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Using MRI on mice bearing a targeted knockout (KO) of the 1,25D3 -MARRS receptor/PDIA3/ERp57 we found that they had decreased body fat relative to their littermate (LM) controls, a condition associated with increased lifespan. Others have found that lower body fat is correlated with decreased lipid droplets in intestinal cells that may be mediated by a factor secreted by germ cells (possibly estradiol). In a reducing environment estradiol competed for binding to the 1,25D3-MARRS receptor/PDIA3/ERp57. A consequence of this was that estradiol stimulated calcium uptake in enterocytes isolated from LM mice. In time course studies, lipid droplets increased in response to 1 nM estradiol from 1-5 D of culture, relative to corresponding controls, while at 6 and 7 D this steroid decreased lipid droplets. Enterocytes from LM or KOs incubated with estradiol for 1-4 D showed the hormone increased lipid droplets. Using the 4 D culture period, 1 and 10 nM estradiol significantly increased the number of lipid droplets in cells from LM mice by 40-60%, compared to equivalent conditions in KO mice. In assessing signal transduction pathways, the hormone increased phospho-Akt levels, but no differences were observed in phospho-mTORC1, or phospho-S6K (although cells from chicks did exhibit a hormone-mediated difference). Finally, the remaining mice (which had stopped reproducing) were allowed to die naturally and lifespan recorded. LM mice lived 687 ± 77 D (without an outlying value) while KO mice lived 740 D ± 80 D. These data suggest the 25D3 -MARRS receptor/PDIA3/ERp57 may contribute to the length of lifespan in mammals.

Steroid hormone receptors localized in the target cell nucleus have been well documented to play a major role in regulating key genes important for organ development and function. These include genes that modulate brain and pituitary, reproductive tract, mammary gland, bone, adipose tissue, metabolic and immune cell functions. Importantly, substantial evidence now demonstrates that steroid receptors also exist outside the nucleus in many organs and cells. Although the nature and functions of many of these receptors is currently being defined, important aspects of extranuclear steroid receptor localization in cell membranes and downstream signaling to intracellular processes that govern normal physiology and disease are emerging. Translation of these basic research findings on membrane steroid hormone receptors to the clinic holds promise for developing new strategies and therapeutic interventions for the benefit of patients afflicted with neurologic, psychiatric, oncologic, cardiovascular and endocrine-metabolic diseases.

As immune defects in amyloid-β (Aβ) phagocytosis and degradation underlie Aβ deposition and inflammation in Alzheimer’s disease (AD) brain, better understanding of the relation between Aβ phagocytosis and inflammation could lead to promising preventive strategies. We tested two immune modulators in peripheral blood mononuclear cells (PBMCs) of AD patients and controls: 1α,25(OH)2-vitamin D3 (1,25D3) and resolvin D1 (RvD1). Both 1,25D3 and RvD1 improved phagocytosis of FAM-Aβ by AD macrophages and inhibited fibrillar Aβ-induced apoptosis. The action of 1,25D3 depended on the nuclear vitamin D and the protein disulfide isomerase A3 receptors, whereas RvD1 required the chemokine receptor, GPR32. The activities of 1,25D3 and RvD1 commonly required intracellular calcium, MEK1/2, PKA, and PI3K signaling; however, the effect of RvD1 was more sensitive to pertussis toxin. In this case study, the AD patients: a) showed significant transcriptional up regulation of IL1RN, ITGB2, and NFκB; and b) revealed two distinct groups when compared to controls: group 1 decreased and group 2 increased transcription of TLRs, IL-1, IL1R1 and chemokines. In the PBMCs/macrophages of both groups, soluble Aβ (sAβ) increased the transcription/secretion of cytokines (e.g., IL1 and IL6) and chemokines (e.g., CCLs and CXCLs) and 1,25D3/RvD1 reversed most of the sAβ effects. However, they both further increased the expression of IL1 in the group 1, sβ-treated cells. We conclude that in vitro, 1,25D3 and RvD1 rebalance inflammation to promote Aβ phagocytosis, and suggest that low vitamin D3 and docosahexaenoic acid intake and/or poor anabolic production of 1,25D3/RvD1 in PBMCs could contribute to AD onset/pathology.

We have used mice with a targeted knockout (KO) of the 1,25D(3)-MARRS receptor (ERp57/PDIA3) in intestine to study rapid responses to 1,25-dihydroxyvitamin D(3) [1,25D(3)] with regards to calcium or phosphate uptake. Western analyses indicated the presence of the 1,25D(3)-MARRS receptor in littermate (LM) mice, but not KO mice. Saturation analyses for [(3)H]1,25D(3) binding revealed comparable affinities for the hormone in lysates from female and male LM, but a reduced B(max) in females. Binding in lysates from KO mice was absent or severely reduced. Enterocytes from KO mice failed to respond to hormone with regard to either ion uptake, while cells from LM mice exhibited an increase in uptake. For calcium uptake, the protein kinase (PK) A pathway mediated the response to 1,25D(3). Enterocytes from LM mice responded to 1,25D(3) with enhanced PKA activity, while cells from KO mice did not, although both cell types responded to forskolin. Calcium transport in LM mice in vivo was greater than in KO mice. Cells from LM and KO mice had cell surface VDR; however, anti-VDR antibodies had no effect on ion uptake. Unlike chicks, the PKC pathway was not involved in phosphate uptake. As in chicks and rats, intestinal cells from adult male mice lost the ability to respond to 1,25D(3) with enhanced phosphate uptake, whereas in female mice, uptake in cells from adults was greater than that observed in young mice. Finally, when we tested phosphate uptake in vivo, we found that young female mice had a much greater rate of transport than young male mice.

UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.

We have crossed ERp57flx/flx mice with commercially available mice expressing villin-driven cre-recombinase. Enterocytes isolated from 3- to 4-wk-old littermate (LM) male mice responded to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] with enhanced phosphate uptake relative to corresponding controls within 1 min of addition, whereas in cells from targeted knockout (KO) mice, the response was severely blunted. Unlike chick enterocytes, mouse enterocytes did not respond to phorbol ester with enhanced phosphate uptake. However, forskolin, which does not stimulate phosphate uptake in chick intestinal cells, did so in enterocytes isolated from either young male LM or KO mice. Intestinal cells isolated from young female LM mice also responded to 1,25(OH)2D3 with enhanced phosphate uptake within 5 min of hormone addition, whereas cells from KO mice did not. Forskolin also stimulated phosphate uptake in enterocytes from young female KO or LM mice. As with intestinal cells from adult male chickens or rats, cells from adult (8 wk) male LM mice lost the ability to respond to 1,25(OH)2D3 with enhanced phosphate uptake. In contrast, intestinal cells from adult female LM mice did respond with enhanced phosphate uptake within 1 min of steroid hormone addition relative to corresponding controls, and the magnitude of the effect was greater than that observed in enterocytes of young females. Cells isolated from young or adult male or female LM mice failed to respond to 1,25(OH)2D3 with enhanced protein kinase C activity. Finally, we have previously reported that mouse enterocytes have cell surface vitamin D receptor; however preincubation of such cells with anti-vitamin D receptor antibodies demonstrated that the classical receptor is not involved in the rapid 1,25(OH)2D3-stimulated uptake of phosphate.

We undertook studies to determine which isotype(s) of protein kinase C (PKC) is/are activated by ligand binding to the 1,25D(3)-MARRS receptor (ERp57/PDIA3) and subsequent stimulation of phosphate uptake. Isolated intestinal epithelial cells from vitamin D-replete chicks were exposed to 1,25(OH)(2)D(3) for 1, 3, or 5min, thoroughly chilled, homogenized, and P(2) fractions (20,000xg post-nuclear pellet) prepared. Western analyses with anti-pan PKC revealed steroid-stimulated redistribution to P(2) membranes 1min after hormone. Using this time point, cells were treated with vehicle, 130-, 300- or 650-pM hormone. Western blots with anti-PKCalpha exhibited redistribution to membranes in a biphasic dose-response curve: slightly stimulated at the lowest dose, maximal at 300pM 1,25(OH)(2)D(3), and equivalent to control levels at the highest dose, paralleling hormone-mediated phosphate uptake. Westerns with anti PKCbeta also revealed hormone-mediated differences, while those with anti PKCgamma did not. RNAi studies were then performed with siRNA against PKCalpha or PKCbeta. Untransfected cells treated with hormone for 7min exhibited enhanced (32)P uptake relative to vehicle controls. Cells transfected with either active siRNA revealed decreased (32)P uptake in both controls (relative to untransfected controls), and hormone treated cells. However, control and transfected cells treated with hormone had equivalent levels of uptake. Western blot analyses confirmed decreased immunoreactivity in transfected cells. Chemical PKCalpha (safingol) and PKCbeta ([3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione] blockers also confirmed the results from siRNA and demonstrated decreased (32)P uptake in cells treated with 1,25(OH)(2)D(3) plus blockers in comparison with cells treated with 1,25(OH)(2)D(3) alone. Thus, PKCalpha and PKCbeta are both involved in steroid-stimulated phosphate uptake.

1,25 Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D{sub 3}, which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D{sub 3}-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NF{kappa}B). In unstimulated cells, 1,25D{sub 3}-MARRS can be co-immunoprecipitated with antibodies directed at NF{kappa}B, and NF{kappa}B is co-precipitated when antibodies against 1,25D{sub 3}-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D{sub 3}-MARRS and NF{kappa}B begin translocating to the nucleus within minutes of co-stimulation with 1,25D{sub 3} and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D{sub 3}-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NF{kappa}B and other factors with which it may be interacting.

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)2D3 traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)2D3 called 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D3-MARRS expression modulates 1,25(OH)2D3 activity in breast cancer cells. Relative levels of 1,25D3-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D3-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)2D3 in MCF-7 cells, a ribozyme construct designed to knock down 1,25D(3)-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D3-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)2D3 ( IC(50) 56+/-24 nM) compared to controls (319+/-181 nM; P

Milk fat globule membrane (MFGM) is a biopolymer composed primarily of membrane proteins and lipids that surround the fat globules in milk. Although it is considered to have potential as a bioactive ingredient, few feeding studies have been conducted to measure its potential benefits. The aim of this investigation was to determine if dietary MFGM confers protection against colon carcinogenesis compared to diets containing corn oil (CO) or anhydrous milk fat (AMF). Male, weanling Fischer-344 rats were randomly assigned to one of three dietary treatments that differed only in the fat source: (1) AIN-76A diet, corn oil; (2) AIN-76A diet, AMF; and (3) AIN-76A diet, 50% MFGM, 50% AMF. Each diet contained 50 g/kg diet of fat. With the exception of the fat source, diets were formulated to be identical in macro and micro nutrient content. Animals were injected with 1,2-dimethylhydrazine once per week at weeks 3 and 4, and fed experimental diets for a total of 13 weeks. Over the course of the study dietary treatment did not affect food consumption, weight gain or body composition. After 13 weeks animals were sacrificed, colons were removed and aberrant crypt foci (ACF) were counted by microscopy. Rats fed the MFGM diet (n = 16) had significantly fewer ACF (20.9 +/- 5.7) compared to rats fed corn oil (n = 17) or AMF (n = 16) diets (31.3 +/- 9.5 and 29.8 +/- 11.4 respectively; P < 0.05). Gene expression analysis of colonic mucosa did not reveal differential expression of candidate colon cancer genes, and the sphingolipid profile of the colonic mucosa was not affected by diet. While there were notable and significant differences in plasma and red blood cell lipids, there was no relationship to the cancer protection. These results support previous findings that dietary sphingolipids are protective against colon carcinogenesis yet extend this finding to MFGM, a milk fat fraction available as a food ingredient.

Transport of 45Ca from the lumen to the venous effluent was studied in duodena of normal, vitamin D3-replete (+D) chicks perfused through the celiac artery with 130 pM 1,25(OH)2D3 or vehicle. Administration of actinomycin D 3 h prior to perfusion did not alter the unstimulated transport rate or diminish the response to exogenous 1,25(OH)2D3: After 40 min exposure to the seco-steroid, 45Ca in the vascular effluent was 140% of control levels. The anti-microfilament agent cytochalasin b and the ionophore monensin, an inhibitor of Golgi function, similarly failed to suppress 1,25(OH)2D3-stimulated calcium transport. In pilot studies, Golgi and basal-lateral membrane fractions were prepared from duodenal epithelium of vitamin D-deficient (-D) chicks treated with vehicle or 650 pmol of 1,25(OH)2D3in vivo 2 h, 10 h, or 15 h before sacrifice, as well as from +D birds. Analyses of Golgi fractions for cathepsin B (CB) activity revealed a biphasic response with time, increasing to 200% of -D levels 2 h after 1,25(OH)2D3 administration and in equivalent preparations from +D birds. Less pronounced increases in acid phosphatase activity were observed in the same membrane fractions. In basal-lateral membranes, enhanced CB activity was detectable 10 h after 1,25(OH)2D3in vivo, rose to 155% of -D levels at 15 h, and to 245% of controls in fractions from +D birds, whereas acid phosphatase was 75%, 81%, and 125% of controls, respectively, at these times. Levels of CB in whole homogenates from which membranes were isolated rose to 200% of controls at 2 h and 10 h after 1,25(OH)2D3, but declined to 112% of controls in +D chicks. Acid phosphatase levels in the same homogenates were lower than or equivalent to control levels. These findings suggested additional perfusion studies in normal birds. Although vascular perfusion with iodoacetate and 1,25(OH)2D3, or leupeptin and 1,25(OH)2D3, failed to inhibit the effect of the vitamin D metabolite, 1,25(OH)2D3-enhanced calcium transport was abolished by addition of 1 mM leupeptin to the lumenal medium, but not by 1 mM pepstatin. The combined data suggest that lysosomes or their hydrolases may be involved in 1,25(OH)2D3-mediated transport of calcium across intestinal epithelial cells.

Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.

Calcium is an essential ion in all organisms and participates in a variety of structural and functional roles. Calcium (re)absorption occurs in epithelia, including the intestine, kidney, mammary glands, placenta, and gills of fish. Its transport is regulated by a complex array of processes that are mediated by hormonal, developmental, and physiological factors involving the gastrointestinal tract, bone, kidney, and the parathyroids. Here we review the calcium transport mechanisms—paracellular, which is energy independent, and transcellular, which is energy dependent—primarily focusing on the intestine. We provide a new perspective on the facilitated diffusion and vesicular transport models to account for the emerging concepts on transcellular calcium transport. Finally, we discuss how 1,25(OH)2D3 and parathyroid hormone regulate calcium transport.

Endochondral ossification comprises a cascade of cell differentiation culminating in chondrocyte hypertrophy and is negatively controlled by soluble environmental mediators at several checkpoints. Proteinases modulate this control by processing protein signals and/or their receptors. Here, we show that insulin-like growth factor I can trigger hypertrophic development by stimulating production and/or activation of proteinases in some populations of chick embryo chondrocytes. Cell surface targets of the enzymes include 1,25-dihydroxyvitamin D3 membrane-associated rapid response steroid receptor (1,25 D3 MARRS receptor), also known as ERp57/GRp58/ERp60. This protein is anchored to the outer surface of plasma membranes and inhibits late chondrocyte differentiation after binding of 1,25-dihydroxyvitamin D3. Upon treatment with insulin-like growth factor I, 1,25 D3 MARRS receptor is cleaved into two fragments of approximately 30 and 22 kDa. This process is abrogated along with hypertrophic development by E-64 or cystatin C, inhibitors of cysteine proteinases. Cell differentiation is enhanced by treatment with antibodies to 1,25 D3 MARRS receptor that either block binding of the inhibitory ligand 1,25-dihydroxyvitamin D3 or inactivate 1,25 D3 MARRS receptor left intact after treatment with proteinase inhibitors. Therefore, proteolytic shedding of 1,25 D3 MARRS receptor constitutes a molecular mechanism eliminating the 1,25-dihydroxyvitamin D3-induced barrier against late cartilage differentiation and is a potentially important step during endochondral ossification or cartilage degeneration in osteoarthritis.

Our concepts of hormone receptors have, until recently, been narrowly defined. In the last few years, an increasing number of reports identify novel proteins, such as enzymes, acting as receptors. In this review we cover the novel receptors for the hormones atrial naturetic hormone, enterostatin, hepcidin, thyroid hormones, estradiol, progesterone, and the vitamin D metabolites 1,25(OH)2D3 and 24,25(OH)2D3. J. Cell. Biochem. 103: 401–407, 2008. © 2007 Wiley-Liss, Inc.

ERp57/GRp58 is a thiol-protein disulphide oxidoreductase and has been studied in many clinically relevant systems, both as a chaperone protein and as a membrane receptor for the steroid hormone, 1,25(OH)2D3. Our laboratory investigates phenomena associated with rapid, membrane-initiated signaling by steroid hormones synthesized from vitamin D (cholecalciferol). We have recently reported that the cell surface receptor for the metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which we have termed the 1,25D3-MARRS (Membrane Associated, Rapid Response Steroid binding) receptor, is in fact identical to ERp57/GRp58. Here we review the dynamic role ERp57/GRp58/1,25D3-MARRS receptor plays in a variety of cellular processes. Starting with its structure at the DNA and protein levels, we review the available literature about its role as a chaperone protein, in immune function through the assembly of MHC class I molecules, DNA binding, and its function as the 1,25D3-MARRS receptor. Finally, we present the role it may play in relation to important disease states. While ERp57/GR58/1,25D3-MARRS receptor is a pivotal protein in many cell functions, it has yet to be determined whether-and to what extent-these phenomena are regulated by the vitamin D endocrine system. However, 1,25(OH)2D3 is involved in differentiation of certain cancer cells and in muscle function, and ERp57/1,25D3-MARRS protein has been reported to be involved in such processes. Thus, medicinal chemistry aimed at the 1,25D3-MARRS receptor in lymphocytes, cancer cells, bone, intestinal epithelia, and kidney may add to the current therapeutic regimens for various disease states.

Membrane-initiated signaling by steroid hormones is now widely accepted. Current debate is centered upon which protein moieties act as membrane-associated receptors. In this review, we consider evidence for the classical vitamin D receptor (VDR) in this role, as well as the more recently identified 1,25D3-MARRS (membrane-associated, rapid response steroid binding) receptor, also known as ERp57/GRp58. The structure of the 1,25D3-MARRS receptor is discussed, with emphasis on two thioredoxin domains that promote dimerization and ligand binding. We then summarize recent studies on a 24,25(OH)2D3 binding protein--catalase--and how ligand-induced decreases in enzymatic activity produce increased reactive oxygen species that target both the 1,25D3-MARRS receptor--but not the VDR--and the protein kinase C signaling pathway. Finally, we briefly discuss the available literature suggesting that the metabolite 25(OH)D3 may also be biologically active.

We previously reported that diosgenin, a plant-derived steroidal sapogenin, improved memory and reduced axonal degeneration in an Alzheimer's disease mouse model. Diosgenin directly activated the membrane-associated rapid response steroid-binding receptor (1,25D3-MARRS) in neurons. However, 1,25D3-MARRS-mediated diosgenin signaling was only shown in vitro in the previous study. Here, we aimed to obtain in vivo evidence showing that diosgenin signaling is mediated by 1,25D3-MARRS in the mouse brain. Diosgenin treatment in normal mice enhanced object recognition memory and spike firing and cross-correlation in the medial prefrontal cortex and hippocampal CA1. In diosgenin-treated mice, axonal density and c-Fos expression was increased in the medial prefrontal and perirhinal cortices, suggesting that neuronal network activation may be enhanced. The diosgenin-induced memory enhancement and axonal growth were completely inhibited by co-treatment with a neutralizing antibody for 1,25D3-MARRS. Our in vivo data indicate that diosgenin is a memory-enhancing drug and that enhancement by diosgenin is mediated by 1,25D3-MARRS-triggered axonal growth.

There is increasing evidence that the vitamin D metabolite, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) has endocrine actions. In the current work, we report that an endogenous binding protein for 24,25(OH)2D3 is catalase, based on sequence analysis of the isolated protein. An antibody (Ab 365) generated against equivalent protein recognized bovine catalase and a 64 kDa band in subcellular fractions of chick intestine. A commercially available anti-catalase antibody reduced specific [3H]24,25(OH)2D3 binding in subcellular fractions of chick intestine by greater than 65%, relative to the same fractions treated with an unrelated antibody (Ab 099). The same commercially available anti-catalase was able to block the inhibitory actions of 24,25(OH)2D3 on 32P uptake in isolated intestinal epithelial cell suspensions. We subsequently characterized binding of steroid to commercially available catalase, and found that between 0 and 5 nM of enzyme added to subcellular fraction P2 (20,000g, 10-min post-nuclear pellet) resulted in a linear increase in the amount of [3H]24,25(OH)2D3 specifically bound. Additional studies indicated that 25(OH)D3 was an effective competitor for binding, whereas 1,25(OH)2D3 only poorly displaced [3H]24,25(OH)2D3. Saturation analyses with added catalase yielded a physiologically relevant affinity constant (KD=5.6+/-2.7 nM) and a Bmax=209+/-34 fmols/mg protein, comparable to previous studies using purified basal lateral membranes or vesicular fractions. Moreover, in a study on subcellular fractions isolated from chickens of varying ages, we found that in females, both specific [3H]24,25(OH)2D3 binding and catalase activity increased from 7- to 58-week-old birds, whereas in males, elevated levels of both parameters were expressed in preparations of 7- and 58-week-old birds. The data suggest that signal transduction may occur through modulation of hydrogen peroxide production.

We report the presence of a mammalian equivalent of the avian M embrane- A ssociated R apid R esponse, S teroid (1,25D 3 -MARRS)-binding protein specific for 1,25(OH) 2 D 3 in a rat small intestinal cell line, IEC-6, that demonstrates rapid responses to the steroid hormone. Identification of transcript and protein was achieved using RT-PCR with several specific primer sets, Western blot analysis with two separate antibodies recognizing distinct regions of the protein, ribozyme knockdown and immunohistochemistry. Promoter analysis of the 1000-bp upstream region of the 1,25D 3 -MARRS gene in several species revealed the presence of a conserved smad-3 element in the 5′ proximal promoter region, but no classical vitamin D response element (VDRE). Treatment of IEC-6 cells with transforming growth factor β1 (TGFβ1) increased steady-state levels of 1,25D 3 -MARRS (mRNA and protein) approximately two-fold over a 24-h period. In contrast, treatment with 1,25(OH) 2 D 3 failed to significantly change 1,25D 3 -MARRS protein or mRNA levels. Localization studies showed rapid nuclear translocation of a pool of 1,25D 3 -MARRS protein after 1,25(OH) 2 D 3 treatment, suggesting that the protein is subject to membrane-initiated signal pathway activation. Together these data point to complex interactions between the two important 1,25(OH) 2 D 3 sensitive response systems in intestinal cells, 1,25D 3 -MARRS protein and the well-studied nVDR, that together work to fine tune intestinal Ca 2+ absorption in a variety of avian and mammalian species.

Scattered reports in the literature have suggested that the metabolite 25-hydroxyvitamin D3 [25(OH)D3] has biological activity. In the present work, perfusion of isolated duodenal loops of normal chickens with 100 nM 25(OH)D3 resulted in enhanced transport of 45Ca within 2 min relative to the vehicle controls. We then tested the effect of a range of 25(OH)D3 concentrations on 45Ca handling by isolated intestinal cells in time course studies. Following a basal uptake period, cell suspensions from 7-week old chicks were treated either with 25, 100, or 300 nM 25(OH)D3, or the vehicle ethanol (0.01%, final concentration). Both 25 and 100 nM 25(OH)D3 resulted in a significant (P

To study the physiological relevance of membrane-initiated steroid signaling, we investigated the correlation of age in male chickens with the magnitude of responses to 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in duodena from 7-, 14-, 28-, and 58-wk-old birds. Measurements of 1,25-(OH)(2)D(3) (130 pM) responsiveness as a function of age, showed a decreased intestinal Ca(2+) transport. Western analyses of isolated basal lateral membranes indicated a decreased expression of the membrane-associated rapid response binding protein with increasing age. Saturation analyses of [(3)H]1,25-(OH)(2)D(3) binding to basal lateral membranes, revealed an allosteric interaction identified as cooperative binding. A significant increase in K(d) was observed with increasing age, indicating decreasing affinity. Determinations of the number of binding sites yielded a binding capacity of 190-250 fmol/mg protein during growth and maturation, whereas in adulthood (58 wk) saturable binding was no longer observed. Data obtained in parallel analyses of binding of [(3)H]1,25-(OH)(2)D(3) to nuclear fraction vitamin D receptor, in contrast, indicated an absence of cooperative binding and an absence of significant changes in K(d) or binding capacity with age. Membrane-initiated signal transduction by 1,25-(OH)(2)D(3) was assessed by determination of protein kinase C and A activities. Stimulation of protein kinase C activity in response to 1,25-(OH)(2)D(3) decreased with age, whereas no age-correlated changes in steroid-stimulated protein kinase A activities were observed. Thus, in conclusion, our experiments demonstrate that there is a decrease in responsiveness to exogenous 1,25-(OH)(2)D(3) as a function of age in duodena of male chickens, which can be correlated to a decreased affinity for 1,25-(OH)(2)D(3), a reduced expression of membrane-associated rapid response binding protein, and a decreased protein kinase C activity.

In freshwater-adapted rainbow trout, intestinal cells (enterocytes) possess receptors for 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in the basolateral membrane, and respond to treatment with 1,25(OH)(2)D(3) with increased intracellular calcium concentrations. No receptors are found for the antagonizing hormone 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] at the enterocyte basolateral membrane, and it has no effect on enterocyte calcium homeostasis. After acclimation to seawater, however, the enterocyte membrane receptors for 1,25(OH)(2)D(3) are down-regulated and specific binding for 24,25(OH)(2)D(3) appears, which is further up-regulated with time spent in seawater. This shift in receptor expression is concurrent with an increased sensitivity of the enterocytes to 24,25(OH)(2)D(3) and a decreased sensitivity to 1,25(OH)(2)D(3). This results in a partial inhibition of intracellular calcium uptake, which would be beneficial when inhabiting a calcium-rich environment like seawater.

Membrane receptors for steroid hormones affect signaling pathways that modulate nuclear function, influence neuronal activity, ion flow, and the circulatory system. Indeed, 'new' steroid hormones have been identified by their interaction with membrane-initiated signaling systems. A brief summary of the FASEB Summer Research Conference devoted to these topics is presented in this mini-review. In addition, attendees of the meeting propose introduction of the following terminology: membrane-initiated steroid signaling (MISS) and nuclear-initiated steroid signaling (NISS) to replace more inaccurate terms in current use.

The calciotropic hormone, 1,25(OH) 2 vitamin D 3 [1,25(OH) 2 D 3 ], controls the formation of dental and bone mineralized tissues. The role of nuclear 1,25(OH) 2 D 3 receptor has been extensively studied in the diverse secretory cells, i.e., osteoblasts, chondrocytes, ameloblasts, and odontoblasts. A nongenomic pathway also has been characterized and related to the interactions of 1,25(OH) 2 D 3 ligand with a putative cell membrane receptor. This recognition moiety called 1,25(OH) 2 vitamin D 3 membrane-associated, rapid-response steroid-binding [1,25D 3 -MARRS] protein is investigated here in the craniofacial skeleton of human embryos and fetuses. Immunolocalization using a specific Ab099 against chick intestinal basolateral 1,25D 3 -MARRS protein was performed. The data show a complementary expression pattern of the membrane receptor when compared with published data on the nuclear receptor, notably during amelogenesis. In mandible, membrane receptors for 1,25(OH) 2 D 3 were identified in the heterogeno...

The calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has been established to control skeletal tissue formation and biomineralization via the regulation of gene expression. This action involves the well-characterized nuclear 1,25(OH)2D3 receptor. However, it has been recognized that several cellular responses to 1,25(OH)2D3 may not to be related to the exclusive nuclear receptor. Indeed, this secosteroid is able to generate rapid responses that have been proposed to be mediated by interactions of the ligand, which is a putative cell membrane-associated rapid-response steroid (MARRS) binding protein for 1,25(OH)2D3 [1,25D3-MARRS]. The nongenomic pathway of 1,25(OH)2D3 was studied here in detail by immunolocalization of the 1,25D3-MARRS during the specific context of human prenatal development. Western blotting with proteins extracted from 4 week- to 27-week-old embryos was performed, evidencing a 65-kDa molecular species recognized by antibody Ab 099 generated against synthetic peptides corresponding to the N terminus of the 1,25D3-MARRS from chick intestinal basolateral membranes. Based on this biochemical conservation of protein in the human species, the temporospatial expression patterns were established in the craniofacial skeleton at the same ages. Comparative analysis was performed in teeth and bones from early morphogenesis to terminal cell differentiation and extracellular biomineralization. The data show the potential implication of 1,25D3-MARRS in the heterogeneous cell population including ameloblasts, odontoblasts, osteoblasts, and osteoclasts. The epithelial-mesenchymal cascade related to odontogenesis was coincident with a sequence of up- and down-regulation of immunoreactive 1,25D3-MARRS. Biomineralization was associated with a striking up-regulation in the adjoining secretory cells in all tissues. Finally, osteoclasts appeared also to express the 1,25D3-MARRS during these early phases of bone modeling. Previously obtained data of the nuclear vitamin D receptor (VDR) expression and this study on 1,25D3-MARRS suggest the existence of cross-talk between the genomic and nongenomic pathways during human development.

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha,25(OH)(2)D(3). Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha,25(OH)(2)D(3) in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha,25(OH)(2)D(3) during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha,25(OH)(2)D(3), PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha,25(OH)(2)D(3) was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1alpha,25(OH)(2)D(3) membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha,25(OH)(2)D(3)-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1alpha,25(OH)(2)D(3) in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G(i)/G(s) had no effect. However, inhibition of G(q) blocked the effect of 1alpha,25(OH)(2)D(3). The rapid effect of 1alpha,25(OH)(2)D(3) also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1alpha,25(OH)(2)D(3) at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.

1 alpha,25-(OH)(2)D(3) exerts its effects on chondrocytes and enterocytes via nuclear receptors (1,25-nVDR) and a separate membrane receptor (1,25-mVDR) that activates protein kinase C (PKC). 24R,25-(OH)(2)D(3) also stimulates PKC in chondrocytes, but through other membrane mechanisms. This study examined the hypothesis that osteoblasts possess distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) that are involved in the activation of PKC and that receptor expression varies as a function of cell maturation state. 1 alpha,25-(OH)(2)D(3) stimulated PKC in well differentiated (UMR-106, MC-3T3-E1) and moderately differentiated (ROS 17/2.8) osteoblast-like cells, and in cultures of fetal rat calvarial (FRC) cells and 2T3 cells treated with rhBMP-2 to promote differentiation. 24R,25-(OH)(2)D(3) stimulated PKC in FRC and 2T3 cultures that had not been treated to induce differentiation, and in ROS 17/2.8 cells. MG63 cells, a relatively undifferentiated osteoblast-like cell line, had no response to either metabolite. Ab99, a polyclonal antibody generated to the chick enterocyte 1,25-mVDR, but not a specific antibody to the 1,25-nVDR, inhibited response to 1 alpha,25-(OH)(2)D(3). 1 alpha,25-(OH)(2)D(3) exhibited specific binding to plasma membrane preparations from cells demonstrating a PKC response to this metabolite that is typical of positive cooperativity. Western blots of these membrane proteins reacted with Ab99, and the Ab99-positive protein had an Mr of 64 kDa. There was no cross-reaction with antibodies to the C- or N-terminus of annexin II. The effect of 24,25-(OH)(2)D(3) on PKC was stereospecific; 24S,25-(OH)(2)D(3) had no effect. These results demonstrate that response to 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) depends on osteoblast maturation state and suggest that specific and distinct membrane receptors are involved.

Dogma for the past three decades has dictated that parathyroid hormone (PTH) has no direct effect on intestine with regard to calcium or phosphate absorption, but rather that PTH acts to promote the synthesis of a hormonally active form of vitamin D, namely 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, diverse laboratories have each provided some evidence to suggest PTH does indeed have a direct effect on intestine. We will briefly review the evidence for biological effects, biochemical effects, and the presence of intestinal receptors for PTH, and conclude with the implications for biomedical research. J. Cell. Biochem. 86: 29–34, 2002. © 2002 Wiley-Liss, Inc.

Using MRI on mice bearing a targeted knockout (KO) of the 1,25D3 -MARRS receptor/PDIA3/ERp57 we found that they had decreased body fat relative to their littermate (LM) controls, a condition associated with increased lifespan. Others have found that lower body fat is correlated with decreased lipid droplets in intestinal cells that may be mediated by a factor secreted by germ cells (possibly estradiol). In a reducing environment estradiol competed for binding to the 1,25D3-MARRS receptor/PDIA3/ERp57. A consequence of this was that estradiol stimulated calcium uptake in enterocytes isolated from LM mice. In time course studies, lipid droplets increased in response to 1 nM estradiol from 1-5 D of culture, relative to corresponding controls, while at 6 and 7 D this steroid decreased lipid droplets. Enterocytes from LM or KOs incubated with estradiol for 1-4 D showed the hormone increased lipid droplets. Using the 4 D culture period, 1 and 10 nM estradiol significantly increased the number of lipid droplets in cells from LM mice by 40-60%, compared to equivalent conditions in KO mice. In assessing signal transduction pathways, the hormone increased phospho-Akt levels, but no differences were observed in phospho-mTORC1, or phospho-S6K (although cells from chicks did exhibit a hormone-mediated difference). Finally, the remaining mice (which had stopped reproducing) were allowed to die naturally and lifespan recorded. LM mice lived 687 ± 77 D (without an outlying value) while KO mice lived 740 D ± 80 D. These data suggest the 25D3 -MARRS receptor/PDIA3/ERp57 may contribute to the length of lifespan in mammals.

Antisera were raised against the NH2-terminus of the putative basal lateral membrane (BLM) receptor for 1,25-dihydroxyvitamin D3[1,25(OH)2D3; BLM-VDR]. In Western analyses of BLM proteins, antibody (Ab) 099 was monospecific for a 64.5-kDa band. A protein of 64.5 kDa was also labeled by the affinity ligand [14C]1,25(OH)2D3-bromoacetate; label was diminished in the presence of excess unlabeled secosteroid. The monoclonal antibody against the nuclear VDR (9A7) failed to detect an appropriate band in BLM fractions. Preincubation of isolated intestinal cells with Ab 099, but not 9A7, affected the following two 1,25(OH)2D3-mediated signal transduction events: augmented intracellular calcium and protein kinase C activity. Subcellular distribution of Ab 099 reactivity by Western analyses and fluorescence microscopy revealed the highest concentrations in BLM followed by the endoplasmic reticulum. Exposure of isolated intestinal cells to 1,25(OH)2D3for 10 s or vascular perfusion of duodena for 5 min resulted in a time-dependent increase in nuclear localization of the BLM-VDR antigen, as judged by electron microscopy, whereas 24,25-dihydroxyvitamin D3failed to increase antigenic labeling in nuclei. Densitometric quantitation of Western blots of subcellular fractions prepared from isolated intestinal cells treated with vehicle or 1,25(OH)2D3confirmed a hormone-induced increase of putative BLM-VDR in the nucleus. It is concluded that a novel cell surface binding protein for 1,25(OH)2D3has been identified.

1,25-(OH)2D3 (1,25) exerts its effects on growth plate chondrocytes through classical vitamin D (VDR) receptor-dependent mechanisms, resulting in mineralization of the extracellular matrix. Recent studies have shown that membrane-mediated mechanisms are involved as well. 1,25 targets cells in the prehypertrophic and upper hypertrophic zones of the costochondral cartilage growth plate (GC cells), resulting in increased specific activity of alkaline phosphatase (ALP), phospholipase A2 (PLA2), and matrix metalloproteinases (MMPs). At the cellular level, 1,25 action results in rapid changes in arachidonic acid (AA) release and re-incorporation, alterations in membrane fluidity and Ca ion flux, and increased prostaglandin E1 and E2 (PGE2) production. Protein kinase C (PKC) is activated in a phospholipase C (PLC) dependent-mechanism, due in part to the increased production of diacylglycerol (DAG). In addition, AA acts directly on the cell to increase PKC specific activity. AA also provides a substrate for cyclooxygenase (COX), resulting in PGE2 production. 1,25 mediates its effects through COX-1, the constitutive enzyme, but not COX-2, the inducible enzyme. Time course studies using specific inhibitors of COX-1 show that AA stimulates PKC activity and PKC then stimulates PGE2 production. PGE2 acts as a mediator of 1,25 action on the cells, also stimulating PKC activity. The rapid effects of 1,25 on PKC are nongenomic, occurring within 3 min and reaching maximal activation by 9 min. It promotes translocation of PKC to the plasma membrane. When 1,25 is incubated directly with isolated plasma membranes, PKCalpha is stimulated although PKCzeta is also present. In contrast, when isolated matrix vesicles (MVs) are incubated with 1,25, PKCzeta is inhibited and PKCalpha is unaffected. These membrane-mediated effects are due to the presence of a specific membrane vitamin D receptor (mVDR) that is distinct from the classical cytosolic VDR. Studies using 1,25 analogs with reduced binding affinity for the classical VDR, confirm that rapid activation of PKC by 1,25 is not VDR dependent. The membrane-mediated effects of 1,25 are critical to the regulation of events in the extracellular matrix produced by the chondrocytes. MVs are extracellular organelles associated with maturation of the matrix, preparing it for mineralization. MV composition is under genomic control, involving VDR-mechanisms. In the matrix, no new gene expression or protein synthesis can occur, however. Differential distribution of PKC isoforms and their nongenomic regulation by 1,25 is one way for the chondrocyte to control events at sites distant from the cell. GC cells contain 1a-hydroxylase and produce 1,25; this production is regulated by 1,25, 24,25, and dexamethasone. 1,25 stimulates MMPs in the MVs, resulting in increased proteoglycan degradation in mineralization gels, and increased activation of latent transforming growth factor-beta 1 (TGF-beta1).

Steroid hormones, including vitamin D metabolites and estrogens, activate target cells through specific receptors that discriminate among ligands based upon recognition of distinct structural features. For both classes of ligands, cell surface and nuclear receptors co-exist in many target cells. Upon ligand binding, these receptors generate both rapid and long lasting responses. While the structure of the nuclear receptors and their function as transcriptional activators of specific target genes is generally understood, the identity of the membrane receptors remains elusive. Using pharmacological, functional and biochemical approaches, new insights are being gained into nature of the cell surface receptors for both vitamin D metabolites and estrogens.

There are currently two main candidates for the membrane receptor for 1,25(OH)2D3: the 1,25D3-MARRS protein/ERp57; and the classical VDR. The 1,25D3-MARRS protein is essential for hormone-stimulated phosphate and calcium uptake in chick intestinal cells, whereas the VDR is not. The 1,25D3-MARRS protein also shows a high degree of correlation with growth periods in which bone is rapidly formed, whereas the VDR does not. However, in rat enterocytes, both the 1,25D3-MARRS protein and the VDR play a role in the rapid, steroid-mediated uptake of phosphate or calcium. Therefore, the theory that alternate binding sites on the VDR for various analogs account for all membrane-initiated phenomena, is incorrect.

We previously reported that diosgenin, a plant-derived steroidal sapogenin, improved memory and reduced axonal degeneration in an Alzheimer's disease mouse model. Diosgenin directly activated the membrane-associated rapid response steroid-binding receptor (1,25D3-MARRS) in neurons. However, 1,25D3-MARRS-mediated diosgenin signaling was only shown in vitro in the previous study. Here, we aimed to obtain in vivo evidence showing that diosgenin signaling is mediated by 1,25D3-MARRS in the mouse brain. Diosgenin treatment in normal mice enhanced object recognition memory and spike firing and cross-correlation in the medial prefrontal cortex and hippocampal CA1. In diosgenin-treated mice, axonal density and c-Fos expression was increased in the medial prefrontal and perirhinal cortices, suggesting that neuronal network activation may be enhanced. The diosgenin-induced memory enhancement and axonal growth were completely inhibited by co-treatment with a neutralizing antibody for 1,25D3-MARRS. Our in vivo data indicate that diosgenin is a memory-enhancing drug and that enhancement by diosgenin is mediated by 1,25D3-MARRS-triggered axonal growth.

The steroid hormone 1,25-(OH),D, modulates a number of cellular functions through nonnuclear pathways, particularly the stimulation of intestinal calcium transport (transcaltachia). The present studies determined whether 1,25-(OH),D, rapidly enhanced phosphate transport in the perfused duodenal loop of normal chicks. Vascular perfusion with 65 pM 1,25-(OH),D, significantly stimulated the appearance of ““P in the venous effluent within 2-8 min, reaching an average of 160% of control levels by 40 min. Lumenal perfusion with hormone failed to augment levels of 83P in the venous effluent. Vascular perfusion with a range of 1,25-(OH),D:, concentrations yielded an apparent biphasic dose-response curve. Two additional vitamin D metabolites, 24,25(OH),D:, and 25(OH)D,, which initiate transcaltachia, were tested for their effect on phosphate transport. Neither 6.5 n&v 24,25(OH),D:, nor 100 n.v 25(OH)D, stimulated ““P movement from the lumen to the venous effluent. When duodena were vascularly perfused with 65 pM 1,25(OH),D:, and either 6.5 nM 24,25(OH),D:, or 100 nM 25(OH)D,,, enhanced phosphate transport was attentuated or abolished. Phosphate transport was also analyzed at metabolite levels 5-fold lower or higher than those in normal chicks. For 24,25(OH),D,, 1.3 nM metabolite did not augment phosphate transport, although stimulation did occur at 32.5 nM steroid (180 2 0.2% ofcontrols). For 25(OH)D,,, no stimulation was observed at 500 nM metabolite, whereas 20 nM steroid resulted in transport that was 160 ? 0.14% ofcontrols. The presence or absence of lumenal Ca”’ did not influence phosphate transport in duodena vascularly perfused with control medium, 65 pM 1,25-(OH)&, or 6.5 nM 24,25(OH),D,. In contrast, vascular perfusion with 100 n.v 25(OH)D, stimulated phosphate transport when lumenal Ca” was present but not when it was absent. The influence of lumenal P, on transcaltachia was then studied. Although 65 pM 1,25-(OH),D,, 6.5 nM 24,25(OH),D,, and 100 nM 25(OH)D,, initiate transcaltachia in the presence of lumenal P,, the absence of the anion abolished the stimulatory effects of 1,25-(OH),D:, and 24,25(OH),D,, on calcium transport but not those induced by 25(OH)D,,. These data suggest a complex regulation of both calcium and phosphate transport based on relative levels of circulating vitamin D metabolites as well as the ionic content of the lumen. (Endocrinology 137: 2254-2261, 1996)

The aim of this study was to investigate the effects and the mechanism of diosgenin, a famous plant-derived steroidal sapogenin, on memory deficits in Alzheimer's disease (AD) model mice. Diosgenin-treated 5XFAD mice exhibited significantly improved performance of object recognition memory. Diosgenin treatment significantly reduced amyloid plaques and neurofibrillary tangles in the cerebral cortex and hippocampus. Degenerated axons and presynaptic terminals that were only observed in regions closely associated with amyloid plaques were significantly reduced by diosgenin treatment. The 1,25D3-membrane-associated, rapid response steroid-binding protein (1,25D3-MARRS) was shown to be a target of diosgenin. 1,25D3-MARRS knockdown completely inhibited diosgenin-induced axonal growth in cortical neurons. Treatment with a neutralizing antibody against 1,25D3-MARRS diminished the axonal regeneration effect of diosgenin in Aβ(1–42)-induced axonal atrophy. This is the first study to demonstrate that the exogenous stimulator diosgenin activates the 1,25D3-MARRS pathway, which may be a very critical signaling target for anti-AD therapy.

The steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) elicits biological responses by both genomic and nongenomic pathways. This report describes purification of a receptor for 1 alpha,25-(OH)2D3 (VDR) located in the basal-lateral membrane (BLM) of vitamin D-replete chick intestinal epithelium, which is implicated in the nongenomic stimulation of calcium transport (transcaltachia). The BLM-VDR exhibited saturable binding for [3H]1,25-(OH)2D3 (KD = 0.72 x 10(-9)M, Bmax = 0.24 pmol/mg of protein). A 4500-fold purification of the BLM-VDR receptor was achieved. In addition, saturable binding was observed for [3H]24R,25-(OH)2D3 at physiologically relevant levels (KD = 19 x 10(-9) M, Bmax = 2.4 pmol/mg of protein) to a component apparently distinct from the 1 alpha,25-(OH)2D3 BLM-VDR. A functional correlation between the BLM-VDR and transcaltachia was observed in three experimental situations: (i) vitamin D deficiency, which suppresses transcaltachia, resulted in reduced specific [3H]1 alpha,25-(OH)2D3 binding in the BLM-VDR, relative to corresponding fractions from vitamin D-replete chicks; (ii) the BLM-VDR exhibited down-regulation of specific [3H]1 alpha,25-(OH)2D3 binding following exposure to nonradioactive 1 alpha,25-(OH)2D3; and (iii) the relative potencies of two "6-s-cis" analogs of 1 alpha,25-(OH)2D3 to initiate transcaltachia and their ability to compete with [3H]1 alpha,25-(OH)2D3 for binding to the BLM-VDR were parallel. The combined results support the existence of a plasmalemal 1 alpha,25-(OH)2D3 receptor which is a prime candidate for signal transduction in transcaltachia.

The steroid hormone 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] stimulates biological responses via both genomic mechanisms and nongenomic mechanisms (opening of voltage-gated Ca 2+ channels). We report here that the two closed B-ring steroid analogs of 1α,25(OH) 2 D 3 , 1α,25(OH) 2 -7-dehydrocholesterol and 1α,25(OH) 2 -lumisterol 3 , are able to generate the nongenomic response, transcaltachia, without the ability to compete with the natural metabolite for binding to its nuclear receptor. We propose that the nongenomic membrane associated receptor can accept the ligand in its closed "6-s- cis " conformation whereas the nuclear receptor prefers the extended "6-s- trans " conformer.

In spite of decades of research, the exact subcellular pathway for calcium transport in intestine has not been elucidated. In this mini-review, we present three models for vectorial movement of calcium across the cell: facilitated (cytoplasmic) diffusion, vesicular/lysosomal transport, and tunneling through the endoplasmic reticulum compartment. We conclude by offering one way to integrate elements of these three models.

Protein kinase C (PKC) is involved in the rapid 1,25(OH) 2 D 3 -mediated extrusion of calcium in chick intestinal epithelial cells. A previous report demonstrated that PKC a and PKC b redistribution corresponded to the dose- response curve for calcium extrusion. We investigated the role of both PKC a and PKC b in hormone-stimulated calcium extrusion by using siRNA in primary cultures of intestinal cells. The results indicated that there was no change in calcium extrusion in cells transfected with siRNA to PKC a , whereas the siRNA to PKC b significantly decreased calcium extrusion in 1,25(OH) 2 D 3 -treated cells when compared with the corresponding hormone-treated cells in transfected and nontransfected cells ( P < 0.01). Similar results were also found using chemical inhibitors of PKC a (safingol) and PKC b ([3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione]) in intestinal cell suspension. The results demonstrated that PKC b inhibited the extrusion of calcium (resulting in increased calcium uptake) from intestinal cells treated with 1,25(OH) 2 D 3 when compared with vehicle controls ( P < 0.001), the PKC a blocker did not show any difference in calcium extrusion among the treatment groups. Using confocal microscopy, we reduced the hormone exposure to 30 sec in order to view redistribution of PKC a and PKC b . Rapid redistribution of PKC a was found to significantly increase in fluorescence in the apical membrane region after a 30 sec exposure of cells to 300- or 650 pM 1,25(OH) 2 D 3 ( P < 0.01 and 0.001, respectively). By comparison, PKC b immunofluorescence was found to increase significantly in the basal lateral region of the cells, relative to controls after the exposure of cells to 300- ( P < 0.01) or 650 pM ( P < 0.05) seco-steroid. The results suggest that PKC a is the PKC isotype involved in 1,25(OH) 2 D 3 -mediated calcium efflux in intestinal epithelial cells.

The steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) generates biological responses via both genomic and nongenomic mechanisms. This article reports the biological profile of four A-ring diastereomers of this secosteroid (results are expressed as percentage of the response of 1 alpha,25-(OH)2D3. The activity of the compounds, 1 alpha,25-(OH)2D3, 1 alpha,25-(OH)2-3-epivitamin D3, 1 beta,25-(OH)2D3, 1 beta,25-(OH)2-3-epivitamin D3, for in vivo intestinal Ca2+ absorption and bone Ca2+ mobilization and in vitro binding to the nuclear receptor (genomic responses) was, respectively, 100, 2.8, 1 alpha,25-(OH)2-3-epivitamin D3 > 1 beta,25-(OH)2D3 > 1 beta,25-(OH)2-3-epivitamin D3. 1 beta,25-(OH)2D3 was a potent antagonist of 1 alpha,25-(OH)2D3-mediated transcaltachia and 45Ca2+ uptake in ROS 17/2.8 cells but was unable to block the genomic 1 alpha,25-(OH)2D3 induction of chick calbindin-D28k (in vivo), induction of MG-63 cell osteocalcin, and HL-60 cell differentiation. These results suggest that analogs of 1 alpha,25-(OH)2D3 may be synthesized which are selective agonists or antagonists of genomic or nongenomic responses in the vitamin D endocrine system.

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) generates biological responses via both genomic and nongenomic mechanisms. This article addresses activity differences between the 6-s-trans (extended) and the 6-s-cis (steroid-like) conformation of 1,25-(OH)2D3 to initiate these responses. Because of facile interconversion of the 6-s-trans and 6-s-cis conformers of 1,25-(OH)2D3, kinetically competent amounts of both conformers exist to interact with any potential receptors for 1,25-(OH)2D3. We have chemically synthesized 1,25-(OH)2-9,14,19,19,19-pentadeuterio-pre-D3 (1,25-(OH)2-d5-pre-D3), an analog of the 6-s-cis conformation of 1,25-(OH)2D3. We found that 1,25-(OH)2-d5-pre-D3 and 1,25-(OH)2D3 were equivalently active in two nongenomic systems (transcaltachia as measured in the perfused chick intestine and 45Ca2+ uptake through voltage-gated Ca2+ channels in ROS 17/2.8 cells). 1,25-(OH)2-d5-pre-D3 was significantly less active both in binding in vitro to the plasma vitamin D-binding protein (7%) and to the chick (10%) and pig (4%) intestinal nuclear 1,25-(OH)2D3 receptors generating genomic biological responses in vivo (induction of plasma levels of osteocalcin, < 5%) or in cultured cells (inhibition of HL-60 cell differentiation, < 5%; inhibition of MG-63 proliferation, < 2%; and induction of osteocalcin, < 2%). These results suggest that the genomic and nongenomic responses are mediated by separate receptors. Further, the 6-s-cis form (steroid-like conformation) of the natural hormone, 1,25-(OH)2D3, may be selectively responsible for its nongenomic function(s).