Bone and the regulation of global energy balance.

The skeleton, populated by large numbers of osteoblasts and long-lived osteocytes, requires a constant supply of energy-rich molecules to fuel the synthesis, deposition and mineralization of bone matrix during bone modelling and remodelling. When these energetic demands are not met, bone acquisition is suppressed. Recent findings suggest that key developmental signals emanating from Wnt low-density lipoprotein-related receptor 5 and hypoxia-inducible factor pathways impact osteoblast bioenergetics to accommodate the energy requirements for bone cells to fulfil their function. In vivo studies in several mutant mouse strains have confirmed a link between bone cells and global metabolism, ultimately leading to the identification of hormonal interactions between the skeleton and other tissues. The hormones insulin and leptin affect postnatal bone acquisition, whilst osteocalcin produced by the osteoblast in turn stimulates insulin secretion by the pancreas. These observations have prompted additional questions regarding the nature of the mechanisms of fuel sensing and processing in the osteoblast and their contribution to overall energy utilization and homeostasis. Answers to such questions should advance our understanding of metabolic diseases and may ultimately improve management of affected patients. In this review, we highlight recent studies in this field and offer a perspective on the evolutionary implications of bone as a metabolic endocrine organ.

Genetic control of chloroform toxicity in mice.

Mouse strain differences suggest intermediate or multifactorial gentic control of chloroform-induced renal toxicity and death. The chloroform dose lethal to 50 percent of animals was four times higher in C57BL/6J males than in DBA/2J males. Twice as much chloroform accumulated in the kidneys of the sensitive as the resistant strain. First generation offspring were midway between parental strains for both parameters.

Evidence that calcitonin stimulates 1,25-dihydroxyvitamin D production and intestinal absorption of calcium in vivo.

Although it is well established that parathyroid hormone and phosphate are important regulators of 1,25-dihydroxyvitamin D [1,25(OH)2D] production, it remains unclear whether calcitonin affects vitamin D metabolism in vivo. Experiments were performed in the rat to determine the effect of chronic calcitonin infusion (0.2 U X h-1) on plasma levels of vitamin D metabolites and on calcium metabolism. Thyroparathyroidectomized animals fed a calcium-replete or calcium-free diet were studied for as long as 2 wk before they were killed. In control rats, a calcium-free diet alone for 12 d resulted in an increase in 1,25(OH)2D levels from 24 +/- 5 to 139 +/- 37 pg . ml-1, P = 0.025. The infusion of calcitonin also stimulated 1,25(OH)2D levels compared with controls on a regular diet (80 +/- 17 vs. 38 +/- 6 pg . ml-1, P less than 0.05) and on a calcium-free diet (460 +/- 50 vs. 139 +/- 37 pg . ml-1, P less than 0.001). In addition, calcitonin increased plasma calcium levels in animals on a regular diet by 50%; this effect was most likely due to increased intestinal absorption of calcium, because removal of calcium from the diet markedly blunted this effect. In contrast, calcitonin administration did not significantly affect 25(OH)D plasma levels. Collectively, these data suggest that calcitonin and calcium are independent regulators of 1,25(OH)2D production and that calcitonin stimulates intestinal absorption of calcium, by increasing circulating levels of 1,25(OH)2D.

1 alpha,25-dihydroxyvitamin D3 induces maturation of the human monocyte cell line U937, and, in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor.

The monocyte factor, interleukin 1, or other factors homologous with interleukin 1, modulates functions of a variety of cells, including T and B lymphocytes, synovial cells, and chondrocytes. We have reported that a human monocyte cell line, U937, produces interleukin 1 when incubated with a soluble factor from lectin-stimulated T lymphocytes. We have also shown that U937 cells have a specific cytosolic receptor for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3). We now report that 1 alpha,25(OH)2D3(10(-11)-10(-10) M) induces maturational changes in the U937 cells similar to those produced by conditioned medium from lectin-stimulated T lymphocytes (increase in Fc receptors and OKM1 binding and decrease in proliferation), but does not induce monokine production as measured by mononuclear cell factor activity. 1 alpha,25(OH)2D3 is 200-300-fold more effective than 25-hydroxyvitamin D3, which is consistent with the known biological potency of these vitamin D3 metabolites. 1 alpha,25(OH)2D3 and the lymphokine together markedly augment maturational effects and, in addition, augment monokine production. The specificity of the interaction is further demonstrated by the lack of augmentation of monokine production with 1 beta,25-dihydroxyvitamin D3 in the presence of lymphokine. These interactions of a classical hormone and the hormonelike product(s) of the immune system with U937 cells serve as a model for human monocyte/macrophage differentiation and suggest a role for these interactions in some aspects of inflammation.

Useful clinical assays for vitamin D metabolites.

Advances in our understanding of the physiology of vitamin D and its importance in health and disease have depended on the accurate measurement of its metabolites in blood. Assays that were once cumbersome and insensitive are now performed easily, are highly sensitive, reproducible, and relatively inexpensive. The availability of these modern techniques has facilitated the clinical diagnosis and treatment of patients.

The synthetic human parathyroid hormone-related protein is inhibited by a parathyroid hormone antagonist in rats in vivo.

The structure of a novel protein, parathyroid hormone-related protein (PTHrP), secreted by human tumors associated with hypercalcemia has recently been determined. Administration of a synthetic fragment of this protein in vivo reproduces features of the clinical paraneoplastic syndrome of humoral hypercalcemia of malignancy and produces biologic responses closely similar to those obtained with parathyroid hormone (PTH). A PTH antagonist designed to reversibly occupy PTH receptors inhibited major actions of the tumor peptide in vivo, including phosphaturia, urinary cAMP excretion, and increased serum ionized calcium. These studies indicate that PTHrP and PTH mediate their bioactivities through shared receptors in vivo and establish a potential specific mechanism-based approach utilizing PTH antagonists for the therapy of tumor-associated hypercalcemia.

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

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

Subcellular localization and partial purification of the 25-hydroxyvitamin D3 1-hydroxylation reaction in the chick myelomonocytic cell line HD-11.

Hypercalcemia in human granuloma-forming diseases like sarcoidosis results from the endogenous overproduction of 1,25-dihydroxyvitamin D [1,25-(OH)2D] by disease-activated tissue macrophages. The recent identification of an immortalized chick myelomonocytic cell line, HD-11, that constitutively expresses a 25-hydroxyvitamin D (25-OHD) 1-hydroxylation reaction has alleviated dependence on studying primary macrophage cultures with no replicative potential in vitro. In these experiments we established conditions for the maximal expression of the HD-11 cell 25-OHD3-1-hydroxylation reaction and localized this activity to the mitochondrial fraction. On a per cell basis, the activity of HD-11 cell 25-OHD3 1-hydroxylation reaction was comparable to that in primary cultures of chick renal tubular epithelial cells, which express the authentic renal 25-OHD3 1-hydroxylase. Maximal product yield was achieved after incubation of HD-11 cells with 200 nM 25-OHD3 for 3 h. Although adherent monolayers possessed 3- to 4-fold more capacity for hormone production than cells in suspension, suspended cells exhibited easily detectable 25-OHD3 catalytic activity (0.58 +/- 0.08 pmol per 10(6) cells per h; +/- SEM), 50% of which remained solubilized in a sonicate of suspended cells cleared of nuclei and plasma membrane. Subcellular localization disclosed 91% of the residual activity to be concentrated in the mitochondrial subfraction. A detergent-solubilized extract of this mitochondrial subfraction contained 1.9 +/- 0.3 pmol 1,25-(OH)2D3 synthetic capacity per mg protein. The catalytic activity (1-hydroxylase activity) was concentrated 20.2-fold after chromatography on octyl-amino agarose and was associated with 0.054 nmol cytochrome P450 per mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone mineral density in women with type II diabetes mellitus.

Bone mineral density (BMD) was assessed in 28 women with type II diabetes mellitus and compared to 207 age-matched nondiabetic women. Mean BMD, as measured by dual-photon absorptiometry, 1.12 +/- 0.3 g/cm2 (+/- SEM), was similar to the mean BMD of control subjects, 1.06 +/- 0.1 g/cm2. Only 1 of the 28 diabetic patients had a BMD less than 0.95 g/cm2 ("fracture threshold"), whereas 25% of the control subjects had a BMD below that level. When diabetic and control subjects were matched for weight as well as age, the data continued to show similar BMD among both groups. Moreover, the disparity between the proportion of weight-matched controls (25%) and diabetic subjects (1 of 28) with a BMD below the fracture threshold persisted. Among the group of 17 diabetic subjects receiving insulin, there was a positive relationship between BMD and insulin dose. There was no significant relationship between BMD, duration of diabetes, or hemoglobin Alc. Thus, women with type II diabetes are not at increased risk for diminished BMD and may be protected against bone loss.

The effect of oral phosphate administration on major indices of skeletal metabolism in normal subjects.

The effect of brief periods of phosphate administration on indices of human skeletal metabolism was investigated. Thirteen subjects (8 women, 5 men; 19-36 years old) received 2 g of oral phosphate daily for 5 days. Serum phosphorus rose 26% (3.8 +/- 0.1 mg/dl to 4.8 +/- 0.1 mg/dl; p less than .01) while total calcium fell (9.3 +/- 0.1 mg/dl to 8.9 +/- 0.1 mg/dl; p less than .01). Parathyroid hormone levels increased by 50% (14.1 +/- 2.0 pg/ml to 21.5 +/- 1.7 pg/ml; p less than .05) although values remained within the normal range. A persistent phosphaturia (0.64 +/- 0.10 g/g Cr to 1.8 +/- 0.4 g/g Cr; p less than .05) and a 69% fall in urinary calcium (80.8 +/- 10.0 mg/g Cr to 24.6 +/- 6.0 mg/g Cr; p less than .001) were observed. 1,25-dihydroxyvitamin D3 and urinary hydroxyproline concentrations did not change significantly but the bone gamma-carboxyglutamic acid protein (BGP) concentration rose 41% by day 2 (9.6 +/- 1.3 mg/ml to 13.5 +/- 2.2 mg/ml; p less than .005) and remained elevated throughout the study period. These results support the possibility that brief periods of phosphate administration may be useful in the therapy of disorders associated with low bone turnover, such as osteoporosis.

Tumor-induced osteomalacia: clinical and basic studies.

A patient with classic clinical and biochemical features of tumor-induced osteomalacia (hypophosphatemia, phosphaturia, and undetectable serum concentrations of 1,25-dihydroxyvitamin D [1,25(OH)2D]) was studied before and after resection of a benign extraskeletal chondroma from the plantar surface of the foot. Presurgical laboratory evaluation was notable for normal serum concentrations of calcium, intact parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and osteocalcin, increased serum alkaline phosphate activity, and frankly elevated urinary cyclic adenosine monophosphate (cAMP) and pyridinium cross-link excretion. Quantitative histomorphometry showed severe osteomalacia and deep erosions of the cancellous surface by active osteoclasts. After resection, serum 1,25(OH)2D normalized within 24 h, while renal tubular phosphorus reabsorption and serum phosphorus did not normalized until days 2 and 3, respectively; serum Ca declined slightly, and serum intact PTH, osteocalcin, and urinary pyridinium cross-link excretion increased dramatically. Urinary cAMP excretion declined immediately after resection and then began to increase concomitant with the increase in serum intact PTH. A second bone biopsy taken 3 months after resection demonstrated complete resolution of the osteomalacia, increased mineral apposition rate (1.09 mu/day), resorption surface (9.2%), mineralizing surface (71%), and bone formation rate (0.83 mm3/mm2/day), and marked decrease in cancellous bone volume (13.1%) and trabecular connectivity compared with first biopsy. Tumor extracts did not affect phosphate transport in renal epithelial cell lines or 1 alpha-hydroxylase activity in a myelomonocytic cell line. The patient's course suggests that the normal 1,25(OH)2D and phosphorus metabolism is due to a tumor product that may be acting via stimulation of adenylate activity. Increased bone resorption prior to surgical resection suggests that the tumor may also produce an osteoclast activator. The rise in resorption surface and pyridinium cross-link excretion, increase in serum osteocalcin and bone mineralization, normalization of osteoid width, and fall in cancellous bone volume after resection are consistent with healing of osteomalacia by rapid remodeling.

Analysis of osteocalcin expression in transgenic mice reveals a species difference in vitamin D regulation of mouse and human osteocalcin genes.

A line of transgenic mice expressing a human osteocalcin genomic fragment (hOClocus) and a murine MC3T3-E1 cell line containing a stably integrated human osteocalcin promoter construct have been developed to characterize the osteogenic and hormonal regulation of human osteocalcin in vivo and in vitro. In this study, we used these models to demonstrate a species difference in the regulation of the mouse and human osteocalcin genes by vitamin D. Repeated administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to mice carrying the hOClocus transgene resulted in striking increases in serum human osteocalcin, whereas serum mouse osteocalcin levels were unchanged after 24 h and only modestly increased 48 h after the second dose of hormone. 1,25(OH)2D3 increased human calvarial mRNA expression by 1.8-fold and slightly decreased mouse osteocalcin mRNA levels by approximately 1.2-fold. Furthermore, treatment of primary calvarial osteoblasts from these mice with 1,25(OH)2D3 increased human osteocalcin production but inhibited mouse osteocalcin protein accumulation. To investigate further the mechanism for the apparent species difference in vitamin D3 induction of mouse and human osteocalcin, we examined the effect of 1,25(OH)2D3 in an MC3T3-E1 cell line (MC4) containing a stably integrated 3900 bp osteocalcin promoter-luciferase construct. Treatment of MC4 cells with ascorbic acid resulted in parallel increases of the endogenous mouse osteocalcin protein and luciferase reporter activity over a 12-day period. Continuous exposure of MC4 cells to 1,25(OH)2D3 resulted in time-and dose-dependent increases in the activity of the phOC3900 luciferase construct. By contrast, the hormone had no effect on mouse osteocalcin protein concentrations and inhibited its induction by ascorbic acid. However, when cells were treated acutely with 1,25(OH)2D3 at later times during growth in ascorbic acid, the induction of mouse osteocalcin protein was only partially inhibited. In conclusion, our results indicate that common osteogenic signals regulate both mouse and human osteocalcin gene expression, but the mouse gene is resistant to induction by vitamin D. This species difference in vitamin D regulation of osteocalcin appears to result from the failure of 1,25(OH)2D3 to transcriptionally activate the mouse osteocalcin gene.

Detection and developmental changes of the 1,25-(OH)2-D3 receptor concentration in mouse skin and intestine.

We investigated [3H]1,25-dihydroxyvitamin D3-specific binding activity in fetal, neonatal, and adult mouse skin to determine (a) during which stage in development the skin develops the capacity to respond to this hormone and (b) whether the hormone binding activity changed during development and maturation. A macromolecule with properties similar to those of the chick intestinal 1,25-dihydroxyvitamin D3 receptor was detected in the skin and intestine of mouse pups at 17 days of fetal life. 1,25-Dihydroxyvitamin D3-specific binding activity from both tissues sedimented on linear sucrose gradients at 3.5-3.7S and eluted from DNA cellulose at 0.22 M KCl. At earlier stages of fetal life (12-14 days) receptor-like activity was detected in cytosols prepared from whole-mouse fetuses. 1,25-Dihydroxyvitamin D3-specific binding activity was quantitated in the skin and intestine throughout development using a chromatin binding assay. Scatchard analysis of saturation binding data showed that the concentration of binding activity in skin increased rapidly after birth and reached a maximum when the mice were 10-19 days old. By contrast, the binding activity that was detected in the fetal and neonatal whole intestine remained low until the mice were weaned. The affinity (Kd) of the binding activity was similar in skin and intestine at all ages studied. It is concluded that 1,25-dihydroxyvitamin D3-specific binding activity appears in both skin and intestine of the mouse prior to birth and increases in these two tissues during different stages in development.

The effect of cell-matrix interaction on parathyroid hormone (PTH) receptor binding and PTH responsiveness in proximal renal tubular cells and osteoblast-like cells.

The interaction of cells with the surrounding extracellular matrix (ECM) or basement membrane (BM) brings about profound changes in cellular biological responses, such as cell differentiation, proliferation, and gene expression. We studied the effect of ECM on PTH receptor binding and on biological responses mediated by PTH, in two cell preparations: 1) the proximal tubular OK opossum kidney cell line; and 2) MC3T3-E1 cells, a clonal line of nontransformed murine osteoblasts. Cells were plated on either plastic surfaces or on tissue culture dishes coated with specific ECM components. In both cell types plated on collagen-type IV (Col-IV), PTH receptor binding, on day 4 of culture, was markedly diminished, when compared with cells on plastic (approximately 45% inhibition, P < 0.01). In addition, Col-IV dose dependently inhibited cAMP generation stimulated by PTH (P < 0.001 vs. plastic), whereas cAMP generation by PGE2, cholera toxin, and forskolin was not altered. In Northern blot analysis, a PTH/PTH-related-protein receptor messenger RNA transcript was detected in both the kidney and bone cells. However, only OK cells manifested a decreased abundance of receptor messenger RNA when plated on Col-IV, compared with plastic. The physiological significance of inhibited cAMP production by Col-IV was evaluated by measuring the influence of different matrices on the activity of Na+/H+ exchanger (NHE) in OK cells and cell mitogenic activity in MC3T3-E1 cells (both responses are negatively modulated by cAMP). OK cells plated on Col-IV showed 70% inhibition of NHE, compared with cells plated on plastic (P < 0.01). PTH inhibits NHE activity in cells on plastic but stimulates exchanger activity by 40% in cells plated on Col-IV. In MC3T3-E1 cells grown on plastic, PTH exerts a dose-dependent antiproliferative effect, which is mediated by cAMP. This effect is mitigated when cells are grown on Col-IV (40-50% less antiproliferative effect). In summary, Col-IV, a maj or BM constituent, has a profound inhibitory effect on PTH binding and PTH-mediated biological responses in both kidney tubular cells and osteoblasts. Altered cellular function by Col-IV may be of physiological relevance in states associated with altered composition of BM or expansion of ECM (e.g. diabetes mellitus and interstitial fibrosis).

Regulation of avian calbindin-D28K gene expression in primary chick kidney cells: importance of posttranscriptional mechanisms and calcium ion concentration.

Vitamin D-dependent calcium-binding protein (calbindin-D28K), is regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and several other factors in a tissue-specific manner, but the controlling mechanisms are still poorly understood. In this study we examined the relative contributions of transcriptional and posttranscriptional mechanisms in the 1,25-(OH)2D3 control of calbindin-D28K mRNA expression in primary chick kidney cells and investigated the effect of extracellular Ca2+ on calbindin-D28K gene expression in the presence and absence of hormone. 1,25-(OH)2D3 treatment (10(-8) M) of cells grown in serum-free medium resulted in a marked 20- to 30-fold increase in calbindin-D28K mRNA peaking at 12-18 h, which then rapidly declined to basal levels by 24 h. The abrupt decline in mRNA appeared to be associated with a reduction in size of the calbindin-D28K transcripts. Nuclear run-off assays showed only a slight (1.5-fold) increase in calbindin-D28K gene transcription 2 h after 1,25-(OH)2D3, whereas parallel assays clearly demonstrated a marked (7-fold) induction in the rate of metallothionein gene transcription 2 h after treatment of chick kidney cells with 10 microM zinc. The induction of calbindin-D mRNA by 1,25-(OH)2D3 required ongoing protein synthesis, since it was blocked by cycloheximide. Calbindin-D28K mRNA was stable for 12 h in the presence of actinomycin-D in both vitamin D-deficient and 1,25-(OH)2D3-treated cells. Both basal and 1,25-(OH)2D3-induced calbindin-D28K mRNA were modulated by the extracellular Ca2+, with maximum expression occurring at 1-2 mM. We conclude that 1,25-(OH)2D3 induces kidney calbindin-D28K mRNA by producing a small increase in its transcriptional rate, which is accompanied by pronounced posttranscriptional effects(s). The striking modulation of calbindin-D28K expression by extracellular Ca2+ is consistent with a putative role for this protein in the regulation of this ion in the kidney cell.

1,25-Dihydroxyvitamin D3 induces the synthesis of vitamin D-dependent calcium-binding protein in cultured chick kidney cells.

A 28,000 mol wt vitamin D-dependent calcium-binding protein (CaBP), first isolated from avian intestine, has recently been shown to exist in kidney and other tissues. To study the mechanism regulating the production of renal CaBP, we used primary cultures of chick kidney cells as an in vitro model. Renal cortical tubules isolated from vitamin D-deficient chicks were grown in serum-free, hormone-supplemented medium. Confluent cells were epithelioid and expressed CaBP, as demonstrated by immunocytochemistry and a specific RIA. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] increased cellular CaBP content in a dose-dependent manner (10(-10)-10(-7) M) from basal concentrations of 50-240 ng/mg protein to maximal concentrations of 600-1200 ng/mg protein 48 h after dosing. Cycloheximide (2 microM) inhibited 1,25-(OH)2D3 induction of CaBP, indicating that the mechanism requires new protein synthesis. Western blotting of cell extracts confirmed the identity of the inducible protein as the 28,000 mol wt CaBP. 25-Hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were effective but somewhat less potent inducers of CaBP, whereas vitamin D3 was without significant effect. The activities of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 are attributed to their rapid conversion by kidney cells to 1 alpha-hydroxylated metabolites. The dose-response profiles for two additional bioresponses in these cells, namely induction of 24-hydroxylase and inhibition of 1 alpha-hydroxylase activity, were comparable to those for CaBP induction. To our knowledge, this is the first description of a cell culture system that exhibits 1,25-(OH)2D3-inducible CaBP in vitro. This model should permit the study of certain aspects of the physiology of 1,25-(OH)2D3 and CaBP in kidney that are not possible in vivo.

1,25-Dihydroxyvitamin D induces lipoprotein lipase expression in 3T3-L1 cells in association with adipocyte differentiation.

1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is known to modulate the development of bone and other mesenchymal cell types. Since osteoblasts and adipocytes are thought to arise in bone marrow from a common progenitor, this work examined the effects of 1,25-(OH)2D3 on adipocyte development, and in particular on the expression of lipoprotein lipase (LPL), which is an early marker for the differentiated adipocyte. 3T3-L1 preadipocytes were cultured in the presence of 1,25-(OH)2D3 (10(-9) to 10(-7) M) for up to 7 days. LPL activity was measured in the medium and cell extracts, and LPL messenger RNA levels were measured by Northern blotting. When compared to control cells, 10(-7) M 1,25-(OH)2D3 increased medium LPL activity by 2- to 3-fold and cellular LPL by 1.5-fold. Significant increases in medium and cellular LPL were observed at 10(-9) M and were maximal at 10(-7) M. Along with the increase in LPL activity, there was an increase in LPL messenger RNA by 2-fold at 5 days, and by 5-fold at 7 days. In addition to an increase in LPL, 1,25-(OH)2D3 increased expression of aP2, an adipocyte-specific marker associated with differentiation. After the addition of 1,25-(OH)2D3, there was a decrease in 3T3-L1 cell number, which is consistent with differentiation, and a decrease in vitamin D receptors. Finally, these cells developed a different morphology. 1,25-(OH)2D3-treated cells assumed a rounded appearance, although without detachment from the dish and without the degree of lipid accumulation usually associated with the addition of insulin, isbutylmethylxanthine, and dexamethasone. It is concluded that 1,25-(OH)2D3 induced LPL expression in 3T3-L1 cells through an induction of differentiation-dependent mechanism(s). These findings suggest an important role for 1,25-(OH)2D3 in normal adipocyte differentiation.

Hypoxia induces vascular endothelial growth factor gene transcription in human osteoblast-like cells through the hypoxia-inducible factor-2alpha.

VEGF is produced by osteoblasts and has been postulated to function as an angiogenic stimulus during normal skeletal development and in fracture repair. In this study, we characterized the molecular mechanisms by which experimental hypoxia increases VEGF mRNA in human MG63 osteoblast-like cells. Exposure of MG63 cells to 1% O(2) for 24 h resulted in a four-fold increase in VEGF mRNA. Immunoblotting of nuclear extracts demonstrated a time-dependent increase in the level of the Hif-2alpha protein, which preceded the rise in VEGF mRNA. To determine the effect of hypoxia on VEGF gene transcription, MG63 cells were transiently transfected with a segment of the VEGF promoter construct fused to luciferase and then exposed to 1% O(2). Hypoxia induced VEGF promoter activity five-fold by 24 h. Forced expression of Hif-2alpha, but not Hif-1alpha, increased both basal and hypoxia induced VEGF promoter activity. By contrast, the ability of the VEGF reporter to respond to hypoxia or recombinant Hif-2alpha was abolished in cells transfected with a VEGF promoter construct containing a mutation in the hypoxia response element. In summary, exposure of osteoblast-like cells to hypoxia induces VEGF expression via induction of Hif-2alpha and transcriptional activation of the VEGF promoter.

Extracellular calcium modulates vitamin D-dependent calbindin-D28K gene expression in chick kidney cells.

The effect of extracellular calcium ion (Ca2+) concentration on 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induction of vitamin D-dependent calcium-binding protein (calbindin-D28K) and its mRNA levels was examined in primary chick kidney cells in vitro. When exposed to normal medium Ca2+ (1.0 mM), 1,25-(OH)2D3 increased calbindin-D28K mRNA, as measured by Northern analysis, by 4-10 fold over basal levels by 12 to 24 h after addition of hormone. In the presence of 0.5 mM Ca2+, 1,25-(OH)2D3 induced calbindin-D28K mRNA by only 2 fold, whereas, when cells were exposed to 2 mM Ca2+, the induction was 10-15 fold. This calcium modulation of 1,25-(OH)2D3 induction was also observed at the level of calbindin-D28K protein concentrations as measured by radioimmunoassay. The alterations in medium Ca2+ were not associated with any change in the rate of total RNA or protein synthesis. These studies suggest that both Ca2+ and 1,25-(OH)2D3 participate in the regulation of calbindin-D28K gene expression in the kidney.

A role for nitric oxide in the regulated expression of the 25-hydroxy-vitamin D-1-hydroxylation reaction in the chick myelomonocytic cell line HD-11.

We have recently described the existence of a cytochrome P450-associated, mitochondrial-based 25-hydroxyvitamin D (25-OHD)-1-hydroxylation reaction in the chick macrophage-like cell line HD-11. Considering that this reaction is regulated by the same set of factors (ie. interferon-gamma, lipopolysaccharide, and glucocorticoids) that modulate expression of the macrophage nitric oxide synthase (mac NOS), we investigated the possibility that endogenous nitric oxide (NO) production may be linked to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D) synthesis by HD-11 cells in vitro. To test this hypothesis we investigated the effects excluding from the extracellular medium the essential amino acid L-arginine, substrate for endogenous NO production, on the basal and stimulated expression of the HD-11 cell 25-OHD-1-hydroxylation reaction. Depletion of L-arginine from the extracellular medium for as little as 6 h resulted in a significant decrease (p < 0.02) in basal 1,25-(OH)2D synthesis; after 15 h in an L-arginine-free environment hormone production was reduced to < 10% of basal levels without any adverse affect on cell viability. Reintroduction of L-arginine, but not D-arginine, into the extracellular medium restored 1,25-(OH)2D3 synthetic capacity fully if done after < or = 6 h of incubation in the absence of L-arginine. Competitive inhibition of NOS with Nw-nitro-L-arginine methyl ester (p < 0.002) and Nw-nitro-L-arginine (p < 0.02) significantly inhibited 1,25-(OH)2D synthesis, indicating that macrophage NO generating capacity is functionally linked to endogenous synthesis of the active vitamin D metabolite.