James C. Fleet

Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 176RXXR179/S180 proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene–environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

Vitamin D and cancer: a review of molecular mechanisms

The population-based association between low vitamin D status and increased cancer risk can be inconsistent, but it is now generally accepted. These relationships link low serum 25OHD (25-hydroxyvitamin D) levels to cancer, whereas cell-based studies show that the metabolite 1,25(OH)2D (1,25-dihydroxyvitamin D) is a biologically active metabolite that works through vitamin D receptor to regulate gene transcription. In the present review we discuss the literature relevant to the molecular events that may account for the beneficial impact of vitamin D on cancer prevention or treatment. These data show that although vitamin D-induced growth arrest and apoptosis of tumour cells or their non-neoplastic progenitors are plausible mechanisms, other chemoprotective mechanisms are also worthy of consideration. These alternative mechanisms include enhancing DNA repair, antioxidant protection and immunomodulation. In addition, other cell targets, such as the stromal cells, endothelial cells and cells of the immune system, may be regulated by 1,25(OH)2D and contribute to vitamin D-mediated cancer prevention.

Differentiation-Specific Histone Modifications Reveal Dynamic Chromatin Interactions and Partners for the Intestinal Transcription Factor CDX2

VIDEO ABSTRACT Cell differentiation requires remodeling of tissue-specific gene loci and activities of key transcriptional regulators, which are recognized for their dominant control over cellular programs. Using epigenomic methods, we characterized enhancer elements specifically modified in differentiating intestinal epithelial cells and found enrichment of transcription factor-binding motifs corresponding to CDX2, a critical regulator of the intestine. Directed investigation revealed surprising lability in CDX2 occupancy of the genome, with redistribution from hundreds of sites occupied only in proliferating cells to thousands of new sites in differentiated cells. Knockout mice confirmed distinct Cdx2 requirements in dividing and mature adult intestinal cells, including responsibility for the active enhancer configuration associated with maturity. Dynamic CDX2 occupancy corresponds with condition-specific gene expression and, importantly, to differential co-occupancy with other tissue-restricted transcription factors, such as GATA6 and HNF4A. These results reveal dynamic, context-specific functions and mechanisms of a prominent transcriptional regulator within a cell lineage.

Intestinal Vitamin D Receptor is Required for Normal Calcium and Bone Metabolism in Mice

BACKGROUND & AIMS Vitamin D receptor (VDR)-knockout mice develop severe hypocalcemia and rickets, accompanied by disruption of active intestinal calcium absorption. To specifically study the effects of VDR in intestinal calcium absorption, we investigated whether restoration of intestinal VDR is sufficient to recover the phenotype of VDR-knockout mice. METHODS We generated mice with intestine-specific transgenic expression of human VDR and crossed them to VDR knockout mice. The intestine, kidney, and bone phenotypes of the VDR- knockout mice with intestine-specific expression of human VDR (knockout/transgenic [KO/TG]) were analyzed. RESULTS Transgenic expression of VDR in the intestine of VDR-knockout mice normalized duodenal vitamin D-regulated calcium absorption as well as vitamin D-regulated calcium binding protein D9k and TRPV6 gene expression in the duodenum and proximal colon. As a result, animal growth and the serum levels of calcium and parathyroid hormone were normalized in KO/TG mice. Other phenotypes were revealed when calcium metabolism was normalized in KO/TG mice: serum 1,25 dihydroxyvitamin D levels were higher in KO/TG mice than normal mice owing to reduced renal expression of the vitamin D-degrading enzyme CYP24, urinary calcium excretion was higher and associated with lower renal calcium binding protein D9k and calcium binding protein D28k than normal mice, and bone density and volume increased in KO/TG compared with normal mice owing to increased mineral apposition rate and osteoblast number. CONCLUSIONS Intestinal VDR and vitamin D-regulated intestinal calcium absorption are critical for controlling whole-body calcium metabolism in growing mice. Normalizing intestinal calcium absorption and metabolism reveals essential roles for VDR in control of bone formation and renal control of serum 1,25(OH)2D and urinary calcium excretion.

Molecular actions of vitamin D contributing to cancer prevention.

The population-based relationship between low vitamin D status and increased cancer risk is now generally accepted. While these relationships are between serum 25 hydroxyvitamin D and cancer, cell-based studies show that the metabolite 1,25 dihydroxyvitamin D is biologically active and influences cell biology relevant to cancer through vitamin D receptor-mediated gene transcription. This review examines this paradox and also discusses the cell and gene targets influenced by 1,25 dihydroxyvitamin D that may account for the anti-cancer actions of vitamin D. A review of the literature shows that while vitamin D-induced growth arrest and apoptosis of tumor cells or their non-neoplastic progenitors are plausible mechanisms, other gene targets related to DNA repair and immunomodulation, and other cell targets such as the stromal cells and cells of the immune system, may be regulated by 1,25 dihydroxyvitamin D and contribute to vitamin D mediated cancer prevention.

NHANES Monitoring of Serum 25-Hydroxyvitamin D: A Roundtable Summary

A roundtable to discuss monitoring of serum 25-hydroxyvitamin D [25(OH)D] in the NHANES was held in late July 2009. Topics included the following: 1) options for dealing with assay fluctuations in serum 25(OH)D in the NHANES conducted between 1988 and 2006; 2) approaches for transitioning between the RIA used in the NHANES between 1988 and 2006 to the liquid chromatography tandem MS (LC-MS/MS) measurement procedure to be used in NHANES 2007 and later; 3) approaches for integrating the recently available standard reference material for vitamin D in human serum (SRM 972) from the National Institute of Standards and Technology (NIST) into the NHANES; 4) questions regarding whether the C-3 epimer of 25-hydroxyvitamin D3 [3-epi-25(OH)D3] should be measured in NHANES 2007 and later; and 5) identification of research and educational needs. The roundtable experts agreed that the NHANES data needed to be adjusted to control for assay fluctuations and offered several options for addressing this issue. The experts suggested that the LC-MS/MS measurement procedure developed by NIST could serve as a higher order reference measurement procedure. They noted the need for a commutability study for the recently released NIST SRM 972 across a range of measurement procedures. They suggested that federal agencies and professional organizations work with manufacturers to improve the quality and comparability of measurement procedures across all laboratories. The experts noted the preliminary nature of the evidence of the 3-epi-25(OH)D3 but felt that it should be measured in 2007 NHANES and later.

Molecular mechanisms for regulation of intestinal calcium absorption by vitamin D and other factors

Optimal intestinal calcium (Ca) absorption is necessary for the protection of bone and the prevention of osteoporosis. Ca absorption can be represented as the sum of a saturable pathway and a non-saturable pathway that is primarily dependent upon luminal Ca concentration. While models have been proposed to describe these transport components, significant gaps still exist in our understanding of these processes. Habitual low intake of Ca up-regulates the saturable transport pathway, a process mediated by increased renal production of 1,25 dihydroxyvitamin D (1,25(OH)2D). Consistent with this, low vitamin D status as well as deletion/mutation of the vitamin D receptor (VDR) or 25 hydroxyvitamin D-1α hydroxylase (CYP27B1) genes limit Ca absorption by reducing the saturable pathway. There is some evidence that non-saturable Ca absorption in the ileum is also regulated by vitamin D status, but the mechanism is unclear. Treatment with a number of hormones can regulate Ca absorption in vivo (e.g. parathyroid hormone (PTH), thyroid hormone, growth hormone (GH)/insulin-like growth factor I (IGF-1), estrogen, testosterone). However, some of these actions are indirect (i.e. mediated through the regulation of vitamin D metabolism or signaling), whereas only a few (e.g. estrogen, IGF-1) have been shown to persist in the absence of vitamin D signaling.

Rapid, Membrane-Initiated Actions of 1,25 Dihydroxyvitamin D: What Are They and What Do They Mean?

Vitamin D is a conditionally required nutrient traditionally thought to influence physiology as the metabolite 1,25-dihydroxyvitamin D [1,25(OH)(2) D] by binding to the vitamin D receptor (VDR) and stimulating the transcription of genes through direct VDR-DNA interactions. However, over the past 15 y research has demonstrated that 1,25(OH)(2) D, as well as other steroid hormones, can rapidly stimulate ion fluxes and activate protein kinases by transcription-independent mechanisms. This review summarizes recent research on the rapid actions of 1,25(OH)(2) D and identifies questions that remain to be answered in this area.

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-1after 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 μM) 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 D9kand 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.

GATA Factors Regulate Proliferation, Differentiation, and Gene Expression in Small Intestine of Mature Mice

BACKGROUND & AIMS GATA transcription factors regulate proliferation, differentiation, and gene expression in multiple organs. GATA4 is expressed in the proximal 85% of the small intestine and regulates the jejunal-ileal gradient in absorptive enterocyte gene expression. GATA6 is co-expressed with GATA4 but also is expressed in the ileum; its function in the mature small intestine is unknown. METHODS We investigated the function of GATA6 in small intestine using adult mice with conditional, inducible deletion of Gata6, or Gata6 and Gata4, specifically in the intestine. RESULTS In ileum, deletion of Gata6 caused a decrease in crypt cell proliferation and numbers of enteroendocrine and Paneth cells, an increase in numbers of goblet-like cells in crypts, and altered expression of genes specific to absorptive enterocytes. In contrast to ileum, deletion of Gata6 caused an increase in numbers of Paneth cells in jejunum and ileum. Deletion of Gata6 and Gata4 resulted in a jejunal and duodenal phenotype that was nearly identical to that in the ileum after deletion of Gata6 alone, revealing common functions for GATA6 and GATA4. CONCLUSIONS GATA transcription factors are required for crypt cell proliferation, secretory cell differentiation, and absorptive enterocyte gene expression in the small intestinal epithelium.