The 1,25D3 -MARRS receptor/PDIA3/ERp57 and lifespan.

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.

Intestinal cell calcium uptake and the targeted knockout of the 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) receptor/PDIA3/Erp57.

We have crossed ERp57(flx/flx) mice with commercially available mice expressing villin-driven cre-recombinase. Lysates of intestinal epithelial cells were prepared from knock-out (KO) mice and littermates (LM) and used in Western blot analyses with Ab099 against the N terminus of the 1,25D(3)-MARRS (membrane-associated, rapid response steroid-binding) receptor: LM mice exhibited one positive band, which was absent in preparations from KO mice. Saturation analyses of cell lysates with [(3)H]1,25D(3) revealed negligible binding in preparations from either female or male KOs. Lysates from female and male LM mice had similar affinities but different numbers of binding sites. Isolated enterocytes were tested for steroid-stimulated calcium uptake. Treatment of cells from female or male LM mice with 1,25D(3) elicited enhanced calcium uptake in females and males within 5 min. Intestinal cells from KO mice exhibited a severely blunted or completely absent response to hormone. Confocal microscopy of intestinal cells revealed the presence of cell surface vitamin D receptors. However, antibodies to the vitamin D receptor failed to block 1,25D(3)-stimulated calcium uptake. In chick enterocytes we have found that the PKA pathway mediates calcium uptake. The time course for activation of PKA in mouse enterocytes paralleled that for enhanced calcium uptake and for LM females reached 250% of controls within 5 min, and 150% of controls in cells prepared from LM males. Enterocytes from female or male KO mice failed to exhibit steroid hormone-stimulated PKA activity, but did respond to forskolin with enhanced calcium uptake. We conclude that the 1,25D(3)-MARRS receptor is of central importance to steroid hormone-stimulated calcium uptake in mammalian intestinal cells.

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells.

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<0.05). Reduction in 1,25D3-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)2D3. Knockdown of 1,25D3-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D3-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)2D3 in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D3-MARRS expression or activity as anticancer agents.

Nuclear translocation of the 1,25D3-MARRS (membrane associated rapid response to steroids) receptor protein and NFkappaB in differentiating NB4 leukemia cells.

1,25 Dihydroxyvitamin D(3) (1,25D(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(3), which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D(3)-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NFkappaB). In unstimulated cells, 1,25D(3)-MARRS can be co-immunoprecipitated with antibodies directed at NFkappaB, and NFkappaB is co-precipitated when antibodies against 1,25D(3)-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D(3)-MARRS and NFkappaB begin translocating to the nucleus within minutes of co-stimulation with 1,25D(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(3)-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NFkappaB and other factors with which it may be interacting.

Novel hormone "receptors".

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)(2)D(3) and 24,25(OH)(2)D(3).

Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in 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.

Endocrine regulation of calcium transport in epithelia.

1. Calcium (re)absorption occurs in epithelia, including the intestine, kidney, mammary glands, placenta and gills (in the case of fish). 2. Calcium is transported across epithelia by two transport mechanisms, paracellular and transcellular, and the movement is regulated by a complex array of transport processes that are mediated by hormonal, developmental and physiological factors involving the gastrointestinal tract, bone, kidney and the parathyroids. 3. Clear understanding of the calcium transport pathways and their endocrine regulation is critical for minimizing various metabolic and health disorders at different physiological stages. Here, we first briefly review the calcium transport mechanisms before discussing in detail the endocrine factors that regulate calcium transport in the epithelia.

Membrane receptors for vitamin D metabolites.

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.

Calcium uptake and membrane trafficking in response to PTH or 25(OH)D3 in polarized intestinal epithelial cells.

Cell culture techniques providing retention of the polarized enterocyte morphology has allowed, for the first time, comparison of parathyroid hormone (PTH)- and 25-hydroxyvitamin D(3) [25(OH)D(3)]-induced (45)Ca uptake with membrane trafficking events discerned using confocal microscopy. Treatment of cells with 65 pM bPTH(1-34) promoted enhanced (45)Ca uptake between 1 and 10 min after peptide. The protein kinase A (PKA) antagonist, RpcAMP inhibited hormone-mediated uptake. At the microscopic level, cells labeled with the endocytic tracking dye FM1-43 revealed increased punctate staining 50-550s after hormone. Pretreatment of cells with RpcAMP abolished this pattern of staining. The calcium indicator dye fluo-3 AM revealed faint punctate labeling in controls, with increased bands of punctate labeling in the apical region of the cells after peptide hormone, and ultimately the basal region. Parallel studies conducted with the metabolite 25(OH)D(3) resulted in a slower stimulation of (45)Ca uptake 5-10 min after steroid, which was also inhibited by preincubation with RpcAMP. Cells labeled with FM1-43 and then treated with steroid showed no change in distribution of fluorescence during the 10 min incubation period. Confocal microscopy with fluo-3 revealed intense apical fluorescence--that after steroid --streamed to a perinuclear position, and ultimately the basal area. Uniformly diffuse staining, which would indicate cytoplasmic calcium transport, was observed only in controls. Membrane trafficking and compartmentalized calcium appear to be integral to agonist mediated cation transport.

Regulation of expression of 1,25D3-MARRS/ERp57/PDIA3 in rat IEC-6 cells by TGF beta and 1,25(OH)2D3.

We examined the transcriptional regulation of expression of the redox-sensitive Membrane-Associated-Rapid Response, Steroid-binding (1,25D(3)-MARRS) protein specific for 1,25(OH)(2)D(3) in a rat small intestinal cell line, IEC-6, that demonstrates rapid responses to 1,25(OH)(2)D(3). 1,25D(3)-MARRS binds and is activated by 1,25(OH)(2)D(3), but is not itself up-regulated by treatment with 1,25(OH)(2)D(3), nor is there a Vitamin D response element (VDRE) in its proximal promoter. We previously reported that transforming growth factor beta (TGFbeta) increased steady state levels of 1,25D(3)-MARRS transcript and protein approximately two-fold [Rohe B, Safford SE, Nemere I, Farach-Carson, MC. Identification and characterization of 1,25D(3)-membrane-associated rapid response, steroid (1,25D(3)-MARRS)-binding protein in rat IEC-6 cells. Steroids 2005;70:458-63]. To determine if this up-regulation could be attributed to the function of a highly conserved consensus smad 3 binding element present in the proximal promoter of the 1,25D(3)-MARRS gene, we created a promoter-reporter [SEAP] construct that was responsive to TGFbeta (200 pM). Deletion or mutation of the smad 3 element greatly reduced the response of the 1,25D(3)-MARRS promoter to TGFbeta. Subsequent studies found that the smad 3 response element is bound by a protein found in the IEC-6 nuclear extract, most likely smad 3. Interestingly, although 1,25(OH)(2)D(3) alone did not increase expression of the 1,25D(3)-MARRS promoter-reporter, co-treatment of transfected IEC-6 cells with 1,25(OH)(2)D(3) and TGFbeta shifted the dose-response curve to a lower effective concentration (100 pM peptide). We conclude that TGFbeta is a transcriptional regulator of 1,25D(3)-MARRS expression via a functional smad 3 element and that cross-talk with non-classical 1,25(OH)(2)D(3)-stimulated pathways occurs. The findings have broad implications for redox-sensitive signaling phenomena including those that regulate phosphate transport in the intestine.

1,25-dihydroxyvitamin D3 stimulates vesicular transport within 5 s in polarized intestinal epithelial cells.

Controversy remains regarding whether the seco-steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances calcium and phosphate movement across the intestinal epithelial cell by facilitated diffusion or a vesicular transport mechanism. In this study we investigated whether membrane trafficking, as judged by confocal microscopy, was sufficiently rapid in comparison to hormone-stimulated uptake of phosphate (32P). Primary cultures of chick intestinal cells were established overnight either in Petri dishes (uptake studies) or chambered coverslips (confocal microscopy). Addition of 130 pM 1,25(OH)2D3 resulted in an apparent increase in 32P uptake within 1 min, relative to controls, that was statistically significant from 3-10 min of incubation. Using the endocytic marker dye, FM1-43, confocal microscopy revealed a profound decrease in membrane-associated fluorescence (apical> basal) within 10 s of hormone treatment, a return of fluorescence at 15-65 s, followed by another round of decreasing and increasing fluorescence. Between 3-9 min of incubation, fluorescence intensity increased 50% (apical region) and 20% (basal region) over control conditions. An antibody (Ab 099) directed against a putative membrane receptor for 1,25(OH)2D3 (1,25D3-MARRS) inhibited both 32P uptake, and changes in fluorescence. In addition, the protein kinase C (PKC) inhibitor, calphostin C, inhibited both 32P uptake and the observed 1,25(OH)2D3-mediated changes in fluorescence. At the microscopic level, calphostin C pretreatment abolished the very rapid redistribution of the endocytic marker dye, although a slight increase in fluorescence was still observed. We conclude that 1,25(OH)2D3-stimulated vesicular trafficking is mediated by the 1,25D3-MARRS protein, implicates a PKC signaling mechanism, and occurs in a time frame that is commensurate with a role in ion transport.

The 1,25D3-MARRS protein: contribution to steroid stimulated calcium uptake in chicks and rats.

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.

Identification and characterization of 1,25D3-membrane-associated rapid response, steroid (1,25D3-MARRS)-binding protein in rat IEC-6 cells.

We report the presence of a mammalian equivalent of the avian Membrane-Associated Rapid Response, Steroid (1,25D3-MARRS)-binding protein specific for 1,25(OH)2D3 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 1,25D3-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 beta1 (TGFbeta1) increased steady-state levels of 1,25D3-MARRS (mRNA and protein) approximately two-fold over a 24-h period. In contrast, treatment with 1,25(OH)2D3 failed to significantly change 1,25D3-MARRS protein or mRNA levels. Localization studies showed rapid nuclear translocation of a pool of 1,25D3-MARRS protein after 1,25(OH)2D3 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)2D3 sensitive response systems in intestinal cells, 1,25D3-MARRS protein and the well-studied nVDR, that together work to fine tune intestinal Ca2+ absorption in a variety of avian and mammalian species.

Expression of a 1,25-dihydroxyvitamin D3 membrane-associated rapid-response steroid binding protein during human tooth and bone development and biomineralization.

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.

Effect of growth and maturation on membrane-initiated actions of 1,25-dihydroxyvitamin D(3). I. Calcium transport, receptor kinetics, and signal transduction in intestine of male chickens.

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.

Environmental salinity regulates receptor expression, cellular effects, and circulating levels of two antagonizing hormones, 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, in rainbow trout.

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.

Identification and characterization of 1,25D3-membrane-associated rapid response, steroid (1,25D3-MARRS) binding protein.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) operates through pharmacologically distinct nuclear receptor-mediated and plasma membrane-initiated mechanisms. The nuclear receptor is well described, but the membrane receptor identity remains unproven. A 66 kDa protein from chick intestinal basolateral membranes was isolated previously and identified as a candidate receptor (now termed 1,25D(3)-MARRS). A chicken cDNA library was screened for clones encoding the N-terminal sequence of 1,25D(3)-MARRS. An exact match was found with an insert containing an open coding region for the full-length candidate 1,25D(3)-MARRS protein. Analysis reveals a 5' untranslated region, a precursor translation product with methionine start site, a signal peptide and a translation product of 505 amino acids prior to translation termination site. Prosite analysis predicts potential sites for phosphorylation by casein kinase II cAMP-dependent kinase, protein kinase C, and tyrosine kinase and an N-myristoylation site with high probability of occurrence. Additionally, two conserved domains capable of interacting with Rel homology domains (RHD) are present. Oligonucleotide primers sets designed to amplify unique regions of the sequence produced amplimers of the predicted size from both chicken and rat intestinal cells. Transcription-translation produced a protein that was recognized in Western blot analysis by Ab099, a polyclonal antibody recognizing the N-terminus of the 66 kDa MARRS protein.

Vectorial Transcellular Calcium Transport in Intestine: Integration of Current Models.

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.

Evidence for distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in osteoblasts.

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.

Diosgenin-induced cognitive enhancement in normal mice is mediated by 1,25D3-MARRS.

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.