Calcium and 1,25(OH)2D: interacting drivers of epidermal differentiation

Both calcium and 1,25(OH)(2)D promote the differentiation of keratinocytes in vitro. The autocrine or paracrine production of 1,25(OH)(2)D by keratinocytes combined with the critical role of the epidermal calcium gradient in regulating keratinocyte differentiation in vivo suggest the physiologic importance of this interaction. The interactions occur at a number of levels. Calcium and 1,25(OH)(2)D synergistically induce involucrin, a protein critical for cornified envelope formation. The involucrin promoter contains an AP-1 site essential for calcium and 1,25(OH)(2)D induction and an adjacent VDRE essential for 1,25(OH)(2)D but not calcium induction. Calcium regulates coactivator complexes that bind to the Vitamin D receptor (VDR). Nuclear extracts from cells grown in low calcium contain an abundance of DRIP(205), whereas calcium induced differentiation leads to reduced DRIP(205) and increased SRC 3 which replaces DRIP in its binding to the VDR. In vivo models support the importance of 1,25(OH)(2)D-calcium interactions in epidermal differentiation. The epidermis of 1alphaOHase null mice fails to form a normal calcium gradient, has reduced expression of proteins critical for barrier function, and shows little recovery of the permeability barrier when disrupted. Thus in vivo and in vitro, calcium and 1,25(OH)(2)D interact at multiple levels to regulate epidermal differentiation.     

Vitamin D regulated keratinocyte differentiation

The epidermis is the largest organ in the body. It is comprised primarily of keratinocytes which are arranged in layers that recapitulates their programmed life cycle. Proliferating keratinocytes are on the bottom-the stratum basale. As keratinocytes leave the stratum basale they begin to differentiate, culminating in the enucleated stratum corneum which has the major role of permeability barrier. Calcium and the active metabolite of vitamin D, 1,25(OH)(2)D(3), play important roles in this differentiation process. The epidermis has a gradient of calcium with lowest concentrations in the stratum basale, and highest concentrations in the stratum granulosum where proteins critical for barrier function are produced. Vitamin D is made in different layers of the epidermis, but 1,25(OH)(2)D(3) is made primarily in the stratum basale. Together calcium and 1,25(OH)(2)D(3) regulate the ordered differentiation process by the sequential turning on and off the genes producing the elements required for differentiation as well as activating those enzymes involved in differentiation. Animal models in which the sensing mechanism for calcium, the receptor for 1,25(OH)(2)D(3), or the enzyme producing 1,25(OH)(2)D(3) have been rendered inoperative demonstrate the importance of these mechanisms for the differentiation process, although each animal model has its own phenotype. This review will examine the mechanisms by which calcium and 1,25(OH)(2)D(3) interact to control epidermal differentiation.     

Effects of calcium and of the Vitamin D system on skeletal and calcium homeostasis: lessons from genetic models

Targeted deletion of genes encoding the 1,25-dihydroxyVitamin D [1,25(OH)(2)D]-synthesizing enzyme, 25 hydroxyVitamin D-1alpha-hydroxylase [1alpha(OH)ase or CYP27B1], and of the nuclear receptor for 1,25(OH)(2)D, the Vitamin D receptor (VDR), have provided useful mouse models of the inherited human diseases, Vitamin D-dependent rickets types I and II. We employed these models and double null mutants to examine the effects of calcium and of the 1,25(OH)(2)D/VDR system on skeletal and calcium homeostasis. Optimal dietary calcium absorption required both 1,25(OH)(2)D and the VDR. Skeletal mineralization was dependent on adequate ambient calcium but did not directly require the 1,25(OH)(2)D/VDR system. Parathyroid hormone (PTH) secretion was also modulated primarily by ambient serum calcium but the enlarged parathyroid glands which the mutants exhibited and the widened cartilaginous growth plates could only be normalized by the combination of calcium and 1,25(OH)(2)D, apparently independently of the VDR. Optimal osteoclastic bone resorption and osteoblastic bone formation both required an intact 1,25(OH)(2)D/VDR apparatus. The results indicate that calcium cannot entirely substitute for Vitamin D in skeletal and mineral homeostasis but that the two agents have discrete and overlapping functions.     

Duodenal expression of the epithelial calcium transporter gene TRPV6: is there evidence for Vitamin D-dependence in humans?

Intestinal absorption of dietary calcium is regulated by 1,25-dihydroxycholecalciferol (1,25(OH)(2)D(3)) in humans and in experimental animals but interspecies differences in responsiveness to 1,25(OH)(2)D(3) are found, possibly due to differences in the promoters of genes for intestinal calcium transport proteins or of the Vitamin D receptor (VDR). The epithelial calcium transporter, known as ECAC2 or CAT1, the product of the TRPV6 gene expressed in proximal intestinal enterocytes, is the first step in calcium absorption and studies in mice have shown that its expression is Vitamin D-dependent. In contrast in man, we showed that duodenal TRPV6 mRNA expression was independent of blood 1,25(OH)(2)D(3), although in Caco-2 cells, 1,25(OH)(2)D(3)-dependent changes have been demonstrated. We sought to explain these findings. A consensus Vitamin D response element in the mouse gene is absent in the human gene. We re-analysed our duodenal expression data according to a CDX2-site polymorphism in the VDR promoter. Mean TRPV6 expression was the same, but there was evidence of different responsiveness to 1,25(OH)(2)D(3). In the GG genotype group, but not the AG, duodenal TRPV6 expression increased with 1,25(OH)(2)D(3). We postulate that lower levels of expression of VDR in the GG group produce greater sensitivity to 1,25(OH)(2)D(3).     

Vitamin D and skin cancer: a problem in gene regulation

The skin is the major source of Vitamin D(3) (cholecalciferol), and ultraviolet light (UV) is critical for its formation. Keratinocytes, the major cell in the epidermis, can further convert Vitamin D(3) to its hormonal form, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] (calcitriol). 1,25(OH)(2)D(3) in turn stimulates the differentiation of keratinocytes, raising the hope that 1,25(OH)(2)D(3) may prevent the development of malignancies in these cells. Skin cancers (squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanomas) are the most common cancers afflicting humans. UV exposure is linked to the incidence of these cancers-UV is thus good and bad for epidermal health. Our focus is on the mechanisms by which 1,25(OH)(2)D(3) regulates the differentiation of keratinocytes, and how this regulation breaks down in transformed cells. Skin cancers produce 1,25(OH)(2)D(3), contain ample amounts of the Vitamin D receptor (VDR), and respond to 1,25(OH)(2)D(3) with respect to induction of the 24-hydroxylase, but fail to differentiate in response to 1,25(OH)(2)D(3). Why not? The explanation may lie in the overexpression of the DRIP complex, which by interfering with the normal transition from DRIP to SRC as coactivators of the VDR during differentiation, block the induction of genes required for 1,25(OH)(2)D(3)-induced differentiation.     

Vitamin D and multiple sclerosis

Vitamin D is a principal regulator of calcium homeostasis. However, recent evidence has indicated that vitamin D can have numerous other physiological functions including inhibition of proliferation of a number of malignant cells including breast and prostate cancer cells and protection against certain immune mediated disorders including multiple sclerosis (MS). The geographic incidence of MS indicates an increase in MS with a decrease in sunlight exposure. Since vitamin D is produced in the skin by solar or UV irradiation and high serum levels of 25-hydroxyvitamin D (25(OH)D) have been reported to correlate with a reduced risk of MS, a protective role of vitamin D is suggested. Mechanisms whereby the active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) may act to mediate this protective effect are reviewed. Due to its immunosuppressive actions, it has been suggested that 1,25(OH)(2)D(3) may prevent the induction of MS.     

Vitamin D and autoimmune diabetes

The biologically active form of vitamin D, 1,25(OH)(2)D(3), is a potent modulator of the immune system as well as a regulator of bone and mineral metabolism. Vitamin D-deficiency in infancy and vitamin D receptor gene polymorphisms may be risk factors for insulin-dependent Diabetes mellitus (IDDM). 1,25(OH)(2)D(3) and its analogs significantly repress the development of insulitis and diabetes in the non-obese diabetic (NOD) mouse, a model of human IDDM. 1,25(OH)(2)D(3) may modulate IDDM disease pathogenesis by repression of type I cytokines, inhibition of dendritic cell maturation, and upregulation of regulatory T cells. The function of vitamin D as a genetic and environmental determining factor for IDDM, the protective role of 1,25(OH)(2)D(3) and its analogs in a mouse model of IDDM, and the possible mechanisms by which this protection occurs will be reviewed.     

1alpha,25(OH)2-vitamin D3 inhibits HGF synthesis and secretion from MG-63 human osteosarcoma cells

Several mesenchymally derived cells, including osteoblasts, secrete hepatocyte growth factor (HGF). 1alpha,25(OH)(2)-vitamin D(3) [1,25(OH)(2)D(3)] inhibits proliferation and induces differentiation of MG-63 osteoblastic cells. Here we show that MG-63 cells secrete copious amounts of HGF and that 1,25(OH)(2)D(3) inhibits HGF production. MG-63 cells also express HGF receptor (c-Met) mRNA, suggesting an autocrine action of HGF. Indeed, although exogenous HGF failed to stimulate cellular proliferation, neutralizing endogenous HGF with a neutralizing antibody inhibited MG-63 cell proliferation; moreover, inhibiting HGF synthesis with 1,25(OH)(2)D(3) followed by addition of HGF rescued hormone-induced inhibition of proliferation. Nonneutralized cells displayed constitutive phosphorylation of c-Met and the mitogen-activated protein kinases mitogen/extracellular signal-regulated kinase (MEK) 1 and extracellular signal-regulated kinase (Erk) 1/2, which were inhibited by anti-HGF antibody. Constitutive phosphorylation of Erk1/2 was also abolished by 1,25(OH)(2)D(3). Addition of HGF to MG-63 cells treated with neutralizing HGF antibody induced rapid phosphorylation of c-Met, MEK1, and Erk1/2. Thus endogenous HGF induces a constitutively active, autocrine mitogenic loop in MG-63 cells. The known antiproliferative effect of 1,25(OH)(2)D(3) on MG-63 cells can be accounted for by the concomitant 1,25(OH)(2)D(3)-induced inhibition of HGF production.     

Vitamin D: A millenium perspective

Vitamin D is one of the oldest hormones that have been made in the earliest life forms for over 750 million years. Phytoplankton, zooplankton, and most plants and animals that are exposed to sunlight have the capacity to make vitamin D. Vitamin D is critically important for the development, growth, and maintenance of a healthy skeleton from birth until death. The major function of vitamin D is to maintain calcium homeostasis. It accomplishes this by increasing the efficiency of the intestine to absorb dietary calcium. When there is inadequate calcium in the diet to satisfy the body's calcium requirement, vitamin D communicates to the osteoblasts that signal osteoclast precursors to mature and dissolve the calcium stored in the bone. Vitamin D is metabolized in the liver and then in the kidney to 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. 1,25(OH)(2)D receptors (VDR) are present not only in the intestine and bone, but in a wide variety of other tissues, including the brain, heart, stomach, pancreas, activated T and B lymphocytes, skin, gonads, etc. 1,25(OH)(2)D is one of the most potent substances to inhibit proliferation of both normal and hyperproliferative cells and induce them to mature. It is also recognized that a wide variety of tissues, including colon, prostate, breast, and skin have the enzymatic machinery to produce 1,25(OH)(2)D. 1,25(OH)(2)D and its analogs have been developed for treating the hyperproliferative disease psoriasis. Vitamin D deficiency is a major unrecognized health problem. Not only does it cause rickets in children, osteomalacia and osteoporosis in adults, but may have long lasting effects. Chronic vitamin D deficiency may have serious adverse consequences, including increased risk of hypertension, multiple sclerosis, cancers of the colon, prostate, breast, and ovary, and type 1 diabetes. There needs to be a better appreciation of the importance of vitamin D for overall health and well being.     

Nuclear lipid microdomains regulate nuclear vitamin D3 uptake and influence embryonic hippocampal cell differentiation

Despite recent advances in the understanding of the role of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) in the CNS, the mechanism of action remains obscure. We demonstrate that some 1,25-(OH)(2)D(3) receptor (VDR) is localized in the cell nucleus in specialized microdomains enriched in sphingomyelin and cholesterol; the integrity of these microdomains is necessary for embryonic hippocampal cell differentiation. Sphingomyelinase (SMase) treatment reduces both VDR and labeled 1,25-(OH)(2)D(3) content in nuclear microdomains. We have previously shown that HN9.10e embryonic hippocampal cells differentiate when incubated with 100 nM 1,25-(OH)(2)D(3) in the presence of 10% fetal calf serum, while serum deprivation induces cell death. In this study, we have investigated whether conditions that alter lipid content of nuclear microdomains modify 1,25-(OH)(2)D(3)-induced differentiation. Serum deprivation activates SMase and modifies the composition of nuclear microdomains, which lose the 1,25-(OH)(2) vitamin D(3) receptor. The incubation of serum-deprived cells with 100 nM 1,25-(OH)(2)D(3) prevents differentiation. However, treatment with 400 nM 1,25-(OH)(2)D(3) during serum withdrawal increases the lipid content of the nuclear microdomains, allows the interaction of 1,25-(OH)(2)D(3) with its receptor, and results in differentiation. These results suggest the presence of VDR in nuclear microdomains is necessary for 1,25-(OH)(2)D(3)-induced differentiation in embryonic hippocampal cells.     

Vitamin D regulated keratinocyte differentiation: role of coactivators

1,25 Dihydroxyvitamin D (1,25(OH)(2)D) regulates the differentiation of keratinocytes. 1,25(OH)(2)D raises intracellular free calcium (Cai) as a necessary early step toward stimulating differentiation. 1,25(OH)(2)D induces the calcium sensing receptor (CaR) in keratinocytes and enhances the calcium response of these cells. Activation of the CaR by calcium increases intracellular free calcium by a mechanism involving phospholipase C (PLC) cleavage of phosphatidylinositolbisphosphate into inositoltrisphosphate (IP(3)) and diacylglycerol (DG). 1,25(OH)(2)D induces the family of PLCs. PLC-gamma1 has a DR6 VDRE in its promoter which binds and is activated by VDR/RAR rather than VDR/RXR. The involucrin gene, which encodes a critical component of the cornified envelope, contains a DR3 VDRE in its promoter that acts in conjunction with a nearby AP-1 site. The sequential regulation of these genes is critical for the differentiation process. In undifferentiated keratinocytes, the VDR binds preferentially to the DRIP complex of coactivators. However, with differentiation DRIP 205 is no longer produced, and the VDR switches partners to the SRC family (SRC2 and 3). These studies suggest that at least part of the sequential activation of genes required during keratinocyte differentiation is regulated by the change (availability) of these different coactivator complexes.     

Recent advances in our understanding of 1,25-dihydroxyvitamin D(3) regulation of intestinal calcium absorption

Calcium is required for many cellular processes including muscle contraction, nerve pulse transmission, stimulus secretion coupling and bone formation. The principal source of new calcium to meet these essential functions is from the diet. Intestinal absorption of calcium occurs by an active transcellular path and by a non-saturable paracellular path. The major factor influencing intestinal calcium absorption is vitamin D and more specifically the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). This article emphasizes studies that have provided new insight related to the mechanisms involved in the intestinal actions of 1,25(OH)(2)D(3). The following are discussed: recent studies, including those using knock out mice, that suggest that 1,25(OH)(2)D(3) mediated calcium absorption is more complex than the traditional transcellular model; evidence for 1,25(OH)(2)D(3) mediated active transport of calcium by distal as well as proximal segments of the intestine; 1,25(OH)(2)D(3) regulation of paracellular calcium transport and the role of 1,25(OH)(2)D(3) in protection against mucosal injury.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus, especially those with antinuclear Abs and increased disease activity, had decreased 1,25(OH)(2)D(3) levels, suggesting that vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)(2)D(3) on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)(2)D(3), although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity, including 1 alpha-hydroxylase, 24-hydroxylase, and the vitamin D receptor, each of which was regulated by 1,25(OH)(2)D(3) and/or activation. Importantly, 1,25(OH)(2)D(3) up-regulated the expression of p27, but not of p18 and p21, which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)(2)D(3) may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders.