The 1 , 25 dihydroxyvitamin D 3-membrane-associated , rapid response steroid-binding receptor

Abstract Introduction In this review, we discuss the evidence for a novel receptor, vitamin D metabolite, 1,25-dihydroxyvitamin D 3 . The protein, which we have termed as the membrane-associ-ated, rapid response steroid-binding (1,25D 3 -MARRS) receptor, is iden-tical to ERp57/PDIA3. This protein has been extensively studied for its role in calcium and phosphate uptake in intestinal cells and more recently has been found to be implicated in cancer and Alzheimer’s disease. In addition, we present a more complete biochemical characterisation of this protein using co-immunoprecipita-tion and chromatin immunoprecipi-tation studies. Conclusion The 1,25D 3 -MARRS protein has both cytoplasmic and nuclear motifs and redistribution within the cell. New evidence shows its potential involve-ment through genomic action. Its involvement in disease states appears primarily through its chaperone action, post-translational modifica-tion of vital proteins and ability to modulate oxidative stress. Introduction


Introduction
The field of steroid research has evolved significantly during the past 60 years focussing on the concept that lipophillic signalling agents lack membrane receptors and act solely through gene transcription.It is now widely accepted that steroid hormones have membrane receptors and can elicit responses that are rapid or 'pre-genomic'.Some of these receptors are the classical nuclear transcription factors that have been modified with a fatty acyl chain (e.g.palmitoylation) to promote association with the plasma membrane.However, in several cases of steroid research, novel receptors have been identified.This review will describe one such novel receptor, the membrane-associated, rapid response steroid-binding (1,25D 3 -MARRS) receptor for the steroid hormone 1,25-dihydroxyvitamin D 3 (1,25D 3 ).

Discussion
The authors have referenced some of their own studies in this review.These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees associated to the institution in which they were performed.All human subjects, in these referenced studies, gave informed consent to participate in the studies.Also, animal care was in accordance with the institutional guidelines.
Figure 1 presents a schematic presentation of the metabolism of vitamin D to 25-hydroxyvitamin D 3 (25D 3 ) in the liver.This metabolite is hydroxylated in the kidney either to 1,25D 3 when an animal is calcium, phosphate or 1,25D 3 deficient or to 24,25D 3 in a replete animal.1,25D 3 is best known for its stimulatory activity, for example, it increases calcium and phosphate absorption in the intestine 1,2 , while 24,25D 3 is known for its inhibitory activity and can cause hypophosphataemia in vivo 3 .
The 1,25D 3 -MARRS receptor: rapid responses for calcium and phosphate uptake 1,25D 3 has been shown to stimulate calcium uptake in intestinal cells from rats 4 , chicks 5 and mice 1 .Each of these has been shown to require the 1,25D 3 -MARRS receptor, most recently shown in mice that were genetically engineered to have a targeted knockout of the gene in their intestine 1 .Similarly, phosphate uptake has been shown to depend on the functional presence of the 1,25D 3 -MARRS receptor in these three species 2,6,7 .In 2004, our laboratory published results 6 using chick intestinal cells and two independent techniques to study the requirement of the 1,25D 3 -MARRS receptor in hormonestimulated phosphate uptake.Using a highly specific polyclonal antibody reagent to the N-terminus (generated by the multiple antigenic peptide procedure), we pre-incubated the intestinal cells with this reagent and subsequently, demonstrated that while 1,25D 3 treated cells exhibited a significant increase in phosphate uptake relative to controls, those treated with a 1/500 dilution of Ab 099, created against 1,25D 3 -MARRS, failed to respond to hormone with increased phosphate uptake.The independent techniques used cells transfected with either a ribozyme directed against the 1,25D 3 -MARRS receptor or the control ribozyme.Those cells transfected with the ribozyme against the receptor were found to have decreased protein levels as demonstrated by the Western blot analysis and exhibited a failed response to the 1,25D 3 -MARRS receptor with increased phosphate uptake relative to the controls and cells treated with the control ribozyme.Protein kinase C (PKC), which mediated the phosphate uptake response in chick intestinal cells, also showed a failed response to stimulation by hormone in the cells transfected with active ribozyme.Finally, we demonstrated a decreased [ 3 H]1,25D 3 binding to the 1,25D 3 -MARRS receptor in cells transfected with the active ribozyme, while the classical vitamin D receptor (VDR) was found to be unaffected in its [ 3 H]1,25D 3 binding.
More recently, we used mice bearing a targeted knockout of the 1,25D 3 -MARRS receptor in intestine that was produced by crossing the intestinal cells with mice bearing villin-driven cre-recombinase.For calcium uptake studies, we found that this protein was required for hormone-stimulated uptake.We also discovered that as in chick intestinal cells 5 , the protein kinase A (PKA) pathway also mediated the calcium uptake 1 .The 1,25D 3 -MARRS receptor was similarly required for the 1,25D 3 -mediated phosphate uptake 2 ; however, the PKC pathway was not involved.We also tested whether the 1,25D 3 -MARRS receptor knockout had observable effects on calcium absorption in vivo, where we administered 45 CaCl 2 by oral gavage, followed by a 5 min absorption and collection of blood by cardiac puncture.The knockout mice had less calcium absorption when compared with the littermates as determined by the serum 45 Ca 8 .

Receptor isolation and characterisation
The need for a membrane receptor to mediate the rapid or 'pre-genomic' effects of the steroid hormone 1,25D 3 , was postulated by Nemere and Szego 4 when they observed rapid (within 5 min) 1,25D 3 -stimulated calcium uptake commensurate with lysosomal enzyme release in rat enterocytes.Since then, several investigators have demonstrated that 1,25D 3 stimulates changes in phospholipid metabolism within the brush border membranes of chick 9,10 and rat 11 enterocytes that are independent of 'genomic' effects as well as 1,25D 3 -stimulated calcium uptake that is independent of transcription or translation events in mouse mammary glands 12 and chick duodena 13 .1,25D 3 also stimulates signalling events in rat enterocytes 14 and porcine parathyroid cells 15 specific to the plasma membrane that induces phospholipid metabolism to increase concentrations of chemical messengers, such as inositol triphosphate, diacylglycerol, phosphatidic acid and/or monoacylglycerol within seconds.These representative examples of non-nuclear or pre-genomic actions, along with the identification of a protein that specifically binds 1,25D 3 in the basal lateral membrane of chick enterocytes to facilitate the calcium uptake 16,17 , has led to the identification of a plasma membrane receptor, now termed the 1,25D 3 -MARRS receptor.
As a novel receptor for the steroid hormone 1,25D 3 , the 1,25D 3 -MARRS receptor has been identified through sequence analysis as a protein that is distinct from both the nuclear VDR (nVDR) and the vitamin D binding protein 18 .It was first identified as a 66 kD membrane protein found at the basal lateral membrane of chick duodena that specifically binds the 1,25D 3 in a manner that is not only saturable 16,17,[19][20][21][22] , but also conserved in the plasma membranes of cells and vesicles across several species 6,23 , including mouse, rat, human and fish 19,24 and is found in several tissues, such as the intestine, bone, teeth, parathyroids, muscle, kidneys and the brain [25][26][27][28][29][30][31][32] .The 1,25D 3 -MARRS receptor has been shown to facilitate various cell signalling events, such as 1,25D 3 -stimulated activation of the PKC pathway, cyclic adenosine monophosphate (cAMP) pathway, mitogen-activated protein kinase (MAPK) pathway and endocytic/ lysosomal transport pathway.The 1,25D 3 -MARRS receptor contains several consensus sequences that shed light on the membrane-initiated steroid signalling (MISS) events that are involved in 'pre-genomic' cellular effects that include an N-myristoylation site, seven PKC sites, a cAMP-dependent kinase site, two thioredoxin folds, six casein kinase II sites and three tyrosine kinase sites.The N-myristoylation site of the 1,25D 3 -MARRS receptor acts as a signal peptide that targets the receptor for insertion into the plasma membrane.The binding of 1,25D 3 to the 1,25D 3 -MARRS receptor at the plasma membrane, is shown to act through a G-protein (G aq ) 33 and the PKC pathway [34][35][36] or the cAMP pathway 21,22,28 to initiate signal transduction events that enhance calcium uptake and/or transport 21,22 , calcium extrusion 28 or phosphate uptake and/or transport 6,32,36,37 .The 1,25D 3 -MARRS receptor also contains two thioredoxin folds that facilitate its ability to interact with calcium binding proteins, such as calnexin and calreticulin to modulate intracellular calcium concentrations (Figure 2).The binding of 1,25D 3 to the 1,25D 3 -MARRS receptor can either activate the MAPK pathway through tyrosine kinase phosphorylation to elicit responses that are also PKC-dependent 33,[38][39][40][41] , or possibly through the Wnt signalling pathway through its casein kinase II site activity.While these characteristics of the 1,25D 3 -MARRS receptor clearly establishes its involvement in 1,25D 3 -stimulated 'pre-genomic' events, the 1,25D 3 -MARRS receptor also contains consensus sequences such as two Rel homology domains, a SMAD3 consensus sequence, a nuclear retention motif and an endoplasmic reticulum retention motif 6,18,23,42 , which demonstrates that the 1,25D 3 -MARRS receptor is also integral in 1,25D 3 -stimulated cell signalling that involve the endocytic/ lysosomal transport pathway to mediate cell events that can integrate 'pre-genomic' signalling events with longer-term genomic responses [22][23][24] .

Chromatin immunoprecipitation and co-immunoprecipitation studies
While MISS events may occur independently of nuclear-initiated steroid signalling (NISS), the 1,25D 3 -MARRS receptor is able to bind to DNA through its Rel homology domain interaction sites 6 (as well as a SMAD3 consensus sequence 23,42 ) and has been shown to translocate to the nucleus 43,44 .Using the chromatin immunoprecipitation studies, we have recently described the 1,25D 3 -MARRS receptor interactions with several genomic sequences both prior to 1,25D 3 exposure in murine enterocytes and within 5 min of hormone addition (see Table 1).Prior to 1,25D 3 exposure, the 1,25D 3 -MARRS receptor interacts with the Homer homologue 1  Investigators have shown that the 1,25D 3 -MARRS receptor has both nuclear and endoplasmic reticulum retention motifs 6 that can facilitate its translocation from the plasma membrane to the Golgi Apparatus 25 , the endoplasmic reticulum where it is shown to be identical to the endoplasmic reticulum p57 (Erp57) 6 or the nucleus 16,17,23,27 to elicit genomic cellular events, which occur through a series of vesicular transport events, with the involvement of cytoskeletal components such as microtubules, microfilaments, and intermediate filaments.While Nemere and Szego 4 have previously identified tubulin as a key cytoskeletal component in 1,25D 3 -mediated calcium uptake, we have recently identified both actin and keratin as proteins that specifically interact with the 1,25D 3 -MARRS receptor before and after exposure to hormone in chick enterocytes.We performed co-immunoprecipitation (co-IP) studies and identified actin and keratin through mass spec-troscopic analysis as proteins that associate with the 1,25D 3 -MARRS receptor.Co-IP of both actin and keratin was also confirmed by the Western blot analysis (data not shown).Using confocal microscopy, we investigated 1,25D 3 -stimulated changes in intracellular redistribution of the 1,25D 3 -MARRS receptor and simultaneous involvement of cytoskeletal actin (F-actin) in chick enterocytes.Using immunofluorescent labelling techniques, we demonstrated that 1,25D 3 induced an increase in intracellular 1,25D 3 -MARRS receptor within 5 min (Figure 3A) with observed cyclical changes in F-actin (Figure 3B, Figure 4).We also found 1,25D 3 stimulated intracellular redistribution of keratin that paralleled intracellular redistribution of the 1,25D 3 -MARRS receptor (Figure 5).We can thus conclude from this evidence that both microfilaments and intermediate filaments are involved in intracellular, 1,25D 3 -stimulated redistribution of the 1,25D 3 -MARRS receptor.

Role of 1,25D 3 -MARRS receptor on select disease states
Vitamin D is a multifunctional hormone and plays some critical roles in diseases and metabolic conditions.While many of its genomic roles associated to the classical VDR have been extensively investigated and will be the subject of future research, we have outlined some of the critical roles of vitamin D that are associated in relation to its membrane receptor, ERp57/1,25D 3 -MARRS protein.This pivotal role is primarily associated with one of the fundamental processes in biological systems: the ability of proteins to fold into a pre-defined and functional conformation.However, when such pre-defined and functional conformation deviates, the disease state arises, or in utero lethality occurs in the case of a homozygous deletion of ERp57/1,25D 3 -MARRS receptor gene 45,46 .Given that the ERp57/1,25D 3 -MARRS receptor is critical for post-translational modi- fication of proteins, in addition to its role as a molecular chaperone, and its requirement in the major histocompatability complex class I assembly and regulation of gene expression, it is highly probable that the protein is involved in some of the disease states, such as prion neurotoxicity, Alzheimer's disease, and cancer, among others.Moreover, its involvement in oxidative stress (reviewed in 47,48 ) makes it even more likely to be associated with such disease states.Here, we discuss some of the recent literatures related to the association of 1,25D 3 -MARRS with select disease states that were not covered in a book chapter we contributed recently 47,48 .

In cancer
Research has shown that 1,25D 3 inhibits growth of several cancer types, including breast cancer.It is primarily its classical receptor that has been implicated in the modulation of cancer by 1,25D 3 , while the role of its membrane counterpart has not been investigated in detail.One recent study on Michigan Cancer Foundation-7 (MCF-7) breast cancer cells suggested 1,25D 3 -MARRS receptor expression to interfere with the growth inhibitory activity of  1,25D 3 , possibly through the nVDR 49 .In contrast, siRNA and genome-wide studies suggested that the anti-proliferative effects of 1,25D 3 in MCF-7 breast tumour cell lines do not rely on classical vitamin D pathways per se 50 .
In a mouse model of skin carcinogenesis, long-term biological response generated by chronic dosing with a non-genomic-selective vitamin D steroid was observed 51 .They showed that both 1,25D₃ and 1α,25(OH)₂-lumisterol, a conformationally restricted analogue that can generate only non-genomic responses, are effective inhibitors of ultraviolet radiation (UVR) damage in an immunocompetent mouse (Skh:hr1) model susceptible to UV-induced tumours.Both 1,25D₃ and 1α,25(OH)₂-lumisterol significantly reduced UVR-induced cyclobutane pyrimidine dimer (it can be highly mutagenic if not repaired prior to cell division and can lead to UV-induced immunosuppression making them potentially carcinogenic), apoptotic sunburn cells and immunosuppression.Furthermore, these compounds inhibited skin tumour development, both papillomas and squamous cell carcinomas, in mice in vivo.They attributed these non-genomic physiological responses to VDR associated with plasma membrane caveolae.While we have refuted this theory and the reason why 1,25D 3 -MARRS receptor is actually involved in this regard in some of our previous works 47,[52][53][54][55] , a recent study with VDR and ERp57 in the photoprotective effects of 1,25D 3 against UVR-induced DNA damage in human skin fibroblasts, indicated that the VDR and ERp57 are directly associated outside the nucleus in which they mediate at least some of the non-genomic actions of 1,25D 3 56 .In prostate cancer LNCaP cells, 1,25D 3 decreased invasiveness of cells by interaction with a putative membrane-associated receptor, which activated membrane-initiated signalling via the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) MAPK signalling pathway 57 .Treatment with 1,25D 3 evoked a dosedependent activation of the JNK/SAPK MAPK signalling pathways within 10 min, which demonstrated membraneinitiated signalling of 1,25D 3 in LNCaP cells.Furthermore, treatment with 1,25D 3 decreased LNCaP cell invasiveness by approximately 20% after 48 h.Using an inhibitor (SP600125) for the JNK/SAPK MAPK signalling pathway in combination with 1,25D 3 on LNCaP cells, the inhibitory action of 1,25D 3 on invasiveness was eliminated 56 .
In a study, tissue cores from 164 gastric cancer patients were stained for ERp57 58 .ERp57 staining was significantly decreased in cancer patients and metastases were compared with both normal gastric mucosa and metaplasias.The reduced ERp57 expression also correlated with greater depth of tumour invasion and advanced stage of the disease.Kaplan-Meier survival analysis determined that tumours with the highest quartile of ERp57 expression were statistically associated with longer post-operative survival, while a Cox proportional hazard analysis showed that maintenance of ERp57 expression was associated with longer post-operative survival.These results may provide useful prognostic information for gastric cancer patients.
These results also suggested that the role of 1,25D 3 MARRS receptor in cancer patients may be modulated through multiple pathways and that it may be dependent on type, cell line or stage of metastasis.Further research should examine the potential for pharmacological or natural agents that modify 1,25D 3 -MARRS expression or activity as anticancer agents.

In Alzheimer's disease
Alzheimer's disease is one of the major diseases associated with ERp57 in which β-amyloids are the major components of the plaque observed in the brains of its patients, usually the elderly.The β-amyloids, though formed naturally, are cleared by the endoplasmic reticulum chaperone proteins, such as ERp57 during post-translational modification.Studies suggest that a chaperone declines, like ERp57, are one of the two central defects in Alzheimer's disease 59 , and deposits of β-amyloids are observed only in the elderly 60,61 .Interestingly, the expression and activity of ERp57/1,25D3-MARRS receptor also declines in the intestine and kidney with age 5,21,22 .
In a recent study, 1,25D 3 MARRS receptor enhanced cerebral clearance of human β-amyloid peptide from mouse brain across the blood-brain barrier involving both genomic and non-genomic pathways 62 .Because forskolin enhanced β-amyloid elimination from the mouse brain 62 , it is more likely to act through ERp57 than the classical VDR.In another study, the structure function results provided evidence that 1,25D 3 activation of VDR-dependent genomic and non-genomic signalling work in combination to recover disregulated innate immune function in patients with Alzheimer's disease 63 .
Recently, certain phytochemicals have been found to be associated with ERp57 function.One example is diosgenin, which is a plant-derived steroidal sapogenin.Mice treated with diosgenin significantly reduced amyloid plaques and neurofibrillary tangles in the cerebral cortex and hippocampus 64 .More importantly, 1,25D₃-MARRS receptor was shown to be a target of diosgenin and 1,25D₃-MARRS receptor knockdown completely inhibited diosgenininduced axonal growth in cortical neurons.On the other hand, catechin (found widely in green tea, cinnamon and cocoa among others), particularly the galloylated one, is found to inhibit ERp57 binding to other macromolecular ligands 65 .This may be of greater importance in designing new polyphenol-based ERp57specific inhibitors that may be useful in ameliorating disease states where ERp57 is known to aggravate the condition.
Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) In addition, there are other reports where ERp57 is involved.One such finding is the requirement of ERp57 for platelet function, aggregation, haemostasis and thrombosis 66,67 .Using enzyme activity function blocking antibodies, Holbrook et al. 67 demonstrated a role for ERp57 in platelet aggregation, dense granule secretion, fibrinogen binding, calcium mobilisation and thrombus formation under arterial conditions.A rabbit antibody generated against the protein strongly inhibited in a functional assay and strongly inhibited platelet aggregation 65 .In another finding, ERp57 was suggested to play an early adaptive response in toxin-mediated stress-associated with Parkinson's disease 68 , for which environmental factors are one among the causes.ERp57 is also implicated in heart failure.The protein is significantly upregulated in both animal and human models of right and left heart failure and appears to work through Src, a downstream target of c-Src kinase; and it was found that Src expression and phosphorylation were markedly upregulated in the failing ventricles 69 .For a brief discussion about the role of ERp57 on a few other diseases, such as diabetes, Wolcott Rallison syndrome and prion neurotoxicity, please refer to other recent reviews 47,48 .

Conclusion
1,25D 3 mediated rapid transport of calcium and phosphate ions in the intestine cells involving the 1,25D3-MARRS receptor.Given that the 1,25D 3 -MARRS protein has both cytoplasmic and nuclear motifs and redistribution within the cell, evidence has emerged for its potential involvement through genomic action.Its involvement in disease states appears primarily through its chaperone action, post-translational modification of vital proteins and the ability to modulate oxidative stress.

Figure 1 :
Figure 1: Schematic representation of the major metabolites of vitamin D. Vitamin D is hydroxylated at C-25 in the liver and then either metabolised to 1,25D 3 in the kidney or 24,25D 3 .1,25D3 is a metabolite that stimulates calcium and phosphate absorption, and hence is produced during deficient states.In the replete states, 24,25D 3 is produced and it acts as an inhibitor of the stimulatory action of 1,25D 3 .

Figure 2 :
Figure 2: Two models of the 1,25D 3 -MARRS receptor.1,25D 3 is predicted to bind near the N-terminus as indicated in both the space filling and ribbon models.

F:
Sterling TM, Khanal RC, Nemere I.The 1,25 dihydroxyvitamin D 3 -membrane-associated, rapid response steroid-binding receptor Feb 01;1(1):4.(HOMER1) gene and the protein disulphide isomerase-3 (PDIA3) gene, which facilitate changes in intracellular calcium.While HOMER1 helps regulate intracellular calcium by coupling surface receptors, such as glutamate, IP3 ryanodine receptors, or cytoplasmic proteins such as PI3 kinase, PDIA3 act as a chaperone protein to mediate calcium sequestration by modulating the activity of calnexin and calreticulin.After the 1,25D 3 exposure, the 1,25D 3 -MARRS receptor interacts with the neutralised-like protein 1B (NEURL1B) gene, zinc transporter 1 (SLC30A1) gene and/or the G protein-coupled receptor 180 (GPR180) gene.NEURL1B encodes the sequence of E3 ubiquitin protein ligase that targets proteins for degradation, and SLC30A1 encodes the sequence for a zinc transporter that inhibits calcium channel activity.The Search Tool for the Retrieval of Interacting Genes/ Proteins (STRING) analysis predicted GPR180 to functionally interact with either the choroidermia-like gene (CHML) or TBC1 domain 8 (TBC1D8) gene to activate Rab family proteins or lysosomal-associated membrane protein 3 (LAMP3) gene, which further implicates the 1,25D 3 -MARRS receptor in 1,25D 3 -stimulated vesicular transport.

Table 1 . DNA iden fi ed from DNA-1,25D 3 -MARRS complexes obtained using chroma n-immunoprecipita on experiments of chick enterocytes exposed to vehicle or hormone. DNA isolates were sequenced and iden fi ed using ExPASy Bioinforma cs Resource Portal.
* The predicted functional partners of the DNA isolates were identified using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) 9.0 Database.Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY)