Differential effects of protease inhibitors on 1,25-dihydroxyvitamin D3 receptors

We describe herein two different effects of protease inhibitors and substrates on receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) obtained from the intestinal mucosa of vitamin D-deficient chicks: inhibition of binding of 1,25(OH)2D3 to its receptor and stabilization of the receptor. Both L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, block [3H]1,25(OH)2D3 binding to the receptor. Fifty per cent inhibition of binding occurs at 20 microM TPCK, and 100% inhibition at 100-200 microM; TLCK is about 25-fold less effective. At higher concentrations (10-100 mM), the chymotrypsin substrates N alpha-p-tosyl-L-arginine methyl ester and tryptophan methyl ester and the cathepsin B inhibitor leupeptin also inhibit [3H] 1,25(OH)2D3 binding to its receptor. Different inhibitors and substrates interact with the receptor differently: TPCK (20 microM) and N alpha-p-tosyl-L-arginine methyl ester (10 mM) are reversible, noncompetitive inhibitors, L-tryptophan methyl ester (20 mM) is a reversible competitive inhibitor, and phenylmethylsulfonyl fluoride (300 microM) shows no effect on [3H]1,25(OH)2D3 binding to its receptor. The most stable form of unoccupied 1,25(OH)2D3 receptors from chick intestinal mucosa was that obtained from a low salt chromatin preparation (t 1/2 = 6.0 h). The presence of KCl drastically decreased receptor stability (t 1/2 = 1.8 h); and the addition of 2.5 mM CaCl2 further reduced their stability. Phenylmethylsulfonyl fluoride and Trasylol inhibited the KCl-induced receptor instability, but did not prevent the additional instability in the presence of CaCl2. In summary, TPCK and TLCK exert direct effects on the 1,25(OH)2D3 receptor molecule, independent of their protease inhibitor function. These compounds may prove useful as covalent affinity labels for the receptor. On the other hand, phenylmethylsulfonyl fluoride and Trasylol stabilize 1,25(OH)2D3 receptors, probably via inhibition of KCl-activated nuclear protease(s). This receptor stabilization will be advantageous in receptor assays and/or purification procedures.     

Salt-induced activation of 1,25-dihydroxyvitamin D3 receptors to a DNA binding form

In this report we examine the DNA-cellulose binding and sedimentation properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors from rat intestine and cultured human mammary cancer cells (MCF-7) extracted in nonactivating (low salt) buffers. Receptors prepared in hypotonic buffer had low DNA binding (13%) compared to receptors extracted with 0.3 M KCl (50%). Treatment of low salt receptor preparations with KCl significantly increased (approximately 3-fold) DNA-binding (activation), demonstrating that receptors can be "activated" in vitro. Activated receptors eluted from DNA-cellulose at 0.18 M KCl. Sedimentation analysis followed by DNA-cellulose binding indicated that activated receptors are approximately 3.2 S and unactivated receptors 5.5 S in size. These results suggest that dissociation of an aggregated moiety may lead to receptor activation. Treatment of unactivated receptor with RNase did not alter DNA binding or sedimentation properties of the aggregated receptor. Treatment of unactivated receptor complexes with heat did not increase DNA binding, and molybdate did not block subsequent salt activation. In summary these results suggest that 1,25(OH)2D3 receptors undergo a salt-induced activation step similar to that described for other steroid receptor systems. However, 1,25(OH)2D3 receptors differ from other steroid receptors in not exhibiting heat activation nor having salt activation blocked by molybdate.     

DNA binding property of vitamin D3 receptors associated with 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3

Using [3H]-26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (F6-1,25-(OH)2D3), we have examined its ability to bind to the 1,25-(OH)2D3 receptor, and the ability of the resulting complex to bind DNA. The binding sites for [3H]F6-1,25-(OH)2D3 in the chick intestinal receptor represented a limited number of saturable sites for which 1,25-(OH)2D3 competes. 1,25-Dihydroxyvitamin D3 is three times more active than F6-1,25-(OH)2D3 in displacing [3H]F6-1,25-(OH)2D3. By affinity chromatography using DNA-Sephadex, the [3H]F6-1,25-(OH)2D3 receptor complex eluted from the column in a single peak at 0.14 M KCl, while [3H]-1,25-(OH)2D3 receptor complex eluted at 0.13 M KCl. These results indicate that F6-1,25-(OH)2D3 and 1,25-(OH)2D3 recognize the same binding site of the receptor and that the F6-1,25-(OH)2D3 receptor complex binds DNA more tightly than the 1,25-(OH)2D3 receptor complex. We suggest that the higher binding affinity for DNA may contribute to the greater biological activity of F6-1,25-(OH)2D3.     

Immunochemical detection of unique proteolytic fragments of the chick 1,25-dihydroxyvitamin D3 receptor. Distinct 20-kDa DNA-binding and 45-kDa hormone-binding species

We have characterized proteolytic fragments of the chick intestinal 1,25-dihyroxyvitamin D3 (1,25-(OH)2D3) receptor, produced through either exogenous or endogenous protease action, utilizing a variety of physical and functional assays coupled to immunoblot detection methodology. Treatment of intestinal cytosol with increasing concentrations of trypsin resulted in a progressive diminishment of the 60-kDa receptor concomitant with the appearance of a 20-kDa fragment reactive by Western blot analysis to an anti-1,25-(OH)2D3 receptor monoclonal antibody. Cleveland analysis supported the receptor-origin of this 20-kDa fragment: a common immunoreactive species of 12 kDa could be generated by Staphylococcus aureus V8 protease treatment of the intact 60-kDa receptor as well as the 20-kDa proteolytic product. The 20-kDa fragment did not bind hormone but was capable of interacting with DNA-cellulose in a fashion identical to that of the 60-kDa receptor and, therefore, may contain the functional DNA-binding domain of the chick 1,25-(OH)2D3 receptor. Thus, this fragment likely represents the complement of a larger hormone-bound fragment that we have previously described (Allegretto, E. A., and Pike, J.W. (1985) J. Biol. Chem. 260, 10139-10145). In contrast to the exogenous effect of trypsin, incubation of cytosol resulted in the time-dependent formation of an endogenous protease-derived fragment of 45 kDa. Cleveland analysis was consistent with the 60-kDa receptor derivation of the 45-kDa fragment. This species retained the hormone-binding site and the antibody determinant but was devoid of DNA-binding activity. Moreover, it generated neither the trypsin-dependent 20-kDa fragment nor the V8 protease-dependent 12-kDa species and, therefore, was derived from the opposite end of the receptor molecule. These data have facilitated the construction of a schematic model of the chick receptor in which the immunoreactive epitope is located between the functional domains for hormone binding and DNA binding.     

Identification of 1,25-dihydroxyvitamin D3 receptors and activities in muscle

Cytosols from cultured myoblast cells (G-8 and H9c2) prepared in high salt (0.3 M KCl) possesses receptor like proteins for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) that sediment in the 3.2 S region of sucrose gradients. These receptors were characterized as having high affinity (Kd less than 0.1 nM) for 1,25-(OH)2D3 and are in low capacity (less than 80 fmol/mg of cytosol protein). Analog competition for receptor binding revealed that 1,25-(OH)2D3 was more potent than 24,25-(OH)2D3, or 25-(OH)2D3 for displacement of 1,25-(OH)2[3H]D3 from these 3.2 S region sedimenting receptors. Furthermore, the receptor proteins had affinity for DNA and eluted from Sephacryl S-200 as a macromolecule with Stokes radius (Rs) of 32 A. High salt cytosol from collagenase-dispersed skeletal muscle cells was also found to possess a 3.2 S 1,25-(OH)2D3 receptor-like protein. The 1,25-(OH)2D3 receptor concentration in both G-8 and H9c2 myoblast lines was found to down-regulate by 50-70% when cells were stimulated to differentiate to myotubes by lowering fetal calf serum to 5% of the medium. Moreover, we demonstrated that 1,25-(OH)2D3 can inhibit DNA synthesis and cell proliferation of the G-8 myoblast cells in a dose-dependent manner. 1,25-(OH)2D3 was more potent at inhibiting cell proliferation in cells grown in 5% serum than in 20% serum. The data suggest that 1,25-(OH)2D3 can act directly on muscle myoblast via a 1,25-(OH)2D3 receptor that is similar to those found in intestine and bone. The data support the possibility that muscle is a target tissue for 1,25-(OH)2D3 and the hormone may act to initiate terminal differentiation of myoblast cells.     

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.     

Retinoic acid modulation of 1,25(OH)2 vitamin D3 receptors and bioresponse in bone cells: species differences between rat and mouse

Retinoic acid (RA) caused a reduction in the level of 1,25(OH)2D3 receptors to 1/3 of control in rat osteoblast-like cells (ROB) while increasing the receptor level to 3-fold the control in mouse osteoblast-like cells (MOB). Scatchard analysis of receptor binding indicated that there was no change in affinity for 1,25(OH)2D3. The changes in receptor levels required time to develop and were dose-dependent. RA also modulated the ability of cells to respond to 1,25(OH)2D3 as measured by the induction of the enzyme 25(OH)D3-24 hydroxylase. Induction of enzyme activity by 1,25(OH)2D3 closely paralleled receptor level established by RA pretreatment. In MOB, the up-regulation of the receptor occurred despite the action of RA to inhibit DNA, RNA and protein synthesis. However, RA stimulation of 1,25(OH)2D3 receptor levels was blocked by the addition of cycloheximide or actinomycin D, indicating that the up-regulation required protein and RNA synthesis. The opposite effect of RA on mouse and rat cells suggests that important species-dependent factors modulate the action of retinoids on mammalian cells.     

Biochemical evidence for 1, 25-dihydroxyvitamin D receptor macromolecules in parathyroid,pancreatic, pituitary,and placental tissues

DNA-cellulose chromatography has been recently employed in our laboratory as an extremely effective and sensitive technique with which to identify macromolecules which specifically bind 1,25-dihydroxyvitamin D (1,25-(OH)₂D). Chromatography of cytosols prepared from rachitic chick intestine, parathyroid gland, pancreas, pituitary gland, and normal rat placenta all demonstrate binding components for 1,25-(OH)₂D which interact with the DNA affinity ligand under low salt conditions (< 0.15M), and can be eluted as a single macromolecular peak during a linear KCl gradient between 0.25–0.30M. Further, sucrose gradient analysis of these DNA-cellulose purified components under high salt conditions (0.3M KCl) indicates a common sedimentation coefficient of 3.3S. Since the receptor properties of this macromolecule in rachitic chick intestine have been previously characterized, it seems likely that these components in the parathyroid, pituitary, pancreas, and placenta represent typical receptors for 1,25-(OH)₂D.     

Unoccupied and in vitro and in vivo occupied 1,25-dihydroxyvitamin D3 intestinal receptors. Multiple biochemical forms and evidence for transformation

The data presented herein indicate that the chick intestinal 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor which is localized in the chromatin fraction of a low salt homogenate (Walters, M. R., Hunziker, W., and Norman, A. W. (1980) J. Biol. Chem. 255, 6799-6805) can exist in three distinct biochemical forms. The three 1,25(OH)2D3 receptor forms depended on the absence or presence of ligand and additionally whether the ligand was acquired in vitro (4 degrees C incubation for 3-4 h) or in vivo (13 nmol of 1,25(OH)2D3 administered intramuscularly 2 h prior to sacrifice). The receptor forms were distinguished by their relative KCl extractabilities from the target tissue chromatin preparation and from a reconstituted nontarget tissue (liver) chromatin preparation, as well as their relative elution from DNA-cellulose columns when applied as a mixture. In all cases the rank order "affinity" of the receptor for chromatin or DNA was: unoccupied 1,25(OH)2D3 receptors less than in vivo occupied receptors less than in vitro occupied receptors. These changes in DNA-binding "affinity" occurred without a major change in overall surface charge of the receptor molecules as evaluated by co-elution of all three receptor forms from DEAE-Sepharose columns. Similarly, these changes in DNA-binding characteristics were not accompanied by changes in the apparent molecular weights of these receptor species (91,900 +/- 3300; 99,700 +/- 9400; 93,100 +/- 5600, respectively) as assessed by Sephacryl S-200 gel filtration chromatography in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, included throughout these experiments to protect from proteolytic damage. These results represent the first demonstration of biochemical heterogeneity in the 1,25(OH)2D3 receptor system and suggest the existence of a two-step transformation process for the 1,25(OH)2D3 receptor.     

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.     

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.     

Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).     

1,25-Dihydroxyvitamin D3 induces 25-hydroxyvitamin D3-24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity receptor for the hormone

A consequence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) action in kidney is the enhanced production of 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). We have studied this apparent induction phenomenon in two established mammalian cell lines of renal origin. A porcine kidney cell line, LLC-PK1, was found to possess typical receptors for 1,25-(OH)2D3 which sediment at 3.3 S and bind to immobilized DNA. Saturation analysis of LLC-PK1 cell cytosol revealed an equilibrium binding constant (Kd) for 1,25-(OH)2D3 of 7.8 X 10(-11) M and a concentration of 5400 binding sites/cell. In the presence of serum, intact LLC-PK1 cells also internalize and bind 1,25-(OH)2D3. In contrast, a monkey kidney cell line, LLC-MK2, was found to contain a negligible concentration of the 1,25-(OH)2D3 receptor by all criteria examined. However, both renal cell lines respond to 1,25-(OH)2D3 with a 2- to 20-fold increase in basal levels of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity. Incubation of viable cell suspensions with 25-hydroxy[26,27-3H]vitamin D3 (0.5 microM) at 37 degrees C for 30 min followed by subsequent analysis of lipid extracts via high performance liquid chromatography was carried out to assess 24,25-(OH)2[3H]D3 formation. Enzyme induction was found to be specific for 1,25-(OH)2D3 in both cell lines with half-maximal stimulation of 24-hydroxylase activity observed at 0.2 and greater than or equal to 1.0 nM 1,25-(OH)2D3 in LLC-PK1 and LLC-MK2, respectively. The response in LLC-PK1 was more rapid (1-4 h) than in LLC-MK2 (4-8 h) following 1,25-(OH)2D3 treatment of cultures in situ. In both cell lines, actinomycin D abolished the 1,25-(OH)2D3-dependent increase in 24-hydroxylase activity. Our results suggest that the high affinity 1,25-(OH)2D3 receptor may not be required for 1,25-(OH)2D3-dependent induction of renal 24-hydroxylase activity. Alternatively, LLC-MK2 cells could contain an atypical form of the 1,25-(OH)2D3 receptor protein which retains functionality but escapes detection by standard binding techniques.     

The role of the vitamin D receptor and ERp57 in photoprotection by 1α,25-dihydroxyvitamin D3

UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.     

Previtamin D3 with a trans-fused decalin CD-ring has pronounced genomic activity

Deletion of C19 in the structure of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] does not substantially alter the biological potency but prevents the conversion between the vitamin and the previtamin form. Hence, this modification allows the study of locked previtamin and vitamin forms. The locked 19-nor-1,25(OH)2-previtamin D3 analog (19-nor-previtamin D) had a low biological activity and was a rather weak activator of the genomic signal transduction pathway. 19-Nor-trans-decalin-1,25(OH)2-vitamin D3 (19-nor-TD-vitamin D), characterized by the presence of a trans-fused decalin CD-ring system, was 10-fold more potent than the parent compound and was a potent activator of the genomic signal transduction pathway. Surprisingly, the previtamin, 19-nor-trans-decalin-1,25(OH)2-previtamin D3 (19-nor-TD-previtamin D), was as potent as 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation and represents the first previtamin structure with pronounced vitamin D-like activity. Furthermore, this compound interacted as efficiently as 1,25(OH)2D3 with the vitamin D receptor (VDR), retinoid X receptor (RXR), coactivators, and DNA, which illustrated its potent ability to activate the genomic signal transduction pathway. Analysis of the transactivation potency of 12 VDR point mutants after stimulation with 19-nor-TD-previtamin D revealed that this analog used the same contact points within the receptor as did 1,25(OH)2D3. This could be confirmed by modeling analysis of this compound in the ligand binding pocket of VDR. In conclusion, a previtamin D3 analog is presented with genomic activities equivalent to 1,25(OH)2D3.     

Monoclonal antibodies to the porcine intestinal receptor for 1,25-dihydroxyvitamin D3: interaction with distinct receptor domains

Monoclonal antibodies to different domains of the porcine intestinal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been produced. A nuclear extract enriched in the 1,25-(OH)2D3 receptor was prepared from small intestinal mucosa of young pigs. The receptor was purified an additional 6600-fold by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration high-performance liquid chromatography, and DEAE-Sepharose chromatography, with an overall yield of 23% and an average purity of 24%. A BALB/c mouse immunized with this material developed serum polyclonal antibodies to the 1,25-(OH)2D3 receptor, as demonstrated by a change in sedimentation of the porcine receptor on sucrose gradients. Spleen cells from this animal were fused with mouse myeloma cells (P3-NSI/1-Ag4-1, SP2/0-Ag14), and 24 hybridomas secreting antibodies to the 1,25-(OH)2D3 receptor were identified by both a radiometric immunosorbent assay and an immunoprecipitation assay. Twenty-one hybridoma lines were cloned by limiting dilution and further characterized as subclass IgG1 antibodies with the exception of one which is an IgA. All but two of the antibodies cross-react with the 1,25-(OH)2D3 receptor from both mammalian (human, monkey, and rat) and avian (chicken) intestine; two antibodies recognize only porcine intestinal receptor. All antibodies are unreactive to the vitamin D serum transport protein. Eight of the antibodies bind denatured receptor on an immunoblot. A solid-phase competition assay was used to identify four groups of antibodies that bind to distinct epitopes on the 1,25-(OH)2D3 receptor. One antibody from each of the four groups was used to examine the effect of antibody binding on the DNA-binding activity of the receptor-hormone complex. One antibody completely inhibited the binding of the 1,25-(OH)2D3 receptor complex to DNA-cellulose, suggesting that the epitope for this antibody may be located in the polynucleotide binding domain of the protein. Antibodies from two additional groups only slightly perturbed DNA binding, while one had no effect, suggesting that these antibodies bind to receptor epitopes distant from the region of the polypeptide directly involved in polynucleotide binding. These antibodies that are directed to several different binding sites on the 1,25-(OH)2D3 receptor provide important new tools to probe the biochemistry and topology of the 1,25-(OH)2D3 receptor and to investigate its role in mediating target tissue response to hormone.     

Hormonal responses to 1,25-dihydroxyvitamin D3 in cultured mouse osteoblast-like cells--modulation by changes in receptor level

The level of 1,25(OH)2D3 receptors in cultured mouse osteoblast-like (OB) cells is modulated by the rate of cell proliferation. We have studied two 1,25(OH)2D3-induced bioresponses to ascertain whether the changes in receptor levels during growth in culture alter cell responsiveness. Nuclear receptor levels were high (127 fmol/100 micrograms DNA) in rapidly dividing (log) cells and low (25 fmol/100 micrograms DNA) in quiescent (confluent) cells. The bioresponses we studied were induction of 25(OH)D3-24-hydroxylase activity (24-hydroxylase) and inhibition of collagen synthesis. The basal levels of 24-hydroxylase were low and similar in cells at log growth phase and confluence. At a maximal induction dose of 13 nM, 1,25(OH)2D3 induced a three-fold rise in enzyme activity at long growth phase, but only caused less than two-fold rise at confluence. The half-maximal dose (ED50) was slightly shifted from 0.6 nM to 0.8 nM. Daily measurement of 1,25(OH)2D3 receptor levels and maximal induction of 24-hydroxylase activity throughout the culture cycle showed a strong correlation between receptor abundance and enzyme induction. The basal level of collagen synthesized by cells in log growth phase was approximately 5% and increased to approximately 8% at confluence. Maximal inhibition of collagen synthesis by 1,25(OH)2D3 reached 80% of control levels in log cells, but was only 40% of control in confluent cells. The ED50 was approximately 0.1 nM in the log cells and increased to approximately 1 nM at confluence. Daily assay of 1,25(OH)2D3 receptor levels and 1,25(OH)2D3 responses during the culture cycle indicated a correlation between changes in receptor level and the extent of inhibition of collagen synthesis. These changes in bioresponse at various growth phases did not occur in rat OB cells where the 1,25(OH)2D3 receptor levels were independent of cell proliferation. The results indicate that cell proliferation rate, via change in receptor levels, determines the magnitude and sensitivity of the cellular responses to 1,25(OH)2D3.