1,25-Dihydroxyvitamin D3 and the vitamin D analogue KH1060 induce hyperproliferation in normal mouse epidermis. A BrdUrd/DNA flow cytometric study

1,25-dihydroxyvitamin D3 (calcitriol) affects differentiation and proliferation of epidermal keratinocytes in vitro and in vivo. We have studied the topical effects of calcitriol (0.08-2.0 micrograms/ml) and of a new vitamin D analogue, the epi-20-analogue KH1060 (0.4-2.0 micrograms/ml) on epidermal proliferation in normal hairless mice. Epidermis was examined at intervals from 4 h to 8 days after a single-dose application. The mitotic rate was assessed by the stathmokinetic method and hyperplasia was scored in histological sections. Cell cycle parameters were measured by bivariate bromodeoxyuridine (BrdUrd)/DNA flow cytometry on isolated epidermal basal cells after pulse-labelling with BrdUrd. Both calcitriol and KH1060 induced a dose- and time-dependent increase in the mitotic rate and in hyperplasia, the latter drug being the most effective. Calcitriol and KH1060 induced changes in the cell cycle traverse compatible with the regenerative reaction seen after other hyperplasiogens, but with an additionally increased accumulation of cells in the G2 phase. This is similar to that seen after topical application of retinoic acid to mouse skin. Our results are thus in contrast to the anti-proliferative effects of calcitriol observed in vitro and following treatment of the hyperproliferative disease psoriasis with calcitriol as well as other vitamin D analogues.     

Selective biological response by target organs (intestine, kidney, and bone) to 1,25-dihydroxyvitamin D3 and two analogues

The hormonally active form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] stimulates biological responses related to calcium homeostasis, cell differentiation, and immunomodulation in many target cells, including leukemic cells. Most of these responses are dependent upon 1 alpha,25(OH)2D3 interaction with a nuclear receptor protein. Structural analogues of 1 alpha,25(OH)2D3 might allow for separation of biological function, avoiding adverse calcemic effects. This report quantitates intestinal calcium absorption, bone calcium resorption, induction of intestinal and renal calcium-binding protein (CaBP), and occupancy of the intestinal and renal nuclear 1 alpha,25(OH)2D3 receptor in vitamin D-deficient chicks after a single dose of 1 alpha,25(OH)2D3, 1 alpha,25-dihydroxyvitamin-16-ene-23-yne-D3 (analogue V), or 22-[m-(dimethylhydroxymethyl)phenyl]-23,24,25,26,27- pentanor-1 alpha-hydroxy-vitamin D3 (analogue EV). The interaction of these compounds with chick intestinal nuclear 1 alpha,25(OH)2D3 receptor and chick plasma vitamin D-binding protein was determined in vitro; analogues V and EV bound 68% and 62% [1 alpha,25(OH)2D3 receptor] and 8% and 13% (vitamin D-binding protein), respectively, as well as 1 alpha,25(OH)2D3 (100%). 1 alpha,25(OH)2D3 doses (0.075-1.2 nmol) generated responses in intestinal calcium absorption, bone calcium resorption, intestinal CaBP, and renal CaBP. When analogue V (1.2-300 nmol) was administered, increases in bone calcium resorption and renal CaBP were noted. However, a significant response in intestinal calcium absorption and intestinal CaBP appeared only after a 300-nmol dose. Unoccupied nuclear 1 alpha,25(OH)2D3 receptor in the intestine and kidney was determined in vivo after doses of 1 alpha,25(OH)2D3, analogue V, or analogue EV. Doses (0.25-6.0 nmol) of 1 alpha,25(OH)2D3 and analogue EV reduced unoccupied receptor to 24% and 59% (intestine) and to 13% and 41% (kidney), respectively. Analogue V (6.0-600 nmol) decreased unoccupied receptor in the kidney. In the intestine analogue V (300-600 nmol) reduced unoccupied receptor only to 75%. These results confirm that some vitamin D analogues can generate selective biological responses and different levels of target organ receptor occupancy.     

A memorial. Professor Sylvester E. Berki, 1930-1996

1 alpha,25-Dihydroxyvitamin D3[1 alpha,25(OH)2D3], an active form of vitamin D, has roles in many biological phenomena such as calcium homeostasis and bone formation, which are thought to be mediated by the 1 alpha,25(OH)2D3 receptor (VDR), a member of the nuclear hormone receptor superfamily. However, the molecular basis for the actions of 1 alpha,25(OH)2D3 in bone formation, its role during development and VDR genetic polymorphisms for predicting bone mineral density are uncertain. To investigate the functional role of VDR, we generated mice deficient in VDR by gene targeting. We report here that in VDR null mutant mice, no defects in development and growth were observed before weaning, irrespective of reduced expression of vitamin D target genes. After weaning, however, mutants failed to thrive, with appearance of alopoecia, hypocalcaemia and infertility, and bone formation was severely impaired as a typical feature of vitamin D-dependent rickets type II (refs 8, 9). Unlike humans with this disease, most of the null mutant mice died within 15 weeks after birth, and uterine hypoplasia with impaired folliculogenesis was found in female reproductive organs. These defects, such as alopoecia and uterine hypoplasia, were not observed in vitamin D-deficient animals. The findings establish a critical role for VDR in growth, bone formation and female reproduction in the post-weaning stage.     

Biologic activity of dihydroxylated 19-nor-(pre)vitamin D3

Vitamin D3 and its hydroxylated metabolites are normally in thermal equilibrium with their previtamin D isomers. To evaluate the biologic activity of 1 alpha, 25-dihydroxyprevitamin D3, we synthesized 19-nor analogs of 1 alpha, 25-dihydroxy(pre)vitamin D3 because the absence of a C19 methylene group prevents the isomerization of these analogs. The affinity of 1 alpha, 25-(OH)2D3-19-nor-D3 for the intestinal vitamin D receptor and plasma vitamin D binding protein was mildly decreased [30 and 20% of the affinity of 1 alpha, 25-(OH)2D3, respectively], but the affinity of 1 alpha, 25-(OH)2-19-nor-previtamin D3 was only 1 and 6% of that of 1 alpha, 25-(OH)2D3 for the receptor and DBP, respectively. The in vitro effects on human promyeloid leukemia (HL-60 cell) differentiation and osteocalcin secretion by human osteosarcoma (MG-63) cells by 1 alpha, 25-(OH)2-19-nor-D3 were nearly identical to those of 1 alpha-25-(OH)2D3, whereas 19-nor-previtamin D3 showed poor activity (2%). The in vivo calcemic effects of both analogs, studied in vitamin D-deficient chicks treated for 10 consecutive days with the analogs, showed no activity of the previtamin D3 analog and reduced calcemic effects (< or = 10%) of 1 alpha, 25-(OH)2-19-nor-D3. We conclude that the previtamin D form of 1 alpha, 25-(OH)2D3 has lost most of its biologic activity in vitro and in vivo.     

1alpha,25-dehydroxyvitamin D3 synergism toward transforming growth factor-beta1-induced AP-1 transcriptional activity in mouse osteoblastic cells via its nuclear receptor

The present study demonstrates 1alpha,25-dehydroxyvitamin D3 (1alpha-25-(OH)2D3) synergism toward transforming growth factor (TGF)-beta1-induced activation protein-1 (AP-1) activity in mouse osteoblastic MC3T3-E1 cells via the nuclear receptor of the vitamin. 1alpha-25-(OH)2D3 synergistically stimulated TGF-beta1-induced expression of the c-jun gene in the cells but not that of the c-fos gene. We actually showed by a gel mobility shift assay 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced AP-1 binding to the 12-(O-tetradecanoylphorbol-13-acetate response element (TRE). 1alpha-25-(OH)2D3 markedly stimulated the transient activity of TGF-beta1-induced AP-1 in the cells transfected with a TRE-chloramphenicol acetyltransferase (CAT) reporter gene. Also, a synergistic increase in TGF-beta1-induced CAT activity was observed in the cells cotransfected with an expression vector encoding vitamin D3 receptor (VDR) and the reporter gene. However, the synergistic CAT activity was inhibited by pretreatment with VDR antisense oligonucleotides. In addition, in a Northern blot assay, we observed 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced expression of the c-jun gene in the cells transfected with the VDR expression vector and also found that the synergistic action was clearly blocked by VDR antisense oligonucleotide pretreatment. The present study strongly suggests a novel positive regulation by 1alpha-25-(OH)2D3 of TGF-beta1-induced AP-1 activity in osteoblasts via "genomic action."     

Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine

BACKGROUND: Type 1 diabetes is characterized by the presence of an autoimmune memory, responsible for the destruction of even syngeneic islet grafts. This recurrence of autoimmunity is partly responsible for the need of extensive immunosuppression in pancreas and islet transplantation in type 1 diabetic patients. The aim of the study was to evaluate the capacity of a 20-epi-analog of vitamin D3, KH1060, both alone and in combination with cyclosporine (CsA) to prevent diabetes recurrence in syngeneic islet grafts in nonobese diabetic (NOD) mice. METHODS: Spontaneously diabetic NOD mice grafted with syngeneic islets (n=500) under the kidney capsule were treated with KH1060, CsA, or a combination of both drugs from the day before transplantation until recurrence or 60 days after transplantation. RESULTS: Vehicle-treated mice showed a recurrence of diabetes in 100% of cases (n=17) within 4 weeks. Treatment with high doses of CsA (15 mg/kg/day) or KH1060 (1 microg/kg/2 days) significantly prolonged islet survival (60 days and 50 days, respectively, versus 9.5 days in controls; P<0.001 and P<0.0001). Mice treated with subtherapeutical doses of both drugs combined (KH1060 0.5 microg/kg/2 days + CsA 7.5 mg/kg/day) had significant prolongation of graft survival (48 days; P<0.001) and more importantly, four of five mice that were still normoglycemic 60 days after transplantation showed no recurrence after discontinuation of all treatment. Histology of the grafts of control and combination-treated mice demonstrated that graft infiltration and islet destruction were less severe in grafts of combination-treated mice. Cytokine mRNA analysis in the grafts 6 days after transplantation revealed a clear suppression of interleukin-12 and T helper 1 cytokines and higher levels of interleukin-4 in combination-treated mice. CONCLUSIONS: KH1060, an analog of 1,25(OH)2D3, delays autoimmune disease recurrence after syngeneic islet transplantation in NOD mice, both alone and especially in combination with CsA, possibly restoring tolerance to beta cells in 30% of cases.     

Nonhypercalcemic analogs of vitamin D stimulate creatine kinase B activity in osteoblast-like ROS 17/2.8 cells and up-regulate their responsiveness to estrogens

We have reported that pretreatment with 1 alpha, 25(OH)2D3(1, 25) up-regulates responsiveness and sensitivity to 17 beta estradiol (E2) in osteoblast-like cells, as measured by parallel stimulation of [3H]thymidine incorporation into DNA and the specific activity of creatine kinase BB (CK). Increased responsiveness was correlated with increased E2 receptor concentration. In this study, we have extended these observations to new nonhypercalcemic analogs of 1,25. We compared the analogs hexafluoro vitamin D3 (FL), and the side chain modified derivatives: EB 1089 (EB), CB 1093 (CB) and MC 1288 (MC) with 1,25 and 25 (OH)D3(25 D3). Treatment with 30 nM E2 for 4 h stimulated CK activity in ROS 17/2-8 cells by 40%; there was no further increase after 3 daily additions of E2. Treatment by 3 daily additions, at 1 nM, of all analogs except 25 D3 caused a 2-3-fold increase in CK specific activity. This schedule of treatment also upregulated the response to 4 h exposure to 30 nM E2 by 30-70% above the response to vitamin D analogs alone, and by up to 2 fold compared to E2 without pretreatment. At 1 pM, the analogs doubled CK activity, and, except for 1,25, upregulated the response to E2 to levels characteristic of each analog. Pretreatment with vitamin D analogs also increased the sensitivity to E2 by lowering the dose for a comparable response to E2 by one or two orders of magnitude. Stimulation of specific activity of CK by the analogs was paralleled by increases in the steady state level of mRNA for CKB, but not in its half life. Whereas pretreatment by vehicle followed by E2 for 2 h was unable to increase CKB mRNA, pretreatment with the analogs made possible detection of mRNA responsiveness to E2. These results add to the evidence for the interaction of estrogens and antiestrogens with vitamin D metabolites in regulation of bone growth in vitro. They also strengthen the potential for treatment of bone loss, as occurs in postmenopausal osteoporosis, by a combination of nonhypercalcemic vitamin D analogs and estrogens.     

Evidence for a 1 alpha,25-dihydroxyvitamin D3 receptor/binding protein in a membrane fraction isolated from a chick tibial fracture-healing callus

Previous biological studies have implicated two vitamin D metabolites, 1 alpha,25(OH)2-vitamin D3[1 alpha,25(OH)2-D3] and 24R,25(OH)2-vitamin D3 [24R,25(OH)2D3] in the process of skeletal fracture-healing. While a nuclear receptor for 1 alpha,25(OH)2D3 is known to be present in osteoblast and absent in osteoclast cell lines, no systematic study has been carried out on the callus tissue which is formed during fracture-healing. The present report shows that a binding protein/receptor for 1 alpha,25(OH)2D3 resides both in a postnuclear membrane fraction and in a high speed cytosol fraction of the callus tissue obtained 10 days after imposition of a tibial fracture. The dissociation constant, KD, for 1 alpha,25(OH)2D3 was 0.83 +/- 0.34 M and 0.66 +/- 0.38 nM respectively, for the membrane and cytosol fractions. Results from a panel of steroid competition assays indicate that both receptor/binding proteins greatly prefer 1 alpha-hydroxylated ligands as compared to 1 alpha-deoxy or 24-hydroxylated ligands. The presence of 1 alpha,25(OH)2D3 receptors in the fracture-healing callus is consistent with the known biological effects of the metabolite on the fracture-healing process.     

Partial prevention of active Heymann nephritis by 1 alpha, 25 dihydroxyvitamin D3

The hormone 1 alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3) has potent immunosuppressive effects in vitro. Recent publications also described a protective effect of the hormone in various animal models of immune-mediated diseases. To test its in vivo activity we induced active Heymann nephritis in Lewis rats that were either untreated or treated with 1,25(OH)2D3 or its synthetic 20-epi analogue, KH1060. Treatment with cyclosporine A (CsA) was used as an immunosuppressive control. In this nephrotic model the administration of 1,25(OH)2D3 (0.5 microgram/kg body weight) given on alternate days during the first 13 days after active immunization significantly reduced the proteinuria as measured by weeks 7-9. This reduction was comparable to the reduction observed in rats treated with CsA (20 mg/kg) on alternate days. A second series of experiments with 1,25(OH)2D3 confirmed these findings. The level of autoantibodies was found to be significantly suppressed during the treatment time in the CsA (20 mg/kg) group, whereas the limit of significance (P = 0.06) was reached in the 1,25(OH)2D3 (0.5 microgram/kg) group. The size of the immune deposits also was found to be substantially smaller in the groups that developed less proteinuria. The administration of 1,25(OH)2D3 transiently increased the mean serum calcium concentration with 2.5 mg/dl above the pretreatment values, and the urinary calcium excretion by a factor of 3-5 during the short treatment time. Treatment with the analogue KH1060 did not reduce the proteinuria significantly. Our experiments add evidence to the hypothesis that 1,25(OH)2D3 in pharmacological doses has immunosuppressive potency.     

Vitamin D receptor expression, 24-hydroxylase activity, and inhibition of growth by 1alpha,25-dihydroxyvitamin D3 in seven human prostatic carcinoma cell lines

Although prostatic cancer is often viewed as an androgen-dependent malignancy, a number of other hormones including 1alpha, 25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] are now recognized to modulate its growth and differentiated phenotype. Seven different continuous human prostatic carcinoma cell lines were examined for the presence of biologically active receptors for 1alpha,25(OH)2D3. All seven lines were found to contain mRNA for the vitamin D receptor using an RNase protection assay. Six of the seven cell lines were found to have high-affinity saturable binding sites for 1alpha,25(OH)2D3. The seventh line was found to contain vitamin D receptors by sucrose gradient analysis. All seven lines were found to express 24-hydroxylase activity by a HPLC assay that measures the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3. 24-Hydroxylase activity was up-regulated in all seven cell lines by preincubation with 1alpha,25(OH)2D3. In the presence of fetal bovine serum, the growth of four of the seven cell lines was inhibited. In the majority of cell lines growth inhibition was related not only to the number of receptors per cell, but also in inverse proportion to the 24-hydroxylase activity of each cell line. The ubiquitous presence of vitamin D receptor and 24-hydroxylase activity in human prostatic carcinoma cells suggests new alternatives for the pharmacological treatment of advanced prostatic cancer and implies that chemoprevention strategies could also make use of this endocrine axis.     

Synthesis, stereochemistry, and biological activity of 1alpha,23,25-trihydroxy-24-oxovitamin D3, a major natural metabolite of 1alpha,25-dihydroxyvitamin D3

The C(23) epimers of 1alpha,23,25(OH)3-24-oxovitamin D3, a major natural metabolite of the secosteroid hormone, 1alpha,25(OH)2D3, were chemically synthesized for the first time. The metabolite was synthesized by palladium coupling of the appropriate CD ring analog with an A ring enyne. Various approaches from quinic acid to the A ring precursors were explored, and a new route to the A ring enyne from quinic acid was developed. The C(23) stereochemistry of the natural 1alpha,23,25(OH)3-24-oxovitamin D3 produced in neonatal human keratinocytes was determined to be S on the basis of the 1H NMR and the HPLC data. The biological activity of 1alpha,23(S), 25(OH)3-24-oxovitamin D3 in primary cultures of bovine parathyroid cells was determined by comparing the potency of this metabolite to that of 1alpha,25(OH)2D3 in suppression of parathyroid hormone (PTH) secretion. The results indicate that 1alpha,23(S), 25(OH)3-24-oxovitamin D3 potently suppressed PTH secretion even at concentrations as low as 10(-)12 M and is equipotent with 1alpha, 25(OH)2D3. The high activity of 1alpha,23(S),25(OH)3-24-oxovitamin D3 cannot be explained on the basis of its affinity for the vitamin D receptor as this metabolite was found to be 10 times less effective than radioinert 1alpha,25(OH)2D3 in blocking the uptake and receptor binding of [3H]-1alpha,25(OH)2D3 in intact parathyroid cells. Further studies are required to explain the molecular basis for the activity of 1alpha,23(S),25(OH)3-24-oxovitamin D3 in its ability to suppress PTH secretion. In summary, our present study indicates that the C(23) stereochemistry of the natural 1alpha,23, 25(OH)3-24-oxovitamin D3 is S and this metabolite is equipotent to 1alpha,25(OH)2D3 in suppressing PTH secretion.     

The role of vitamin D derivatives and retinoids in the differentiation of human leukaemia cells

The capabilities of 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), and two novel vitamin D analogues, EB1089 and KH1060, to induce the differentiation of two established leukaemia cell lines, U937 and HL-60, were assessed alone or in combination with the retinoid compounds, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA). The vitamin D derivatives acted to increase the differentiation of U937 and HL-60 cell cultures in a dose-dependent manner, as determined by nitroblue tetrazolium (NBT) reduction, with EB1089 and KH1060 being more effective than the native hormone. As an additional index of leukaemic cell differentiation, induction of expression of the phenotypic cell surface antigen, CD14, and the beta2-integrins, CD11b and CD18 by the vitamin D and retinoid compounds were monitored using fluorescence activated cell sorting (FACS) analyses. Following 96-hr treatment of U937 and HL-60 cells with 5 x 10(-10) M of the vitamin D derivatives, a striking increase in CD14 antigen expression was apparent, indicating the promotion by these compounds of a monocyte/macrophage lineage of cells. CD11b and CD18 antigen expression were also raised above control levels. In contrast, both retinoid compounds used at the higher concentration of 1 x 10(-8) M were not effective inducers of CD14 antigen expression. However, CD11b and CD18 were both readily increased in U937 and HL-60 cell cultures. Treatment of U937 cell cultures with the vitamin D compounds and the retinoids resulted in cooperative effects on induction of differentiation, with correlation by both NBT reduction and FACS analyses of CD14 antigen expression. The presence of 9-cis RA or ATRA appeared to contribute to the further increase of CD14 in these cells. HL-60 cell cotreatment with these compounds also displayed enhanced cooperative effects in phagocytic function by NBT reduction. However, analysis of CD14 revealed a dramatic diminution in HL-60 cells treated with the combinations of the vitamin D derivatives and the retinoids. Assessment of HL-60 cell morphology treated with these combinations demonstrated the presence of a mixed population of monocytes and granulocytes. CD11b and CD18 antigen expression was also enhanced in both cell lines with cotreatment. The ability of EB1089 and KH1060 to induce leukaemic cell differentiation may provide an additional option for therapeutic use alone or together with other differentiation agents such as 9-cis RA or ATRA.     

17Beta-estradiol antagonizes effects of 1alpha,25-dihydroxyvitamin D3 on interleukin-6 production and osteoclast-like cell formation in mouse bone marrow primary cultures

In mouse bone marrow primary cultures, the formation of osteoclast-like, i.e. tartrate-resistant acid phosphatase (TRAP)- and calcitonin receptor-positive multinucleated cells (MNC), when induced by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), can be suppressed by 17beta-estradiol (17beta-E2), whereas 17alpha-E2 is without any effect. 17beta-E2, above 10(-11) M, significantly reduced 1alpha,25(OH)2D3-mediated TRAP+ MNC formation in cultured bone marrow cells from both female and male mice. The estrogen at 10(-8) M suppressed the peak response to the vitamin D sterol by 50%. 17beta-E2 significantly suppressed basal and 1alpha,25(OH)2D3-stimulated cellular production of interleukin (IL)-6. IL-6 alone, although bone marrow cells in hormone-free culture produced appreciable amounts of the cytokine, did not induce any TRAP+ MNC. Therefore, the changes in IL-6 production induced by the hormones could not be the sole determinant for the extent of TRAP+ MNC formation. However, the stimulatory effect of 1alpha,25(OH)2D3 on osteoclastogenesis nevertheless can be significantly reduced by a neutralizing monoclonal anti-IL-6 antibody. In the presence of 10(-8) M 17beta-E2, the anti-IL-6 monoclonal antibody does not achieve any further suppression of 1alpha,25(OH)2D3-related osteoclast-like cell formation. Our data suggest that induction of osteoclastogenesis by 1alpha,25(OH)2D3 is partially dependent on IL-6 signaling and can be modulated by 17beta-E2 through interference with IL-6 receptor activation, in addition to inhibition of IL-6 production by marrow stromal cells.     

The integration of object properties

Growth plate cartilage cell express receptors for, and are affected by both IGF-I and 1 alpha, 25(OH)2D3. The studies were undertaken to investigate interaction between these two hormone systems, that is, (i) to study effects of 1 alpha, 25(OH)2D3 on IGF-type 1 receptors (IGFIR), on IGF-I stimulated cell replication, colony formation, and on alkaline phosphatase activity (AP), and conversely, (ii) to study the effect of IGF-I on vitamin D receptor (VDR) expression on 1 alpha, 25(OH)2D3 stimulated growth parameters and on AP activity. Freshly isolated rat tibial chondrocytes were grown in monolayer cultures, (serum-free) or in agarose stabilized suspension cultures (0.1% FCS). Vitamin D receptor and IGFIR were visualized by immunostaining with the monoclonal antibody (mAb) 9A7 gamma and mAb alpha IR3, respectively, and quantitated by RT-PCR for mRNA and by Scatchard analysis using [3H]-1,25(OH)2D3 and [125I]-alpha IR3. Cell proliferation was measured by [3H]-thymidine incorporation, growth curves in monolayer cultures, and by colony formation in agarose-stabilized suspension cultures. IGF-I dose-dependently increased [3H]-thymidine incorporation. 1 alpha, 25(OH)2D3, but not 1 beta, 25(OH)2D3 was stimulatory at low ((10-12 M) and slightly inhibitory at high (10-8 M) concentrations. The effect of IGF-I was additive to that of 1 alpha, 25 (OH)2D3 [IGF-I 60 ng/ml, 181 +/- 12.7; 1 alpha, 25(OH)2D3 10(-12) M, 181 +/- 9.8%, IGF-I + 1 alpha, 25(OH)2D3, 247 +/- 16.7%, P < 0.05 by ANOVA] and specifically obliterated by polyclonal IGF-I antibody (AB-1). Interaction could also be confirmed in suspension cultures. IGFIR mRNA and [125I]-alphaIR3 binding was increased by low (10(-12) m) but not by high (10(-8) M) concentrations of 1 alpha, 25(OH)2D3. Homologous up-regulation by IGF-I (60 ng/ml) was specifically inhibited by AB-1 and markedly amplified by coincubation with 1 alpha, 25(OH)2D3 (10(-12)m). Immunostaining with alpha IR3 showed specific IGFIR expression in rat growth cartilage, but not liver tissue. Stimulation of chondrocytes with 1 alpha, 25(OH)2D3 or IGF-I suggested some increase of receptor expression in single cells, but the predominant effect was increased recruitment of receptor positive cells, Vitamin D receptor expression was markedly stimulated (fourfold) by IGF-I (60 ng/ml), but not IGF-II and inhibited by actinomycin D. This study shows that IGF-I and 1 alpha, 25(OH)2D3 mutually up-regulate their respective receptors in growth plate chondrocytes. In parallel, they have additive effects on cell proliferation and colony formation suggesting independent effector pathways.     

Vitamin D derivatives inhibit the mitogenic effects of IGF-I on MCF-7 human breast cancer cells

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and four novel synthetic analogues (EB1089, KH1060, KH1230 and CB1093) on IGF-I-stimulated growth of MCF-7 human breast cancer cells have been determined. A significant time- and dose-dependent inhibition of IGF-I-stimulated cell growth was seen with EB1089, such that after 7 days of treatment with 10(-8) M EB1089, the mitogenic effect of IGF-I (30 ng/ml) was negated. Comparison with 1,25(OH)2D3 showed the synthetic analogues to be more potent. The anti-oestrogen ICI 182,780 similarly inhibited IGF-I-stimulated growth of these cells and in combination with EB1089 exerted additional inhibitory effects. Retinoids (all-trans-retinoic acid or the isomer 9-cis-retinoic acid) were less effective in limiting MCF-7 cell responsiveness to IGF-I but, in combination with EB1089, a co-operative effect was achieved. Using radioligand-binding techniques, we observed that 1,25(OH)2D3 and EB1089 down-regulated the levels of 125I-IGF-I binding to MCF-7 cell membranes. Scatchard analysis showed that EB1089 decreased maximal binding approximately 2-fold. Vitamin D derivatives were also demonstrated to reduce IGF-I receptor expression in MCF-7 cells by Western analysis. Our findings demonstrate that vitamin D derivatives limit responsiveness of MCF-7 cells to the mitogenic effects of IGF-I, which may be mediated by reduction of IGF-I receptor expression.     

Comparison of 6-s-cis- and 6-s-trans-locked analogs of 1alpha,25-dihydroxyvitamin D3 indicates that the 6-s-cis conformation is preferred for rapid nongenomic biological responses and that neither 6-s-cis- nor 6-s-trans-locked analogs are preferred for genomic biological responses

The hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] generates biological responses via both genomic and rapid, nongenomic mechanisms. The genomic responses utilize signal transduction pathways linked to a nuclear receptor (VDRnuc) for 1alpha,25(OH)2D3, while the rapid responses are believed to utilize other signal transduction pathways that may be linked to a putative membrane receptor for 1alpha,25(OH)2D3. The natural seco steroid is capable of facile rotation about its 6,7 single carbon bond, which permits generation of a continuum of potential ligand shapes extending from the 6-s-cis (steroid like) to the 6-s-trans (extended). To identify the shape of conformer(s) that can serve as agonists for the genomic and rapid biological responses, we measured multiple known agonist activities of two families of chemically synthesized analogs that were either locked in the 6-s-cis (6C) or 6-s-trans (6T) conformation. We found that 6T locked analogs were inactive or significantly less active than 1alpha,25(OH)2D3 in both rapid responses (transcaltachia in perfused chick intestine, 45Ca2+ influx in ROS 17/2.8 cells) and genomic (osteocalcin induction in MG-63 cells, differentiation of HL-60 cells, growth arrest of MCF-7 cells, promoter transfection in COS-7 cells) assays. In genomic assays, 6C locked analogs bound poorly to the VDRnuc and were significantly less effective than 1alpha,25(OH)2D3 in the same series of assays designed to measure genomic responses. In contrast, the 6C locked analogs were potent agonists of both rapid response pathways and had activities equivalent to the conformationally flexibile 1alpha,25(OH)2D3; this represents the first demonstration that 6-s-cis locked analogs can function as agonists for vitamin D responses.     

New 1 alpha,25-dihydroxy vitamin D3 analogues with side chains attached to C-18: synthesis and biological activity

A new class of analogues of 1 alpha,25-dihydroxy vitamin D3 has been synthesised, in which the side chain (C-23 to C-27) has been removed and where new hydroxylated side chains have been attached to the C-18 methyl group. These analogues show antiproliferative activity in U937 and HaCaT cells comparable to that of 1 alpha,25-dihydroxy vitamin D3. Lack of calcemic activity makes these analogues potentially useful in the treatment of proliferative diseases.     

Measurement of vitamin D3 metabolites in smelter workers exposed to lead and cadmium

OBJECTIVES: To investigate the effects of lead and cadmium on the metabolic pathway of vitamin D3. METHODS: Blood and urinary cadmium and urinary total proteins were measured in 59 smelter workers occupationally exposed to lead and cadmium. In 19 of these workers, the plasma vitamin D3 metabolites, (25-hydroxycholecalciferol (25 OHD3), 24R, 25-dihydroxycholecalciferol (24R,25(OH)2D3) and 1 alpha,25-dihydroxycholecalciferol (1 alpha, 25(OH)2D3)) were measured together with blood lead. Vitamin D3 metabolites were measured by radioimmunoassay, (RIA), lead and cadmium by atomic absorption spectrophotometry, and total proteins with a test kit. RESULTS: Ranges for plasma 25(OH)D3, 24R,25(OH)2D3 and 1 alpha,25(OH)2D3 were 1.0-51.9 ng/ml, 0.6-5.8 ng/ml, and 0.1-75.7 pg/ml, respectively. Ranges for blood lead were 1-3.7 mumol/l, (21-76 micrograms/dl), blood cadmium 6-145 nmol/l, and urinary cadmium 3-161 nmol/l. Total proteins in random urine samples were 2.1-32.6 mg/dl. Concentrations of lead and cadmium in blood showed no correlation (correlation coefficient -0.265) but there was a highly significant correlation between blood and urinary cadmium. Concentrations for 24R,25(OH)2D3 were depressed below the normal range as blood and urinary cadmium increased, irrespective of lead concentrations. High cadmium concentrations were associated with decreased plasma 1 alpha,25(OH)2D3 when lead concentrations were < 1.9 mumol/l and with above normal plasma 1 alpha,25(OH)2D3 when lead concentrations were > 1.9 mumol/l, Kruskal-Wallis analysis of variance (K-W ANOVA) chi 2 = 10.3, p = 0.006. Plasma 25(OH)D3 was negatively correlated with both urinary total proteins and urinary cadmium, but showed no correlation with plasma 24R,25(OH)2D3, 1 alpha,25(OH)2D3, blood lead, or blood cadmium. CONCLUSION: Continuous long term exposure to cadmium may result in a state of equilibrium between blood and urinary cadmium. Cadmium concentrations in blood could be predicted from the cadmium concentration of the urine, (regression coefficient +0.35 SE 0.077). Exposure to cadmium alone decreased the concentrations of 1 alpha,25(OH)2D3 and 24R,25(OH)2D3, whereas exposure to both cadmium and lead increased the concentrations of 1 alpha,25(OH)2D3. It has been suggested that cadmium and lead interact with renal mitochondrial hydroxylases of the vitamin D3 endocrine complex. Perturbation of the vitamin D metabolic pathway by cadmium may result in health effect, such as osteoporosis or osteomalacia, risks which are possibly increased in the presence of lead.