UMR-106-01 osteoblast-like cells respond to high concentrations of parathyroid hormone (PTH) in vitro by decreasing thymidine incorporation, a marker of DNA synthesis and cell proliferation. This response is different from in vivo conditions, such as primary and secondary hyperparathyroidism, in which high PTH levels are associated with an increased number of osteoblasts. When the response of UMR-106-01 cells to PTH is evaluated in vitro, however, these cells are exposed to only a single hormone. The present study was designed to evaluate the combined effects of two hormones, PTH and insulin, on the DNA synthesis of UMR-106-01 cells. PTH is known to decrease and insulin to increase thymidine incorporation by UMR-106-01 cells. To examine the interaction of these hormones, acute studies, defined as a 24 h exposure to hormone, and chronic studies, defined as a 7 day exposure to hormone, were performed. Both acute and chronic exposure to 10(-9) M PTH decreased thymidine incorporation by UMR-106-01 cells, with suppression ranging from 27 to 81% (P < 0.05). Both acute and chronic exposure to 10(-8) M insulin (INS) increased thymidine incorporation by UMR-106-01 cells; this ranged from 26 to 58% (P < 0.05). However, chronic exposure to 10(-9) M PTH followed by an acute exposure to 10(-8) M INS resulted in a 710% increase in thymidine incorporation (P < 0.01). Reversing the sequence by chronically exposing UMR-106-01 cells to 10(-8) M INS followed by acute exposure to 10(-9) M PTH resulted in a 53% decrease in thymidine incorporation (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
High levels of interleukin-6 (IL-6) have been detected in synovial fluid from patients with inflammatory arthropathies associated with local bone resorption, suggesting a role for IL-6 as a local regulator of bone resorption and remodeling. In the present study we examined the effects of IL-6 on [3H]thymidine ([3H]TdR) incorporation, collagen synthesis, and alkaline phosphatase activity in UMR-106-01 rat osteoblastic osteosarcoma cells. IL-6 stimulated a dose-dependent increase in [3H]TdR incorporation that was maximal at 1000 U/ml (-147% of basal, p less than 0.005) in osteoblastlike cells that were in a logarithmic phase of growth. The increase in [3H]TdR incorporation was maximal between 12 and 24 h and was neutralized by pretreatment with the polyclonal rabbit antibody to IL-6. IL-6 also increased cell number and the secretion of prostaglandin E2 in UMR-106-01 cells in logarithmic growth phase. The stimulation of [3H]TdR incorporation and release of PGE2 into the culture medium by IL-6 was inhibited by indomethacin. A 24 h exposure of the osteoblastlike cells to 1000 U/ml of IL-6 reduced [3H]proline incorporation into collagenase-digestible (CDP) protein to 73% of control values (p less than 0.01). Noncollagen protein (NCP) synthesis was inhibited to 80% of control values (p less than 0.01) by 1000 U/ml of IL-6. The inhibitory effect was relatively greater on CDP than on NCP and consequently resulted in a decrease in the percentage of collagen synthesis. Alkaline phosphatase activity was not altered in these cells after a 24 h exposure to 1-1000 U/ml of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)
While calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, the compartmentalization and regulated release of calcium is cell type-specific. We employed thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase activity which block the transport of Ca2+ into intracellular stores, to characterize free Ca2+ compartmentalization in UMR 106-01 osteoblastic osteosarcoma cells. Each drug elicited transient increases in cytosolic free Ca2+ ([Ca2+]i), followed by a stable plateau phase which was elevated above the control [Ca2+]i. The release of Ca2+ from intracellular stores was coupled to an increased plasma membrane Ca2+ permeability which was not due to L-type Ca2+ channels. Thapsigargin and tBuHQ emptied the intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The results of sequential and simultaneous additions of thapsigargin and tBuHQ indicate that both drugs depleted the same Ca2+ store and inhibited the same Ca(2+)-ATPase activity.
Tumor necrosis factor (TNF-alpha) has been shown to play an important role in local control of bone remodeling. The interaction of TNF-alpha and PTH was evaluated in UMR-106-01 cells, a phenotypic osteoblastic osteosarcoma cell line. We examined the influence of TNF-alpha on the two signal transduction systems triggered by PTH in UMR-106-01 cells, adenylate cyclase and free cytosolic calcium ([Ca2+]i). cAMP generation was inhibited in TNF-alpha-pretreated cells by 69, 61, 34, and 21% at PTH concentrations of 0.1, 1, 10, and 100 nM, respectively. Inhibition was seen at TNF-alpha doses of 100-1500 units/ml after a minimum incubation time of 12 h. TNF-alpha inhibition of the PTH-stimulated increase in [Ca2+]i was even more pronounced: treated cells showed no change in baseline [Ca2+]i after stimulation with 40 nM PTH. Treatment with TNF-alpha was also found to inhibit both arms of the PTH response in the nontransformed osteoblastic cell line, MC3T3-E1. TNF-alpha treatment did not alter cAMP generation in response to PGE2. TNF-alpha inhibition of the PTH-stimulated cAMP response was reversed completely by addition of cholera toxin (5 micrograms/ml) and partially by forskolin (10 microM) but not pertussis toxin (100 and 500 ng/ml). Scatchard analysis using PTHrP revealed that TNF-alpha treatment reduced the number of receptors but had no effect on KD. These findings suggest that TNF-alpha inhibits the osteoblastic response to PTH at least in part because of a reduction in receptor number. Further investigation is indicated to provide insight into the interaction of calciotropic hormones and cytokines in vivo.
The effect of four different neuropeptides and norepinephrine (NE) on cyclic AMP formation in four different osteoblastic cell lines and in isolated neonatal mouse calvarial bone cells has been examined. In the rat osteosarcoma cell line UMR-106-01, vasoactive intestinal polypeptide (VIP, 0.001-1 microM), calcitonin gene-related peptide (CGRP, 0.3-30 nM), and NE (0.1-300 microM), but not neuropeptide Y (NPY, 0.001-1 microM) or substance P (SP, 0.1-10 microM), caused a dose-dependent stimulation of cyclic AMP formation. The stimulatory effects were synergistically potentiated by forskolin (0.1-3 microM). The effects of NE and VIP were time dependent, with an optimal effect seen at 5 minutes. The amount of cyclic AMP accumulated in cells stimulated with NE and VIP was in the same range. The amplitude of the cyclic AMP response induced by CGRP was smaller than that caused by VIP and NE. In the human osteosarcoma cell line Saos-2, NE (0.1 microM) and VIP (0.3 microM) stimulated cyclic AMP formation, and the effect was synergistically potentiated by forskolin. In the absence of forskolin, no effect of CGRP (30 nM) could be seen in the Saos-2 cells, but in the presence of forskolin (3 microM) a stimulatory effect was observed. SP and NPY did not change basal cyclic AMP levels in Saos-2 cells. In the osteoblastic osteosarcoma cell line of rat, ROS 17/2.8, NE (0.1 microM) caused a significant stimulatory action on cyclic AMP formation that was synergistically potentiated by forskolin (3 microM), VIP, CGRP, and SP did not affect the cellular content of cyclic AMP in ROS 17/2.8.(ABSTRACT TRUNCATED AT 250 WORDS)
The calcium and phospholipid-dependent protein kinase C (PKC) system appears to play an important role in mediating hormonal effects in various tissues including bone. Accordingly, we characterized PKC activity in the UMR-106-01 rat osteosarcoma osteoblastlike cell line and examined its hormonal regulation. UMR-106-01 cells were found to possess a classic, phorbol ester-activated PKC system, which was highly calcium and phospholipid dependent. A 30 s exposure to 10 nM bovine parathyroid hormone (PTH) (1-34) increased cytosolic and membrane-bound PKC activity by 12 and 157%, respectively, resulting in a 2.2-fold increase in the membrane-bound to cytosolic (MB/C) activity ratio (all p less than 0.01). The MB/C activity ratio was highest at 20 min, exhibiting a 2.8-fold increase over the control values (p less than 0.01). In contrast, 10 nM insulin increased cytosolic PKC activity but decreased membrane-bound activity, resulting in a 61% decrease in the MB/C activity ratio at 20 min (p less than 0.02). Moreover, insulin reduced PTH stimulation of the PKC activity ratio by 42 and 62% at 30 s and 20 min, respectively (p less than 0.02). Thus, PTH and insulin have opposing effects on the PKC activity ratio in UMR-106-01 cells.
The effects of ACE-011 on safety, pharmacokinetics, and bone biomarkers were evaluated in healthy, postmenopausal women. Our data indicate that ACE-011 results in a sustained increase in biomarkers of bone formation and reduction in markers of bone resorption. The activin type IIA receptor (ActRIIA) is the high-affinity receptor for activin. ACE-011 is a dimeric fusion protein consisting of the extracellular domain of the human ActRIIA linked to the Fc portion of human IgG1. ACE-011 binds to activin, preventing activin from binding endogenous receptors. A randomized, double-blind, placebo-controlled study was conducted to evaluate the safety and tolerability of ACE-011. Forty-eight healthy, postmenopausal women were randomized to receive either a single dose of ACE-011 or placebo and were followed for 4 mo. Dose levels ranged from 0.01 to 3.0 mg/kg intravenously and from 0.03 to 0.1 mg/kg subcutaneously. Safety and pharmacokinetic (PK) analyses and the biological activity of ACE-011, as assessed by markers of bone turnover, and follicle stimulating hormone (FSH) levels were measured. No serious adverse events (AEs) were reported. AEs were generally mild and transient. The PK of ACE-011 was linear over the dose range studied, with a mean half-life of 24-32 days. The absorption after subcutaneous dosing was essentially complete. ACE-011 caused a rapid and sustained dose-dependent increase in serum levels of bone-specific alkaline phosphatase (BSALP) and a dose-dependent decrease in C-terminal type 1 collagen telopeptide (CTX) and TRACP-5b levels. There was also a dose-dependent decrease in serum FSH levels consistent with inhibition of activin. ACE-011 is a novel agent with biological evidence of both an increase in bone formation and a decrease in bone resorption. ACE-011 may be an effective therapy in a variety of diseases involving bone loss.
Stephen Hough passed away on December 5(th) 2014 after a short but heroic battle against cancer. He was Emeritus Professor, Division of Endocrinology Department of Medicine, University of Stellenbosch and Tygerberg Academic Hospital, South Africa. He was the former Chairman of the Department of Internal Medicine and former Head of the Division of Endocrinology. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Low levels of physical activity or sun exposure and limitations to physical functioning (or disability) have been identified as possible risk factors for hip fracture. However, these factors are closely related and data on their independent and joint association with risk of hip fracture are limited. A total of 158 057 individuals aged ≥45 years sampled from the general population of New South Wales, Australia, from the prospective 45 and Up Study completed a baseline postal questionnaire in 2006-9 including data on physical activity (Active Australia questionnaire); sun exposure (usual time outdoors); and physical functioning (Medical Outcomes Score-Physical Functioning; scored 0-100). Incident first hip fractures were ascertained by linkage to administrative hospital data (n = 293; average follow-up 2.3 years). The Relative Risk (RR) of hip fracture was estimated using Cox proportional hazards. Poorer physical functioning, lower physical activity and less time outdoors were positively related to each other at baseline and individually associated with significantly increased hip fracture risk. However, physical activity and time outdoors were not significantly related to hip fracture risk after adjustment for baseline physical functioning or when analysis was restricted to those with no or mild baseline physical limitation. In contrast, physical functioning remained strongly related to hip fracture risk after adjustment for the other two factors; compared with the group without limitation (100) the RR of hip fracture among those with mild (75-95), moderate (50-70), severe (25-45) and greatest (0-20) level of physical limitation were 1.38 (95%CI 0.88-2.14), 2.14 (1.29-3.53), 3.87 (2.31-6.44), and 5.61 (3.33-9.42), respectively. The findings suggest that limitation in physical functioning, but not physical activity or time outdoors, is strongly related to hip fracture risk. The apparent increased risk of hip fracture previously described for low physical activity or sun exposure may be, at least in part, due to uncontrolled confounding.
The fate of 125I-labeled calcitonin and calcitonin receptors in BEN and UMR 106-06 cells was studied after binding of the ligand. At 37 degrees C but not at 4 degrees C, 125I-labeled salmon calcitonin bound to both cell types and was internalized as evidenced by increasing resistance to removal by acid pH. This process was dependent on de novo protein synthesis. Cells were pretreated with salmon calcitonin and washed with acidified buffer to release cell-surface bound hormone and to allow assessment of cell-surface receptor concentration. It was found that initially there was a temperature- and time-dependent calcitonin-induced loss of binding capacity after exposure to salmon calcitonin, suggestive of endocytosis of the calcitonin-receptor complex. In the continued presence of calcitonin, receptors were lost only at the same rate as the normal turnover of calcitonin receptors as assessed in the presence of cycloheximide. These data are consistent with the presence of two functional populations of calcitonin receptors, only one of which is induced to internalize by ligand binding.
The ability of the new nitrogen-containing bisphosphonate disodium-1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphona te (EB-1053) to inhibit osteoclastic resorption was examined in vitro and in vivo. Results were compared to those obtained with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (pamidronate or APD). In vitro, when tested in osteoclast precursor-dependent systems (fetal mouse metacarpals and a coculture system), EB-1053 suppressed 45Ca release effectively and was found to be about 10 times more potent than pamidronate (ED50 = 2.5 x 10(-7) versus 2.5 x 10(-6) M, respectively). The EB-1053-inhibited osteoclastic resorption could be reversed by treatment with parathyroid hormone (PTH). In vivo, daily subcutaneous injections of EB-1053 to young growing rats for 7 days increased metaphyseal bone mass in tibiae dose dependently. In these experiments EB-1053 was about 50 times more potent than pamidronate. These studies show that EB-1053 is a very potent bisphosphonate that has potential use in the treatment of skeletal disorders.
A group of 5-day-old mice were injected intraperitoneally with (3-amino-1-hydroxypropylidine)-1,1-bisphosphonate (APD). Morphologic changes were observed in vitally stained osteoclasts on parietal bones 3 days later, and these were judged to be degenerative. At this time significantly increased numbers of nuclei per osteoclast and total numbers of osteoclast nuclei were observed. However, at 4 days after the injection of APD, the total numbers of osteoclasts were significantly reduced relative to controls. When parietal bones were maintained in culture, APD reduced osteoclast numbers and inhibited cell-mediated 45Ca2+ release. Exposure of bones to parathyroid hormone increased the number of osteoclasts counted 1 day later. This effect was not blocked by APD. Calcitonin prevented the reduction in osteoclast numbers due to APD in vitro. We conclude that APD has a direct effect on resorbing mouse osteoclasts.
Bisphosphonates are drugs that suppress osteoclast-mediated bone resorption and are used with increasing frequency in the treatment of skeletal disorders. Therapeutic regimens are largely based on pharmacodynamic information because of difficulties in obtaining and interpreting pharmacokinetic data. We describe here the application of a permanently cannulated rat model, previously used in other areas of endocrine research, to the simultaneous study of pharmacokinetic and pharmacodynamic properties of the newly developed bisphosphonate EB-1053 [1-hydroxy-3-(1-pyrrolidinyl)propylidene-1,1-bisphosphonate]. Two groups of five rats each received daily intravenous injections of [14C]EB-1053 (0.025 and 0.1 mg/day, respectively); a third group (n = 7) received only normal saline injections and served as control. Treatment was given for at least 20 days. A fourth group (n = 3) received IV injections of the bisphosphonate on three separate occasions. Following IV administration, EB-1053 was rapidly cleared from the circulation. Urinary excretion of radioactivity reached about 55% of the daily administered dose within 48 h and remained at this level during the whole treatment period, indicating continuing retention of the bisphosphonate. Bone resorption, assessed biochemically as the hydroxyproline to creatinine ratio in urine, was suppressed effectively with both doses used. Suppression reached a maximum around day 4 and remained at the same level until the end of treatment. Accumulation of the bisphosphonate in the skeleton was therefore not associated with a cumulative effect on bone resorption. This strongly suggests that in treatment planning a distinction should be made between surface-bound and hence biologically active bisphosphonate from the drug which is incorporated in bone during bone turnover.(ABSTRACT TRUNCATED AT 250 WORDS)
Although analogs and metabolites of vitamin D have been tested for their calciotropic activity, very little information has been available concerning the effects of these compounds on gene expression. In this study one analog of vitamin D, 1,25,28-trihydroxyvitamin D2 [1,25,28-(OH)3D2], and one metabolite, 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3], were tested for their effect on intestinal calbindin-D9K mRNA and protein as well as for their effect on intestinal calcium absorption and bone calcium mobilization. These compounds were also evaluated for their ability to compete for rat intestinal 1,25-(OH)2D3 receptor sites and to induce differentiation of human leukemia (HL-60) cells as indicated by reduction of nitro blue tetrazolium. In vivo studies involved intrajugular injection of 12.5 ng 1,25-(OH)2D3 or test compound to vitamin D-deficient rats and sacrifice after 18 h. 1,25,28-Trihydroxyvitamin D2 had no effect on intestinal calcium absorption, bone calcium mobilization, or intestinal calbindin-D9K protein and mRNA. Competitive binding to 1,25-(OH)2D3 receptors was 0.8% of that observed using 1,25-(OH)2D3. However, 20- and 40-fold higher doses of 1,25,28-(OH)3D2 (250 and 500 ng) resulted in significant inductions in calbindin-D9K protein and mRNA (3.5 to 7.4-fold), although doses as high as 800 ng were found to have no effect on intestinal calcium absorption or bone calcium mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)
We previously showed that OCT, an analog of 1,25-(OH)2D3 with little calcemic activity, can decrease PTH mRNA levels in normal rats and inhibit PTH secretion in cultured bovine parathyroid cells with the same potency as 1,25-(OH)2D3 and that in normal rats fed a normal calcium diet, administration of OCT (500 ng) for 5 days did not increase plasma Ca. Thus, to determine if PTH suppression by OCT contributes to its lack of calcemic activity and to further characterize the effects of OCT on Ca metabolism, we performed several studies in parathyroidectomized (PTX) rats. PTX rats, maintained on a normal diet (0.9% Ca), received daily injections of vehicle, 1,25-(OH)2D3 (200 ng/day), or OCT (200 ng/day) for 6 days. Plasma Ca was measured daily. Plasma Ca in control rats stayed between 6.60 and 7.40 mg/dl, whereas Ca increased to 12.9 +/- 0.42 mg/dl in 1,25-(OH)2D3-treated rats and to 9.53 +/- 0.35 mg/dl in OCT-treated rats after 6 days. With a Ca-deficient diet, control rats maintained a plasma Ca between 4.25 and 4.60 mg/dl, but Ca increased to 13.7 +/- 0.24 mg/dl with 1,25-(OH)2D3 and to 7.29 +/- 0.17 mg/dl with OCT. Since the elevation in Ca by OCT was similar with both diets, OCT appears to act primarily on bone. PTX rats were infused with PTH (1.84 micrograms/kg/day) via an Alzet pump to achieve normal plasma Ca and then treated daily with either vehicle or OCT (200 ng/day). After 6 days, OCT increased serum Ca to 10.7 +/- 0.21 mg/dl over a control value of 8.58 +/- 0.29 mg/dl.(ABSTRACT TRUNCATED AT 250 WORDS)
In this study we assessed whether osteogenic cells respond in a differential manner to changes in surface roughness depending on their maturation state. Previous studies using MG63 osteoblast-like cells, hypothesized to be at a relatively immature maturation state, showed that proliferation was inhibited and differentiation (osteocalcin production) was stimulated by culture on titanium (Ti) surfaces of increasing roughness. This effect was further enhanced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we examined the response of three additional cell lines at three different maturation states: fetal rat calvarial (FRC) cells (a mixture of multipotent mesenchymal cells, osteoprogenitor cells, and early committed osteoblasts), OCT-1 cells (well-differentiated secretory osteoblast-like cells isolated from calvaria), and MLO-Y4 cells (osteocyte-like cells). Both OCT-1 and MLO-Y4 cells were derived from transgenic mice transformed with the SV40 large T-antigen driven by the osteocalcin promoter. Cells were cultured on Ti disks with three different average surface roughnesses (Ra): PT, 0.5 microm; SLA, 4.1 microm; and TPS, 4.9 microm. When cultures reached confluence on plastic, vehicle or 10(-7) M or 10(-8) M 1,25(OH)2D3 was added for 24 h to all of the cultures. At harvest, cell number, alkaline phosphatase-specific activity, and production of osteocalcin, transforming growth factor beta1 (TGF-beta1) and prostaglandin E2 (PGE2) were measured. Cell behavior was sensitive to surface roughness and depended on the maturation state of the cell line. Fetal rat calvarial (FRC) cell number and alkaline phosphatase-specific activity were decreased, whereas production of osteocalcin, TGF-beta1, and PGE2 were increased with increasing surface roughness. Addition of 1,25(OH)2D3 to the cultures further augmented the effect of roughness for all parameters in a dose-dependent manner; only TGF-beta1 production on plastic and PT was unaffected by 1,25(OH)2D3. OCT-1 cell number and alkaline phosphatase (SLA > TPS) were decreased and production of PGE2, osteocalcin, and TGF-beta1 were increased on SLA and TPS. Response to 1,25(OH)2D3 varied with the parameter being measured. Addition of the hormone to the cultures had no effect on cell number or TGF-beta1 production on any surface, while alkaline phosphatase was stimulated on SLA and TPS; osteocalcin production was increased on all Ti surfaces but not on plastic; and PGE2 was decreased on plastic and PT, but unaffected on SLA and TPS. In MLO-Y4 cultures, cell number was decreased on SLA and TPS; alkaline phosphatase was unaffected by increasing surface roughness; and production of osteocalcin, TGF-beta1, and PGE2 were increased on SLA and TPS. Although 1,25(OH)2D3 had no effect on cell number, alkaline phosphatase, or production of TGF-beta1 or PGE2 on any surface, the production of osteocalcin was stimulated by 1,25(OH)2D3 on SLA and TPS. These results indicate that surface roughness promotes osteogenic differentiation of less mature cells, enhancing their responsiveness to 1,25(OH)2D3. As cells become more mature, they exhibit a reduced sensitivity to their substrate but even the terminally differentiated osteocyte is affected by changes in surface roughness.
Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in alpha-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.
1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is an important regulator of bone metabolism involved in both formation and resorption. Traditionally it was assumed that vitamin D receptors are intracellular. Recent data indicate that vitamin D may also act through a membrane receptor, specifically raising intracellular calcium and inositol 1,4,5 triphosphate. The present study was undertaken to explore further the mechanism(s) of vitamin D-induced bone resorption in cultured bone. 1,25(OH)2D3 induced a dose-dependent increase of calcium efflux from cultured bone. This increase was completely obliterated by inhibition of protein kinase C (PKC) with either staurosporine or calphostin C. In cultured rat calvariae, 1,25(OH)2D3 also induced a dose-dependent translocation of PKC from cytosol to membrane. The activation of PKC by 1, 25(OH)2D3 occurred following a 30-s incubation, peaked at 1 minute, and disappeared by 5 minutes. 1,25(OH)2D3 did not increase cAMP production in similarly cultured calvaria. These results suggest that the action of 1,25(OH)2D3 on calcium flux from cultured bone is mediated, in part, via activation of PKC.
The actions of a novel vitamin D3 analog calcipotriol (MC 903), on human bone-derived cells were compared to those of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Both calcipotriol and 1,25-(OH)2D3 inhibited the proliferation of human osteoblast-like cells in a dose-dependent manner (10(-10)-10(-6) M), an effect observed at different cell densities. Lower concentrations of either agent exerted no marked effect on the growth of the cells compared to untreated cultures. Calcipotriol and 1,25-(OH)2D3 were equipotent in stimulating the activity of alkaline phosphatase and the synthesis of osteocalcin in human osteoblast-like cells. The stimulation of alkaline phosphatase activity and osteocalcin synthesis by both compounds was evident by 24 h and was increased progressively up to 96 h in a dose-dependent manner over the concentration range of 10(-10)-10(-6) M. The increment in both proteins was dependent on cell density and was attenuated at higher cell densities. In contrast to these actions, neither calcipotriol nor 1,25-(OH)2D3 (10(-14)-10(-6) M) affected the synthesis of prostaglandin E2. These studies indicate that calcipotriol and 1,25-(OH)2D3 exhibit a similar spectrum of activity on human osteoblast-like cells in vitro.
The bisphosphonate alendronate is a potent inhibitor of bone resorption by its direct action on osteoclasts. In addition, there is some data suggesting that alendronate could also inhibit bone resorption indirectly by interacting with osteoblasts. Parathyroid hormone-related protein (PTHrP) produced by osteoblasts and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are regulators of bone remodeling, which have interrelated actions in these cells. In this study, we assessed whether alendronate can affect PTHrP expression in the presence or absence of 1,25(OH)2D3 in human primary osteoblastic (hOB) cells from trabecular bone. Cell total RNA was isolated, and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out using human PTHrP-specific primers. PTHrP in the hOB cell-conditioned medium was analyzed by a specific immunoradiometric assay. We found that PTHrP mRNA and secreted PTHrP were maximally inhibited by 10(-8) - 10(-6) M of 1,25(OH)2D3 treatment within 8-72 h in hOB cells. Alendronate (10(-14) - 10(-8) M) modified neither PTHrP mRNA nor PTHrP secretion, although it consistently abrogated the decrease in PTHrP production induced by 1,25(OH)2D3 in these cells. On the other hand, alendronate within the same dose range did not affect either the vitamin D receptor (VDR) mRNA or osteocalcin secretion, with or without 1,25(OH)2D3, in hOB cells. The inhibitory effect of alendronate on the 1,25(OH)2D3-induced decrease in PTHrP in these cells was mimicked by the calcium ionophore A23187 (5 x 10-6 M), while it was eliminated by 5 x 10(-5) M of nifedipine. Furthermore, although alendronate alone failed to affect [Ca2+]i in these cells, it stimulated [Ca2+]i after pretreatment of hOB cells with 10(-8) M of 1,25(OH)2D3, an effect that was abolished by 5 x 10(-5) M of nifedipine. These results show that alendronate disrupts the modulatory effect of 1,25(OH)2D3 on PTHrP production in hOB cells. Our findings indicate that an increase in calcium influx appears to be involved in the mechanism mediating this effect of alendronate.
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to induce maturational changes in the human promyelocytic leukemia cell line HL-60 and in the human monocytic cell line U-937. Changes in cytosolic calcium have been reported to regulate cellular processes. We used the fluorescent dye Quin 2 to examine the effects of vitamin D metabolites on cytosolic calcium levels in HL-60 and U-937 cells. 1,25-(OH)2D3 (20 nM) increases cytosolic calcium by 24% over a 5-min period in HL-60 but not in U-937 cells. 1,25-(OH)2D3 (0.2 nM and 4 nM) has no effect on cytosolic calcium levels in either cell type. 24,25-(OH)2D3 (20 nM) has no effect on cytosolic calcium in HL-60 cells. Nifedipine (1 mM) has no effect on cytosolic calcium levels over 30 min and likewise does not block the 1,25-(OH)2D3-induced increase in cytosolic calcium in HL-60 cells. However, chelation of extracellular calcium with EGTA (10 mM) blocks the 1,25-(OH)2D3-induced increment in cytosolic calcium, but does not block the 1,25-(OH)2D3-induced maturational changes in HL-60 cells. The data suggest that 1,25-(OH)2D3 but not 24,25-(OH)2D3 increases cytosolic calcium in HL-60 cells within 5 min and the increment is due to increased influx of calcium. 1,25-(OH)2D3 modifies membrane permeability to calcium independent of calcium channels sensitive to nifedipine. Finally, 1,25-(OH)2D3-induced maturational changes in HL-60 cells can take place without an increase in cytosolic calcium.
PTH and 1,25(OH)(2)D each exert dual anabolic and catabolic skeletal effects. We assessed the potential interaction of PTH and 1,25(OH)(2)D in promoting skeletal anabolism by comparing the capacity of exogenous, intermittently injected PTH(1-34) to produce bone accrual in mice homozygous for the 1 alpha(OH)ase-null allele [1 alpha(OH)ase(-/-) mice] and in wildtype mice. In initial studies, 3-mo-old wildtype mice were either injected once daily (40 microg/kg) or infused continuously (120 microg/kg/d) with PTH(1-34) for up to 1 mo. Infused PTH reduced BMD, increased the bone resorption marker TRACP-5b, and raised serum calcium but did not increase serum 1,25(OH)(2)D. Injected PTH increased serum 1,25(OH)(2)D and BMD, raised the bone formation marker osteocalcin more than did infused PTH, and did not produce sustained hypercalcemia as did PTH infusion. In subsequent studies, 3-mo-old 1 alpha(OH)ase(-/-) mice, raised on a rescue diet, and wildtype littermates were injected with PTH(1-34) (40 microg/kg) either once daily or three times daily for 1 mo. In 1 alpha(OH)ase(-/-) mice, baseline bone volume (BV/TV) and bone formation (BFR/BS) were lower than in wildtype mice. PTH administered intermittently increased BV/TV and BFR/BS in a dose-dependent manner, but the increases were always less than in wildtype mice. These studies show that exogenous PTH administered continuously resorbs bone without raising endogenous 1,25(OH)(2)D. Intermittently administered PTH can increase bone accrual in the absence of 1,25(OH)(2)D, but 1,25(OH)(2)D complements this PTH action. An increase in endogenous 1,25(OH)(2)D may therefore facilitate an optimal skeletal anabolic response to PTH and may be relevant to the development of improved therapeutics for enhancing skeletal anabolism.
Purified chick duodenal brush border membrane vesicles (BBMV) were used to assess the effect of vitamin D on intestinal Ca2+ transport and membrane stability. BBMV preparations are right-side-out as judged by a nine-fold increase in accessibility of lactoperoxidase to core material actin in the presence of Triton X-100. Freshly prepared BBMV from vitamin D-deficient chicks support both sodium-dependent glucose transport and Ca2+ uptake. In vivo treatment with 1,25(OH)2D3 results in an 85% increase in the Vmax of Ca2+-uptake from 2.2 to 3.9 nmol/min/mg protein. The Km of Ca2+-uptake (0.9 mM) is independent of the vitamin D status of the chick. The majority of BBMV derived from vitamin D-replete chicks were destabilized and rendered incapable of supporting either sodium-dependent glucose uptake or Ca2+ uptake if they were held at 0-4 degrees C for 2 to 24 h. In 40 separate experiments, 80% of membranes derived from vitamin D-replete chicks showed characteristics of destabilization, whereas only 24% of all control membranes exhibited a lack of viability.
The 25(OH)D3/1,25(OH)2D3 24-hydroxylase (24-hydroxylase) displays an induction profile responsive to vitamin D (D) abundance and is hence only observed in normal extracellular Ca2+ concentrations. However, the participation of Ca2+ in the expression of the 24-hydroxylase gene in vivo is not known. The present studies investigate the role played by the circulating Ca2+ and the D3 and/or 1,25(OH)2D3 status on the 1,25(OH)2D3-mediated inducibility of the 24-hydroxylase gene in rat duodenum. Hypocalcemic D-depleted rats were supplemented with calcium alone to normalize serum Ca2+ without normalizing the D3 status or were acutely or chronically supplemented with D3 or 1,25(OH)2D3. Messenger RNA for the 24-hydroxylase was undetectable in the intestine of hypocalcemic D-depleted rats, and short- or long-term calcium supplementation was completely unsuccessful in inducing its expression. By contrast, acute 1,25(OH)2D3 administration led to significant increases in the levels of expression of the gene which was independent of the calcium intake, the prevailing circulating Ca2+, and the D3 or 1,25(OH)2D3 status. Moreover, 24-hydroxylase gene expression was only found to respond to acutely administered 1,25(OH)2D3, the mRNA levels being unaltered following continuous exposure to physiological or pharmacological doses of the hormone for 7 days. Time-course studies revealed, however, that induction of the gene was clearly apparent early in the 1,25(OH)2D3 supplementation course but gradually faded by 3 days to return to basal uninduced levels by 7 days, suggesting the presence of intestinal adaptation mechanism(s) which down-regulated the responsiveness in the continuous presence of 1,25(OH)2D3. Our data show the lack of effect of calcium alone or in combination with 1,25(OH)2D3 on the in vivo induction of the 24-hydroxylase gene expression in rat intestine. By rapidly reacting to surges in 1,25(OH)2D3 concentrations, the 24-hydroxylase efficiently controls the amount of 1,25(OH)2D3 in intestine as the first step in the biotransformation pathway aimed at the irreversible clearance of the secosteroid hormone.
CaBP-D28 mRNA expression in rat heart, testis, and lung was assessed by polymerase chain reaction (PCR). The animal model used was the hyperinduced vitamin D-treated rat (100 ng 1,25-dihydroxyvitamin D subcutaneously, daily for 7 days). For the PCR studies, two pairs of 20 mer oligonucleotide primers (designated 1-4 according to their position on the coding strand, but with primers 3 and 4 in reverse orientation) derived from the rat CaBP-D28 cDNA sequence were tested in various combinations. Optimal conditions were established using a 1:100 dilution of cDNA from normal rat kidney. Bands of the predicted sizes of 869 (1, 3), 994 (1, 4), 725 (2, 3), and 850 (2, 4) nucleotide base pairs resulted, but with varying intensities: 2,4 approximately 1,3 > 1,4 > 2,3. Repeat PCR (recycling after 1:100 dilution and readdition of reagents and primers with at least one different primer) provided strong additional amplification, particularly with the 1,4/2,4 combination. Under these conditions, mixing experiments showed that CaBP-D28 transcripts were detectable at 10(-7)- to 10(-9)-fold lower levels of expression than in D+ kidney. When RNA was isolated and cDNA generated from test tissues from 4 individual vitamin D-stimulated (D+) and vitamin D-deficient (D-) rats, repeat PCR (1,4/2,4 primer combination) provided no evidence of significant CaBP-D28 mRNA expression in the nonclassic target tissues, in contrast to strong bands in both the D- kidney (undiluted) and D+ kidney (1:100 dilution) preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
We report here the first association between vitamin D-resistant rickets, alopecia, and type 1 diabetes in a child with compound heterozygous mutations in the VDR gene. Transfection studies suggest dissociated effects of VDR gene mutations on the regulation of genes involved in vitamin D metabolism and dendritic cell maturation.
Whereas vitamin D may play a role in the immune tolerance process, no patient has been reported to associate hereditary vitamin D-resistant rickets (HVDRR) and an autoimmune disease, and no attempt has been made to delineate the outcome of mutations of the vitamin D receptor (VDR) on the transcription of genes controlling immune tolerance.
The VDR gene was analyzed in a child with vitamin D-resistant rickets, total alopecia, and early childhood-onset type 1 diabetes. Patient's fibroblasts and COS-7 cells transfected with wildtype or mutant VDRs were studied for ligand-binding capacity, transactivation activity using two gene promoters [CYP-24, a classical 1,25(OH)2D3-responsive gene, and relB, a critical NF-kappaB component for regulation of dendritic cell differentiation], VDR-RXR heterodimers association to CYP 24 VDREs by gel mobility shift assays, and co-activator binding by Glutathione-S-transferase pull-down assays.
Two novel compound heterozygous mutations (L263R and R391S) were identified in the VDR ligand-binding domain in this child. Both mutations significantly impaired VDR ligand-binding capacity but had dissociated effects on CYP-24 and RelB promoter responses to vitamin D. CYP 24 response binding to SRC-1 and RXR-heterodimer binding to CYP24 VDREs were abolished in L263R mutants but normal or partially altered in R391S mutants. In the opposite, RelB responses to vitamin D were close to normal in L263R mutants but abolished in R391S mutants.
We report the first clinical association between HVDRR, total alopecia, and early childhood-onset type 1 diabetes. Mutations in the VDR ligand-binding domain may hamper the 1,25(OH)2D3-mediated relB responses, an effect that depends on the site of the VDR mutation and cannot be anticipated from VDR ligand-binding ability or CYP-24 response. Based on these results, we propose to survey the immune function in patients with HVDRR, including those with moderate features of rickets.
Hereditary vitamin D--resistant rickets (HVDRR) is a genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young boy who exhibited the typical clinical features of HVDRR but without alopecia.
The patient's VDR was studied using cultured dermal fibroblasts, and the recreated mutant VDR was analyzed in transfected cells.
The patient's fibroblasts were resistant to 1,25-dihydroxyvitamin D [1,25(OH)2D3], exhibiting only a slight induction of 24-hydroxylase gene expression when treated with 1 microM 1,25(OH)2D3 x [3H]1,25(OH)2D3 binding was absent in cell extracts from the patient's fibroblasts. Sequence analysis of the VDR gene uncovered a unique 5-bp deletion/8-bp insertion in exon 4. The mutation in helix HI of the ligand-binding domain deletes two amino acids (H141 and T142) and inserts three amino acids (L141, W142, and A143). In transactivation assays, the recreated mutant VDR was 1000-fold less active than the wildtype (WT) VDR. In glutathione S-transferase (GST) pull-down assays, the mutant VDR bound GST-retinoid X receptor (RXR) weakly in the absence of 1,25(OH)2D3; however, the binding did not increase with increasing concentrations of ligand. The mutant VDR did not bind to GST-vitamin D receptor interacting protein (DRIP) 205 at concentrations up to 1 microM 1,25(OH)2D3. We also examined effects of the three individual mutations on VDR transactivation. Only the insertion of A143 into the WT VDR disrupted VDR transactivation to the same extent observed with the natural mutation.
We describe a novel insertion/substitution mutation in helix Hl of the VDR ligand-binding domain (LBD) that abolishes ligand binding and result in the syndrome of HVDRR. This is the first time an insertion/substitution has been found as the defect-causing HVDRR.
Paricalcitol is a less hypercalcemic vitamin D analog that has been shown to suppress secondary hyperparathyroidism and to prevent the associated histomorphometric changes in bone. In this study, we show that paricalcitol also ameliorates the renal insufficiency-induced loss of bone mineral and the mechanical competence of bone.
Renal bone disease is a common consequence of chronic renal insufficiency and the associated secondary hyperparathyroidism (SH). Paricalcitol [19-nor-1,25(OH)(2)D(2)] has been shown to ameliorate SH and prevent renal failure-induced histomorphometric changes in bone with minimal calcemic and phosphatemic activity. However, information about its efficacy on restoration of bone structural strength is lacking. In this study, we explored the effects of paricalcitol treatment on bone structure and strength in a model of advanced renal disease.
Forty-five 8-week-old rats were randomly assigned to either surgical 5/6 nephrectomy (NTX) or Sham-operation. After a 15-week postoperative disease progression period, the NTX rats were further allocated to uremic control (NTX) and treatment (NTX + paricalcitol) groups, the latter of which received paricalcitol for the subsequent 12 weeks. After 27 weeks, the animals were killed, plasma samples were collected, and both femora were excised for comprehensive analysis of the femoral neck and midshaft (pQCT and biomechanical testing).
High mortality that exceeded 30% was observed in both NTX groups. NTX induced over a 13-fold increase in plasma PTH, whereas this increase was only 5-fold after paricalcitol treatment. At the femoral neck, NTX was associated with an 8.1% decrease (p < 0.05) in vBMD and a 16% decrease in breaking load (p < 0.05) compared with the Sham group, whereas paricalcitol treatment completely prevented these changes. At the femoral midshaft, the NTX resulted in a 6.6% decrease in cortical BMD (p < 0.01 versus Sham), and this change was also prevented by paricalcitol.
Paricalcitol administration prevented renal insufficiency-associated decreases in BMD in the femoral neck and the femoral midshaft and restored bone strength in the femoral neck. Therefore, paricalcitol can efficiently ameliorate renal insufficiency-induced loss of bone mineral and mechanical competence of bone.
Parathyroid hormone (PTH) is a major stimulus for the renal production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Elevated arterial blood ionized calcium ([Ca2+]) depresses serum 1,25-(OH)2D3 in nonparathyroidectomized rats even when serum PTH is maintained at high levels by infusion. However, suppression by [Ca2+] of endogenous PTH, causing the fall in 1,25-(OH)2D, cannot be excluded. To determine whether [Ca2+] regulates 1,25-(OH)2D3 in the absence of a variation in PTH, we parathyroidectomized (PTX) rats (post-PTX calcium levels less than 7.0 mg/dl), inserted arterial and venous catheters, and then replaced PTH using an osmotic pump. We varied [Ca2+] by infusing either 75 mM sodium chloride (control), 0.61 mumol/min of EGTA (EGTA), or calcium chloride at 0.61 mumol/min (low calcium) or 1.22 mumol/min (high calcium) for 24 h 5 days after surgery. Blood was then drawn from the rat through the arterial catheter. Compared with the control, [Ca2+] fell with EGTA, remained constant with the low-calcium infusion, and rose with the high-calcium infusion. 1,25-(OH)2D3 was correlated inversely with [Ca2+] in all four groups together (r = -0.635, n = 34, p less than 0.001), within the control group alone (r = -0.769, n = 11, p less than 0.002), and within the EGTA group alone (r = -0.774, n = 10, p less than 0.003). Serum phosphorus, PTH, and arterial blood pH were not different in any group, and none correlated with serum 1,25-(OH)2D3. We conclude that 1,25-(OH)2D3 levels are regulated by [Ca2+] independently of serum PTH, phosphorus, and acid-base status, all of which support the hypothesis that [Ca2+] is a principal regulator of serum 1,25-(OH)2D3 in the rat.
To examine the role of lipid metabolism in the growth and function of osteoblast-like cells, we studied ROS 17/2.8 osteosarcoma cells and primary cultures of rat calvarial osteoblasts during growth in a serum-free medium supplemented by purified human lipoproteins or by liposomes. Increase in ROS cell number was measured in sparse (1-5 X 10(3)/cm2) cultures over 6-8 days. Liposomes (0-300 micrograms/ml) and high (HDL), low (LDL), and very low density (VLDL) lipoprotein fractions (0-300 micrograms apoprotein) markedly stimulated cell growth. Cells plated at 5 X 10(3)/cm2 achieved growth rates in the presence of LDL or HDL comparable to 10% fetal bovine serum. Serum-free culture with exogenous lipid maintained the response of cell cyclic AMP accumulation to parathyroid hormone. Cyclic AMP response to parathyroid hormone was enhanced by glucocorticosteroid, and was attenuated by 1,25-dihydroxyvitamin D (1,25(OH)2D) with an EC50 (10(-10) M) comparable to that previously observed in serum-cultured cells (J. Biol. Chem. 258:736, 1985). 1,25(OH)2D also increased the alkaline phosphatase activity in ROS cells cultured in lipid-supplemented serum-free culture. Lipoproteins or liposomes also markedly enhanced the proliferative response of sparse cultures of normal rat osteoblasts to polypeptide mitogens.
Calbindin-D28K is a member of a superfamily of calcium binding proteins that share a common avidity for the divalent calcium ion. The ambient concentration of calcium in the blood circulation is thought to orchestrate the release of parathyroid hormone and calcitonin and to govern the activity of renal 1-hydroxylase and thereby synthesis of 1,25-dihydroxyvitamin D3. We report here the results of experiments designed to assess the possible contribution of dietary calcium status upon calbindin-D28K gene expression in the intestine of vitamin D-deficient chicks. The actions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and dietary calcium intake upon intestinal calbindin-D28K and calbindin-D28K mRNA were monitored by ELISA and dot-blot hybridization analyses, respectively. Vitamin D3-deficient chicks were fed either a calcium-supplemented diet (3% w/w) or a diet containing low calcium (0.4% w/w). These dietary manipulations evoked a highly significant change in serum calcium status. However, the levels of calbindin-D28K protein and its corresponding mRNA were unaffected. Administration of 1,25-(OH)2D3 (1-16 nmol per animal) to both "normocalcemic" and hypocalcemic vitamin D-deficient chicks resulted in an equivalent stimulation of duodenal calbindin-D28K accumulation of calbindin-D28K mRNA. Intestinal calbindin-D28K was stimulated 20- to 28-fold (above control levels) by 6-8 nmol 1,25-(OH)2D3 in both dietary treatment groups when measured 48 h after the single injection. Hence, despite the existence of a relatively large difference in serum calcium levels, the molecular actions of 1,25-(OH)2D3 in the vitamin D-deficient animal are apparently well insulated from serum calcium chemistry. These observations support the notion that, in the absence of vitamin D3, the calcium ion per se is unable to modulate the calbindin-D28K gene in vivo.
The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through its ability to regulate cellular programs of gene expression. Although RXR appears to participate as a heterodimeric partner with the VDR, absolute evidence for its role remains equivocal in vivo. To test this role and to investigate the requirement for comodulator interaction, we identified VDR- and retinoid X receptor (RXR)-interacting LXXLL peptides and examined whether these molecules could block vitamin D and 9-cis retinoic acid (9-cis RA) response. We used a mammalian cell two-hybrid system to screen a series of nuclear receptor (NR)-reactive LXXLL peptides previously identified through phage display screening for hormone-dependent reactivity with either VDR or RXR. Three categories of peptides were identified: those reactive with both VDR and RXR, those selective for RXR, and those unreactive to either receptor. Peptide fusion proteins were then examined in MC3T3-E1 cells for their ability to block induction of the osteocalcin (OC) promoter by 1,25(OH)2D3 or stimulation of a retinoic acid response element-thymidine kinase (RARE-TK) reporter by 9-cis-RA. Peptides that interacted with both VDR and RXR blocked 1,25(OH)2D3-dependent transcription by up to 75%. Control LXXLL sequences derived from Src-1 and Grip also suppressed 1,25(OH)2D3-induced transactivation; peptides that interacted with RXR blocked 9-cis-RA-induced transcription. Interestingly, two RXR-interacting peptides were also found to block 1,25(OH)2D3 response effectively. These studies support the idea that comodulator recruitment is essential for VDR- and RXR-mediated gene expression and that RXR is required for 1,25(OH)2D3-induced OC gene transcription. This approach may represent a novel means of assessing the contribution of RXR in various endogenous biological responses to 1,25(OH)2D3.
The purpose of this article has been to describe recent evidence that supports the idea(62) that 1,25-(OH)2D3 acts mechanistically like that of other steroid hormones. This evidence includes the finding that a clear structural interrelationship exists between the VDR and other members of the steroid receptor gene family, the observation that the VDR is required for gene promoter transactivation, and the identification of VDREs that act in cis to mediate 1,25-(OH)2D3 response. The VDR has been found to bind in vitro specifically to these functional DNA sites. Current evidence, however, indicates that the receptor may interact at these sites not as a monomer or homodimer but rather as a heterodimer with a protein whose identity remains unknown. Future studies with regard to the mechanism of vitamin D action must be aimed at gaining additional insight into the nature of VDREs, acquiring further detail about the interaction of the VDR with these elements, identifying factors that facilitate VDR DNA binding, and determining the biochemical mechanism by which the binding of receptor to these elements leads to modulation of common transcriptional machinery. In addition, 1,25-(OH)2D3 acts to suppress a number of genes, for example collagen, calcitonin, and parathyroid hormone. Efforts to elucidate these actions are currently underway, but the mechanism by which attenuation of response occurs remains largely uncharacterized. Finally, it is possible that additional mechanisms of vitamin D action may exist. Each of these areas offers a considerable challenge to future research.
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.
Bone cells isolated from the Hyp mouse, the murine homologue for hypophosphatemic vitamin D-resistant rickets, produce abnormal bone when transplanted to either normal or phosphate-supplemented Hyp mice. To assess whether correction of the bone formation by mutant cells transplanted into either normal or Hyp mice could be achieved in the presence of supraphysiologic serum concentrations of 1.25-dihydroxyvitamin D3 (1.25-(OH)2D3), recipient mice of both genotypes were infused continuously with 1.25-(OH)2D3 (0.2 micrograms/kg/day). Bone nodules present in transplants recovered after 14 days were characterized by measuring the osteoid thickness and volume. Administration of 1.25-(OH)2D3 to Hyp mice corrected the defective bone formation by normal cells but not by pair-transplanted Hyp cells, despite normalization of serum phosphate levels and 3-fold increases in serum 1.25-(OH)2D3. The osteoid thickness and volume in Hyp transplants into 1.25-(OH)2D3-treated Hyp mice were, however, markedly reduced down to values observed for Hyp transplants into recipient normal mice. Administration of 1.25-(OH)2D3 to normal mice improved further bone formation by mutant cells without affecting that by pair-transplanted normal cells. Administration of 24.25-(OH)2D3 (1 microgram/kg/day) combined with 1.25-(OH)2D3 to recipient mice of both genotypes prevented the sharp fall in serum 24.25-(OH)2D3 but was not more beneficial than 1.25-(OH)2D3 alone for improving bone formation by transplanted Hyp cells. These observations demonstrate an abnormal response of the mutant cells to the extracellular environment and support the concept of an intrinsic osteoblast defect in the Hyp mouse.
Recruitment of osteoclasts from monocytic precursors is modulated by local signals. We previously showed that monoblastic differentiation in U937 cells is stimulated by 1,25-(OH)2D3 and cAMP in series. We investigate here the combined effects of these agents to stimulate differentiation of osteoclast-like cells from mouse marrow. Cells from mouse marrow were harvested and cultured in alpha-MEM with 10% fetal bovine serum. The appearance of tartrate-resistant acid phosphatase-containing multinuclear cells was measured after 8 days in culture by cytochemical staining. Continuous exposure of cultures to 10 nM 1,25-(OH)2D3 positively stimulated development of these cells after 8 days (101 +/- 3 cells per well, n = 74). No osteoclast-like cells were found when 1,25-(OH)2D3 was added for the first 4 days followed by 4 days more with no treatment. PGE2 (1 microM) as a single agent added during the last 4 days of culture was not able to recruit osteoclast-like cells. However, cultures exposed to 1,25-(OH)2D3 during the first 4 days and 1 microM PGE2 during the second 4 days developed osteoclast-like cells at 8 days [66 +/- 8% of the formation seen with 1,25-(OH)2D3 alone, p less than 0.05]. Dibutyryl cAMP (1 microM to 3 mM) was also not effective used as a single agent, but was able to stimulate formation of TRAP-positive multinuclear cells when 1,25-(OH)2D3 preceded its addition to culture medium. cAMP analogs therefore mimicked the effect of 1 microM PGE2, but these experiments do not allow us to assign the PGE2 action entirely to activation of cAMP second messenger.(ABSTRACT TRUNCATED AT 250 WORDS)
The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and its analogue 22-oxa-1,25(OH)2D3 (22-oxacalcitriol) (OCT) on calcium and bone metabolism were examined in an animal model of hypercalcemia with continuous infusion of parathyroid hormone-related peptide (PTHrP), to determine whether active vitamin D could counteract the skeletal action of PTHrP in addition to its reported effect in suppressing the production of PTHrP in cancer cells. Parathyroid glands were removed from 8-week-old Sprague-Dawley rats to eliminate the confounding effects of endogenous PTH. Animals were then continuously infused with human PTHrP(1-34) at a constant rate via osmotic minipumps for 2 weeks, and at the same time treated orally or intravenously with OCT or 1,25(OH)2D3 four to nine times during the 2-week period. Under these conditions, OCT and, surprisingly, 1,25(OH)2D3 alleviated hypercalcemia in a dose-dependent manner. 1,25(OH)2D3 and OCT suppressed the urinary excretion of deoxypyridinoline, although they did not affect renal calcium handling, suggesting that the antihypercalcemic effect is attributable to the inhibition of bone resorption. These active vitamin D compounds also counteracted the effects of PTHrP at the proximal renal tubules, as reflected by a decrease in phosphate excretion. Histomorphometric analysis of bone revealed a dose-related decrease in parameters of bone resorption. These results suggest that 1,25(OH)2D3 as well as OCT has the potential to alleviate hypercalcemia, at least in part, through the inhibition of bone resorption in hypercalcemic rats with constant PTHrP levels. We propose that the main function of active vitamin D in high bone-turnover states is to inhibit bone resorption, and this may have important implications for the understanding of the role of active vitamin D in the treatment of metabolic bone diseases, such as osteoporosis.
EB 1089 is a vitamin D analog that is less potent than 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in its calcemic action but more potent in its antiproliferative action. We characterized the interaction of 1,25(OH)2D3 and EB 1089 with renal 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway, and compared the effects of 1,25(OH)2D3 and EB 1089 on induction of 24-hydroxylase mRNA in mouse kidney and intestine. 1,25(OH)2D3 and EB 1089 were competitive inhibitors of 24-hydroxylase activity. However, the Ki for 1,25(OH)2D3 (5.2 +/- 2.5 nM) was significantly lower than that for EB 1089 (286 +/- 59 nM). In the kidney, the time course and extent of 24-hydroxylase mRNA induction, relative to 18S rRNA, was similar for 1,25(OH)2D3 and EB 1089 with a peak response at approximately equal to 6 h that was sustained for at least 16 h. In the intestine, however, induction of 24-hydroxylase mRNA, relative to 18S rRNA, was approximately 50% lower for EB 1089 than for 1,25(OH)2D3 at 3 h (p < 0.05) and 6 h (p < 0.05) while at 16 h 24-hydroxylase mRNA was no longer detectable. Moreover, while both 1,25(OH)2D3 and EB 10898 elicited a similar dose-dependent induction of 24-hydroxylase mRNA in the kidney (EC50 = 0.4 +/- 0.13 and 0.3 +/- 0.08 ng/g for EB 1089 and 1,25(OH)2D3, respectively), the EC50 for EB 1089 (6.6 +/- 1.7 ng/g) was significantly higher than that for 1,25(OH)2D3 (0.9 +/- 0.32 ng/g) in the intestine (p < 0.01). EB 1089 was also less effective than 1,25(OH)2D3 in the induction of intestinal but not renal calbindin-D9k mRNA. To determine the mechanism for tissue-specific differences in potency, we determined the binding affinity of 1,25(OH)2D3 and EB 1089 for the vitamin D receptor. In the kidney, Kd values for 1,25(OH)2D3 (0.40 +/- 0.95 nM) and EB 1089 (0.48 +/- 0.04 nM) were not different. However, in the intestine, the Kd for EB 1089 (1.43 +/- 0.19 nM) was significantly higher than that for 1,25(OH)2D3 (0.85 +/- 0.06 nM; p < 0.05). Our results demonstrate that: (i) EB 1089 has a 50-fold lower affinity than 1,25(OH)2D3 for renal 24-hydroxylase, suggesting that it is more resistant to catabolism by the C-24 oxidation pathway; and (ii) EB 1089 and 1,25(OH)2D3 exhibit tissue-specific differences in vitamin D receptor-mediated responses in vivo that may be ascribed, at least in part, to differences in binding affinities for the vitamin D receptor.
Transport of 45Ca from the lumen to the venous effluent was studied in duodena of normal, vitamin D3-replete (+D) chicks perfused through the celiac artery with 130 pM 1,25(OH)2D3 or vehicle. Administration of actinomycin D 3 h prior to perfusion did not alter the unstimulated transport rate or diminish the response to exogenous 1,25(OH)2D3: After 40 min exposure to the seco-steroid, 45Ca in the vascular effluent was 140% of control levels. The anti-microfilament agent cytochalasin b and the ionophore monensin, an inhibitor of Golgi function, similarly failed to suppress 1,25(OH)2D3-stimulated calcium transport. In pilot studies, Golgi and basal-lateral membrane fractions were prepared from duodenal epithelium of vitamin D-deficient (-D) chicks treated with vehicle or 650 pmol of 1,25(OH)2D3 in vivo 2 h, 10 h, or 15 h before sacrifice, as well as from +D birds. Analyses of Golgi fractions for cathepsin B (CB) activity revealed a biphasic response with time, increasing to 200% of -D levels 2 h after 1,25(OH)2D3 administration and in equivalent preparations from +D birds. Less pronounced increases in acid phosphatase activity were observed in the same membrane fractions. In basal-lateral membranes, enhanced CB activity was detectable 10 h after 1,25(OH)2D3 in vivo, rose to 155% of -D levels at 15 h, and to 245% of controls in fractions from +D birds, whereas acid phosphatase was 75%, 81%, and 125% of controls, respectively, at these times.(ABSTRACT TRUNCATED AT 250 WORDS)
The treatment of choice for pseudo-vitamin D deficiency rickets (PDDR), caused by mutations in the 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1; 1alpha-OHase) gene, is replacement therapy with 1,25(OH)2D3. We have previously engineered an animal model of PDDR by targeted inactivation of the 1alpha-OHase gene in mice. Replacement therapy was performed in this model. The 1alpha-OHase-/- mice and heterozygote controls were treated with 500 pg of 1,25(OH)2D/g body weight/day for 2 weeks, followed by 100 pg of 1,25(OH)2D3/g body weight/day for an additional 3 weeks before death at 8 weeks of age. Blood biochemistry analysis revealed that the rescue treatment corrected the hypocalcemia and secondary hyperparathyroidism. The daily injections of 1,25(OH)2D3 induced strong expression of CYP24, the 25-hydroxyvitamin D 24-hydroxylase gene. Bone histology and histomorphometry confirmed that the rickets and osteomalacia were cured. The rescue regimen also restored the biomechanical properties of the bone tissue within normal parameters. These results show that chronic treatment with the active 1,25(OH)2D3 metabolite is effective to rescue the PDDR phenotype of 1alpha-OHase mutant mice.
Impaired bone formation due to defective osteoblast function, as reflected in a decreased serum osteocalcin (OC) concentration in the patients with diabetes, has been implicated in the development of diabetic osteopenia. The role of hyperglycemia in this decrease in serum OC concentration was investigated. 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), an active form of vitamin D3, stimulated OC secretion from the human osteosarcoma cell line MG-63 in a dose-dependent manner. Exposure of the cells to high concentrations of glucose for 7 days significantly impaired 1,25(OH)2D3-induced OC secretion as compared with that observed with cells maintained under normal glucose (5.5 mM) or high mannitol conditions. The inhibitory effect of glucose was in a dose-dependent manner up to 55 mM. High glucose (55 mM) also attenuated the 1,25(OH)2D3-induced increase in OC mRNA abundance in MG-63 cells, suggesting that the inhibition of the 1,25(OH)2D3-induced increase in OC secretion by exposure to a high concentration of glucose was, at least in part, mediated at the transcriptional level. High glucose significantly decreased the number of 1,25(OH)2D3 receptors in MG-63 cells, without any change in the dissociation constant for 1,25(OH)2D3; this effect was not mimicked by high mannitol, indicating specificity for glucose. These observations suggest that a high glucose concentration significantly impairs the ability of osteoblastic cells to synthesize OC in response to 1,25(OH)2D3 by reducing 1,25(OH)2D3 receptor number, and that impaired cell function caused by sustained exposure to high glucose contributes to the defect in bone formation observed in the patients with diabetic osteopenia.
To evaluate the osteoblastic function in patients with multiple pituitary hormone deficiencies (M-PHD) and with isolated growth hormone deficiency (I-GHD), bone cells were cultured and the effects of 10(-8) M 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) on parameters of cell proliferation, osteoblastic differentiation, and local paracrine regulation were measured. Three days of 1,25(OH)2D3 treatment increased alkaline phosphatase activity and osteocalcin release but inhibited [3H]thymidine incorporation in all cell cultures from patients as well as from controls. In addition, 1,25(OH)2D3 increased the release of both total and active transforming growth factor-beta (TGF-beta) in bone cells from controls by, respectively, 4.9- and 3.2-fold and in bone cells from I-GHD by 5.1- and 1.5-fold, respectively. However, in bone cells from M-PHD, the stimulation of total TGF-beta release was significantly lower (1.3-fold) than in control and I-GHD cells, and active TGF-beta release was not stimulated at all. One year of supplementation with human growth hormone did not improve this deficient TGF-beta release in bone cells from M-PHD. We conclude that cultured bone cells from I-GHD and M-PHD show a normal response to 1,25(OH)2D3 regarding cell proliferation and osteoblastic differentiation, which implicates a normal 1,25(OH)2D3-receptor function. In cells from controls and I-GHD, 1,25(OH)2D3 enhanced both total and active TGF-beta release. However, bone cells from M-PHD showed a deficient TGF-beta response to 1,25(OH)2D3. These results suggest that the regulation of TGF-beta production is a major paracrine factor involved in hypopituitarism.
We evaluated in normal and hypophosphatemic (Hyp) mice whether changes in serum levels of osteocalcin in response to dietary phosphate supplementation, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) administration were related to perturbations in calcium phosphate homeostasis. In normal mice, serum osteocalcin levels were not altered by phosphate supplementation. In contrast, phosphate supplementation in Hyp mice led to a 2-fold decrease in serum osteocalcin to normal levels after 3 days and to an increase in osteocalcin levels after 14 days. The decrease in osteocalcin was associated with normophosphatemia, severe hypocalcemia, and marked increases in circulating 1,25(OH)2D3 levels, whereas the increase in osteocalcin levels was associated with normophosphatemia and no change in serum calcium and 1,25(OH)2D3. Administration of PTH decreased serum osteocalcin in both genotypes. Infusion of 1,25(OH)2D3 for 3 days elicited increases in serum osteocalcin and calcium levels in normal mice, whereas in Hyp mice it produced significant decreases in osteocalcin levels and no change in serum calcium. However, with a more prolonged infusion of 1,25(OH)2D3, hypercalcemia and increases in serum osteocalcin were induced in mutant mice. Our results suggest that the abnormal osteocalcin response of Hyp mice is not directly attributable to an osteoblast dysfunction but is secondary, at least in part, to perturbations in factors that modulate the osteoblast activity, especially serum calcium and/or PTH.
We examined the role of caveolae and caveolin-1 in the mechanism of 1alpha,25(OH)(2)D(3) action in growth plate chondrocytes. We found that caveolae are required for rapid 1alpha,25(OH)(2)D(3)-dependent PKC signaling, and caveolin-1 must be present based on studies using chondrocytes from Cav-1(-/-) mice.
1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] regulates endochondral ossification in part through membrane-associated mechanisms, including protein kinase C (PKC) signaling activated by a membrane-associated 1alpha,25(OH)(2)D(3)-binding protein, ERp60. We tested the hypothesis that caveolae are required for 1alpha,25(OH)(2)D(3) action and play an important role in regulating chondrocyte biology and growth plate physiology.
Rat costochondral chondrocytes were examined for caveolae by transmission electron microscopy of cultured cells and of cells in situ. Western blots and confocal microscopy were used to detect caveolae proteins including caveolin-1 (Cav-1) and 1alpha,25(OH)(2)D(3) receptors. Caveolae cholesterol was depleted with beta-cyclodextrin (CD) and effects of 1alpha,25(OH)(2)D(3) on PKC, DNA synthesis, alkaline phosphatase, and proteoglycan production determined. Chondrocytes from Cav-1(-/-) and C57BL/6 wildtype mice were also treated with 1alpha,25(OH)(2)D(3). Epiphyses and costochondral junctions of 8-week-old male Cav-1(-/-) and wildtype mice (N = 8) were compared by histomorphometry and microCT. Data were analyzed by ANOVA and Bonferroni for posthoc comparisons.
Growth zone chondrocytes had caveolae and Cav-1, -2, and -3. Resting zone chondrocytes, which do not exhibit a rapid 1alpha,25(OH)(2)D(3)-dependent increase in PKC activity, also had these caveolins, but caveolae were larger and fewer in number. ERp60 but not VDR co-localized with Cav-1 in plasma membranes and in lipid rafts. CD-treatment blocked 1alpha,25(OH)(2)D(3) effects on all parameters tested. The Cav-1(-/-) cells did not respond to 1alpha,25(OH)(2)D(3), although 1alpha,25(OH)(2)D(3) increased PKC, alkaline phosphatase, and [(35)S]-sulfate incorporation in wildtype C57BL/6 cells. Histology and microCT showed that Cav-1(-/-) growth plates were longer and had more hypertrophic cells in each column. Growth plate changes were reflected in the metaphysis.
The membrane-mediated effects of 1alpha,25(OH)(2)D(3) require caveolae and Cav-1, and Cav-1 deficiency results in altered growth plate physiology.
1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] functions in vertebrate organisms as a primary regulator of calcium and phosphorus homeostasis, an activity that is achieved through direct actions on gene expression in intestine, kidney, and bone. Recent studies have identified novel genes such as TRPV5, TRPV6, and RANKL whose products are integral to the maintenance of extracellular calcium. The objective of this progress report/review is to describe our recent results that identify the mechanisms of 1,25(OH)(2)D(3) action on the expression of TRPV6 and RANKL. A series of molecular, cellular, and in vivo studies have been conducted to define the molecular mechanisms that control the expression of TRPV6 and RANKL. Cell culture-based assays, chromatin immunoprecipitation (ChIP) and ChIP-DNA microarray (ChIP-chip) methods, and a series of molecular techniques were used to identify and characterize upstream regions of mouse and human TRPV6 and RANKL genes. We discovered that these genes were regulated by at least five separate enhancer regions. In the TRPV6 gene, these enhancers were all located within 5 kb of the transcriptional start site (TSS), and each contained one or more vitamin D regulatory elements (VDREs). In the RANKL gene, these regulatory regions span over 80 kb of upstream sequence, the most distal 76 kb from the TSS. This regulatory region is central to the regulation of RANKL expression in vitro and in vivo. Our studies identified key regulatory regions within the TRPV6 and RANKL genes that are essential for their individual expression in the intestine and bone, respectively.
The purpose of this work was to test the hypothesis that reduced responsiveness of target organs to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is associated with osteoporosis. Peripheral blood mononuclear (PBM) cells have been previously shown to be a valid model for the action of 1,25(OH)2D3 on its classic target organs in various pathologic and physiologic situations. The responsiveness of lymphocytes to the hormone can be assessed by the extent of inhibition it exerts on the proliferative response to mitogenic lectins. A group of 39 postmenopausal women, at least 10 years after the menopause, participated in the study. Osteoporosis, defined as the presence of at least one nontraumatic vertebral crush fracture, was diagnosed in 19 subjects. Mitogenesis of PBM cells stimulated by phytohemagglutinin and cultured for 72 h in the presence or absence of 1,25-(OH)2D3 (0.03-1 nmol/liter) was assessed by [3H]thymidine incorporation during a 4 h pulse. The maximal inhibitory effect of 1,25-(OH)2D3 at saturating concentration (1 nM/liter) was 74.6 +/- 2.8% (mean +/- SEM) for normal compared to 65.3 +/- 2.9% for osteoporotic women (P = 0.015). The geometric mean of the ED50 values of 1,25-(OH)2D3 was 60% higher in the osteoporotic than in the normal group (P = 0.035). Our data are consistent with the notion that reduced responsiveness of target organs to 1,25-(OH)2D3 is associated with osteoporosis.
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.
A 35-year-old white male with rheumatoid arthritis who had developed hypercalcemia, hypercalciuria, and nephrolithiasis was found to be abnormally sensitive to vitamin D as a result of lack of regulation of circulating 1,25-dihydroxyvitamin D (1,25-(OH)2D). An increase in daily intake of vitamin D from 10 micrograms (400 units) per day to 50 micrograms (2000 units) per day produced an abnormal elevation in serum 1,25-(OH)2D, hypercalcemia, and hypercalciuria which were corrected by prednisone. Serum 25-hydroxyvitamin D initially was abnormally low, and increased with vitamin D to values which were in the low normal range. There were significant positive correlations between serum 1,25-(OH)2D (p less than .05) and serum calcium and between serum 1,25-(OH)2D and urinary calcium (p less than .05). Serum immunoreactive parathyroid hormone, initially in the lower range of normal, decreased further during hypercalcemia. A radiograph of the chest, gallium scan, and serum angiotensin-converting enzyme activity were normal. No granulomas or evidence of lymphoma were found in biopsies of the liver and of several lymph nodes. It is concluded that the abnormal calcium metabolism in this patient resulted from increased circulating 1,25-(OH)2D and that the defect in vitamin D metabolism was not related to sarcoidosis, other granulomatous disease, Hodgkin's disease, or lymphoma. The relationship, if any, of the abnormal metabolism of vitamin D and calcium to rheumatoid arthritis remains to be established.