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.     

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.     

Specific binding protein for 1,25-dihydroxyvitamin D3 in bovine mammary gland

Specific binding proteins for 1,25-dihydroxyvitamin D₃ were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[³H]vitamin D₃ with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (K_d = 4.2 × 10^?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D₃, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and vitamin D₃ did not effectively compete with 1,25-dihydroxyvitamin D₃ for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D₃. In vitro subcellular distribution of 1,25-dihydroxy[³H]vitamin D₃ demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[³H]vitamin D₃ was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[³H]vitamin D₃ was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D₃ in this tissue.     

On the specificity of vitamin D compounds binding to chick pig intestinal 1,25-dihydroxyvitamin D3 receptor

The binding activity of four vitamin D metabolites and/or analogs for the intestinal 1,25-dihydroxyvitamin D3 receptor was evaluated after incubation at 25 degrees C for 1 h or at 0-4 degrees C for 18 h. The incubation conditions, which had no effect on the binding of 1,25-dihydroxyvitamin D3, had a dramatic effect on the binding of 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 and a small but reproducible effect on 24,25-dihydroxyvitamin D3 binding to receptor. Affinities 10- to 20-fold higher were obtained for 25-hydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3, and affinities 3-fold higher were obtained for 24,25-dihydroxyvitamin D3 at the 0-4 degrees C/18-h incubation. A comparison of intestinal receptor from chick and pig with nine vitamin D compounds showed no major differences between the two species. The relative affinity of the vitamin D analogs to compete with tritiated 1,25-dihydroxyvitamin D3 for the receptor in pig nuclear extract, expressed as ratios of the molar concentration required for 50% binding of the tritiated 1,25-dihydroxyvitamin D3 compared to nonradioactive 1,25-dihydroxyvitamin D3, are as follows: 1,25-dihydroxyvitamin D3 (1) = 1,25-dihydroxyvitamin D2 = 24-homo-1,25-dihydroxyvitamin D3 greater than 1,24,25-trihydroxyvitamin D3 (4) greater than 25-hydroxyvitamin D3 (21) = 10-oxo-19-nor-25-hydroxyvitamin D3 = 1 alpha-hydroxyvitamin D3 (37) greater than 24,25-dihydroxyvitamin D2 (257) much much greater than vitamin D3 (greater than 10(6)).     

The chick intestinal cytosol binding protein for 1,25-dihydroxyvitamin D3: A study of analog binding

The structural features of 1,25-dihydroxyvitamin D₃ that permit its high affinity binding to a 3.7 S protein from chick intestinal cytosol were determined in a series of binding and competition experiments analyzed by sucrose density gradient centrifugation. Optimal binding to the 3.7 S protein was achieved when both 1α- and 25-hydroxyls were present in the vitamin D₃ molecule. Modification of the side chain by the introduction of a methyl on C-24 and a double bond on C-22,23 (1,25-dihydroxyvitamin D₂) did not alter the binding of 1,25-dihydroxyvitamin D₃, but significantly diminished the binding of 25-hydroxyvitamin D₃. However, introduction of a hydroxyl on C-24 decreased the ability of either 1,25-dihydroxyvitamin D₃ or 25-hydroxyvitamin D₃ to compete, especially when the 24-hydroxyl was in the S configuration. These results reveal that the 3.7 S protein requires specific ligand structural features for binding and suggest that metabolite discrimination by the chick intestinal receptor system is likely located in the 3.7 S cytosol protein.     

A specific binding protein for 1,25-dihydroxyvitamin D3 in rat intestinal cytosol.

In the presence of 0.3 M potassium chloride and 0.5 mM dithiothreitol, rat intestinal cytosol contains two binding proteins for 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃)¹ having sedimentation coefficients of 3.2S and 5–6S. The 3.2S protein is specific for 1,25-(OH)₂D₃ as determined by competition analysis, whereas the 5–6S protein binds 25-hydroxyvitamin D₃ (25-OH-D₃) exclusively.     

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.     

Apparent nuclear localization of unoccupied receptors for 1,25-dihydroxyvitamin D3

Previous studies have demonstrated that unoccupied 1,25-dihydroxyvitamin D₃ receptors are associated with crude chromatin under hypotonic conditions $\text{in}\text{vitro}$. The data presented herein show that unoccupied 1,25-dihydroxyvitamin D₃ receptors appear to be associated with chromatin prior to solubilization by dilution/homogenization in both high and low salt buffers. Additionally the unoccupied receptors are recovered nearly quantitatively from purified nuclei. These results suggest that unoccupied 1,25-dihydroxyitamin D₃ receptors may be localized within nuclei $\text{in}\text{vivo}$.     

Effect of vitamin D3 administration on serum 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and osteocalcin in vitamin D-deficient elderly people

In elderly institutionalized people, confined to bedroom and receiving no vitamin D supplementation, the frequency of vitamin D deficiency is found very high. Systematic administration of vitamin D has, therefore, been proposed to correct vitamin D deficiency. Within this context, we studied 40 elderly institutionalized subjects (mean age 80.5 + 7.2 yr) with low 25(OH)D3 concentrations (4.4 + 1.8 micrograms/l). Sixteen of them (Group I) had low serum calcium concentrations (less than 2.3 mmol/l) and 24 (Group II) had normal serum calcium concentrations (from 2.3 to 2.6 mmol/l). As hypocalcemia has been shown to regulate 1,25(OH)D3 production independent of PTH in animals and in humans, we compared their respective responses to the administration of vitamin D3. Subjects received a total dose of 15 mg (600,000 IU) of vitamin D3 divided into 3 i.m. injections at one month intervals and were explored before therapy and one and 6 months after the last dose of vitamin D3. The treatment induced a similar marked rise in 25(OH)D3 levels (from 4.1 + 1.7 to 24.4 + 8.7 micrograms/l for group I and from 5.1 + 1.8 to 27.2 + 8.0 micrograms/l for group II) in both groups but increased the 1,25(OH)2D3 concentrations only in group I (from 22.9 + 6.9 to 32.6 + 11.3 ng/l). Meanwhile serum calcium concentrations rose in group I (to low normal range i.e. 2.31 + 0.07 mmol/l) and were unaffected in group II. These results suggest that hypocalcemia is a potent stimulator of renal 1-hydroxylase in elderly people. Furthermore, a transient significant (P less than 0.01) increase in serum osteocalcin (from 10.6 + 4.1 to 14.1 + 5.9 micrograms/l) could be observed in group I which demonstrates for the first time that the osteocalcin response of osteoblasts to stimulation by 1,25(OH)2D3 is retained in very old people.     

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.     

Biological activity assessment of the 26,23-lactones of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 and their binding properties to chick intestinal receptor and plasma vitamin D binding protein

The binding of the natural and unnatural diastereoisomers 25-hydroxyvitamin D3-26,23-lactone and 1,25 dihydroxyvitamin D3-26,23-lactone to the vitamin D-binding protein (DBP) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] chick intestinal receptor have been investigated. Also, the biological activities, under in vivo conditions, of these compounds, in terms of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM), in the chick are reported. The presence of the lactone ring in the C23-C26 position of the seco-steroid side chain increased two to three times the ability of both 25(OH)D3 and 1,25(OH)2D3 to displace 25(OH)[3H]D3 from the D-binding protein; however, the DBP could not distinguish between the various diastereoisomers. In contrast, the unnatural form (23R,25S) of the 25-hydroxy-lactone was found to be 10-fold more potent than the natural form, and the unnatural (23R,25S)1,25(OH)2D3-26,23-lactone three times more potent than the natural 1,25-dihydroxy-lactone in displacing 1,25(OH)2[3H]D3 from its intestinal receptor. While studying the biological activity of these lactone compounds, it was found that the natural form of the 25-hydroxy-lactone increased the intestinal calcium absorption 48 h after injection (16.25 nmol), while bone calcium mobilization was decreased by the same dose of the 25-hydroxy-lactone. The 1,25-dihydroxyvitamin D3-26,23-lactone in both its natural and unnatural forms was found to be active in stimulating ICA and BCM. These results suggest that the 25-hydroxy-lactone has some biological activity in the chick and that 1,25(OH)2D3-26,23-lactone can mediate ICA and BCM biological responses, probably through an interaction with 1,25-(OH)2D3 specific receptors in these target tissues.     

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.     

Regulation of cellular retinol binding protein type II by 1,25-dihydroxyvitamin D3.

Previously we purified and sequenced an 18-kDa chick duodenal protein that was modulated by 1,25-dihydroxyvitamin D3. The N-terminus of this protein has striking sequence homology to cellular retinol binding protein type II (CRBP II). Furthermore, this purified chick protein binds retinol. Antibodies have now been generated to the chick protein and used for immunoblot analysis to demonstrate that the chick protein has molecular weight, tissue distribution, and subcellular localization similar to rat CRBP II. These antibodies also cross-reacted with rat CRBP II. Antibodies to rat CRBP II cross-react with the chick protein. Northern analysis using a cDNA probe for rat CRBP II showed a single 860 base pair mRNA in both chick and rat intestinal RNA preparations. These results demonstrate that the 1,25-dihydroxyvitamin D3 modulated protein in chick embryonic organ culture is chick CRBP II. Pulse-chase experiments in chick embryonic duodenal organ culture strongly suggest that 1,25-dihydroxyvitamin D3 markedly decreases the synthesis of CRBP II, while not changing the degradation rate. The concentration of 1,25-dihydroxyvitamin D3 required for the decrease in CRBP II synthesis is approximately that required to stimulate calcium uptake into embryonic chick duodenal organ cultures.     

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dehydroxy[3H-26, 27] vitamin D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D₃ binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$]vitamin D₃ (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (K_d) of 7.1 × 10^?11m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 10³ receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D₃ binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 10⁸m^? min^? and 7.1 × 10^?3 min^?, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$] vitamin D₃ a truly high affinity binding site.     

1,25-Dihydroxyvitamin D3 increases serum levels of the vitamin K-dependent bone protein

1,25-dihydroxyvitamin D₃ increases serum levels of bone Gla protein (BGP). The maximal increase occurs 12 h after injection and is given by 350 ng 1,25(OH)₂D₃ per 180 g body weight. In both 2 and 11 month-old male rats, the maximal increase is about 3 times the normal level, while in 2 month old female rats, the maximal increase is 2 times the normal level. These effects of 1,25(OH)₂D₃ in rats parallel the previously described effects of the vitamin on BGP secretion by rat osteosarcoma cells in culture.BGP is the first bone-specific protein whose synthesis in animals is dramatically increased by 1,25(OH)₂D₃. The possible functions of BGP in the biological actions of 1,25(OH)₂D₃ on bone are discussed.     

Differential effects of 1,25-dihydroxyvitamin D3 upon intestinal vitamin D3-dependent calbindin (a 28,000-dalton calcium binding protein) and its mRNA in D-replete and D-deficient chickens

The effect of vitamin D3 status upon the responsiveness of chick intestinal epithelium to exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was studied. Intestinal calbindin [A recent consensus decision was made to redesignate the vitamin D-dependent calcium binding protein as "calbindin-D28K" (R.H. Wasserman (1985) in Vitamin D: Chemical, Biochemical, and Clinical Update (Norman, A.W., Schaefer, K., Grigoleit, H.-G., and Herrath, D.V., Eds.), pp. 321-322, de Gruyter, Berlin/New York).] protein and intestinal calbindin mRNA were quantitated in birds which had been raised on a vitamin D3-deplete (-D) or on a vitamin D3-replete (+D) diet. 1,25(OH)2D3 stimulated intestinal calbindin mRNA levels in -D chickens in a proportional dose-dependent manner, when measured at both 12 and 48 h after administration of the hormone. A first increase was observed with 1,25(OH)2D3 concentrations between 0.065 and 0.65 nmol. The maximal stimulation achieved by 1,25(OH)2D3 (6.5-18 nmol) in -D tissue was approximately 10-fold over the calbindin mRNA levels present in vehicle-treated birds. The increase of calbindin mRNA in -D birds was associated with a similar dose-dependent increase in calbindin protein in 1,25(OH)2D3-treated -D birds after 12 or 48 h. In +D intestine, while exogenous 1,25(OH)2D3 also increased calbindin mRNA levels in a dose-dependent fashion, the maximal stimulation observed after 5 h (1.2- to 2-fold) was clearly less than that observed in -D intestine. In contrast to -D birds, intestinal calbindin levels in +D birds were decreased by administration of exogenous 1,25(OH)2D3. Administration of 32.5 to 65 nmol 1,25(OH)2D3 resulted in an approximately 1.8-fold repression compared to vehicle-treated birds. This differential responsiveness between +D and -D intestines with respect to 1,25(OH)2D3 was not explained either by differences in the uptake in the chromatin fractions of these tissues or by metabolism of radiolabeled 1,25(OH)2D3. Dietary withdrawal of vitamin D3 led to a gradual decline in ambient intestinal calbindin levels, while intestinal sensitivity to 1,25(OH)2D3 was restored. These findings suggest that vitamin D3 status regulates intestinal responsiveness to the seco-steroid 1,25(OH)2D3.     

Multiple sites of action of the vitamin D endocrine system: FSH stimulation of testis 1,25-dihydroxyvitamin D3 receptors

1,25-Dihydroxyvitamin D [1,25(OH)2D] receptors exist in numerous unexpected tissues. These include, for example, rat lung, heart, testis, and uterus, but not prostate and bladder. The issues of 1,25(OH)2D effects on and receptor location in the testis were addressed by (a) physiological and pharmacological manipulations of tubule cell types and (b) histological examination of testes of vitamin D-deficient rats. FSH treatment in hypophysectomized adult rats increased 1,25(OH)2D receptor levels by 135% (P less than 0.01). Busulfan treatment reduced testis receptor levels by 35% (P less than 0.05) after 35 days (maximum effect), and the effect was reversed after recovery (85 d). Cryptorchidism for 5 or 50 days resulted in modest (33%, P less than 0.05) or substantial (79%, P less than 0.001) reductions in receptor levels. Only the FSH treatment and 50 days cryptorchidism reduced receptor levels in the residual tissue. The testes of vit. D-deficient rats showed incomplete spermatogenesis and degenerative changes. Although interpretation is complicated by the intricate communication among testis cell types, these data suggest that the Sertoli cell is a primary site of action of 1,25(OH)2D in the testis. Moreover, these data indicate that 1,25(OH)2D receptor function in the testis relates to germ cell division/maturation, although this may be an indirect effect via the Sertoli cells.