Association of 1,25-dihydroxyvitamin D3 with cultured 3T6 mouse fibroblasts. Cellular uptake and receptor-mediated migration to the nucleus

The uptake of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by intact cells was investigated using the cultured embryonic 3T6 mouse fibroblast as a model. Suspended cells, incubated for 60-90 min in serum-containing culture medium supplemented with 1,25-(OH)2D3 (2 nM), maximally accumulate hormone which becomes bound to a typical vitamin D 3.3 S receptor protein. Incubation of cells with varying concentrations of 1,25-(OH)2D3 reveals the presence of 21,000 receptor molecules/3T6 cell, with an apparent uptake constant of 6-8 X 10(-10) M at 37 degrees C. This value contrasts with the equilibrium dissociation constant (Kd) for 1,25-(OH)2D3 binding of 6 X 10(-11) M as determined at 2 degrees C in disrupted cell cytosol. The distribution of unoccupied (R0) receptors is predominantly (greater than 85%) cytosolic in the hormone-deprived state (1,25-(OH)2D3 less than 0.05 nM), whereas exposure to 1,25-(OH)2D3 (2 nM) leads to almost complete nuclear localization of the occupied receptor at both 2 and 37 degrees C. This phenomenon was similarly supported through reconstitution of receptor and purified 3T6 nuclei in vitro in which binding also occurs at 2 degrees C. The majority (65%) of intact cell-formed receptor-nuclear complexes can be solubilized by micrococcal nuclease treatment, suggesting the participation of DNA in the acceptor binding site for the 1,25-(OH)2D3 receptor. Consistent with these data, DNA-binding of receptor also occurred in vitro at 2 degrees C and was a characteristic of both occupied (Rs) and unoccupied receptors. However, elution of the latter occurred at reduced ionic strength, implying that the hormone does physically alter the receptor protein. This binding was also sensitive to prior ethidium bromide saturation of DNA-cellulose, but not phosphocellulose. Although the biologic effects of the 1,25-(OH)2D3 hormone in 3T6 fibroblasts are as yet unknown, the present findings support previous work with 1,25-(OH)2D3 receptors and suggest that this cell represents a good model for the study of nuclear events associated with the molecular action of 1,25-(OH)2D3.     

1,25-Dihydroxyvitamin D3 target cells in rat mammary gland

In autoradiograms of mammary glands of rats on days 18 and 20 of pregnancy and day 6 of lactation after injection of 3H 1,25-(OH)2 vitamin D3, a nuclear concentration of radioactivity is observed in alveolar and ductal cells, as well as in cells of the epidermis of the nipple. Myoepithelial cells and connective tissue cells do not concentrate the hormone in their nuclei. The nuclear radioactivity appears specific, since 1,25-(OH)2 vitamin D3 but not 25-(OH)2 vitamin D3 prevents the uptake. The results suggest the existence of nuclear receptors for 1,25-(OH)2 vitamin D3 in mammary tissues and thus complements previous biochemical evidence showing the presence of cytoplasmic receptors for the hormone in mouse mammary glands; in addition, our results allow the identification of the cell types possessing the receptors.     

Nuclear and cytoplasmic receptors for 1,25-dihydroxycholecalciferol in intestinal mucosa

The apparent hormonal form of cholecalciferol, 1,25-dihydroxycholecalciferol (1,25-(OH)₂-CC), was incubated with intestinal mucosa homogenates and whole intestinal tissue, $\text{in}\text{vitro}$. After 40–70 min, 1,25-(OH)₂-CC was specifically associated with the nuclear chromatin fraction. This sterol remains bound to the cytosol fraction at 0°C and a dramatic movement to the nuclear chromatin occurs at 37°C indicating that the subcellular localization of the sterol is temperature dependent. Isolated intestinal cytosol, previously incubated with 1,25-(OH)₂-CC, is required for transportation of the hormone to the intestinal chromatin fraction; cytosol fractions from other tissues are ineffective mediators of this sterol migration. It is concluded that the intestinal cytosol contains a specific receptor that functions to transport 1,25-(OH)₂-CC to the nucleus, its probable site of action.     

Immunochemical studies on the putative plasmalemmal receptor for 1, 25(OH)(2)D(3). I. Chick intestine

Antisera were raised against the NH(2)-terminus of the putative basal lateral membrane (BLM) receptor for 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3); BLM-VDR]. In Western analyses of BLM proteins, antibody (Ab) 099 was monospecific for a 64.5-kDa band. A protein of 64.5 kDa was also labeled by the affinity ligand [(14)C]1, 25(OH)(2)D(3)-bromoacetate; label was diminished in the presence of excess unlabeled secosteroid. The monoclonal antibody against the nuclear VDR (9A7) failed to detect an appropriate band in BLM fractions. Preincubation of isolated intestinal cells with Ab 099, but not 9A7, affected the following two 1,25(OH)(2)D(3)-mediated signal transduction events: augmented intracellular calcium and protein kinase C activity. Subcellular distribution of Ab 099 reactivity by Western analyses and fluorescence microscopy revealed the highest concentrations in BLM followed by the endoplasmic reticulum. Exposure of isolated intestinal cells to 1,25(OH)(2)D(3) for 10 s or vascular perfusion of duodena for 5 min resulted in a time-dependent increase in nuclear localization of the BLM-VDR antigen, as judged by electron microscopy, whereas 24, 25-dihydroxyvitamin D(3) failed to increase antigenic labeling in nuclei. Densitometric quantitation of Western blots of subcellular fractions prepared from isolated intestinal cells treated with vehicle or 1,25(OH)(2)D(3) confirmed a hormone-induced increase of putative BLM-VDR in the nucleus. It is concluded that a novel cell surface binding protein for 1,25(OH)(2)D(3) has been identified.     

Pyloric gastrin-producing cells and pyloric sphincter muscle cells are nuclear targets for 3H 1,25(OH)2 vitamin D3. Studied by autoradiography and immunohistochemistry

Autoradiographic studies were conducted to identify and characterize target cells for 1,25(OH)2 vitamin D3 in the pyloric region of rats and mice. After injection of 3H 1,25(OH)2 vitamin D3, nuclear concentration of radioactivity was observed in nuclei of duodenal epithelium and certain cells of pyloric glands, while most of the epithelial cells in the pyloric and gastric glands did not show nuclear labeling. In combined immunohistochemical studies, cells in the pyloric glands that showed nuclear concentration of radioactivity, were stained in their cytoplasm with antibodies to gastrin. Also, cells of the pyloric sphincter muscle showed nuclear labeling, in contrast to cells of the duodenal muscularis, which remained unlabeled under the conditions of the experiments. The results indicate that the cells with nuclear radioactivity contain receptors for 1,25(OH)2 vitamin D3 and suggest that gastrin secretion and pyloric muscle functions are regulated by a direct action of 1,25(OH)2 vitamin D3 on these cells.     

1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K)

A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process.     

1,25 (OH)2 vitamin D3 sites of action in the brain. An autoradiographic study

After injection of 3H 1,25 (OH)2 vitamin D3 to adult rats and mice, under normal or vitamin D deficient diet, the hormone was found to be accumulated in nuclei of neurons in certain brain regions. Nuclear concentration was prevented or diminished, when excess unlabeled 1,25 (OH)2 vitamin D3 was injected before 3H 1,25 (OH)2 vitamin D3, while excess 25 (OH) vitamin D3 did not prevent nuclear labeling. Highest nuclear concentration of 3H 1,25 (OH)2 vitamin D3 is observed in certain neurons in the nucleus interstitialis striae terminalis, involving its septo-preoptic pars dorsolateralis and its anterior hypothalamic-thalamic portion, and in the nucleus centralis of the amygdala, all constituting a system of target neurons linked by a component of the stria terminalis. Nuclear concentration of 3H 1,25 (OH)2 vitamin D3 is also found in neurons in the periventricular nucleus of the preoptic-hypothalamic region, including its extensions, the parvocellular paraventricular and arcuate nucleus, in the ventromedial nucleus, supramammillary nucleus, reticular nucleus of the thalamus, ventral hippocampus, caudate nucleus, pallium, in the midbrain-pontine central gray, dorsal raphe nucleus, parabrachial nuclei, cranial motor nuclei, substantia gelatinosa of the sensory nucleus of the trigeminus, Golgi type II cells of the cerebellum, and others. The extensive distribution of target neurons suggests that 1,25 (OH)2 vitamin D3 regulates the production of several aminergic and peptidergic messengers, and influences the activity of certain endocrine-autonomic, sensory and motor systems.     

Solubilization of 1,25-dihydroxyvitamin D3 receptor with pyridoxal 5'-phosphate from hen intestinal mucosa

1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) receptor was solubilized in cytosol fractions upon homogenization of hen intestinal mucosa with pyridoxal 5'-phosphate contained in a low ionic strength buffer. Pyridoxal 5'-phosphate did not inhibit the binding of 1,25-(OH)2D3 to its receptor. The receptor solubilized with pyridoxal 5'-phosphate was similar to the KCl-solubilized receptor in its binding affinity to the hormone and sedimentation coefficient. A majority (greater than 90%) of the mucosal 1,25-(OH)2D3 receptors were obtained as associating with crude chromatin which was prepared with a low ionic strength buffer, and this fraction of the receptor was solubilized with pyridoxal 5'-phosphate. Ten millimolar pyridoxal 5'-phosphate was as effective as approx 0.2 M KCl in solubilizing the receptor from the crude chromatin. Pyridoxal 5'-phosphate also showed a potency to dissociate the 1,25-(OH)2D3-receptor complex previously bound to DNA-cellulose. Pyridoxal 5'-phosphate-related compounds such as pyridoxamine 5'-phosphate and pyridoxal did not show this potency. These results suggest that pyridoxal 5'-phosphate reduced the interaction of 1,25-(OH)2D3 receptor with its nuclear binding components without inhibiting the binding of the receptor to the hormone.     

Interaction between 1,25-dihydroxyvitamin D3 receptors and intestinal nuclei. Binding to nuclear constituents in vitro

The molecular action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is thought to involve its localization within the nucleus of target cells, a process mediated by intracellular receptors. This report probes both the association between chick intestinal 1,25(OH)2D3 receptors and purified homologous nuclei and the interaction between this receptor and nucleic acids. 1,25(OH)2D3 receptors bound to purified nuclei in a apparently saturable manner (Kd = 2.2-4.8 X 10(-10) M) under conditions of intermediate ionic strength and constant protein concentration. Nuclear binding was hormone-dependent; whereas receptor-hormone complex (Rs) binds to nuclei under the ionic conditions employed here (greater than 70%), hormone-free (R0) receptors do not bind (less than 10%). Binding was localized to the nuclear chromatin fraction and was extremely sensitive to KCl concentration both in the incubation medium and during postincubation treatment of nuclei. The interaction appeared to be temperature-independent, suggesting the lack of a classic activation event characteristic of most steroid receptors. Partial digestion of intestinal nuclei with DNase I eliminated subsequent receptor binding by greater than 95%, pointing to the involvement of DNA in the binding interaction. In turn, receptors were found to bind to both DNA and RNA, a characteristic independent of receptor aggregation, but sensitive to disruption with increasing ionic strength buffers. Elution of both Rs and R0 from DNA appeared identical (0.28 M KCl), whereas the strength of interaction with RNA was much less (0.12 M KCl). Thus, while there appeared to be a fundamental difference between R0 and Rs, such that only the binding of receptor-hormone complex to nuclei was allowed under the conditions employed here, this characteristic was not observed during DNA binding. Nevertheless, the possibility exists that the in vivo interaction between 1,25(OH)2D3 receptor and nuclei involves DNA and that this nuclear constituent may be the ultimate site of action of this unique sterol hormone.     

Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3

Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3.     

Receptors for 1,25-dihydroxyvitamin D3 in chick pancreas: a partial physical and functional characterization

1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) receptors from the rachitic chick pancreas have been partially characterized. Analyses of these receptors by isokinetic gradient centrifugation and analytical gel filtration reveal a sedimentation coefficient (S) of 3.3-3.7, a molecular weight (Mr) of 58,500-68,000, and a calculated Stokes molecular radius (Rs) of 34-36 A. Polyethylenimine-ammonium sulfate precipitation of pancreatic cytosol partially purifies aporeceptor and reduces nonspecific binding (in part, 5.8S DBP), thus providing material more amenable to kinetic analyses, Binding studies incorporating this fractionated cytosol reveal an equilibrium dissociation constant (K4) of approximately 0.112 nM at 2 degrees C for the 1,25-(OH)2D3-receptor interaction. Competition studies further demonstrate a particular preference for 1,25-(OH)2D3 over 1,24(R),25-trihydroxyvitamin D3, 24(R),25-dihydroxyvitamin C3, and 25-hydroxyvitamin D3. The pancreatic receptor also binds to immobilized group-selective affinity ligands such as DNA, cibacron blue, and heparin, and can be eluted as a single macromolecular species during standard linear KCl gradients. Its interaction with these ligands supports the premise that the 1,25-(OH)2D3 receptors' fundamental mode of action is at the level of the cellular genome. Salt-dependent nuclear uptake and chromatin localization studies with this receptor in vitro also support this potential site of action. Significantly, a physiologic dose of 1,25-(OH)2[3H]D3 to rachitic chicks leads to the in vivo formation of a receptor-hormone complex as identified by DNA-cellulose chromatography. These observations provide further evidence that the pancreatic protein is a biologically relevant component of the chick pancreas which functions to accumulate hormone intracellularly under physiologic situations.     

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.     

Nuclear receptors for 1,25(OH)2 vitamin D3 in thymus reticular cells studied by autoradiography

After injection of 3H 1,25(OH)2 vitamin D3 to rats fed a vitamin D-deficient diet, nuclear concentration and retention of radioactivity exists in reticular cells of the thymus medulla and cortex, as well as outer cells of developing Hassal's corpuscles. Lymphocytes do not show nuclear concentration of radioactivity. Nuclear concentration in reticular cells is prevented by prior injection of excess 1,25(OH)2 vitamin D3. The results indicate that reticular-endothelial cells contain nuclear receptors for 1,25(OH)2 vitamin D3 and suggest that effects of 1,25(OH)2 vitamin D3 on immune response and lymphocyte differentiation are indirect and mediated through genomic modulation of reticular cell functions such as messenger secretion.     

The role of vitamin K in the interaction of 1,25-dihydroxyvitamin D3 receptors with DNA

Vitamin K deficiency in rats caused a rise of in vivo occupied 1,25(OH)2D3 receptor level in chromatin of the intestinal mucosa and a marked (2-2.5-fold) increase of intestinal cytosolic 1,25(OH)2D3-receptor complex binding with heterologous DNA, whereas maximum binding capacity and equilibrium dissociation constant of cytosolic 1,25 (OH)2D3 receptors did not change. Preincubation of renal and intestinal cytosol of vitamin K-deficient rats with microsomal vitamin K-dependent gamma-carboxylating system reduced sharply 1,25(OH)2D3-receptor complex binding with DNA. In rats treated by vitamin K antagonist along with a low calcium diet, no dramatic decrease of occupied 1,25(OH)2D3 receptors occurred after the animals were maintained with a high calcium diet. No such effect was observed in vitamin K-replete rats. The data demonstrate vitamin K-dependent Ca-sensitive qualitative modification of 1,25(OH)2D3 receptor dropping its binding performance to DNA.     

Are the receptors of 1,25-dihydroxyvitamin D3 vitamin K dependent?

Alimentary deficiency of vitamin K in rats causes a decrease in the level of in vivo occupied nuclear 1,25 (OH)2D3 receptors in small intestinal mucosa and an 2-2.5-fold increase in the ability of cytosolic 1,25 (OH)2D3-receptor complexes to bind to heterologous DNA. The 1,25 (OH)2D3 binding by the receptors is thereby unaffected. Preincubation of kidney and intestinal cytosol of rats with the secondary K-avitaminosis induced by vitamin K antagonist with the microsomal vitamin K-dependent gamma-carboxylation system sharply decreases the binding of the 1.25 (OH)2D3-receptor complexes to DNA. In rats treated with the vitamin K antagonist in combination with a low calcium diet, the subsequent maintenance on a high calcium diet does not cause, in contrast with vitamin K-repleted animals, a sharp decrease of the level of the in vivo occupied 1,25 (OH)2D3 receptors. In vitro Ca2+ cations decrease the binding of the 1,25 (OH)2D3-receptor complexes to DNA only in vitamin K-repleted rats (ED50 = 2.5 x 10(-6) M). The existence of a vitamin K-dependent Ca-sensitive mechanism regulating the binding of the 1,25 (OH)2D3 receptor to DNA has been postulated for the first time.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

Localization of 1,25-dihydroxyvitamin D3 in intestinal nuclei in vivo

Autoradiography of frozen sections of intestinal tissue taken from rachitic chickens given a single intravenous dose of 1,25-dihydroxy[23,24³H]vitamin D₃ (650 pmol, 78 Ci/ mmol) has been carried out. Specific localization of label in the nuclei of intestinal villi and crypt cells could be demonstrated at 2.5 to 6 h postinjection. In contrast, no concentration or localization of radioactivity could be detected in intestinal muscle, liver, and skeletal muscle. These results strongly support the concept that the function of 1,25-dihydroxyvitamin D₃ in intestine is mediated by a nuclear mechanism.     

A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells.

1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.     

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts

The rapid, non-genomic actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(-/-) osteoblasts isolated from wild-type and VDR null mice to study the increase in intracellular calcium ([Ca(2+)](i)) and activation of protein kinase C (PKC) induced by 1,25(OH)(2)D(3). Within 1 min of 1,25(OH)(2)D(3) (100 nM) treatment, an increase of 58 and 53 nM in [Ca(2+)](i) (n = 3) was detected in VDR(+/+) and VDR(-/-) cells, respectively. By 5 min, 1,25(OH)(2)D(3) caused a 2.1- and 1.9-fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated-MBP(4-14) in VDR(+/+) and VDR(-/-) osteoblasts. The 1,25(OH)(2)D(3)-induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)(2)D(3) treatment resulted in the same degree of translocation of PKC-alpha and PKC-delta, but not of PKC-zeta, from cytosol to plasma membrane in both VDR(+/+) and VDR(-/-) cells. These experiments demonstrate that the 1,25(OH)(2)D(3)-induced rapid increases in [Ca(2+)](i) and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)(2)D(3) in osteoblasts.     

Identification of lamin B and histones as 1,25-dihydroxyvitamin D3-regulated nuclear phosphoproteins in HL-60 cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3)-induced differentiation of HL-60 leukemia cells is accompanied by a number of cellular changes including regulation of oncogene expression and induction of terminal differentiation. We investigated the mechanism by which 1,25-(OH)2D3 induces these changes. We detected 10 nuclear phosphoproteins, designated p66, p45, p36, p33, p32, p27, p22, p19, p18 and p17, that show alterations in phosphorylation within 6-40 h of 1,25-(OH)2D3 treatment. When phosphorylation reactions were performed with isolated nuclei (in vitro), three of these proteins were phosphorylated in a calcium and phospholipid dependent manner: p66, p36, and p19 P66 was phosphorylated in response to 1,25-(OH)2D3 and purified in a manner similar to that used for nuclear lamins. Western blot analysis of 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels confirmed its identity as lamin B. Phosphorylation of p17 and p18 decreased following 1,25-(OH)2D3 treatment. We separated p17 and p18 by SDS-PAGE and obtained N-terminal amino acid sequence to identify these phosphorproteins as histones H2b and H3, respectively. P19 and p22 were both DNA-cellulose binding proteins whose phosphorylation was altered by 1,25-(OH)2D3 treatment. Increased phosphorylation of p27 was detected using 2-dimensional SDS-PAGE. Phosphorylation of nuclear proteins in the intact cell (in vivo), revealed increases in p66, p45, p36, and p33 phosphorylation and a decrease in p17 phosphorylation following 1,25-(OH)2D3 treatment. We detected an increase in phosphorylation of p32, which was extracted with salt from nuclei and migrated on SDS-PAGE similar to histone H1. Thus, we have identified 1,25-(OH)2D3-sensitive nuclear phosphoproteins, including lamin B and several histones. We have also detected and characterized several less abundant nuclear DNA binding phosphoproteins whose phosphorylation was affected by 1,25-(OH)2D3.