Stimulation of arachidonic acid metabolism in primary cultures of osteoblast-like cells by hormones and drugs

The effects of parathyroid hormone (PTH), dihydroxycholecalciferol (1,25-(OH)2 D3), thrombin, epidermal growth factor (EGF) and 12-o-tetradecanoylphorbol-13-acetate (PMA) on the biosynthesis and release of arachidonic acid metabolites were studied in primary cultures of osteoblast-like cells isolated from 18-day-old chick embryo calvaria. Cells were labelled with (14C)-arachidonic acid for 30 h. The radioactive eicosanoids were extracted from the cell culture media after a further 30 h stimulation period and analysed on a PRP-1 column by HPLC. The radioactive products were characterized by co-elution of (3H) standard prostanoids. Osteoblasts showed a basal release of the prostanoids 6-keto-PGF1 alpha, TXB2, PGF2 alpha, PGE2, PGD2 and PGB2, the latter being the most abundant one. Indomethacin (10(-5) M) effectively inhibited the basal release, but not that of an as yet unidentified compound. The release of prostanoids was stimulated by PTH (2 U/ml), thrombin (0.4 NIH/ml), EGF (50 ng/ml) and PMA (25 ng/ml), the latter being by far the most potent one. 1,25-(OH)2D3 was found to slightly inhibit the prostanoid release. These results indicate: (1) primary cultures of osteoblasts synthesize several prostaglandins, thromboxane B2 and one unidentified product. (2) the action on bone of PTH and the various drugs tested may be, at least partly, mediated by an increased prostaglandin production by osteoblasts. Clearly this does not apply to 1,25-(OH)2D3.     

Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts

1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse bone marrow cells and osteoblasts, 1,25(OH)₂D₃ induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than 10^?9 M of 1,25(OH)₂D₃. In the presence of bone morphogenetic protein 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)₂D₃ (greater than 10^?11 M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)₂D₃. In addition, the expressions of RANKL mRNA and proteins were induced by 1,25(OH)₂D₃ in osteoblasts, and further upregulated by BMP-2. In mouse bone marrow cell cultures without 1,25(OH)₂D₃, BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)₂D₃.     

Synthesis and biological evaluation of a 3-positon epimer of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71)

1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ (ED-71), an analog of active vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], possesses a hydroxypropoxy substituent at the 2β-position of 1,25(OH)₂D₃. ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)₂D₃. Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)₂D₃, an epimer of 1,25(OH)₂D₃ at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)₂D₃ at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D₃ analogs were found to be 1,25(OH)₂D₃ > ED-71 ≥ 3-epi-1,25(OH)₂D₃  3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

Increased biological potency of hexafluorinated analogs of 1,25-dihydroxyvitamin D3 on bovine parathyroid cells

1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is known to be involved in regulating the proliferation of parathyroid cells and PTH synthesis through reactions involving its nuclear receptor. We evaluated the effects of 1,25-(OH)₂D₃ and its hexafluorinated analog, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D₃ (26,27-F₆-1,25-(OH)₂D₃), on parathyroid cells. The 1,25-(OH)₂D₃ and 26,27-F₆-1,25-(OH)₂D₃ each inhibited [³H]thymidine incorporation and ornithine decarboxylase (ODC) activity, which is important in cell proliferation, in primary cultured bovine parathyroid cells. The inhibitory effect of 26,27-F₆-1,25-(OH)₂D₃ on PTH secretion from parathyroid cells was significantly more potent than that of 1,25-(OH)₂D ₃ between 10⁻¹¹ M and 10⁻⁸ M. Study of 26,27-F₆-1,25-(OH)₂D₃ metabolism in parathyroid cells in vitro elucidated its slower degradation than that of 1,25-(OH)₂D₃. After 48 h of incubation with [1β-³H]26,27-F₆-1,25-(OH)₂D₃, two HPLC peaks, one for [1β-³H]26,27-F₆-1,25-(OH)₂D₃, and a second larger peak for [1β-³H]26,27-F₆-1,23(S),25-(OH)₃D₃, were detected. No metabolites were detected after the same period of incubation with 1,25-(OH)₂[26,27-³H]D₃. We observed that 26,27-F₆-1,23(S),25-(OH)₃D₃ was as potent as 1,25-(OH)₂D₃ in inhibiting the proliferation of parathyroid cells.Data suggest that the greater biological activity of 26,27-F₆-1,25-(OH)₂D₃ is explained by its slower metabolisms and by the retention of the biological potency of 26,27-F₆-1,25-(OH)₂D₃ even after 23(S)-hydroxylation.     

PTH(7-84) inhibits PTH(1-34)-induced 1,25-(OH)2D3 production in murine renal tubules

To elucidate whether PTH(7-84), a degradation product of PTH(1-84), which inhibits PTH(1-84)-induced bone resorption, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7-84) on PTH(1-34)-induced production of 1,25-(OH)₂D₃ in primary cultured murine renal tubules.Neonatal mouse renal tubules cultured in serum-free MEM for 7 days were treated with PTH(1-34) and/or PTH(7-84). Three hours after addition of 25-OHD₃ (10⁻⁶ M), 1,25-(OH)₂D₃ was determined. PTH(1-34) stimulated the conversion of 25-OHD₃ to 1,25-(OH)₂D₃, and PTH(7-84) dose-dependently inhibited this process. Real-time PCR revealed that PTH(1-34) increased the expression level of 1α-hydroxylase mRNA, whereas PTH(7-84) did not affect the expression level 1α or 24-hydroxylase mRNA.These in vitro data suggest that PTH(7-84) elicits an antagonistic effect in renal tubules through receptors different from the type I PTH/PTHrP receptor. This may at least partly account for the decreased serum level of 1,25-(OH)₂D in patients with severe primary hyperparathyroidism with renal failure.     

The Paracrine Feedback Loop Between Vitamin D3 (1,25(OH)2D3) and PTHrP in Prehypertrophic Chondrocytes

The endocrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH‐related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)₂D₃ and PTH. This was investigated in ATDC5 cells treated with 10^?8 M 1,25(OH)₂D₃ or PTHrP, Col2‐pd2EGFP transgenic mice, and primary Col2‐pd2EGFP growth plate chondrocytes isolated by FACS, using RT‐qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)₂D₃ receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)₂D₃ decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α‐ and 24‐hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)₂D₃ treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate. 1,25(OH)₂D₃ decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)₂D₃ by increasing VDR production. In light of the role of 1,25(OH)₂D₃ and PTHrP in modulating chondrocyte differentiation, 1,25(OH)₂D₃ in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration. J. Cell. Physiol. 229: 1999–2014, 2014. © 2014 Wiley Periodicals, Inc.

     

1,25-dihydroxyvitamin D3 and macrophage colony-stimulating factor-1 synergistically phosphorylate talin

Macrophage colony stimulating factor (CSF-1) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are potent inducers of macrophage differentiation. Both appear to modulate protein phosphorylation, at least in part, through protein kinase C (PKC) raising the question as to whether they concurrently impact on macrophage-like cells. In this regard, we utilized the CSF-1 dependent murine macrophage-like line BAC 1.25F5. CSF-1 treatment of these cells for 30 min leads to particular phosphorylation of a 165 kDa protein, the putative CSF-1 receptor, and a 210 kDa moiety. 1,25(OH)₂D₃ exposure for 24 h prior to addition of CSF-1 enhances phosphorylation of the 165 kDa species and, especially, the 210 kDa protein. Phosphorylation of the latter protein is 1,25(OH)₂D₃ dose- and time-dependent and the molecule is specifically immunoprecipitated with a rabbit polyclonal anti-talin antibody. Experiments with okadaic acid show that the enhanced phosphorylation of talin does not result from serine phosphatase inhibition. CSF-1 and 1,25(OH)₂D₃, alone or in combination, do not increase talin protein expression. The tyrosine kinase inhibitor, genestein, blocks 1,25(OH)₂D₃/CSF-1 induced phosphorylation of the putative CSF-1 receptor but has no effect on talin phosphorylation which occurs exclusively on serine. In contrast to genestein, staurosporin, an inhibitor of PKC, inhibits phosphorylation of talin. Moreover, exposure of 1,25(OH)₂D₃ pretreated cells to phorbol 12-myristate 13-acetate (PMA) in place of CSF-1 also prompts talin phosphorylation. Finally, 1,25(OH)₂D₃ enhances ³[H]PDBu binding, indicating that the steroid increases PMA receptor capacity. Thus, CSF-1 and 1,25(OH)₂D₃ act synergistically via PKC to phosphorylate talin, a cytoskeletal-associated protein.     

Regulation of the CYP27B1 5′-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

Autoradiographic studies with 3H 1,25 (OH)2 vitamin D3 and 3H 25 (OH) vitamin D3 in rat parathyroid glands

Summary After injection of radiolabeled 1,25 (OH)₂ vitamin D₃, nuclear concentration of radioactivity is observed in parenchymal cells of the parathyroid gland in pregnant, adult male, and 10-day male neonatal rats. In competition studies with unlabeled 1,25 (OH)₂ vitamin D₃, but not with 25 (OH) vitamin D₃, nuclear uptake is prevented. Experiments with ³H 25 (OH) vitamin D₃, in contrast to ³H 1,25 (OH)₂ vitamin D₃, do not show nuclear concentration in cells of the parathyroid. The results of the autoradiographic studies suggest the presence of receptors for a direct effect of 1,25 (OH)₂ vitamin D₃ on the parathyroid gland for modulation of parathyroid hormone secretion.     

Lead inhibits 1,25-dihydroxyvitamin D-3 regulation of calcium metabolism in osteoblastic osteosarcoma cells (ROS 17/2.8)

We have determined the dose-response of 1,25-dihydroxyvitamin D-3 (1,25-(OH)₂D₃) on the intracellular free calcium-ion concentration ([Ca²⁺¹]_i) in the osteoblastic osteosarcoma cells, ROS 17/2.8, using ¹⁹F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorephenoxy)ethane-N, N, N′, N′-tetraacetic acid (5F-BAPTA). The dose-response demonstrated an inverted U-shaped relationship with maximal elevation of [Ca²⁺]_i at doses of 1 to 10 nM 1,25-(OH)₂D₃. At 10 nM, 1,25-(OH)₂D₃ elevated the [Ca²⁺]_i from a control level of 118±4 nM to a peak value of 237±8 nM within 40 min. 1,25-(OH)₂D₃ also increased the intial rate of Ca²⁺ influx into ROs 17/2.8 cells, measured by ⁴⁵Ca uptake, with a dose-response relationship which paralleled its effects on [Ca²⁺]_i. Treatment of ROS 17/2.8 cells with Pb²⁺ at 1 and 5 μM significantly increased [Ca²⁺]_i but significantly reduced the 1,25-(OH)₂D₃-induced elevation of [Ca²⁺]i. Simultaneous treatment of naive cells with 1,25-(OH)₂D₃ and Pb²⁺ produce little reduction of 1,25-(OH)₂D₃ and Pb²⁺ produce little reduction of 1,25-(OH)₂D₃-induced ⁴⁵Ca uptake while 40 min treatment with Pb²⁺ before addition of 1,25-(OH)₂D₃ significantly reduced the 1,25-(OH)₂D₃-induced increase in ⁴⁵Ca influx. These findings suggest that Pb²⁺ acts by inhibiting 1,25-(OH)₂D₃-activation of Ca²⁺ channels and interferes with 1,25-(OH)₂D₃ regulation of Ca²⁺ metabolism in osteoblastic bone cells.     

Involvement of endogenously produced 1,25-dihydroxyvitamin D-3 in the growth and differentiation of human keratinocytes

In this study, we investigated the possibility that cultured keratinocytes from normal human adult skin produce 1,25-dihydroxyvitamin D-3 (1,25(OH)₂D₃, a biologically active form of vitamin D-3) from 25-hydroxyvitamin D-3 [25(OH)D₃], and that 1,25(OH)₂D₃ endogenously produced by keratinocytes is involved in the self regulation of their growth and differentiation. To determine whether 1,25(OH)₂D₃ is produced from 25(OH)D₃ by skin keratinocytes, 25(OH)[³H]D₃ was added to keratinocyte cultures and incubated for 1 h and 5 h. The intracellular and extracellular metabolites were analyzed by three chromatographic systems. The three chromatograms revealed that the major metabolite produced from 25(OH)₂D₃ was 1,25(OH)₂D₃. Most of the 1,25(OH)₂D₃ endogenously produced from 25(OH)D₃ remained within the cells. To examine the time course of 1,25(OH)₂D₃ production, the amount of 1,25(OH)[³H]D₃ was measured at 15 min, 1 h, 5 h and 10 h, being at a maximum 1 h after the addition of 25(OH)D₃. These data indicate that keratinocytes rapidly convert 25(OH)D₃ to 1,25(OH)₂D₃ and that 1,25(OH)₂D₃ is not released into the medium. To determine whether endogenously produced 1,25(OH)₂D₃ is involved in the regulation of growth and differentiation of normal human keratinocytes, we examined the effects of 1,25(OH)₂D₃ and 25(OH)D₃ on their growth and differentiation. Keratinocyte growth was inhibited to 52.6% and 23.4% by 10^?8 M and 10^?7 M 1,25(OH)₂D₃ and to 80.5% and 23.9% by 10^?8 M and 10^?7 M 25(OH)D₃, respectively. Differentiation of these cells was evaluated by quantifying the number which express involucrin, a precursor protein of cornified envelope. The population of involucrin expressing cells (differentiated cells) increased from 6.2% to 14.5% by 2.5·10^?7 M 1,25(OH)₂D₃, and to 11.8% by 2.5·10^?7 M 25(OH)D₃. These results clearly indicate that 25(OH)D₃ is as effective on human keratinocytes as 1,25(OH)₂D₃ in inhibiting growth and inducing differentiation, although to a slightly lesser extent than 1,25(OH)₂D₃. The possibility that the effect of 25(OH)D₃ is mediated through binding to the 1,25(OH)₂D₃ receptor can be excluded, since a competitive binding assay revealed that the affinity of 25(OH)D₃ for the 1,25(OH)₂D₃ receptor in a cytosolic extract of keratinocytes was 100-times lower than that of 1,25(OH)₂D₃. Thus, these results suggest that 1,25(OH)₂D₃ endogenously produced in keratinocytes from 25(OH)D₃ is involved in the regulation of their growth and differentiation in vitro.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3

The most biologically active metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D₃ (25(OH)D₃) can affect osteoblast function via conversion to 1,25(OH)₂D₃, however, it is largely unknown whether 25(OH)D₃ can affect primary osteoblast function on its own. Furthermore, 25(OH)D₃ is not only converted to 1,25(OH)₂D₃, but also to 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D₃ itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)₂D₃ can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D₃ and 1,25(OH)₂D₃ by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)₂D₃ synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D₃. We demonstrated that 24R,25(OH)₂D₃ increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)₂D₃ strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D₃, 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)₂D₃, but also to 24R,25(OH)₂D₃ by enhancing osteoblast differentiation. This suggests that 25(OH)D₃ can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)₂D₃, but also via conversion to 24R,25(OH)₂D₃. Whether 25(OH)D₃ has direct actions on osteoblast function needs further investigation.     

1,25-Dihydroxyvitamin D3 and 22-oxa-1,25-dihydroxyvitamin D3 in vivo nuclear receptor binding in developing bone during endochondral and intramembranous ossification

Target cells for ³H-labeled 1, 25(OH)₂ vitamin D₃ [1,25(OH)₂D₃, vitamin D] and its analog ³H-labeled 22-oxa-1, 25(OH)₂ vitamin D₃ (OCT) have been identified during endochondral and intramembranous ossification in developing, undecalcified, unembedded bone, using thaw-mount autoradiography. Two-day-old neonatal rats were injected with [³H]1,25(OH)₂D₃ or [³H]OCT; after 2 h leg, spine, and head were frozen and sectioned. In the epiphyseal-metaphyseal region specific nuclear concentrations of [³H]1,25(OH)₂D₃ and [³H]OCT were observed in identical cell populations, being low in cells of the articular and resting zone, intermediate in the proliferating zone, and highest in hypertrophic chondrocytes and in osteoblasts and precursor cells. In the primary spongiosa intertrabecular spaces there were a large number of cells with nuclear labeling — probably osteoblasts and precursor cells. In contrast, in the secondary spongiosa intertrabecular spaces, apparent blood-forming cells were mostly unlabeled. Osteoblasts along bone spicules and compact bone in long bones, vertebrae, and head also showed strong nuclear labeling, as did cells of the periosteum. These data suggest that 1,25(OH)₂D₃ and OCT regulate development, differentiation, and activities of chondrocytes and osteoblasts, including differentiation of resting chondrocytes into proliferating and hypertrophic chondrocytes that involve chondroclastic enlargement of lacunae and trans-differentiation of surviving hypertrophic chondrocytes; differentiation of stroma cells into osteoblasts; and in periosteum and other regions of intramembranous ossification differentiation of precursor cells and osteoblasts. Nuclear receptor binding and their selective and hierarchical distribution during cell differentiation appear to correspond to multiple genomic effects toward growth, regeneration and repair. The findings indicate a physiological significance and therapeutic potential of 1,25(OH)₂D₃ and in particular of its less hypercalcemic analog OCT.On leave from the University of North Carolina     

Studies on the mode of action of calciferol: Comparison of the biochemical properties of an antiserum and the chick intestinal receptor both specific for 1,25-dihydroxyvitamin D3

The structural requirements for the interaction of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] with an anti-1,25(OH)₂D₃ antiserum and with the natural cytosolic receptor for 1,25(OH)₂D₃ isolated from chick intestine have been evaluated quantitatively. The antiserum was raised in a rabbit against a 1,25(OH)₂D₃-hemisuccinate derivative which was linked to bovine serum albumin at the C-3 position of the steroid. For these cross-reaction studies structural analogs of 1,25(OH)₂D₃ were used in competitive protein binding assays; their ability to interact with the binding proteins was expressed as relative competitive index (RCI) values where the RCI of 1,25(OH)₂D₃ is defined to be 100. The results indicate that the 25-hydroxyl group is the most important hydroxyl for the interaction of 1,25(OH)₂D₃ with this antiserum. The absence of this hydroxyl group decreases the RCI value to 0.2. Lack of the hydroxyl at carbon-3 or carbon-1 decreases the RCI value to 33 or 25, respectively, indicating that the specificity of this antiserum for the A ring is much lower than for the side chain. The high specificity for the side chain is underlined by the fact that insertion of an additional hydroxyl group at C-24 or C-26 of 1,25(OH)₂D₃ decreases the binding affinity to the antiserum markedly. The chick intestinal mucosal receptor shows a comparable high specificity for the side chain of 1,25(OH)₂D₃, but an even higher specificity for the A ring in comparison to the antiserum. With the intestinal receptor, the 3-hydroxyl is only 1/ 10th as important as the 1-hydroxyl group and the 25-hydroxyl group for the binding process. Scatchard analysis showed a K_D value of 1.7 × 10^?10m for the antiserum and 2.3 × 10^?10m for the chick intestinal mucosal receptor for the equilibrium binding of 1,25(OH)₂D₃ at 2 °C. The association rate constant at 2 °C was determined to be 5.8 × 10⁷ M^?1 min^?1 for the antiserum and 0.55 × 10⁷ M^?1 min^?1 for the receptor, indicating a 10-fold more rapid association of 1,25(OH)₂D₃ to the antiserum in comparison to the receptor. Furthermore, the dissociation process was found to be slower for the chick intestinal receptor (dissociation rate constant 3.6 × 10^?5 min^?1 versus 21.0 × 10^?5 min^?1).     

Vitamin D Up-Regulates the Vitamin D Receptor by Protecting It from Proteasomal Degradation in Human CD4+ T Cells

The active form of vitamin D₃, 1,25(OH)₂D₃, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4⁺ effector T cells. The biological actions of 1,25(OH)₂D₃ are mediated by the vitamin D receptor (VDR) and are believed to correlate with the VDR protein expression level in a given cell. The aim of this study was to determine if and how 1,25(OH)₂D₃ by itself regulates VDR expression in human CD4⁺ T cells. We found that activated CD4⁺ T cells have the capacity to convert the inactive 25(OH)D₃ to the active 1,25(OH)₂D₃ that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)₂D₃ does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4⁺ T cells. Furthermore, 1,25(OH)₂D₃ induces a significant intracellular redistribution of the VDR. We show that 1,25(OH)₂D₃ stabilizes the VDR by protecting it from proteasomal degradation. Finally, we demonstrate that proteasome inhibition leads to up-regulation of VDR protein expression and increases 1,25(OH)₂D₃-induced gene activation. In conclusion, our study shows that activated CD4⁺ T cells can produce 1,25(OH)₂D₃, and that 1,25(OH)₂D₃ induces a 2-fold up-regulation of the VDR protein expression in activated CD4⁺ T cells by protecting the VDR against proteasomal degradation.     

Effects of clomiphene and tamoxifen in vivo on the bone-resorbing effects of parathyroid hormone and of high oral doses of calcitriol (1,25(OH)2D3) in rats with intact ovarian function consuming low calcium diet

Two experiments were undertaken to study the abilities of clomiphene citrate (20 mg/kg body wt/wk s.c.) and tamoxifen citrate (20 mg/kg body wt/wk s.c.) to slow bone resorption mediated by (a) endogenous parathyroid hormone (PTH) and (b) exogenous calcitriol (1,25(OH)₂D₃) in vivo in rats with intact ovarian function. Groups of rats with ⁴⁵Ca-labelled bones were fed a low-calcium (0.01% Ca) diet to stimulate secretion of PTH. Neither clomiphene nor tamoxifen slowed the mobilization of ⁴⁵Ca from femoral bone or prevented the reduction in bone calcium induced by feeding this diet. Moreover these drugs did not depress the urinary excretion of ⁴⁵Ca or hydroxyproline. These observations indicated that clomiphene and tamoxifen did not inhibit PTH-mediated bone resorption. Administering calcitriol (50 ng/day) orally for 14 days raised plasma calcium, increased urinary ⁴⁵Ca and its specific activity and decreased femur ⁴⁵Ca: all these responses were similar in animal, receiving calcitriol alone and calcitriol with clomiphene or tamoxifen. The femur ⁴⁵Ca values (dpm × 10⁻³) were: (means ± SD, n = 8) placebo, 1901 ± 127; 1,25(OH)₂D₃, 1727 ± 96⁷; clomiphene + 1,25(OH)₂D₃, 1694 ± 93⁷; tamoxifen + 1,25(OH)₂D₃, 1664 ± 61⁷. (⁷ = P < 0.01). Thus neither clomiphene nor tamoxifen prevented calcitriol-mediated bone resorption in vivo in the rat.     

Studies on the mode of action of calciferol: Subcellular localization of 1,25-dihydroxyvitamin D3 in chicken parathyroid glands

As a further means of evaluating 1,25-dihydroxyvitamin D₃-parathyroid gland interaction and its relation to calcium homeostasis, a comparative study of the subcellular localization of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]in the parathyroid glands, intestinal mucosa, kidney, and liver of rachitic chickens has been carried out. Only in the chromatin fraction from parathyroids and intestinal mucosa could there be demonstrated selective and specific localization of the 1,25(OH)₂D₃. The chromatin-bound picomoles of 1,25(OH)₂D₃ (per gram of tissue) was in the ratio (mucosa:parathyroids:kidney:liver) of 1.0:0.23:0.11:0.17 2 h after an intracardial injection of 290 pmol of [³H]1,25(OH)₂D₃. This same ratio after a 30-min (23 °C) homogenate incubation with 1 × 10^?8m [³H]1,25(OH)₂D₃ was 1.0:1.0:0.10:0.03. Analogous results were obtained when reconstituted chromatin and cytosol fractions from the different tissues were compared for chromatin localization efficiency. This chromatin localization of 1,25(OH)₂D₃ in the parathyroid glands was temperature dependent. In addition, parathyroid glands were found to contain 3.0–3.5 S cytoplasmic and KCl-extractable chromatin receptors specific for 1,25(OH)₂D₃.     

1,25-Dihydroxyvitamin D3 increases type 1 interleukin-1 receptor expression in a murine T cell line

The biologically active metabolite of vitamin D₃, 1,25 (OH)₂ D₃, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and ¹²⁵I-interleukin 1α, we show that 1,25 (OH)₂ D₃ (5,50 nM) enhanced ¹²⁵I-interleukin 1α binding up to almost 2-fold over control. This 1,25 (OH)₂ D₃ effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of ¹²⁵I-interleukin 1α binding data demonstrated that 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D₃, 25 (OH) D₃, also augmented ¹²⁵I-interleukin 1α binding but at steroid levels 2–3 log orders greater than 1,25 (OH)₂ D₃. This observation, combined with the presence of high-affinity ³H-1,25 (OH)₂ D₃ receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)₂ D₃-enhanced ¹²⁵I-interleukin 1α binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)₂ D₃ enhanced interleukin 1 receptor expression. Northern blots hybridized with a ³²P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)₂ D₃ enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)₂ D₃ pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1α). These findings further support 1,25 (OH)₂ D₃'s role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.