This study explores the effects of chronic administration of vitamin D(3) compounds on several biological functions in mice. Knowledge of long-term tolerability of vitamin D(3) analogs may be of interest in view of their potential clinical utility in the management of various pathologies such as malignancies, immunological disorders and bone diseases. Four unique vitamin D(3) analogs (code names, compounds V, EO, LH and LA) and 1,25-dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)) were administered i.p. for 55 weeks to Balb/c mice. Each analog had previously been shown to have potent in vitro activities. After 55 weeks of administration, the mice had a profound decrease in their serum levels of interleukin-2 (IL-2). Likewise, several analogs depressed serum immunoglobulin G concentrations (compounds LH and LA), but levels of blood lymphocytes and splenic lymphocyte subsets (CD4, CD8 and CD19) were not remarkably depressed. The percent of committed myeloid hematopoietic stem cells was 4- to 5-fold elevated in the bone marrow of the mice that received analogs LH and V; nevertheless, their peripheral blood white and red cell counts and platelets were not significantly different in any of the groups. The mice that received 1,25(OH)(2)D(3) had a decrease in bone quantity and quality with a decrease in cross-sectional area and cortical thickness, and a 50% reduction in both stiffness and failure load compared with the control group. In contrast, the cohort that received a fluorinated analog (compound EO) developed bones with significantly larger cross-sectional area and cortical thickness as well as stronger mechanical properties compared with the control group. At the conclusion of the study, body weights were significantly decreased in all experimental mice. Their blood chemistries were normal. Extensive gross and microscopic autopsy analyses of the mice at the conclusion of the study were normal, including those of their kidneys. In conclusion, the vitamin D(3) analogs were fairly well tolerated. They did suppress immunity as measured by serum IL-2 and may provide a means to depress the immune response after organ transplantation and for autoimmune diseases. Use of these analogs prevented the detrimental effects of vitamin D(3) administration on mechanical and geometric properties of bone, while one analog (compound EO) actually enhanced bone properties. These results suggest that long-term clinical trials with the analogs are feasible.
EA Smith, EP Frankenburg, SA Goldstein, K Koshizuka, E Elstner, J Said, T Kubota, M Uskokovic, and HP Koeffler
M R Haussler, C A Haussler, P W Jurutka, P D Thompson, J-C Hsieh, L S Remus, S H Selznick, and G K Whitfield
Vitamin D plays a major role in bone mineral homeostasis by promoting the transport of calcium and phosphate to ensure that the blood levels of these ions are sufficient for the normal mineralization of type I collagen matrix in the skeleton. In contrast to classic vitamin D-deficiency rickets, a number of vitamin D-resistant rachitic syndromes are caused by acquired and hereditary defects in the metabolic activation of the vitamin to its hormonal form, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or in the subsequent functions of the hormone in target cells. The actions of 1,25(OH)2D3 are mediated by the nuclear vitamin D receptor (VDR), a phosphoprotein which binds the hormone with high affinity and regulates the expression of genes via zinc finger-mediated DNA binding and protein–protein interactions. In hereditary hypocalcemic vitamin D-resistant rickets (HVDRR), natural mutations in human VDR that confer patients with tissue insensitivity to 1,25(OH)2D3 are particularly instructive in revealing VDR structure/function relationships. These mutations fall into three categories: (i) DNA binding/nuclear localization, (ii) hormone binding and (iii) heterodimerization with retinoid X receptors (RXRs). That all three classes of VDR mutations generate the HVDRR phenotype is consistent with a basic model of the active receptor as a DNA-bound, 1,25(OH)2D3-liganded heterodimer of VDR and RXR. Vitamin D responsive elements (VDREs) consisting of direct hexanucleotide repeats with a spacer of three nucleotides have been identified in the promoter regions of positively controlled genes expressed in bone, such as osteocalcin, osteopontin, β3-integrin and vitamin D 24-OHase. The 1,25(OH)2D3 ligand promotes VDR-RXR heterodimerization and specific, high affinity VDRE binding, whereas the ligand for RXR, 9-cis retinoic acid (9-cis RA), is capable of suppressing 1,25(OH)2D3-stimulated transcription by diverting RXR to form homodimers. However, initial 1,25(OH)2D3 liganding of a VDR monomer renders it competent not only to recruit RXR into a heterodimer but also to conformationally silence the ability of its RXR partner to bind 9-cis RA and dissociate the heterodimer. Additional probing of protein–protein interactions has revealed that VDR also binds to basal transcription factor IIB (TFIIB) and, in the presence of 1,25(OH)2D3, an RXR-VDR-TFIIB ternary complex can be created in solution. Moreover, for transcriptional activation by 1,25(OH)2D3, both VDR and RXR require an intact short amphipathic α-helix, known as AF-2, positioned at their extreme C-termini. Because the AF-2 domains participate neither in VDR-RXR heterodimerization nor in TFIIB association, it is hypothesized that they contact, in a ligand-dependent fashion, transcriptional coactivators such as those of the steroid receptor coactivator family, constituting yet a third protein–protein interaction for VDR. Therefore, in VDR-mediated transcriptional activation, 1,25(OH)2D3 binding to VDR alters the conformation of the ligand binding domain such that it: (i) engages in strong heterodimerization with RXR to facilitate VDRE binding, (ii) influences the RXR ligand binding domain such that it is resistant to the binding of 9-cis RA but active in recruiting coactivator to its AF-2 and (iii) presents the AF-2 region in VDR for coactivator association. The above events, including bridging by coactivators to the TATA binding protein and associated factors, may position VDR such that it is able to attract TFIIB and the balance of the RNA polymerase II transcription machinery, culminating in repeated transcriptional initiation of VDRE-containing, vitamin D target genes. Such a model would explain the action of 1,25(OH)2D3 to elicit bone remodeling by stimulating osteoblast and osteoclast precursor gene expression, while concomitantly triggering the termination of its hormonal signal by inducing the 24-OHase catabolizing enzyme.
Journal of Endocrinology (1997) 154, S57–S73
I. R. Dickson and P. M. Maher
When growth cartilage from rachitic chicks was cultured in the presence of the calcium-regulating hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), collagen resorption was increased and collagen synthesis decreased compared to control cultures containing no hormone. The minimum concentration of the hormone that caused a statistically significant inhibition of collagen synthesis was 10 −8 mol/l. Collagen synthesis by growth cartilage from normal chicks was also reduced by 1,25-(OH)2D3, showing that it was not an abnormal response of vitamin D-depleted tissue. 25-Hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 also inhibited collagen synthesis by cultures of growth cartilage but only at higher metabolite concentrations. 1,25-Dihydroxyvitamin D3 (10−7 mol/l) did not significantly inhibit collagen synthesis by cultures of articular fibrocartilage and of sternal cartilage, tissues that do not calcify physiologically. The minimum concentration of 1,25-(OH)2D3 (10−9 mol/l) necessary to cause decreased collagen synthesis by embryonic chick calvaria was lower than the value obtained with growth cartilage; this suggests that bone cells may be more sensitive to the hormone in this respect than are growth cartilage chondrocytes. These findings provide evidence of a direct role of 1,25-(OH)2D3 in the control of endochondral bone formation which is consistent with its primary role in the maintenance of plasma calcium homeostasis.
J. Endocr. (1985) 105, 79–85
C Menendez, M Lage, R Peino, R Baldelli, P Concheiro, C Dieguez, and FF Casanueva
Leptin, the product of the ob gene, is secreted into the circulation by white adipose tissue; its major role being to participate in the regulation of energy homeostasis. Plasma leptin levels are mainly determined by the relative adiposity of the subject; however, the great dispersion of values for any given body mass index and the noteworthy gender-based differences indicate that other factors are operating. Steroid hormones actively participate in the regulation of leptin secretion; however, non-steroid nuclear hormones have either not been studied or have provided contradictory results. In order to understand the role of hormones of the non-steroid superfamily such as 3,5,3'-tri-iodothyronine (T(3)), vitamin D(3) and retinoic acid (RA) in the control of leptin secretion, in the present work doses of 10(-9), 10(-8) and 10(-7) M of these compounds have been studied on in vitro leptin secretion. The organ culture was performed with omental adipose tissue samples from healthy donors (n=28). T(3) was devoid of effect at any dose studied, while an inhibition of leptin secretion was observed with 9-cis-RA (slight) and all-trans-RA (potent). Interestingly, vitamin D(3) exerted a powerfully inhibitory role at the doses studied, and its action was synergistic with all-trans-RA. In conclusion, in vitro leptin secretion by human adipose tissue is negatively controlled by either RA or vitamin D(3). The clinical significance of leptin regulation by this superfamily of nuclear receptors remains to be ascertained.
P-M Bourlon, A Faure-Dussert, B Billaudel, B Ch J Sutter, G Tramu, and M Thomasset
The pancreatic B cell is equipped with specific receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and contains vitamin D-dependent calcium binding proteins (calbindin-D). Insulin secretion is impaired by vitamin D deficiency and is restored by 1,25-(OH)2D3 (concomitantly with an improved calcium handling within B cells) but the effect of 1,25-(OH)2D3 on the pancreatic B cell via calbindin-D is unclear. Therefore we examined the relationship between calbindin-D28K or calbindin-D9K and the activity of the endocrine pancreas in normal (N), four week vitamin D-deficient (−D) and one week 1,25-(OH)2D3-replete (+D) rats. Calbindin-D9K was not found in the pancreas, neither in the islets nor in the exocrine part, of any of the groups of rats (N, −D, or +D). Surprisingly, total islet calbindin-D28K content was increased by vitamin D deficiency and partly restored by 1,25-(OH)2D3. Calbindin-D28K immunostaining was observed only on A and B cells in the endocrine part of the pancreas, the greatest staining being found in A cells. This difference in staining density was increased by vitamin D deficiency and decreased by 1,25-(OH)2D3 treatment. In vitro, 1,25-(OH)2D3 also produced a negative influence on calbindin-D28K staining in A cells, as demonstrated using pieces of pancreas incubated with the steroid for 2 h. No significant influence on labeling intensity of B cell calbindin-D28K could be shown. Plasma insulin and islet insulin release in response to 10 mm arginine stimulation were decreased in −D rats and enhanced in +D rats towards N values. In contrast, plasma glucagon and the amount of glucagon secretion, stimulated in vitro by 10 mm arginine or by low (1·7 mm) glucose concentration, was increased in −D rats and attenuated by 1,25-(OH)2D3.
Thus there appears to be no relationship between the steady state level of B cell calbindin-D28K and the regulation of insulin secretion by 1,25-(OH)2D3 in vitamin D-deficient rats. However there is a correlation between A cell calbindin-D28K and glucagon secretion, which are both negatively regulated by 1,25-(OH)2D3. The predominance of calbindin-D28K in A cells raises the question as to how A and B cells interact and the role of calbindin-D28K in calcium handling.
Journal of Endocrinology (1996) 148, 223–232
D. R. Shamley, R. Buffenstein, G. Veale, and J. M. Pettifor
The effects of vitamin D3 deficiency on the ontogeny of calcium-binding proteins (CaBPs) and the vitamin D receptor in the duodenum, kidney and cerebellum of the mouse were examined. Maternal vitamin D status did not affect the time of appearance of the fetal 28 kDa CaBP (CaBP-D28k) in the cerebellum, kidney and duodenum, and the 9 kDa CaBP (CaBP-D9k) in the intestine and kidney. Vitamin D receptor was undetectable in all fetal tissues, regardless of maternal vitamin D status, at all stages of gestation examined. Thus it appears that maternal vitamin D status does not affect the ontogeny of CaBP-D9k or CaBP-D28k in the mouse fetus. The factors that influence the appearance of calbindins in the fetus are unclear.
Journal of Endocrinology (1993) 139, 473–478
J. D. Wark and V. Gurtler
1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) selectively enhances prolactin gene expression in GH4C1 clonal rat pituitary tumour cells. Because this effect requires extracellular Ca2+, we studied the effect of 1,25-(OH)2D3 on another Ca2+-dependent process, agonist-induced hormone secretion. Pretreatment with 1,25-(OH)2D3 (1 nmol/l) caused at least 25-fold sensitization of GH4C1 cells to the voltage-sensitive Ca2+ channel agonist BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate) as a prolactin secretagogue. This inductive effect of 1,25-(OH)2D3 followed a similar time-course to the enhancement of prolactin production. 1,25-(OH)2D3 had no effect on basal or BAY K 8644-induced 45Ca2+ uptake. The Ca2+-selective divalent cation ionophore 11,19,21-trihydroxy-4,6,8,12,14,18,20-heptamethyl-9-oxo-22-(tetrahydro-5 methyl-5-tetra hydro-5-(1-hydroxyethyl)-5-methyl-2-furanyl)-10,16-docosadienoic acid (ionomycin; 12 nmol/l–1·2 μmol/l) caused no significant increase in prolactin secretion in the absence of 1,25-(OH)2D3, but in cells treated with 1,25-(OH)2D3-(1 nmol/l), it increased prolactin secretion by 73% at 12 nmol/l and by a maximum of 98% at 0·12 μmol/l. These data demonstrate that vitamin D markedly enhances the responsiveness of GH4C1 functional pituitary tumour cells to two secretagogues which acts primarily through Ca2+-dependent mechanisms. They support the proposal that 1,25-(OH)2D3 acts in this cultured cell model either by effecting a redistribution of intracellular Ca2+ or by increasing the response of a Ca2+ -sensitive effector system, but not by enhancing agonist-induced Ca2+ uptake.
J. Endocr. (1988) 117, 293–298
M. C. d'Emden and J. D. Wark
Vitamin D may regulate pituitary function, as there are selective effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on gene expression in clonal pituitary tumour cells, and on TRH-induced TSH release in normal rat pituitary cells in vitro. The role of Ca2+ in 1,25-(OH)2D3-enhanced TSH release from primary rat pituitary cell cultures was investigated. Pretreatment with 10 nmol 1,25-(OH)2D3/l for 24 h augmented KCl (3–60 mmol/l)-induced TSH release over 1 h at all KCl concentrations greater than 7·5 mmol/l (P< 0·001), with a 76% enhancement of TSH release induced by 30 mmol KCl/l (P<0·001). The Ca2+ channel antagonist nifedipine (10 nmol/l–10 μmol/l) caused a concentration-dependent inhibition of KCl (60 mmol/l)-induced TSH secretion. Pretreatment with 1,25-(OH)2D3 enhanced KCl-induced release at all concentrations of nifedipine (P<0·001). The Ca2+ selective divalent cation ionophore ionomycin (1 nmol/l–1 μmol/l), and the Ca2+ channel agonist BAY K 8644 (10 nmol/l–1 μmol/l) increased prolactin secretion but did not increase TSH release, and 1,25-(OH)2D3 had no effect. At an extracellular Ca2+ concentration of less than 500 nmol/l, TRH-induced TSH release was observed only after treatment with 1,25-(OH)2D3 (P<0·01). As the extracellular Ca2+ concentration was increased, greater increments of TRH-induced TSH release were observed following pretreatment with 1,25-(OH)2D3 (P<0·01). However, the effect of 1,25-(OH)2D3 in the thyrotroph was independent of the pretreatment extracellular Ca2+ concentration. We have shown that 1,25-(OH)2D3 acts selectively on the thyrotroph to enhance in-vitro responsiveness to TRH and KCl. These data suggest that the action of 1,25-(OH)2D3 in the thyrotroph is to enhance intracellular signal transduction. They further support a permissive or regulatory role of vitamin D in the normal pituitary gland.
Journal of Endocrinology (1989) 121, 441–450
Y. Nys, R. Bouillon, H. Van Baelen, and J. Williams
The concentration of 25-hydroxyvitamin D3-binding protein (DBP) was measured, by immunodiffusion, in the blood of chickens from embryonic stages to sexual maturity. Low levels of DBP and 1,25-(OH)2D3 were detectable in the blood of chick embryos from the 12th and 17th day of incubation respectively and stayed at the same low levels until hatching. The blood concentration of DBP doubled between the 1st and 5th days of life, then increased slowly and reached the mean level of the adult male at 7–8 weeks of age. The concentration of DBP was independent of vitamin D status in growing chickens. A large increase was observed in DBP blood levels in hens just before sexual maturity. This change, and those observed in moulting hens, followed the variations in plasma concentrations of oestradiol more closely than those of progesterone or testosterone. Moreover, a large increase in plasma DBP levels was induced in immature chickens by oestradiol (0·5 mg/day), but not by testosterone or progesterone. Finally, the experimental suppression of egg shell formation and the associated decrease in 1,25-(OH)2D3 plasma levels had no effect on plasma DBP concentrations. However, 1,25-(OH)2D3 and DBP levels were higher in hens laying shell-less eggs than in immature pullets. The increases in DBP levels at hatching, in immature pullets treated with oestrogens, in hens laying uncalcified eggs and at the onset of egg production were associated with increases in 1,25-(OH)2D3, suggesting a relationship between the levels of DBP and 1,25-(OH)2D3 in the blood.
J. Endocr. (1986) 108, 81–87
M Depreter, J Vandesompele, M Espeel, F Speleman, and F Roels
Peroxisomes are ubiquitous organelles required for several metabolic functions. Their dysfunction is responsible for a group of human inherited disorders. In the search for endogenous factors regulating the peroxisomal compartment in normal liver, we treated female rats with dehydroepiandrosterone (DHEA) and 25-hydroxycholecalciferol for 1 and 6 days. Relative transcription levels of 39 selected genes were evaluated by real-time quantitative RT-PCR analysis. Catalase (peroxisomal marker)-specific activity was assayed in total liver homogenate and peroxisomes were visualized by catalase localization. DHEA induced peroxisome proliferation and raised catalase specific activity. Expression levels of 16 (of which 11 were peroxisomal) genes were altered. Pex 11, acyl-CoA oxidase,l - andd -multifunctional enzyme, thiolase 1, phytanoyl-CoA hydroxylase, 70 kDa peroxisomal membrane protein and very long chain acyl-CoA synthetase were upregulated, three others were downregulated. Vitamin D caused downregulation of six genes. Administration of vitamin D to peroxisomal disorder patients may be contraindicated. The adrenocortical hormone DHEA is a potential natural regulator of the peroxisomal compartment. Its therapeutic use in X-linked adrenoleukodystrophy, some other beta-oxidation defects and classical Refsum should be considered.