a novel, synthetic 1,3-diglucuronide form of calcitriol (1,3-diG) could target the proximal colon and upregulate genes involved in Ca absorption (i.e. Trpv6, S100g). 13-week-old female C57BL6/J mice were fed AIN93 G diet containing increasing levels of one of the two compounds for 2 weeks (delivering 0, 0.25, 0.5, 1, or 2 ng calcitriol equivalent per day). Both compounds induced a dose-dependent upregulation of Cyp24a1 and Trpv6 gene expression in the proximal colon. 1,3-diG also induced S100g gene expression in the proximal colon. Duodenal expression of Trpv6 was upregulated at higher doses of 1,3-diG but not Sg leaf. These data suggest that both glycosylated and glucuronidated calcitriol could be used to target the proximal colon but that dosing must be optimized to limit systemic effects that could cause hypercalcemia. Future studies will test the translational potential of these compounds to determine if they can increase Ca absorption at proximal colon and whether this can help protect bone.

Calcium and vitamin D intake maintained from preovariectomy independently affect calcium metabolism and bone properties in Sprague Dawley rats.

UNLABELLED: The interaction of habitual Ca and vitamin D intake from preovariectomy to 4 months postovariectomy on bone and Ca metabolism was assessed. Higher Ca intake suppressed net bone turnover, and both nutrients independently benefitted trabecular structure. Habitual intake of adequate Ca and ~50 nmol/L vitamin D status is most beneficial. INTRODUCTION: Dietary strategies to benefit bone are typically tested prior to or after menopause but not through menopause transition. We investigated the interaction of Ca and vitamin D status on Ca absorption, bone remodeling, Ca kinetics, and bone strength as rats transitioned through estrogen deficiency. METHODS: Sprague Dawley rats were randomized at 8 weeks to 0.2 or 1.0 % Ca and 50, 100, or 1,000 IU (1.25, 2.5, or 25 µg) vitamin D/kg diet (2 × 3 factorial design) and ovariectomized at 12 weeks. Urinary (45)Ca excretion from deep-labeled bone was used to assess net bone turnover weekly. Ca kinetics was performed between 25 and 28 weeks. Rats were killed at 29 weeks. Femoral and tibiae structure (by µCT), dynamic histomorphometry, and bone Ca content were assessed. RESULTS: Mean 25(OH)D for rats on the 50, 100, 1,000 IU vitamin D/kg diet were 32, 54, and 175 nmol/L, respectively. Higher Ca intake ameliorated net bone turnover, reduced fractional Ca absorption and bone resorption, and increased net Ca absorption. Tibial and femoral trabecular structures were enhanced independently by higher Ca and vitamin D intake. Tibial bone width and fracture resistance were enhanced by higher vitamin D intake. Dynamic histomorphometry in the tibia was not affected by either nutrient. A Ca × vitamin D interaction existed in femur length, tibial Ca content, and mass of the soft tissue/extracellular fluid compartment. CONCLUSIONS: Adequate Ca intake and serum 25(OH)D level of 50 nmol/L provided the most benefit for bone health, mostly through independent effects of Ca and vitamin D.

Using genomics to understand intestinal biology.

The use of microarrays to evaluate the transcriptome has transformed our view of biology. In addition to the focused, hypothesis-testing studies that we have traditionally conducted in cell biology, we are now able to see global changes within the entire system of the cell in response to a treatment. By examining a biological question under multiple complementary perturbations model systems (e.g. yeast, C. Elegans) have revealed new complexity that would have been impossible to see on a gene-by-gene approach. Unfortunately, beyond the use of transcript profiles to define the molecular signature of diseases (e.g. cancer), transcriptomics has not been extensively used to study intestinal biology. This review will provide a roadmap for effective use of gene expression profiling for biological research and will review some of the microarray work that has been done to better understand the nature of intestinal development and enterocyte differentiation.

DASH without the dash (of salt) can lower blood pressure.

While the debate regarding the effectiveness of dietary sodium restriction raged, the Dietary Approaches to Stop Hypertension (DASH) research group showed that a diet rich in fruits, vegetables, and low-fat dairy products can reduce blood pressure in the general population and people with Stage I hypertension. The original DASH diet did not require either sodium restriction or weight loss--the two traditional dietary tools to control blood pressure--to be effective. A recent study from the DASH research group now finds that coupling the original DASH diet with sodium restriction is more effective than either dietary manipulation alone.

Increased vitamin D receptor level enhances 1,25-dihydroxyvitamin D3-mediated gene expression and calcium transport in Caco-2 cells.

Altered vitamin D receptor (VDR) level has been proposed to explain differences in intestinal responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We tested whether the enterocyte VDR level influences 1,25(OH)2D3-mediated gene expression and transepithelial calcium (Ca) transport in the human intestinal cell line Caco-2. Cells were stably transfected with a human metallothionein (hMT) IIA promoter-human VDR (hVDR) complementary DNA (cDNA) transgene that overexpressed hVDR in response to heavy metals. In MTVDR clones, induction of 25-hyroxyvitamin D3-24-hydroxylase (24-OHase) messenger RNA (mRNA) expression by 1,25(OH)2D3 (10(-9) M, 4 h) was correlated to metal-induced changes in nuclear VDR level (r2 = 0.99). In MTVDR clones, basal VDR level was 2-fold greater and 1,25(OH)2D3-mediated Ca transport (10(-7) M, 24 h) was 43% higher than in parental Caco-2 cells. Treatment of MTVDR clones with Cd (1 microM, 28 h) increased VDR level by 68%, significantly enhanced 1,25(OH)2D3-mediated Ca transport by 24%, and increased accumulation of calbindin D9K mRNA by 76% relative to 1,25(OH)2D3 alone. These observations support the hypothesis that the enterocyte VDR level is an important modulator of intestinal responsiveness to 1,25(OH)2D3.

Leptin and bone: does the brain control bone biology?

The means by which obesity leads to high bone density and protects individuals from osteoporosis is not known. The study of bone biology in two mouse models of obesity, leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, points to a role for leptin in the control of bone density. When leptin action is missing in these mice, bone density is high. This is true despite concurrent hypogonadism and hypercortisolism, two strong proresorptive signals that would normally lead to low bone density. Curiously, leptin does not have a direct effect on osteoblasts, which suggests the existence of a central, neuroendocrine pathway that controls bone mass.

Specific 1,25(OH)2D3-mediated regulation of transcellular calcium transport in Caco-2 cells.

Calcium transport in the apical-to-basolateral (A-to-B) or B-to-A direction was examined in cells treated with 10 nM 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3, calcitriol] for up to 72 h. Net A-to-B calcium transport was positive at all time points and increased from 0.14 +/- 0.06 to 0.50 +/- 0.01 nmol. well-1. min-1 after 72 h of calcitriol treatment. Neither phenol red transport nor transepithelial electrical resistance was altered by calcitriol treatment, suggesting that the increase in net A-to-B calcium transport was not due to paracellular movement. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 (100 nM, 48 h) alters basal or calcitriol-stimulated A-to-B calcium transport. Treatment with the calmodulin antagonist trifluoperazine (50 microM) reduced calcitriol-stimulated A-to-B Ca transport by 56%. The transcription inhibitor actinomycin D inhibited calcitriol-regulated A-to-B calcium transport as well as calbindin D9k and 24-hydroxylase mRNA accumulation. These data demonstrate that calcitriol-mediated A-to-B calcium transport in Caco-2 cells is a specific, transcellular process that requires transcriptional events normally mediated through the vitamin D receptor.

Vitamin D receptors: not just in the nucleus anymore.

Calcitriol, the active metabolite of vitamin D, is a steroid hormone that regulates calcium metabolism and cell differentiation by interacting with its nuclear receptor--the vitamin D receptor (VDR)--and by stimulating gene transcription. During the last decade, calcitriol also has been shown to stimulate rapid signal transduction pathways. This observation supports the hypothesis that a membrane-bound receptor similar to those that mediate peptide hormone biology exists. Recent research provides evidence for such a unique membrane VDR. Future research challenges are to integrate the membrane VDR into existing knowledge of vitamin D biology.

Reciprocal regulation of HFE and NNamp2 gene expression by iron in human intestinal cells.

The newly identified hemochromatosis gene, HFE, and a candidate iron transporter gene, Nramp2, have been proposed as key factors responsible for the regulation of intestinal iron absorption. Although the exact functions of these proteins in intestinal iron absorption are unknown, HFE may be required for the down-regulation of iron absorption that occurs with increasing iron status, and Nramp2 may up-regulate iron absorption when iron status is low. Thus, we examined whether the expression of the HFE and Nramp2 genes are regulated by iron status in the human intestinal cell line Caco-2. HFE mRNA and HFE protein were increased and Nramp2 mRNA was decreased by increasing cellular iron status in Caco-2 cells. This iron-mediated modulation of mRNA levels was specific to iron. Moreover, super-induction of HFE mRNA in the presence of cycloheximide suggests that HFE gene expression may be controlled by a short-lived repressor protein. HFE and Nramp2 mRNA levels also changed in opposite directions during cellular differentiation. This reciprocal modification of the HFE and Nramp2 gene expression during both iron treatment and cell differentiation in Caco-2 cells is consistent with an opposing role for these proteins in homeostatic regulation of human intestinal iron absorption.

Intestinal calcium absorption in the aged rat: evidence of intestinal resistance to 1,25(OH)2 vitamin D.

We investigated the role of circulating 1,25-dihydroxycholecalciferol (1,25(OH)2D) and intestinal resistance to 1,25(OH)2D in the diminished intestinal calcium absorption capacity of the senescent rat. We measured plasma 1,25(OH)2D, total and unoccupied duodenal vitamin D receptor, duodenal calbindin D9k protein (calbindin D), and net dietary calcium absorption in rats at several ages. As expected, circulating 1,25(OH)2D, calbindin D, and net calcium absorption decreased with age. However, no age-related changes were evident in intestinal vitamin D receptor levels. We then measured duodenal calcium absorption from in situ intestinal loops after continuous s.c. infusion of 1,25(OH)2D for up to 6 days and found that despite a marked elevation of plasma 1,25(OH)2D duodenal calcium absorption was significantly lower in old compared with young rats. To assess calcium absorption over a wide physiological range of plasma 1,25(OH)2D, in a dose-response study we altered plasma 1,25(OH)2D by continuous infusion of 1,25(OH)2D (at 0, 4, or 14 ng/100 g BW/day) for 9 days. We found that the slope of the linear regression between plasma 1,25(OH)2D and duodenal Ca transport in old rats was only 46% of that observed in young rats, suggesting an age-related resistance of the duodenal calcium transport process to the hormonal action of 1,25(OH)2D. Collectively, our observations suggest a dual defect in vitamin D metabolism in old animals: one defect related to the low circulating levels of 1,25(OH)2D and a second defect related to a relative intestinal resistance to the action of 1,25(OH)2D, which is apparently not due to a reduction in intestinal vitamin D receptor levels.

The genetics of osteoporosis: vitamin D receptor polymorphisms.

Osteoporosis is a metabolic bone disease characterized by low bone mass and deterioration of bone tissue that leads to bone fragility and an increase in fracture risk. It is a disease with a complex etiology that includes genetic and environmental contributors. Environmental factors that influence bone density include dietary factors-such as intakes of calcium, alcohol, and caffeine-and lifestyle factors-such as exercise and smoking. Ethnic differences in the propensity to nontraumatic bone fracture suggest that genetic factors are important. Recently, common allelic variations in he vitamin D receptor gene have been found to be associated with bone mineral density in racially diverse population groups, as well as in prepubertal girls, young adult and postmenopausal women, and men. However, many studies have not been able to find this association. Additional approaches, such as sib-pair analysis, will probably be necessary in the future to identify the important determinants of osteoporosis.

Identification of Nramp2 as an iron transport protein: another piece of the intestinal iron absorption puzzle.

Although a number of iron-binding proteins have been identified, the roles for specific proteins in mediating iron absorption have not been definitively assigned. Two recent papers report the identification of an iron transport protein that may be responsible for movement of iron from the intestinal lumen into the enterocyte. Coupled with the recent identification of the protein mutated in hemochromatosis, researchers are now establishing a clearer picture of the mechanism of intestinal iron absorption.

Dietary selenium repletion may reduce cancer incidence in people at high risk who live in areas with low soil selenium.

Studies examining the relationship between dietary selenium intake and risk of various cancers have shown that low selenium intake is associated with higher cancer rates. A recent well-controlled intervention trial studied whether selenium supplementation can prevent cancer in subjects who have a history of skin cancer and live in areas of the United States with low soil selenium levels. Selenium supplementation did not reduce skin cancer rates, but the incidence of total, lung, colorectal, and prostate cancers was significantly reduced by the intervention. Although these data need confirmation, they suggest that adequate selenium intake is essential for cancer prevention.

The effects of aging on the bone inductive activity of recombinant human bone morphogenetic protein-2.

We examined the effects of gain on the ectopic bone-forming ability of recombinant human BMP-2 (rhBMP-2) in rats and investigated the mechanism by which aging might affect this type of bone. Bone formation induced after 12 days of sc implantation of 5 micrograms rhBMP-2 was reduced as animals aged from 1-16 months. The osteocalcin messenger RNA levels of implants also declined in aging animals. When the implant period was doubled, 16-month-old rats formed amounts of bone equivalent to those in 3-month-old rats. Increasing the dose of rhBMP-2 increased bone formation in older rats. To get a response comparable to that seen in 1-month-old rats given 5 micrograms rhBMP-2 for 12 days, 3-month-old rats required 30 micrograms rhBMP-2, whereas 16-month-old rats required 60 micrograms. Treatment with either GH or 1,25-dihydroxyvitamin D3 during the 12-day implantation period returned the bone formation in 16-month-olds rats to that in 3-month-old rats. These studies show that aging blunts rhBMP-2 inducted bone formation in rats. We speculate that the decreased response may be due in part to a decrease in the number of mesenchymal stem cells present in order rats or to a change in the responsiveness of these target cells to rhBMP-2.

Discovery of the hemochromatosis gene will require rethinking the regulation of iron metabolism.

The identity of the protein responsible for hemochromatosis, the iron overload disease, has eluded scientists for years. However, a recent report identifies the gene where the hemochromatosis defect lies. It is a gene that encodes a major histocompatibility complex (MHC) class-1-like protein called HLA-H. The mechanism by which an HLA-H defect alters iron metabolism is still unidentified. However, this new discovery will certainly ignite a new wave of study into the physiology of iron metabolism and its regulation.

Bone lead as a risk factor for hypertension in men.

While some have observed a positive relationship between blood lead levels and blood pressure, this relationship has been controversial. A recent study examined the hypothesis that bone lead levels, used as a measure of long-term lead accumulation, are associated with an increased risk of hypertension in men. Using a backward elimination logistic regression model, the researchers found that tibia lead, body mass index, and family history of hypertension contributed to the development of hypertension. An increase in tibia lead from 8 to 37 micrograms per gram of bone mineral was associated with an increased odds ratio of hypertension of 1.5. This is an interesting preliminary finding that requires support from additional experimentation.

1 alpha,25-(OH)2-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro.

Several 1 alpha,25-(OH)2-vitamin D3 (1 alpha,25-(OH)2-D3) analogs have significant antiproliferative effects in vitro but do not elevate serum calcium in vivo. We tested whether the lack of a calcemic response of a vitamin D analog in vivo is due to its inability to stimulate intestinal calcium absorption by examining the effect of several such compounds on transepithelial calcium transport in the human colonic carcinoma cell line Caco-2. The relative stimulations of calcium transport by the four A-ring diastereomers of 1 alpha, 25-(OH)2-D3 (1 alpha,3 beta) and a 3 beta-bromoacetate analog (1 alpha,3 beta-BrAc) of the vitamin following 48-h treatment of cells at 10 nM were 1 alpha,3 beta (=100%), 1 alpha,3 alpha (+45.2%), 1 beta,3 beta (-15.6%), 1 beta,3 alpha (+6.5%), and 1 alpha,3 beta-BrAc (+50.6%). This was similar to the reported affinity of these compounds for the vitamin D receptor (VDR) and suggests that VDR binding predicts calcium transport. In contrast, three noncalcemic, sidechain- or D-ring-modified analogs of vitamin D, 1 alpha,25-(OH)2-16-ene-D3, 1 alpha,25-(OH)2-16-ene-23-yne-D3, and 1 alpha,25,28-(OH)3-D2 (at 10 nM for 48 h), showed a different relationship between VDR affinity (150, 60, and 63% of 1 alpha, 25-(OH)2-D3, respectively) and calcium transport (74.1, 126, and 10%, respectively). Elevated calcium transport was accompanied by higher 24-hydroxylase and calbindin D9k mRNA levels. The data demonstrate that although some vitamin D compounds cannot stimulate calcium transport due to an inability to interact with the VDR (e.g., 1 beta isomers), other factors, e.g., differential cellular metabolism, may account for variations in biological response in vivo to various vitamin D analogs.

Are low-sodium diets appropriate for treated hypertensive men?

Because a low-sodium diet is often recommended as part of the treatment to control hypertension, a recent study examined the relationship between sodium intake, 24-hour urinary sodium excretion, and subsequent cardiovascular disease in treated hypertensive men and women. Contrary to expectations, the men with the lowest urinary sodium excretion had the highest incidence of myocardial infarction. Concerns about the group studied and some of the measurements used raise doubt about the results of this study, especially in light of the weight of data from population studies supporting a link between low blood pressure and low sodium intake. However, should future studies corroborate this one, clinicians will need to re-evaluate the simple notion that severe dietary sodium restriction is always beneficial for people with hypertension.