Modulation of rat calbindin-D28 gene expression by 1,25-dihydroxyvitamin D3 and dietary alteration

We have used a specific cDNA to the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28k) to study the regulation of the expression of this mRNA in rat kidney and brain. The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and dietary alteration on genomic expression were characterized by both Northern and slot blot analysis. Administration of 1,25-(OH)2D3 for 7 days (25 ng/day) to vitamin D-deficient rats resulted in a marked increase in renal calbindin-DmRNA, renal calbindin, and serum calcium. When vitamin D-deficient rats were supplemented for 10 days with calcium (3% calcium gluconate in the water, 2% calcium in the diet) serum calcium levels were similar to the levels observed in the 1,25-(OH)2D3-treated rats. However, in the calcium-supplemented rats the levels of renal calbindin and renal calbindin mRNA were similar to the levels observed in the vitamin D-deficient rats, suggesting that calcium alone without vitamin D does not regulate renal calbindin gene expression in vivo. In dietary alteration studies in vitamin D-replete rats, renal calbindin protein and mRNA increased 2.5-fold in rats fed diets low in phosphate providing evidence that in the rat the nutritional induction of calbindin is accompanied by a corresponding alteration in the concentration of its specific mRNA. Under low dietary calcium conditions, the levels of renal calbindin protein and mRNA were similar to the levels observed in control rats, although 1,25-(OH)2D3 serum levels were markedly elevated, suggesting that factors in addition to 1,25-(OH)2D3 can modulate renal calbindin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hormones and development on the expression of the rat 1,25-dihydroxyvitamin D3 receptor gene. Comparison with calbindin gene expression

We have used specific cDNAs to the rat vitamin D receptor (VDR) and to the mammalian vitamin D-dependent calcium-binding proteins (calbindin-D9k in intestine and calbindin-D28k in kidney) in order to obtain a better understanding of the regulation of the VDR gene and its relationship to calbindin gene expression. Hormonal regulation and development expression of the rat VDR gene were characterized by both Northern and slot blot analyses. Administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 25 ng/day for 7 days) to vitamin D-deficient rats resulted in an increase in calbindin mRNA in intestine and kidney but no change in VDR mRNA in these tissues. Vitamin D-deficient rats responded to dexamethasone treatment (100 micrograms/100 g of body weight/day for 4 days) with a 2.5-fold increase in intestinal VDR mRNA which was accompanied by a 4-fold decrease in intestinal calbindin-D9k mRNA. Developmental studies indicated a pronounced increase in renal VDR mRNA and calbindin-D28k mRNA between birth and 1 week of age. In the intestine, an induction of VDR and calbindin-D9k gene expression was observed at a later time, during the 3rd postnatal week (the period of increased duodenal active transport of calcium). Taken collectively, our data indicate that in the adult rat, target tissue response to hormone is not modified by a corresponding alteration in new receptor synthesis. However, developmental studies indicate that the induction of 1,25(OH)2D3 receptor mRNA is correlated with the induction of calbindin gene expression. Our results also demonstrate that glucocorticoid administration can result in an alteration in intestinal calbindin and VDR gene expression.     

In vitro enzyme activation with calbindin-D28k, the vitamin D-dependent 28 kDa calcium binding protein.

Purified porcine erythrocyte membrane Ca(2+)-ATPase and 3':5'-cyclic nucleotide phosphodiesterase were stimulated in a dose-dependent, saturable manner with the vitamin D-dependent calcium binding protein from rat kidney, calbindin-D28k (CaBP-D28k). The concentration of CaBP-D28k required for half-maximal activation (K0.5 act.) of the Ca(2+)-ATPase was 28 nM compared to 2.2 nM for calmodulin (CaM), with maximal activation equivalent upon addition of either excess CaM or CaBP-D28k. 3':5'-Cyclic nucleotide phosphodiesterase (PDE) also showed equivalent maximum saturable activation by calbindin (K0.5 act. = 90 nM) or calmodulin (K0.5 act. = 1.2 nM). CaBP-D28k was shown to effectively compete with CaM-Sepharose for PDE binding. Immunoprecipitation with CaBP-D28k antiserum completely inhibited calbindin-mediated activation of PDE but had no effect on calmodulin's ability to activate PDE. While the physiological significance of these results remains to be established, they do suggest that CaBP-D28k can activate enzymes and may be a regulator of yet to be identified target enzymes in certain tissues.     

Expression in Escherichia coli of full-length and mutant rat brain calbindin D28. Comparison with the purified native protein

Studies of vitamin D-dependent 28-kilodalton calcium binding protein (calbindin D28) have been hindered by difficulties in purifying large amounts of the protein. In order to overcome this problem, we cloned and expressed a full-length rat brain calbindin D28 cDNA. In addition, we isolated and purified to homogeneity, native rat brain calbindin D28. The isolated native protein has an apparent molecular mass of 27 kDa and properties similar to those of the well-characterized chicken calbindin D28. It has an acidic isoelectric point (approximately 4.5), a high affinity for calcium, and an amino terminus blocked to Edman degradation. The properties of the native and the recombinant proteins were examined by gel electrophoresis, isoelectric focusing, protein sequencing, amino acid composition analysis, and calcium binding assays. We demonstrated that: (i) the authentic and the full-length recombinant proteins have similar molecular weights and isoelectric points; (ii) the proteins have the same amino acid composition; (iii) the proteins bind calcium in a similar manner; (iv) the absence of a blocking NH2-terminal group in the recombinant protein does not appreciably influence the binding of calcium. To further examine the calcium binding properties of this protein, we constructed deletion mutants lacking one or both of the two putative degenerated calcium binding sites (EF hand regions). These deletions resulted in smaller proteins that still bound calcium. The ability to express and purify calbindin D28 and mutants thereof should allow the systematic elucidation of structure-function relationships in this class of calcium binding proteins.     

Calbindin-D28k decreases L-type calcium channel activity and modulates intracellular calcium homeostasis in response to K+ depolarization in a rat beta cell line RINr1046-38

Calbindin-D(28k), acts as a modulator of depolarization induced calcium transients in the pancreatic beta cell. However, specific mechanisms have not been defined. Here we show for the first time that the calcium binding protein calbindin-D(28k) acts by affecting calcium influx through voltage-dependent calcium channels in RIN pancreatic beta cells. Whole-cell patch-clamp recordings revealed that Ca(2+) current amplitudes of calbindin-D(28k) expressing RINr1046-38 beta cells were smaller than the Ca(2+) current amplitudes in control cells in response to depolarizing pulses. The peak current was observed at +20mV and the average amplitude was approximately 50pA in the calbindin expressing cells compared to approximately 250pA in control cells. In calbindin-D(28k) expressing cells, the channels had enhanced sensitivity to Ca(2+) dependent inactivation and currents decayed much more rapidly than in control cells. The Ca(2+) channels affected by calbindin were found to have biophysical properties consistent with dihydropyridine-sensitive L-type calcium channels. In response to depolarizing concentrations of K(+), calbindin expression caused a five-fold decrease in the rate of rise of [Ca(2+)](i) and decay was slower in the calbindin expressing cells. Application of verapamil resulted in a drop in the [Ca(2+)](i) signal to pre-stimulation levels indicating that the Ca(2+) channel responsible for the depolarization evoked Ca(2+) entry, modulated by calbindin, is the L-type. Co-immunoprecipitation and GST pull-down assays indicate that calbindin-D(28k) can interact with the alpha(1) subunit of Ca(v)1.2. We thus conclude that calbindin-D(28k) can regulate calcium influx via L-type calcium channels. Our findings suggest a role for calbindin-D(28k) in the beta cell in modulating Ca(2+) influx via L-type voltage-dependent calcium channels.     

Analysis of rat vitamin D-dependent calbindin-D28k gene expression

We report the use of a cloned cDNA for mammalian calbindin-D28k (28-kDa vitamin D-dependent calcium-binding protein) to study the expression of the rat calbindin gene. Tissue distribution studies, using Northern analysis, indicated that calbindin-D28k-mRNA is detected in rat kidney and brain but is not detected in rat intestine, testes, bone, pancreas, liver, lung, or skeletal muscle. Both rat kidney and brain contain three RNA species (1.9, 2.8, and 3.2 kilobase pairs). The regulation of the gene was characterized by both Northern and slot blot analysis. Hormonal regulation, developmental expression of calbindin-D28k-mRNA, and the effect of dietary alteration were examined. In the kidney all three species of mRNA were dependent on the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) for their induction. The time course of induction of renal calbindin-D28k-mRNA indicated that a significant increase in calbindin-D-mRNA was detectable as early as 2 h following a single injection of 1,25-(OH)2D3 (200 ng/100 g of body weight), reaching a maximum at 12 h. Unlike the kidney high levels of calbindin-D28k-mRNA were observed in the brain of vitamin D-deficient rats. The concentration of calbindin-D28k-mRNA in brain was unchanged after 1,25-(OH)2D3 administration. Developmental studies indicated that calbindin-D-mRNA in rat kidney and brain is present prior to birth but is developmentally regulated in a tissue-specific manner. The most pronounced changes in the abundance of renal calbindin-D28k-mRNA occur between birth and 1 week of age. Unlike the kidney a large increase in brain calbindin-D28k-mRNA occurs at a later time, between 1 and 2 weeks of age (the period of major synapse formation). In dietary alteration studies results of Northern blot analysis indicate that low dietary phosphorus results in increased calbindin-D-mRNA in kidney but not in brain. These studies represent the first analysis of the rat calbindin-D28k gene and its regulation in vivo. Our findings suggest that in rat kidney and brain there are significant differences both in the expression of the gene for calbindin-D28k and its regulation by 1,25-(OH)2D3.     

Calbindin-D9K immunolocalization and vitamin D-dependence in the bone of growing and adult rats

This report presents evidence for the presence of the vitamin D-dependent calcium-binding protein, calbindin-D9K, in bone cells and matrix. In undecalcified frozen sections of growing and adult rat bone, calbindin-D9K was immunohistochemically localized in trabecular bone of the epiphysis and metaphysis and in cortical bone of the diaphysis. It was found within the cytoplasm of osteocytes, of osteoblasts lining the osteoid, and osteoblasts inside the osteoid seams. It was also found in the osteoblast processes and the anastomosed reticulum of the processes connecting the osteocytes with each other. Extracellularly, calbindin-D9K immunoreactivity was present in compact cortical bone in the areas of the mineralized matrix surrounding the osteocyte lacunae, and in the pericanalicular walls containing the cell processes. Calbindin-D9K immunoreactivity was low or absent from the cytoplasm of osteocytes in trabecular bone from severely vitamin D-deficient rats and restored in vitamin D-deficient rats given a single dose of 1,25(OH)2-VitD3. Thus, the synthesis of immunoreactive calbindin-D9K by osteoblasts and osteocytes in trabecular bone is vitamin D-dependent. The presence of immunoreactive calbindin-D9K in the osteocytes and their cell processes suggests that this calcium-binding protein is involved in the calcium fluxes regulating bone calcium homeostasis. Its localization in osteoblasts involved in bone formation and in their cell processes suggests that it has a role in the calcium transport from these cells towards the sites of active bone mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoprotective effects of calbindin-D(28k) against antimycin-A induced hypoxic injury in proximal tubular cells

Intracellular calcium plays an important role on the pathogenesis of hypoxia-induced cellular injury. Calbindin-D(28k), a cytosolic vitamin D-dependent calcium binding protein, can serve as a buffer to limit a surge in intracellular Ca2+ concentration ([Ca2+]i) induced by various stimulations. To evaluate the possible cytoprotective effect of calbindin-D(28k) against hypoxic injury in proximal tubular cells, a plasmid containing calbindin-D(28k) cDNA under the control of CMV immediate-early gene promoter was transfected into the murine proximal tubular epithelial (MCT) cells. The expression of calbindin-D(28k) in the transfected cells was verified with Northern blot analysis, Western blot analysis, and immunofluorescent staining. The non-transfected and transfected MCT cells were subjected to chemical hypoxia induced by antimycin A (10 microM) and glucose deprivation for 30-120 min. The transfection of calbindin-D(28k) reduced lactate dehydrogenase (LDH) release by 41%, 41%, 24%, and 24%, respectively, at 30, 60, 90 and 120 min after hypoxia when compared to the non-transfected cells (all p < 0.05). Cell viability after hypoxic injury was also significantly higher in transfected cells than non-transfected cells. Transfection with the plasmid without calbindin-D(28k) cDNA did not affect LDH release or cell viability after chemical hypoxic injury. [Ca+2]i was measured ratiometrically with fura-2 after exposure to chemical hypoxia. The rate of initial rise in [Ca2+]i and final [Ca+2]i at 30-120 min were significantly lowered in transfected cells. In conclusion, this study demonstrated that transfection of calbindin-D(28k) gene into MCT cells provide protective effects against chemical hypoxic injury probably through its buffering effects on [Ca+2]i.     

A characterization of vitamin D-independent intestinal calcium absorption in the osteopetrotic (op/op) mouse.

Previous work in our laboratory showed that the osteopetrotic (op/op) mouse possesses a vitamin D-independent mechanism of intestinal calcium absorption. This study was performed in an effort to further characterize the mechanism. The vitamin D-deficient op/op mouse absorbed calcium faster than either a vitamin D-deficient or 1, 25-dihydroxyvitamin D(3)-supplemented wild-type mouse. This increased rate of absorption was not found at concentrations of calcium that result in diffusional calcium absorption. Thus, vitamin D-deficient op/op mice had intestinal calcium absorption similar to that of vitamin D-deficient wild-type littermates when increasing levels of calcium were administered. Also, mRNA and protein levels of calbindin-D9k were similar in vitamin D-deficient wild-type and op/op mice as well as in wild-type and op/op mice treated with 1, 25-dihydroxyvitamin D(3). Therefore, the mechanism of vitamin D-independent intestinal calcium absorption in the op/op mouse is distinct from vitamin D-dependent intestinal calcium absorption.     

Amino acid sequence analysis of two mouse calbindin-D9k isoforms by tandem mass spectrometry. Protein modification by internal insertion of a single amino acid

Two forms of calbindin-D9k have sometimes been observed within a single tissue. Sequencing of these proteins has been complicated by the presence of blocked amino termini. Tandem mass spectrometry is a powerful tool for comparing related proteins, and its use does not depend upon an unblocked amino terminus. In the present studies, calbindin-D9k was purified from the intestines of mice (270 animals per purification) by use of gel permeation chromatography and two preparative electrophoresis steps in the presence and absence of EDTA. The purified protein appeared to be homogeneous following electrophoresis under nondenaturing conditions, but two components were identified by sodium dodecyl sulfate-gel electrophoresis and immunoblotting. Two forms of the protein were isolated by reverse-phase high performance liquid chromatography. In each of three preparations, the average ratio of the major:minor isoforms was 2:1. The major form contained 77 amino acids and lacked the amino-terminal serine found in 78-amino acid calbindins from rat and pig. The amino acid sequence was identical with the deduced sequence reported for rat intestinal calbindin-D9k in 73 of 77 positions. In the minor form, a glutamine was found in a location between Lys-43 and Ala-44 of the major form and between the two calcium binding sites of the protein. The minor form was otherwise identical with the major form, including the presence of a blocked amino terminus. The inserted glutamine was located at the site of an intron in the rat calbindin gene, suggesting the possibility that alternative splicing produced the two forms of calbindin-D9k. The functional significance of an inserted amino acid between the two calcium binding sites remains to be explored.     

Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum-Free Medium

The Ca2+-binding proteins parvalbumin (Mr = 12K) and calbindin D28K [previously designated vitamin D-dependent Ca2+-binding protein (Mr = 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum-free cultures of hypothalamic cells from 16-day-old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin-immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti-parvalbumin serum seems to recognize only the metal-bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+-dependent processes associated with neurotransmitter release.     

Calbindin-D28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity

The rate of osteoblast apoptosis is a critical determinant of the rate of bone formation. Because the calcium-binding protein calbindin-D(28k) has anti-apoptotic properties in neuronal cells and lymphocytes, we searched for the presence of this protein in osteoblastic cells and investigated whether it can modify their response to proapoptotic signals. Calbindin-D(28K) was expressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection of rat calbindin-D(28k) cDNA blocked tumor necrosis factor alpha (TNFalpha)-induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell viability and nuclear morphology of cells cotransfected with the green fluorescent protein targeted to the nucleus, whereas transfection of the empty vector had no effect. Calbindin-D(28k) levels in several stably transfected MC3T3-E1 lines were directly related to protection from TNFalpha-induced apoptosis. Purified rat calbindin-D(28k) markedly reduced the activity of caspase-3, a critical molecule for the degradation phase of apoptosis, in a cell-free assay. In addition, cell extracts from MC3T3-E1 cells expressing high levels of calbindin-D(28k) decreased caspase-3 activity, compared with extracts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGTA or addition of other calcium-binding proteins such as calbindin-D(9k), S100, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, calbindin-D(28k) interacts with the active form of caspase-3 as demonstrated by a GST pull-down assay. These results demonstrate that calbindin-D(28k) is a biosynthetic product of osteoblasts with a role in the regulation of apoptosis. They also reveal that the antiapoptotic properties of calbindin-D(28k) may result not only from calcium buffering but also from the ability of the protein to interact with and to inhibit caspase-3 activity, a property that is independent of its calcium binding capability.     

Localization of 28 kDa calbindin in human odontoblasts

Summary The presence of 28 kDa calbindin in human odontoblasts was studied by use of specific antibodies raised against chick duodenal 28 kDa calbindin, in immunofluorescence, immuno-peroxidase, and electron-microscopic labelling experiments.The calbindin-like protein was detected mainly in the cytoplasm of odontoblast cell bodies, in their processes and occasionally in their nuclei. Correspondingly, at the ultrastructural level, immunoreactive material was associated with the cytosol, microfilaments and cilia. These findings suggest that human odontoblasts express a 28 kDa vitamin D-dependent calcium-binding protein, unlike those of rats and mice in which ameloblasts are the only cells immunoreactive for the protein.     

Biological actions and mechanism of action of calbindin in the process of apoptosis

Although it was originally proposed that the major role of calbindin is to facilitate the vitamin D dependent movement of calcium through the cytosolic compartment of the intestinal or renal cell, we found that calbindin also has a major role in different cell types in protecting against apoptotic cell death. Calbindin, which buffers calcium, can inhibit apoptosis induced by different proapoptotic stimuli. Expression of calbindin-D(28k) in neural cell suppressed the proapoptotic actions of presenilin-1, which is causally linked to familial Alzheimer's disease, by preventing calcium mediated mitochondrial damage and the subsequent release of cytochrome c. Calbindin, by buffering intracellular calcium can also protect HEK 293 kidney cells from parathyroid hormone induced apoptosis that was found to be mediated by a phospholipase C dependent increase in intracellular calcium. In addition, cytokine mediated destruction of pancreatic beta cells can be prevented by calbindin. Induction by cytokines of nitric oxide, peroxynitrite and lipid hydroperoxide production was significantly decreased in calbindin expressing beta cells. Thus, calbindin-D(28k), by inhibiting free radical formation, can protect islet beta cells from autoimmune destruction in type 1 diabetes. Calbindin-D(28k) can also protect against apoptosis in bone cells. Calbindin was found to block apoptosis in osteocytic and osteoblastic cells. Our findings suggest that calbindin is capable of directly inhibiting the activity of caspase-3, a common downstream effector of multiple apoptotic signaling pathways, and that this inhibition results in an inhibition of tumor necrosis factor (TNFalpha) and glucocorticoid induced apoptosis in bone cells. Thus, while part of calbindin's protective effect may result from buffering rises in intracellular calcium, other mechanisms of action, such as inhibition of caspase activity, also play a significant role in the prevention of apoptosis by calbindin-D(28k). These findings have implications for the prevention of degeneration in different cell types and therefore could prove important for the therapeutic intervention of many diseases, including diabetes and osteoporosis.     

Calbindin-D32k is localized to a subpopulation of neurons in the nervous system of the sea cucumber Holothuria glaberrima (Echinodermata)

Members of the calbindin subfamily serve as markers of subpopulations of neurons within the vertebrate nervous system. Although markers of these proteins are widely available and used, their application to invertebrate nervous systems has been very limited. In this study we investigated the presence and distribution of members of the calbindin subfamily in the sea cucumber Holothuria glaberrima (Selenka, 1867). Immunohistological experiments with antibodies made against rat calbindin 1, parvalbumin, and calbindin 2, showed that these antibodies labeled cells and fibers within the nervous system of H. glaberrima. Most of the cells and fibers were co-labeled with the neural-specific marker RN1, showing their neural specificity. These were distributed throughout all of the nervous structures, including the connective tissue plexi of the body wall and podia. Bioinformatics analyses of the possible antigen recognized by these markers showed that a calbindin 2-like protein present in the sea urchin Strongylocentrotus purpuratus, corresponded to the calbindin-D32k previously identified in other invertebrates. Western blots with anti-calbindin 1 and anti-parvalbumin showed that these markers recognized an antigen of approximately 32 kDa in homogenates of radial nerve cords of H. glaberrima and Lytechinus variegatus. Furthermore, immunoreactivity with anti-calbindin 1 and anti-parvalbumin was obtained to a fragment of calbindin-D32k of H. glaberrima. Our findings suggest that calbindin-D32k is present in invertebrates and its sequence is more similar to the vertebrate calbindin 2 than to calbindin 1. Thus, characterization of calbindin-D32k in echinoderms provides an important view of the evolution of this protein family and represents a valuable marker to study the nervous system of invertebrates.     

Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of expression of vitamin D-dependent Ca2(+)-binding protein, calbindin-D28K, in the yolk sac

The developing chick embryo acquires calcium from two sources. Until about Day 10 of incubation, the yolk is the only source; thereafter, calcium is also mobilized from the eggshell. We have previously shown that during normal chick embryonic development, vitamin D is involved in regulating yolk calcium mobilization, whereas vitamin K is required for eggshell calcium translocation by the chorioallantoic membrane. We have studied here the biochemical action of 1,25-dihydroxy vitamin D3 in the yolk sac by examining the expression and regulation of the cytosolic vitamin D-dependent calcium-binding protein, calbindin-D28K. Two types of embryos are used for this study, normal embryos developing in ovo and embryos maintained in long-term shell-less culture ex ovo, the latter being dependent solely on the yolk as their calcium source. Our findings are (1) calbindin-D28K is expressed in the embryonic yolk sac, detectable at incubation Days 9 and 14; (2) the embryonic yolk sac calbindin-D28K resembles that of the adult duodenum in both molecular weight (Mr 28,000) and isoelectric point, as well as the presence of E-F hand Ca2(+)-binding structural domains; (3) systemic calcium deficiency caused by shell-less culture of chick embryos results in enhanced expression of calbindin-D28K in the yolk sac during late development; (4) yolk sac calbindin-D28K expression is inducible by 1,25-dihydroxy vitamin D3 treatment in vivo and in vitro; and (5) immunohistochemistry revealed that yolk sac calbindin-D28K is localized exclusively to the cytoplasm of the yolk sac endoderm. These findings indicate that the chick embryonic yolk sac is a genuine target tissue of 1,25-dihydroxy vitamin D3.