1,25-Dihydroxyvitamin D3 analog structure-function assessment of the rapid stimulation of intestinal calcium absorption (transcaltachia).

The possibility is now emerging that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can mediate biologic responses via both genomic and nongenomic pathways. To understand the molecular basis of the nongenomic response of transcaltachia, defined as the 1,25-(OH)2D3-mediated rapid (2-10 minutes) stimulation of calcium transport from the brush border to the basal lateral membrane of the epithelial cell in vitamin D-replete chick intestine, and to address the issue of whether the same receptor for the secosteroid serves as the signal transducer for both genomic and nongenomic pathways, we carried out structure-function studies using seven analogs of 1,25-(OH)2D3 with different affinities for the classic nuclear 1,25-(OH)2D3 receptor as measured by determination in a steroid competition assay of the relative competitive index (RCI). The RCI of 1,25-(OH)2D3 is by definition 100. 1,25-(OH)2D3 initiates transcaltachia within 2-10 minutes of vascular perfusion and yields a biphase response curve. Dose-dependent stimulations of Ca2+ transport by the seven analogs indicates that different structural features are essential for initiating the transcaltachic response as contrasted with binding to the classic nuclear receptor. Vascular perfusion with analogs AT (25-OH-16-ene-23-yne-D3) and Y (25-OH-23-yne-D3), which are known to activate Ca2+ channels but bind very poorly to the classic receptor (RCI less than 0.5), are efficient in stimulating Ca2+ transport. By comparison, compounds BT [1 alpha,24S-(OH)2-22-en-26,27-dehydrovitamin D3] and V (1,25-(OH)2-16-ene-23-yne-D3], which bind very well to the classic nuclear receptor (RCI 75-111) but do not activate Ca2+ channels, are inefficient in stimulating Ca2+ transport. These results indicate that the membrane components that respond to the analogs of 1,25-(OH)2D3 with activation of Ca2+ channels have a different ligand specificity than the classic nuclear receptor.     

Increased vitamin D receptor level enhances 1,25-dihydroxyvitamin D3-mediated gene expression and calcium transport in Caco-2 cells.

Altered vitamin D receptor (VDR) level has been proposed to explain differences in intestinal responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We tested whether the enterocyte VDR level influences 1,25(OH)2D3-mediated gene expression and transepithelial calcium (Ca) transport in the human intestinal cell line Caco-2. Cells were stably transfected with a human metallothionein (hMT) IIA promoter-human VDR (hVDR) complementary DNA (cDNA) transgene that overexpressed hVDR in response to heavy metals. In MTVDR clones, induction of 25-hyroxyvitamin D3-24-hydroxylase (24-OHase) messenger RNA (mRNA) expression by 1,25(OH)2D3 (10(-9) M, 4 h) was correlated to metal-induced changes in nuclear VDR level (r2 = 0.99). In MTVDR clones, basal VDR level was 2-fold greater and 1,25(OH)2D3-mediated Ca transport (10(-7) M, 24 h) was 43% higher than in parental Caco-2 cells. Treatment of MTVDR clones with Cd (1 microM, 28 h) increased VDR level by 68%, significantly enhanced 1,25(OH)2D3-mediated Ca transport by 24%, and increased accumulation of calbindin D9K mRNA by 76% relative to 1,25(OH)2D3 alone. These observations support the hypothesis that the enterocyte VDR level is an important modulator of intestinal responsiveness to 1,25(OH)2D3.     

1,25-Dihydroxy-19-nor-vitamin D(2), a vitamin D analog with reduced bone resorbing activity in vitro

1,25-Dihydroxy-19-nor-vitamin D(2) (19-norD(2)), a new analog of 1,25(OH)(2)D(3), suppresses parathyroid hormone in renal failure patients and in uremic rats but has less calcemic activity than 1,25(OH)(2)D(3). Although 19-norD(2) has high affinity for the vitamin D receptor and similar pharmacokinetics to those of 1,25(OH)(2)D(3), it has much less bone resorbing activity in vivo. The intrinsic activity of 19-norD(2) on osteoclastogenesis and activation of bone resorption in mouse bone marrow cultures was examined to determine the mechanism involved. 19-norD(2) and 1,25(OH)(2)D(3) (10 nM) were equivalent in stimulating the formation and maintenance of large multinucleated, tartrate-resistant acid phosphatase-positive cells. However, the amount of bone resorbed by osteoclasts stimulated by 10 nM 19-norD(2), as measured by pit-forming assays, was reduced 62% compared with 10 nM 1,25(OH)(2)D(3)-stimulated osteoclasts (P < 0. 05). This difference could not be attributed to enhanced catabolism or to downregulated vitamin D receptor. The rate of degradation of 19-norD(2) in cultures was approximately 20% greater than 1, 25(OH)(2)D(3), not enough to account for the different effects on bone resorption. The VDR levels were identical in cultures that were treated with 19-norD(2) and 1,25(OH)(2)D(3). In summary, 19-norD(2) is less effective than 1,25(OH)(2)D(3) in stimulating mouse marrow osteoclasts to resorb bone. The reason for this difference is not clear but seems to involve the late maturation and/or activation of osteoclasts as the number of pits produced by each tartrate-resistant acid phosphatase-positive cell is reduced under stimulation by 19-norD(2) compared with 1,25(OH)(2)D(3).     

Isolation of osteoclasts from fetal rat long bones

Cell isolates containing multinucleate osteoclasts were obtained from longitudinally split fetal rat long bones by treatment with testicular hyaluronidase. The total yield of osteoclasts and the osteoclast enrichment of the isolate were increased if the intact bones were first cultured for 72 h. Even greater enhancement was obtained if the bones were treated with 1,25-dihydroxycholecalciferol [1,25(OH)2D3] during the culture period. This technique resulted in a cell population containing approximately 15% osteoclasts in yields greater than 50 osteoclasts per long bone. The yield of osteoclasts and the percentage of osteoclasts correlated well with the extent of bone resorption induced by 1,25(OH)2D3. The effectiveness of several isolation procedures was compared using the 1,25(OH)2D3-treated long bones. Conventional digestion with 1 mg/ml crude collagenase gave a much poorer yield of osteoclasts than simply agitating the split long bones. Hyaluronidase plus EDTA was not significantly different from EDTA alone. Even with milder procedures, however, the isolated osteoclasts were damaged as judged by their failure to exclude trypan blue. The osteoclasts are obviously very fragile cells. The isolation technique coupled with May-Grunwald-Giemsa staining permitted reliable determination of the median number of nuclei per osteoclast. This parameter was the same in uncultured bones or in bones cultured for 72 h in control media. Treatment with 1,25(OH)2D3 increased the nuclear number. At lower levels of bone resorption, nuclear number did not increase, but it was significantly greater in more highly resorbed bones.     

Relative potencies of 1,25-(OH)(2)D(3) and 19-Nor-1,25-(OH)(2)D(2) on inducing differentiation and markers of bone formation in MG-63 cells.

19-Nor-1,25-(OH)(2)D(2), an analog of 1,25-(OH)(2)D(3), is used to treat secondary hyperparathyroidism because it suppresses parathyroid hormone synthesis and secretion with lower calcemic and phosphatemic activities. 19-Nor-1,25-(OH)(2)D(2) is approximately 10 times less active than 1,25-(OH)(2)D(3) in promoting bone resorption, which accounts in part for the low potency of this analog in increasing serum calcium and phosphorus. Concern that 19-nor-1,25-(OH)(2)D(2) also could be less potent than 1,25-(OH)(2)D(3) on bone formation led to a comparison of the potency of both compounds on osteoblasts. In the human osteoblast-like cell line MG-63, 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) had a similar potency in upregulating vitamin D receptor content and suppressing proliferation. Both sterols caused a similar reduction in DNA content and proliferating cell nuclear antigen protein expression. Time-course and dose-response studies on 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) induction of the marker of bone formation, osteocalcin, showed overlapping curves. The effects on alkaline phosphatase (ALP) activity also were studied in MG-63 cells that had been co-treated with either sterol and transforming growth factor-beta, an enhancer of 1,25-(OH)(2)D(3)-induced ALP activity in this cell line. Transforming growth factor-beta alone had no effect, whereas 1,25-(OH)(2)D(3) and 19-nor-1,25-(OH)(2)D(2) increased ALP activity similarly. These studies demonstrate that 19-nor-1,25-(OH)(2)D(2) has the same potency as 1,25-(OH)(2)D(3) not only in inducing vitamin D receptor content, osteocalcin levels, and ALP activity but also in controlling osteoblastic growth. Therefore, it is unlikely that 19-nor-1,25-(OH)(2)D(2) would have deleterious effects on bone remodeling.     

Actions of 1,25-dihydroxyvitamin D3 on human mesangial cells.

In the present study, we examined specific binding of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by an effects of 1,25(OH)2D3 on human mesangial cells (hMC), obtained from healthy portions of tumor-bearing kidneys. Receptors for 1,25(OH)2D3 were characterized by (1) sucrose density gradient analysis, (2) Scatchard analysis, and (3) DNA affinity of the receptor molecule. Specific binding occurred by a single class of macromolecules, sedimenting with 3.5 S in sucrose density gradients (5% to 20%). Receptors showed high affinity (Kd, 1.39 x 10(-10)), and specific binding capacity (Nmax) of 821 binding sites per cell. 1,25(OH)2D3 (10(-6) to 10(-10)) reduced both DNA synthesis (by [3H]thymidine incorporation) and cell growth (by cell counting) throughout the log-phase and confluence. Further evidence of functional effects of 1,25(OH)2D3 on hMC is provided by ultrastructural studies, which showed rapid increase of electron-dense lysosomal particles in hMC exposed to 1,25(OH)2D3. The data identify actions of 1,25(OH)2D3, a molecule with recently recognized immunoregulatory roles, on hMC. The results are consistent with a role of 1,25(OH)2D3 in control of mesangial cell function.     

Effect of 1,25-dihydroxyvitamin D3 on mouse mammary tumor (GR) cells: evidence for receptors, cellular uptake, inhibition of growth and alteration in morphology at physiologic concentrations of hormone

Mammary glands are target tissues for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We have examined a mouse mammary tumor cell line (GR) for receptors of 1,25(OH)2D3 and have examined alterations in the growth and morphology of these cells in response to 1,25(OH)2D3. GR cells contain a high affinity (Kd approximately 10(-11)), low-capacity receptor with a high specificity for 1,25(OH)2D3. The 1,25(OH)2D3 receptor in GR cells has a sedimentation coefficient of 3.5 and elutes from DEAE cellulose columns with approximately 0.15 M KCl. These properties of the receptor are similar to those reported for other 1,25(OH)2D3 receptors. 1,25(OH)2D3 is internalized by GR cells in situ and specifically bound 1,25(OH)2D3 is found predominantly, if not entirely, in the nucleus as determined by cell fractionation and autoradiographic techniques. The incubation of GR cells in culture for 7 days with 1,25(OH)2D3 markedly alters cell growth. Cell growth is retarded in a dose-dependent manner; physiologic concentrations (10(-10) M) of 1,25(OH)2D3 retard cell growth by approximately 50%. In addition, GR cells incubated with 10(-9) to 10(-8) M 1,25(OH)2D3 undergo marked morphological changes. The incubation of GR cells with other vitamin D metabolites such as 25-hydroxyvitamin D3 (25(OH)D3) at a concentration of 10(-9) M does not significantly alter cell growth or morphology. The presence of high affinity receptors for 1,25(OH)2D3, the specific internalization of 1,25(OH)2D3 predominantly into the nuclei, and the significant effects of physiological concentrations of 1,25(OH)2D3 on cell growth suggest a direct, specific, nuclear effect of 1,25(OH)2D3 on GR cells. The mouse mammary tumor model might be useful in examining the effect of 1,25(OH)2D3 on tumor formation.     

Constitutive expression of a vitamin D 1-hydroxylase in a myelomonocytic cell line: a model for studying 1,25-dihydroxyvitamin D production in vitro

The capacity of the v-myc-transformed, chicken myelomonocytic cell line HD-11 to metabolize 25-hydroxyvitamin D3 (25-OHD3) was examined. HD-11 cells produced and secreted a metabolite of 25-OHD3 that was bound with high affinity by receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. On normal-phase HPLC, this metabolite cochromatographed with authentic 1,25-(OH)2D3 in both hexane- and methylene chloride-based solvent systems. The 25-OHD3 1-hydroxylation reaction was substrate saturable with a Km of 73 nM 25-OHD3 and a maximal velocity of 167 fmol per 10(6) cells per h. This reaction was inhibited by ketoconazole, a recognized inhibitor of cytochrome P450 mixed-function oxidases including the authentic, renal 25-OHD3 1-hydroxylase. On the other hand, HD-11 cell 1,25-(OH)2D3 production was not affected by the antioxidant DPPD, a known inhibitor of free radical-generated 1,25-(OH)2D3. In addition to synthesizing 1,25-(OH)2D3, this monocyte-macrophage cell line also has the potential to be a target for the hormone; HD-11 cells express high-affinity receptor for 1,25-(OH)2D3 (Kin = 0.06 nM).     

Stimulus-specific 1,25(OH)2D3 modulation of TNF and IL-1-beta gene expression in human peripheral blood mononuclear cells and monocytoid cell lines

It is known that 1,25(OH)2D3 inhibits IL-2, IFN-gamma, and GM-CSF mRNA accumulation in activated T cells. While 1,25(OH)2D3 enhances macrophage competence, its effect on cytokine gene expression in monocytes is less well defined. Using Northern blot analysis, we examined the effect of 1,25(OH)2D3 pretreatment on IL-1 beta and TNF gene expression in LPS- and PHA-stimulated human PBMNC and several human myeloid cell lines (U937 and THP1). In PBMNC, preincubation had no effect on the steady-state level of LPS-induced TNF or IL-1 beta mRNA. When PHA was used to stimulate pretreated PBMNC, a 60-80% inhibition of TNF mRNA levels was observed. There was no effect on IL-1 beta mRNA. In U937 cells, 1,25(OH)2D3 preincubation resulted in a 4-fold increase in the level of TNF and IL-1 beta mRNA levels. Pretreatment had no effect on TNF or IL-1 beta gene expression in THP1 cells. The observation that PHA-induced TNF gene expression is modulated by 1,25(OH)2D3 is novel. Possible mechanisms by which 1,25(OH)2D3 preincubation may influence mitogen-specific inducible cytokine gene expression in different cell types are discussed.     

Vector-averaged gravity-induced changes in cell signaling and vitamin D receptor activity in MG-63 cells are reversed by a 1,25-(OH)2D3 analog,EB1089

Skeletal unloading in an animal hindlimb suspension model and microgravity experienced by astronauts or as a result of prolonged bed rest causes site-specific losses in bone mineral density of 1%-2% per month. This is accompanied by reductions in circulating levels of 1,25-(OH)(2)D(3), the active metabolite of vitamin D. 1,25-(OH)(2)D(3), the ligand for the vitamin D receptor (VDR), is important for calcium absorption and plays a role in differentiation of osteoblasts and osteoclasts. To examine the responses of cells to activators of the VDR in a simulated microgravity environment, we used slow-turning lateral vessels (STLVs) in a rotating cell culture system. We found that, similar to cells grown in microgravity, MG-63 cells grown in the STLVs produce less osteocalcin, alkaline phosphatase, and collagen Ialpha1 mRNA and are less responsive to 1,25-(OH)(2)D(3). In addition, expression of VDR was reduced. Moreover, growth in the STLV caused activation of the stress-activated protein kinase pathway (SAPK), a kinase that inhibits VDR activity. In contrast, the 1,25-(OH)(2)D(3) analog, EB1089, was able to compensate for some of the STLV-associated responses by reducing SAPK activity, elevating VDR levels, and increasing expression of osteocalcin and alkaline phosphatase. These studies suggest that, not only does simulated microgravity reduce differentiation of MG-63 cells, but the activity of the VDR, an important regulator of bone metabolism, is reduced. Use of potent, less calcemic analogs of 1,25-(OH)(2)D(3) may aid in overcoming this defect.     

Down-regulation of osteoclast differentiation by daidzein via caspase 3.

Phytoestrogens are plant-derived compounds with estrogen-like activity. Phytoestrogen-rich diets may prevent postmenopausal osteoporosis and these molecules maintain bone mass in ovariectomized animals. We compared the effects of the isoflavone daidzein, which has no action on tyrosine kinases, and 17beta-estradiol on the development and activity of osteoclasts in vitro. Nonadherent porcine bone marrow cells were cultured on dentine slices or on culture slides in the presence of 10-8 M of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], with or without 10(-8) M of daidzein, 10(-8) M of 17beta-estradiol for 9-11 days. Multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells that resorbed bone (osteoclasts) developed in the presence of 1,25(OH)2D3. The number of osteoclasts formed in response to 1,25(OH)2D3 was reduced by 58 +/- 8% by daidzein and 52 +/- 5% by estrogen (p < 0.01); these effects were reversed by 10-6 M of ICI 182,780. The area resorbed by mature osteoclasts was reduced by 39 +/- 5% by daidzein and 42 +/- 6% by estradiol (p < 0.01). Both compounds also inhibited the 1,25(OH)2D3-induced differentiation of osteoclast progenitors (mononucleated TRAP+ cells), 53 +/- 8% by daidzein and 50 +/- 7% by estradiol (p < 0.05). Moreover, daidzein and estradiol promoted caspase-8 and caspase-3 cleavage and DNA fragmentation of monocytic bone marrow cells. Caspase-3 cleavage was reversed by 10-8 M of ICI 182,780. Both compounds up-regulated the expression of nuclear estrogen receptors ER-alpha and ER-beta. Thus, daidzein, at the same concentration as 17beta-estradiol, inhibits osteoclast differentiation and activity. This may be caused by, at least in part, greater apoptosis of osteoclast progenitors mediated by ERs.     

1,25(OH)2D3 receptors and endorgan response in experimental aluminium intoxication

Severe aluminium-induced osteomalacia is refractory to treatment with 1,25(OH)2D3 which frequently causes hypercalcemia. To further explore the mechanisms involved, we have utilized a model of short-term aluminium intoxication in the rat (total: 11 mg elemental aluminium in 3 weeks) to study (a) 1,25(OH)2D3 receptor status in a variety of classical and non-classical target organs for 1,25(OH)2D3; (b) circulating 1,25(OH)2D3 levels; (c) baseline duodenal calcium transport, utilising the Ussing chamber, to investigate the functional significance of receptor status in a classical target organ; and (d) duodenal calcium transport response to exogenously administered 1,25(OH)2D3. Both in the three week model and in the 16 week model (total: 41 mg elemental calcium) increased maximal specific binding capacity for 1,25(OH)2D3 (Nmax), that is, number of unoccupied receptors, was observed in nuclear fractions of all tissues studied. Receptor affinity, the apparent dissociation constant KD, was unchanged. Total binding capacity, measured after displacement of endogenous ligand by Mersalyl, that is, the sum of occupied plus non-occupied receptors, was also increased. Both circulating 1,25(OH)2D3, mucosa-to-serosa calcium flux (Jms) and net calcium flux (Jnet) were reduced under baseline conditions, suggesting the lack of a direct relationship between receptor expression and endorgan response. Following exogenous 1,25(OH)2D3 administration, calcium Jms and Jnet were significantly lower in the aluminium intoxicated animals, with the increment induced in Jnet in aluminium intoxicated animals being 63% of that induced in controls. Our data suggest that resistance to the action of 1,25(OH)2D3 in aluminium intoxication is postreceptor in nature.