Receptors for 1α,25(OH)2D3: Past, Present, and Future

READERS OF THE ARTICLE on page 13531 of the current issue of JBMR may be surprised to find a paper discussing the existence of a second cellular receptor for the steroid hormone 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). This paper, entitled “Identification of a membrane receptor for 1,25-dihydroxyvitamin D3 which mediates rapid activation of protein kinase C,” describes the existence of a membrane receptor in chondrocytes that is linked to the generation of rapid, nongenomic responses. The traditional view has been that all steroid hormones, including 1α,25(OH)2D3, have only one class of receptors, namely those cytosolic/nuclear proteins that bind with exquisite specificity their cognate ligands and then interact in the nucleus with the promoters of genes that are either up- or down-regulated. Yet there is little doubt from the report of Nemere et al. that they believe they are studying in chondrocytes a membrane receptor for 1α,25(OH)2D3. The molecular structures of vitamin D3 and 1α,25(OH)2D3 are closely allied to that of classical steroid hormones. The classic steroid hormones include the progestins, estrogens, androgens, glucocorticoids, and the mineralocorticoids. Technically, vitamin D is a seco-steroid. Seco-steroids are those in which one of the rings of the cyclopentanoperhydrophenanthrene ring structure of the classic steroid has undergone fission of a carbon-carbon bond; in the instance of vitamin D, this is the 9,10 carbon bond of ring B. Those endocrinologists who have been following the development of the vitamin D story over the past three decades will recognize this latest paper as another piece of the still-unfolding vitamin D endocrine system puzzle. Figure 1 presents a timeline describing the evolution in our understanding of the details of steroid hormone interactions with their receptors over the interval from 1960 to the present. This timeline is presented to allow the reader to place in context the emergence of a second receptor for 1α,25(OH)2D3. Prior to 1960, it was not generally accepted that steroid hormones had specific receptors; however, Clara Szego had suggested their existence as early as 1957.2 In a symposium at the Woods Hole Marine Biological Laboratory in 1955, Dr. Szego said “The ‘trigger’ of hormone actions may be considered to be the resultant new biochemical entity formed by the interaction of the hormone and an active receptor site, most likely a protein, of the target cell.” But in the interval 1960–1965, a major breakthrough occurred in the steroid hormone field, which was pioneered by the laboratories of Elwood Jensen and Jack Gorski for estrogen and Isidore Edelman for aldosterone. Collectively, these investigators laid the foundation for the modern era of research on the role of how steroid hormones, in concert with their cognate receptors, regulate gene transcription in selected target cells. Steroid receptor timeline, with emphasis on 1α,25(OH)2D3 receptors. The five cells (A, B, C, D, and E) depict the conceptual milestones over the interval 1965–1998 which have led to the development of our current understanding of steroid hormone receptor action. The essence of the concept is that steroid hormones mediate biological responses in target cells via stereoselective interaction with their cognate receptors. The symbol and related shapes indicate the steroid receptor; indicates any steroid hormone that is a ligand for the steroid receptor (cells A and B) or represents 1α,25(OH)2D3 as a ligand for the 1α,25(OH)2D3 receptors (cells C, D, and E); and the symbol ♦ indicates the steroid hormone 24R,25(OH)2D3. See the text for a detailed discussion. Jensen's group provided some fundamental guidelines to the mechanism of estrogen action. Principally, they emphasized the utility of employing high specific-activity [3H]estradiol to demonstrate the selective uptake of physiological doses of the steroid only by target tissues that were known to be responsive to estradiol.3 Gorski's laboratory first demonstrated that after estradiol dosing of rats in vivo there occurred a rapid stimulation (within 1–2 h after treatment) both of RNA polymerase activity in the rat uterus4 and stimulation of protein synthesis.5 Next, Gorski and Noteboom demonstrated the stereospecific binding of [3H]estradiol to proteins present in the rat uterus.5 Then Gorski's laboratory, using sucrose gradient centrifugation, found that the [3H]estradiol was selectively bound to a protein molecule of 200 kDa, which was separated from other macromolecules of the cell.6 The conclusion from both these papers was that there existed in the rat uterus a receptor with stereospecificity for estradiol. Edelman's laboratory, which was studying the action of aldosterone on the stimulation of sodium transport in toad bladders, reported the selective accumulation of [3H]aldosterone in the nucleus of toad bladder target cells and the aldosterone stimulation of DNA-directed RNA synthesis and its inhibition by actinomycin D.7 Thus, cell A (Fig. 1) presents the 1965 era paradigm that the steroid receptor was likely localized in the cytosolic compartment of target cells and, when occupied by ligand, had the capability of entering the nucleus where it might regulate in some unknown fashion DNA-directed RNA synthesis. The first recognition of the existence of a receptor for 1α,25(OH)2D3 occurred in 1969 by Haussler and Norman who identified in chick intestinal epithelial cell nuclei the presence of a chromosomal receptor protein which bound the biologically active form of vitamin D3.8 This protein could be solubilized from the chromatin by either 0.3 M KCl or high pH and was purified 167-fold over the crude mucosal homogenate. Further, the binding capacity of the receptor for the active metabolite was saturated after the administration of physiological doses of [3H]vitamin D3. In the interval from 1965–1975, there were intensive efforts with regard to all steroid hormones to characterize the details of the ligand interaction with the receptor and to identify the subcellular location of the unoccupied and occupied receptors. Cell B in Fig. 1 illustrates the paradigm for this decade. The notion was that the unoccupied steroid receptor existed principally in the cytosolic compartment of the cell. Then, after occupancy of the receptor by ligand, there existed a requirement for a ligand-dependent activation or transformation of the steroid-receptor complex before translocation to the nucleus.9 It is interesting to note that researchers working on steroid hormones and their receptors in the 1965–1975 era did not generally acknowledge that the active form of vitamin D was actually a steroid hormone. Thus, in the 1976 Scientific American review article that described the cell B paradigm, there was no mention of the discovery of a new steroid hormone derived from vitamin D3.9 With respect to 1α,25(OH)2D3 receptors, additional reports appeared from 1972 to 1976 from several laboratories describing in much more detail the presence in chick intestine of both cytosolic receptors10-13 and nuclear receptors.14-16 One vexing problem concerned whether the unoccupied 1α,25(OH)2D3 receptor was localized in the cytosolic (like the estrogen and progesterone receptors), or nuclear compartment (like the thyroid receptor), or was present simultaneously in both compartments. In the interval of 1982–1984, there was yet another series of advances with respect to the steroid receptor time line which led to the replacement of the paradigm of cell B by the paradigm of cell C (Fig. 1). Over this interval, there was a series of papers that addressed the issue of the subcellular localization of unoccupied receptors for 1α,25(OH)2D3 in chick intestinal epithelial cells. Walters et al. found, using homogenate preparations, that the unoccupied 1α,25(OH)2D3 receptor was partitioned between the nucleus and the cytosol; further, the balance of the partition ratio depended upon the ionic strength of the buffers employed for the studies.17 The implication of this observation is that it is possible to observe the unoccupied 1α,25(OH)2D3 receptor exclusively in either the cytosol fraction or the nucleus, depending upon the ionic strength of the buffers employed. A companion paper then presented data that unoccupied 1α,25(OH)2D3 receptors were associated with chromatin prior to solubilization by dilution/homogenization in both high- and low-salt buffers,18 suggesting that the in vivo localization of the unoccupied receptor was largely nuclear. Also, it was noted that the intestinal receptor occupancy fraction mirrored the serum 1α,25(OH)2D3 levels after either 1α,25(OH)2D3 or 1α(OH)D3 treatment.19 Ultimately, when monoclonal antibodies to the 1α,25(OH)2D3 nuclear receptor became available from the Haussler and Pike laboratory,20 it was possible to study directly the subcellular localization of unoccupied 1α,25(OH)2D3 receptors21-23; the consensus that emerged was that the largest proportion was present in the nucleus in all of the cell types studied. During this same time interval, two back-to-back papers in Nature in 1984 destroyed the cell B paradigm for classical steroid hormones; evidence was presented that unoccupied steroid hormone receptors could be found in the cell nucleus. King and Green demonstrated, through the use of newly available monoclonal antibodies, that the unoccupied estrogen receptor was, in fact, localized in the nucleus of the uterus target cells.24 Prior to the advent of the monoclonal antibodies, the subcellular localization of steroid receptors could only be determined when they were occupied with high specific activity tritiated preparations of steroid hormones. The corollary was that there were no procedures for in situ study of unoccupied receptors. In the second of these papers, Gorski's laboratory reported that when enucleated cell preparations (spheroplasts) of steroid hormone target cells were analyzed, there was no detectable specific binding of [3H]estradiol.25 Their interpretation was that the unoccupied steroid receptor was not normally present in the cytosolic compartment of target cells. Thus, a new paradigm emerged (see cell C, Fig. 1) where the unoccupied steroid receptor was partitioned between the cell nucleus and the cell cytosol. Three related important events in the embryonic field of vitamin D receptors occurred gradually over the era 1975–1985. First, there was acceptance by endocrinologists and the Endocrine Society that 1α,25(OH)2D3 is a steroid hormone. Second, there was appreciation that ligands for cytosolic/nuclear receptors were not restricted to just steroids; thyroxine, 9-cis-retinoic and 9-trans-retinoic acids and others were shown to be potent initiators of similar signal transduction pathways. Third, while the original expectation was that receptors for 1α,25(OH)2D3 would be found associated with target cells in the three primary target organs responsible for calcium homeostasis, namely intestine, bone, and kidney, this turned out to be a very narrow perception of what we now know is the reality of the breadth and diversity of the vitamin D endocrine system. Over 30 different target organs are known to possess cytosolic/nuclear receptors for 1α,25(OH)2D3.26 Thus, the physiological impact of 1α,25(OH)2D3 includes not only calcium homeostasis but also actions in the immune system, cell differentiation (hematopoietic, skin, and brain cells), regulation of peptide hormone secretion (parathyroid hormone, insulin), and the potential for therapeutic action in cancer cells (leukemia, breast, prostate, and colon cells). There are two other significant milestones in the steroid receptor timeline that must be mentioned. One that occurred over ∼1985–1988 was the cloning of all the steroid receptors27 (including the 1α,25(OH)2D3 receptor).28 This provided, for the first time, knowledge of the precise molecular weights of the receptors (a topic about which there had been much debate over the years) and, more importantly, specification of their precise amino acid sequence and the subsequent realization that all these proteins were evolutionarily related. Another, most significant, addition to the steroid receptor time line occurred in 1996 with the publication of back-to-back papers describing for the first time the three-dimensional structure of the ligand binding domain of the retinoic acid receptor (RAR)γ29 and the thyroid receptor.30 The surprising observation was that, at the secondary and tertiary protein structure level, the proteins were very similar. It should remembered that the retinoic (RAR), thyroid (TR), 1α,25(OH)2D3 (VDR), and estrogen (ER) receptors all belong to the same subfamily of the superfamily of steroid receptors,31 and thus might be predicted to display a high degree of structural homology. The ligand-binding domain is composed of a functional three-layered arrangement of 12 α helices which form a quite cloistered ligand binding pocket. Also K. Yamamoto, in collaboration with P. Sigler, reported in 1991 a crystallographic analysis of the interaction of the glucocorticoid receptor's zinc finger region with DNA.32 It is to be anticipated that soon the complete three-dimensional structure of steroid hormone receptors will become available, of course, including the VDR. Thus, the stage is now set in terms of the receptor time line (Fig. 1) for an introduction of the research area which resulted in the publication of the Nemere et al.1 paper under discussion in this editorial. A wide variety of studies have suggested that not all of the actions of 1α,25(OH)2D3 can be explained by receptor-hormone interactions with the genome.33 Rapid actions of 1α,25(OH)2D3 have been observed at both the cellular (e.g. calcium transport across a tissue) and subcellular level (membrane calcium transport, changes in intracellular second messengers). Table 1 summarizes the cell types in which rapid responses to 1α,25(OH)2D3 have been observed and indicates the general nature of the “rapid” response. In with our understanding of the interaction of 1α,25(OH)2D3 with its nuclear and the of details regulation of gene it is at the time of of this that the field of rapid responses is only in a A system in the laboratory of Norman is the of the vitamin chick where of 1α,25(OH)2D3 the of or rapid stimulation of system in the laboratory of the rapid uptake (within through in both it has been that there a membrane receptor for 1α,25(OH)2D3 with ligand binding that are different from those of the receptor; in system, the that the complex mediates the signal transduction of the hormone via of as to the biological of 1α,25(OH)2D3 that not to be by the nuclear receptor for 1α,25(OH)2D3 (see Table 1) include activation of protein kinase C activity in rapid changes in cytosolic levels in primary of a variety of in or cells and their and in levels in the of the these rapid to be not by the nuclear receptor for 1α,25(OH)2D3 but by an signal transduction system which is by a membrane protein for 1α,25(OH)2D3. has been presented that the view that the membrane receptor is on the of the Also, a cell membrane binding protein for 1α,25(OH)2D3, which has been with and suggested to be a membrane has been and purified are by the paradigm by cell D (Fig. 1). is new about cell D is that it indicates the presence of two different receptors for 1α,25(OH)2D3. that with the introduction of the concept of a second receptor for 1α,25(OH)2D3, there a it is to have a to between the classical nuclear 1α,25(OH)2D3 receptor and the newly described membrane receptor Also, the cell D paradigm the concept that steroid nuclear receptors, including the form either or as of their functional which is formed on the promoters of selected one of the in the paradigm of cell D is to whether the protein of the and are whether they are derived from different genes or of the same primary no data have been provided on this however, two have been K. laboratory has data from cells which has been to indicate the existence of membrane for the is to in cells that display there is present in the membrane an membrane protein that will form a functional complex with that has to that yet is how the complex is to the of a rapid response. One possible might from the of studies are on an antibodies to the classical in with a rapid of cells that have been to 1α,25(OH)2D3. The and report that there is a of accumulation the cell membrane as a of at early after 1α,25(OH)2D3 to the of whether the and are different or similar from the laboratories of Norman and have employed of 1α,25(OH)2D3 which are restricted in with the 1α,25(OH)2D3 to their to the rapid of or a variety of The molecule 1α,25(OH)2D3 is very and normally a about the carbon-carbon bond of the B for details Fig. 1 in it is possible to the about the carbon-carbon Thus, a of that possess only a which with the of shapes by the 1α,25(OH)2D3. we found that the could only as for rapid responses and were to bind directly to the in steroid or to responses. have these to that the ligand binding domain of the has a different specificity that of the and that, it is likely that the and are not the same The of steroid hormone stimulation of rapid biological responses is not restricted to the steroid hormone 1α,25(OH)2D3. There is an and for all of the other classical steroid hormones a wide variety of biological responses which to be explained via interaction of the hormone with the cytosolic/nuclear Table a of some of these Also, there are for and reports describing the existence of or studies on membrane receptors The topic has been by a of Also, this general topic has that the first steroid hormone rapid responses has been for of The first rapid responses of steroid hormones will be in The paper under review represents a collaboration from the laboratory of who has pioneered on the rapid actions of 1α,25(OH)2D3 with respect to intestinal and the laboratory of and who have studying the cell and actions of chondrocytes with respect to the rapid and the of 1α,25(OH)2D3 and Nemere to the collaboration an that was a purified of the chick intestinal membrane receptor for and to the collaboration their and working with rat primary which were then of primary cell with several it has been possible to a that cells either from the or the cell The paper presents of data which are to present the report a membrane receptor for 1α,25(OH)2D3, which mediates the rapid and nongenomic regulation of protein kinase C Figure 1 in Nemere the data describing the of the membrane receptor in of and The are upon the specificity of the which was using purified 1α,25(OH)2D3 membrane receptor of derived from chick intestinal cells. this is the first paper this the only of specificity provided is an of Fig. 1 indicates that there are in the between the and the serum for both the and cells. in of the is the that membrane in Fig. and is not more there also to be significant in the cell cytosol and nucleus. Figure in paper presents sodium and analysis of and from of the and The are the it is that a protein of in chick intestinal and as the and from both the and it is not what is the of the additional which are in the derived from both the and membrane But it is possible to have that there are similar proteins present in both the chick intestinal and rat membrane two very important are whether this cell protein as a binding protein and whether this binding protein can actually as a receptor and activation of some form of signal The to these two are provided in and and present the of analysis with and the transformation from the from and first both to present the with the which is by most researchers to a of the proteins ligand and the receptor or the reported data may of the for these it is to what is the of the the not actually and there are no on the or in the text or that the have been of these of these of are addressed in the paper of entitled of receptor from and It should be that the present studies are very to both the of the in the membrane and as as the in the from the cell system. In this the data of the paper is provided in Fig. where the of the to the activation of activity in cell after with 1α,25(OH)2D3 for is In the of 1α,25(OH)2D3 in a stimulation of activity and this is by by the presence of while there was no inhibition from the The are to be for their collaboration and as their their of evidence for a membrane receptor for 1α,25(OH)2D3 in cells. One with to in this system. in the the will and the describing the of to with the Also it will be very interesting to the nature of the signal transduction linked to the that is by 1α,25(OH)2D3 in the the data of the Nemere et al. paper the paradigm of cell D of Fig. which two receptors for 1α,25(OH)2D3. what the with respect to the steroid receptor One is in the paradigm of cell (Fig. 1). In cell a vitamin receptor has been to the this is a receptor for 24R,25(OH)2D3. what are some of the and on this believe that is only a of the activity of the to 1α,25(OH)2D3, as has been suggested by and The view as by and Norman is that both and 1α,25(OH)2D3 are for calcium and biological indicate that there is a of the and serum which are with the after of a chick study an important role for not in the of a which has a Thus, have biological actions and from 1α,25(OH)2D3, then it to the existence of a receptor for 24R,25(OH)2D3. there has been little data on that there is one that evidence for the existence of a receptor with stereospecificity for which is present in the membrane fraction of the of the of form of for the biological of from the study of who a of the the a that could biological of 24R,25(OH)2D3. of the from before development of those was in of of the from these an accumulation of at of the and of there a specific receptor for then should of another problem describing the receptors in the vitamin D endocrine system. we with a and With the possible existence of three vitamin receptors, and one with a second vitamin D metabolite ligand, at the very it will be for when and at to their use of the D and the VDR. course, it will to the the to whether paradigm cells C, D, or or some other are of the biological reality of vitamin metabolite receptors that mediate the responses associated with the vitamin D endocrine system. One of the to the steroid receptor timeline with its five paradigm of 1α,25(OH)2D3 receptors (Fig. 1) this the paper in this issue of is to to the reader that the concept of steroid receptor action in fact, undergone a evolution over the past and that were in place in the cell B are now and by new Thus, the concept of a membrane receptor for 1α,25(OH)2D3 by the laboratories of and to this another and data on the steroid receptor to It that there will be no in the vitamin D endocrine