Prostaglandin E2 binding and cyclic AMP production in isolated bone cells

The binding of prostaglandin E2 (PGE2) to bone cells was studied to provide direct evidence for the existence of specific receptors in bone. Bone cells were isolated by collagenase digestion of fetal and newborn rat calvaria. Isolated cells were incubated with 3H-PGE2 and collected on Millipore filters. Specific binding was determined by subtracting the binding that occurred with 10(-6) M non-radioactive PGE2 and 3H-PGE2 from that with 3H-PGE2 alone. With heterogeneous cell preparations and at PGE2 concentrations from 10(-9) - 1.7 X 10(-8) M at 37 degrees C, specific binding reached steady state within 10 min. Bound 3H-PGE2 was displaced by the addition of increasing amounts of unlabeled PGE2. Inhibition of PGE2 binding was observed with PGE1 and the endoperoxide analog, U44069, but not with PGF2 alpha, a lipopolysaccharide, or 13,14-dihydro 15-keto PGE2. Studies with bone cell populations, obtained by sequential digestions, indicated that an osteoclastic population binds 30-fold more PGE2 than osteoblastic cells. Scatchard analyses revealed that the osteoclastic cells have an affinity constant for PGE2 binding similar to that obtained with heterogeneous populations. However, the PGE2 binding capacity in this osteoclastic population was fivefold greater than in the heterogeneous population. The osteoclastic population responded with an increase in cyclic AMP to lower concentrations of PGE2 than the osteoblastic populations. These studies suggest that differences in the binding capacity of PGE2 receptors exist among bone cell-types and that these differences are reflected in the cellular cyclic AMP response.     

Phorbol ester (TPA) reduces prostaglandin E2-stimulated cAMP production by desensitization of prostaglandin E2 receptors in a clonal osteoblast-like cell line,MOB 3-4

Summary A clonal osteoblast-like cell line, MOB 3-4, increased cAMP production in response to prostaglandin E₂ (PGE₂) (5–500 ng/ml). The purpose of this study was to show the effects of tumor-promoting phorbol ester (e.g., 12-O-tetradecanoylphorbol 13-acetate, TPA) on basal and PGE₂-stimulated cAMP production and the affinity of PGE₂ receptors in the cells. Pretreatment with TPA (1 nM–10 μM) for 30 minutes increased basal cAMP production, whereas it markedly reduced the PGE₂-stimulated cAMP production in the presence of 0.1 mM isobuthylmethyl xanthine. Both the TPA increase and reduction were dose- and time-dependent. However, TPA exerted no effect on forskolinor cholera toxin-stimulated cAMP production. Copretreatment with TPA and H-7, an inhibitor of protein kinase C (PKC), prevented the TPA-induced increase in basal cAMP production, whereas it did not prevent the reduction of the PGE₂-stimulated cAMP production. On the other hand, TPA (0.1–10 μM) decreased³H-PGE₂ binding in a dose- and time-dependent manner. Scatchard analysis revealed that TPA decreased the apparent affinity of PGE₂ receptors without effect on their apparent number. In addition, 1-oleoyl-2-acetylglycerol (12.6 μM), a synthetic diacylglycerol analog, did not mimic the TPA action on³H-PGE₂ binding. Thus, TPA at relatively high concentrations appeared to increase basal cAMP production by a PKC-mediated mechanism, and it appeared to directly act on PGE₂ receptors to decrease their apparent affinity and thereby reduce the PGE₂-stimulated cAMP production in the clonal osteoblast-like MOB 3-4 cell line.     

Effects of tumor necrosis factor alpha on parathyroid hormone-induced increases in osteoblastic cell cyclic AMP

Summary Tumor necrosis factor α (10⁻¹⁰–10⁻⁸M) had no effects on cyclic AMP production by the osteoblastic osteosarcomal cells, Saos-2 and G292, or normal rat calvarial cells. The cytokine did, however, inhibit the parathyroid hormone (PTH)-induced effect on cyclic AMP in the Saos-2 and normal rat osteoblastic cells. This inhibitory effect did not occur on prostaglandin E₂-induced cyclic AMP increases in the osteoblastic cells. Interleukin-1 (10 U/ml −100 U/ml) did not produce any effect on basal levels or PTH-induced cyclic AMP increases in these cells.     

Recombinant interleukin-1 stimulates prostaglandin E2 production by osteoblastic cells: Synergy with parathyroid hormone

Summary Recombinant mouse IL-1 (Interleukin-1) has been shown to be capable of stimulating prostaglandin E₂ (PGE₂) production by isolated rat osteoblastic cells in a dose-dependent manner. The rapidity of the effect (1 hour) and the potency of IL-1 (5×10⁻¹² M) in producing this effect suggest that IL-1 may exert some of its effects on bone via PGE₂. Parathyroid hormone (PTH) appears to have a strong synergistic effect with IL-1. These data further substantiate the role of IL-1 in bone physiology.     

6-keto-prostaglandin E1-stimulated bone resorption in organ culture

Summary Previous investigations have shown that prostaglandin E₂ (PGE₂), 13,14-dihydro-PGE₂, and prostacyclin (PGI₂) are among the most potent prostaglandin stimulators of bone resorption. 6-Ketoprostaglandin F_1α (6-keto-F_1α; also called 6-oxo-prostaglandin F_1α), a metabolite of PGI₂ formed by spontaneous hydrolysis, has little bone resorptive or other biological activity. The present study demonstrated that another metabolite of PGI₂, 6-keto-prostaglandin E₁ (6-keto-PGE₁; also called 6-oxo-prostaglandin E₁), was active in stimulating bone resorption in fetal rat long bone organ culture. 6-Keto-PGE₁ stimulated significant release of previously incorporated⁴⁵Ca over the concentration range of 10⁻⁹ to 10⁻⁵ M. The potency of 6-keto-PGE₁ was one-twelfth that of PGE₂. If 6-keto-PGE₁ is formed by bone or adjacent tissues, or reaches bone through the circulation, it could significantly affect bone mineral metabolism.     

Effects of prostacyclin and prostaglandin E1 (PGE1) on bone resorption in the presence and absence of parathyroid hormone

Summary Prostaglandins have been shown to stimulate osteoclastic bone resorption in organ culture but morphologic studies of isolated osteoclasts have shown a transient calcitonin-like inhibiting effect of these agents. We looked for a dual effect on bone resorption by comparing the early and late effects of prostaglandin E₁ (PGE₁), prostacyclin (PGI₂), 6α-carbaprostaglandin I₂ (C-PGI₂), a carbon substituted analog of PGI₂, and salmon calcitonin (CT) on the release of previously incorporated⁴⁵Ca from fetal rat long bones cultured in the presence of an inhibitor of cyclooxygenase, RO-20-5720. Experiments were performed in both the presence and absence of PTH (400 ng/ml), which was administered 24 hours before addition of prostaglandins or CT. In control cultures not stimulated by PTH, CT (100 mU/ml) produced significant decreases in⁴⁵Ca release at 48, 72, and 96 hours while PGE₁ (10⁻⁶ M), PGI₂ (10⁻⁵), and C-PGI₂ (10⁻⁶ M) each produced significant increases in resorption at 24 through 96 hours. PGE₁ at 10⁻⁵ M, but not 10⁻⁶ M, caused a significant decrease in medium⁴⁵Ca of 21% at 1 and 2 hours. Medium calcium measurements suggest that the change in⁴⁵Ca was due to inhibition of release and not to increased uptake. PGI₂ (10⁻⁵ M) and C-PGI₂ (10⁻⁶ M) caused no significant inhibitory effect. In cultures stimulated by PTH, CT produced significant inhibition of bone resorption of 6 through 96 hours, but no inhibition of bone resorption was noted at either early or late time points with PGE₁, PGI₂, or C-PGI₂. Moreover, the addition of PGI₂ (10⁻⁵M) to PTH-treated cultures actually enhanced⁴⁵Ca release beginning to 6 hours, when PGI₂ alone had no effect upon bone resorption. These results confirm that high concentration of PGI₂ can stimulate bone resorption and show a similar response to a stable analog, C-PGI₂. Moreover, PGI₂ was found to enhance PTH-stimulated bone resorption. A small transient inhibition of⁴⁵Ca release was observed with a high concentration of PGE₁ (10⁻⁵M) in the absence of PTH, which could be due to a transient direct inhibitory action upon osteoclasts.     

The role of glucocorticoids and prostaglandin E2 in the recruitment of bone marrow mesenchymal cells to the osteoblastic lineage: Positive and negative effects

The role of glucocorticoids in bone formation presents a problem because although pharmacological doses in vivo give rise to osteoporosis, physiological concentrations are required for osteoblast (OB) differentiation in vitro. To try and rationalize this dichotomy, we investigated the effect of dexamethasone on the recruitment of OB precursors present in bone marrow. Using the CFU-f assay, we can measure (1) total colony formation; (2) the osteoblastic differentiation of the colonies defined as their ability to express alkaline phosphatase, synthesize collagen, and to calcify; and (3) colony expansion as either average colony surface area or average colony number. In control cultures and in the presence of 10⁻¹⁰–10⁻⁹ M dexamethasone, colony formation and total cell number was maximal, but the addition of PGE₂ had no effect on colony number and very few colonies expressed the OB phenotype. In the presence of 10⁻⁸–10⁻⁷ M dexamethasone, colony numbers and total cell numbers were reduced but were increased by the addition of PGE₂, the average colony cell number and surface area were relatively unchanged and a proportion of the colonies expressed APase, calcified and synthesized collagen. In cultures containing 10⁻⁶–10⁻⁵ M dexamethasone, colony numbers were further reduced but were stimulated by the addition of PGE₂ and some colonies differentiated; however, colony expansion was dramatically reduced by up to 80%. These results suggest that physiological levels of glucocorticoids are necessary for OB differentiation and allow the control of OB recruitment by PGE₂. High levels of glucocorticoids drastically reduce proliferation of the OB precursors leading to glucocorticoid-induced osteoporosis. Received: 8 May 1995 / Accepted: 23 February 1996     

Chloroquine,hydroxystilbamidine, and dapsone inhibit resorption of fetal rat bone in organ culture

Summary Three potential inhibitors of lysosomal enzyme release, chloroquine, hydroxystilbamidine, and dapsone were tested for their effects on the release of previously incorporated⁴⁵Ca and beta (β)-glucuronidase from fetal rat long bones cultured in a chemically defined medium. At concentrations of 10⁻⁵ to 10⁻⁸M, all three agents were able to inhibit the stimulation of bone resorption by parathyroid hormone (PTH) or prostaglandin E₂ (PGE₂). Inhibition was seen at concentrations which did not alter the uptake of (³H)-2-deoxy-glucose or the incorporation of (³H)-thymidine in bone. While the inhibitors blocked the stimulation of β-glucuronidase release by PTH and PGE₂, they could also cause a direct increase in total β-glucuronidase content and release. Hence the usual strong correlation between the release of β-glucuronidase and⁴⁵Ca was no longer seen in the presence of inhibitors. These data indicate that chloroquine, hydroxystilbamidine, and dapsone are potent inhibitors of bone resorption which may act by blocking the release of lysosomal enzymes in cells stimulated by PTH or PGE₂, but may have a different effect on other cell populations.     

Effect of prostaglandin E2 on mineralization of bone nodules formed by fetal rat calvarial cells

The effects of PGE₂ on mineralized bone nodule formation were studied in fetal rat calvarial (RC) cells in vitro. Continuous exposure of RC cells to 3x10^-8M PGE₂ induced a twofold increase in mineralized bone nodule formation and a 1.5-fold increase in alkaline phosphatase (ALPase) activity without affecting RC cell growth. These stimulatory effects were evoked by concentrations of 3x 10^-9-3x10^-6 M PGE₂ and the maximal effect was observed with 3x10^-8 M PGE₂. The in vitro effects of PGE₂ were evident when RC cells were exposed to it on days 8–14 and 8–21, which correspond to the post-confluent culture stage, but no effects were observed when the cells were exposed on days 1–7, the growth stage. The ALPase activity was also higher (1.2–1.4-fold) when 3x10^-8 M PGE₂ was added during the post-confluent stage. In order to determine the effect of PGE₂ during the mineralization phase of bone nodules in the presence of a large population of osteoprogenitor cells, RC cells were exposed to dexamethasone for 7 days before PGE₂ was added during the post-confluent stage. A significantly higher percentage of nodules mineralized were observed with 3x10^-8-3x10^-9 M PGE₂ (1.6-and 1.4-fold, respectively), than in control cultures. Analysis of the mineral-related proteins by EDTA extraction of bone nodules followed by electrophoresis and Stains-All staining revealed an increased total amount of osteopontin extracted from the mineralized matrix after PGE₂ treatment. The osteopontin content was highest after 3x10^-8 M PGE₂, with a 73% increase of the densitometric intensity of the bands, although this increase, reflected the increased number of mineralized bone nodules due to PGE₂. These findings suggest that PGE₂ may increase the proportion of functional osteoblasts able to produce mineralized bone nodules in the population by stimulating differentiation during the postconfluent stage of RC cell culture.     

Parathyroid Hormone,Prostaglandin E2, and 1,25-Dihydroxyvitamin D3 Decrease the Level of Na+-Ca2+ Exchange Protein in Osteoblastic Cells

We previously described Na⁺-Ca²⁺ exchange in osteoblastic rat osteosarcoma cells (UMR-106) and demonstrated that Na⁺-dependent Ca²⁺ transport was inhibited by 24-hour treatment of cells with parathyroid hormone (PTH), prostaglandin E₂ (PGE₂), or 1,25(OH)₂D₃. To determine whether this inhibition of Na⁺-Ca²⁺ exchange is at the level of exchanger protein synthesis we have examined exchanger protein levels using immunoblot analysis. UMR-106 cells were treated for 24 hours with or without PTH, PGE₂, or 1,25(OH)₂D₃. Plasma membrane fractions (7500 g) were obtained and proteins were separated by SDS-PAGE, transferred to nylon membranes, and immunoblotted with a polyclonal antibody to the canine cardiac Na⁺-Ca²⁺ exchanger. In rat cardiac membranes, we detected 125 and 75 kD bands, similar to findings for the canine exchanger. In the osteoblastic UMR cell membranes, a specific band was detected at 90 kD that decreased 65% after treatment of cells with PTH. Inhibition by PTH was dose dependent, was maximal with 10⁻⁷ M PTH, and required 16–24 hour treatment time. Similar inhibition was observed after a 24 hour treatment with 10⁻⁶ M PGE₂ or 10⁻⁸ M 1,25(OH)₂D₃. These results demonstrate the presence of a specific protein in UMR cells that cross-reacts with antibody directed against the cardiac Na⁺-Ca²⁺ exchanger. Thus, the previously reported inhibition of Na⁺-Ca²⁺ exchange activity by calcemic agents in osteoblasts appears to be due to regulation of exchanger protein levels in these osteoblastic cells. Received: 5 February 1996 / Accepted: 18 October 1996     

Inhibitory and stimulatory effects of prostaglandins on osteoclast differentiation

The effect of prostaglandins (PGs) on osteoclast differentiation, an important point of control for bone resorption, is poorly understood. After an initial differentiation phase that lasts at least 4 days, murine monocytes, cocultured with UMR106 osteoblastic cells (in the presence of 1,25-dihydroxyvitamin D₃) give rise to tartrate-resistant acid phosphatase (TRAP) positive osteoclast-like cells that are capable of lacunar bone resorption. PGE₂ strongly inhibits TRAP expression and bone resorption in these cocultures. To examine further the cellular mechanisms associated with this inhibitory effect, we added PGE₂ to monocyte/UMR106 cocultures at specific times before, during, and after this initial 4-day differentiation period. To determine whether this PGE₂ inhibition was dependent on the type of stromal cell supporting osteoclast differentiation, we also added PGE₂ to cocultures of monocytes with ST2 preadipocytic cells. Inhibition of bone resorption was greatly reduced when the addition of PGE₂ to monocyte/UMR106 cocultures was delayed until the fourth day of incubation; when delayed until the seventh day, inhibition did not occur. PGE₂ inhibition of bone resorption was concentration-dependent and at 10⁻⁶ M was also mediated by PGE₁ and PGF_2α. In contrast to its effects on monocyte/UMR106 cocultures, PGE₂ stimulated bone resorption in monocyte/ST2 cocultures. Both ST2 cells and UMR106 cells were shown to express functional receptors for PGE₂. These results show that PGs strongly influence the differentiation of osteoclast precursors and that this effect is dependent not only on the type and dose of PG administered, but also on the nature of the bone-derived stromal cell supporting this process. Received: 12 October 1995 / Accepted: 1 April 1996     

Effect of low-dose infusion of prostaglandin E1 on vecuronium-induced neuromuscular blockade

The effect of low-dose (20 ng·kg⁻¹·min⁻¹) infusion of prostaglandin E₁ (PGE₁) on vecuronium-induced neuromuscular blockade was studied. The study population consisted of 24 elderly patients (65–75 years old) and 24 younger adult patients (25–56 years old). They were randomly assigned to the control and PGE₁ groups. The steady-state dose requirement (SSDR) of vecuronium was derived from ondemand infusion of the drug which produced a stable twitch height of 20% of its baseline reading, and recovery time after steady-state infusion was defined as the time for recovery from twitch height from 25% to 75%. The patients in the PGE₁ group received an infusion of PGE₁ 20 ng·kg⁻¹·min⁻¹, while those in the control group received an infusion of normal saline. The SSDR (23.2±9.1 and 34.2±5.9 μg·kg⁻¹. hr⁻¹, respectively;P=0.02) was significantly less and the recovery time (35.0±9.5 and 19.9±4.2 min, respectively;P=0.01) was significantly longer in the elderly than in the younger patients. However, low-dose infusion of PGE₁ significantly increased the SSDR (23.2±9.1 to 37.4±3.7 μg· kg⁻¹·hr⁻¹;P=0.01) and shortened the recovery time (35.0±9.5 to 23.5±4.0 min;P=0.02) in elderly patients. We concluded that low-dose infusion of PGE₁ is effective in preventing the prolonged action of vecuronium in elderly patients.     

Hormonal and nonhormonal desensitization in isolated bone cells

Summary Prior exposure to PTH markedly decreased the responsiveness of isolated, cultured bone cells to the stimulatory effect of the hormone on cyclic AMP formation. This process of desensitization developed within 30 min, persisted during prolonged incubation of the cells in PTH-free medium, and could not be attributed to enhanced excretion of cyclic AMP from the cells, nor to the extracellular accumulation of an inhibitor of PTH action. Adenylate cyclase activity in a subcellular fraction derived from PTH-treated cells was refractory to PTH and to sodium fluoride. These results indicate that PTH-mediated desensitization reflects, at least in part, impaired cyclic AMP formation. Adenosine and PGE₂, known stimulators of bone cell cyclic AMP formation, elicited agonist-specific desensitization, and also desensitized bone cells to the effects of subsequently added PTH. PTH blunted the cellular response to adenosine, but not to PGE₂. Modest refractoriness to PTH was evident in cells that had been treated previously with the cyclic AMP phosphodiesterase inhibitors IBMX, theophylline, and Bt₂cAMP, whereas treatment with sodium butyrate had no effect. The actions of the inhibitors, like that of PTH, were rapid in onset and long-lasting. Desensitization caused by previous treatment with the phosphodiesterase inhibitors, and with PTH itself, was accompanied by enhanced phosphodiesterase activity in bone cell homogenates. Induction of phosphodiesterase activity may well contribute to desensitization in the bone cell system. Abbreviations used in this paper: HBSS, Hank's balanced salt solution; EBSS, Earle's balanced salt solution; IBMX, 3 isobutyl 1 methyl xanthine; PTH, synthetic bovine parathyroid hormone (amino acids 1-34); PGE₁, prostaglandin E₁; PGE₂, prostaglandin E₂; Bt₂cAMP, N⁶,O²′ dibutyryl cyclic 3′,5′ adenosine monophosphate.     

Monocytes mediate osteoclastic bone resorption by prostaglandin production

Summary Monocytes are frequently found adjacent to active bone resorption surfaces in both physiological and pathological situations and may play a key role in bone resorption. There is strong circumstantial evidence that monocytes are precursors for osteoclasts in vivo, and recently they have been shown to resorb devitalized bone directly. The present study shows that monocytes can also resorb bone by stimulation of osteoclasts. Live fetal rodent bones prelabeled with⁴⁵Ca and cultured for 48–96 h in the presence of human monocytes or monocyte-conditioned medium released 80% more mineral than bones cultured in control medium. Bone matrix sustained comparable resorption as demonstrated by a 2-fold decrement in the extracted dry weights of the bones cultured in monocyte-conditioned medium. Histological examination of the bones cultured with monocytes or monocyte-conditioned medium showed increased osteoclast number and activity when compared with bones cultured in control medium. Known inhibitors of osteoclastic activity (phosphate 6 × 10⁻³M, cortisol 10⁻⁶M, and calcitonin 50 mU/ml) inhibited monocyte-conditioned medium-mediated bone resorption. The monocyte-conditioned medium contained sufficient prostaglandin E to account for the bone resorption. Indomethacin 10⁻⁵M added to the monocyte cultures blocked monocyte-conditioned media-induced bone resorption and prostaglandin release. These experiments suggest that monocytes stimulate osteoclastic bone resorption by prostaglandin production. Monocyte-induced bone resorption is partly reversed by inhibitors of osteoclast function. Monocyte-induced osteoclastic bone resorption may play an important role in physiologic bone remodeling and in bone destruction that occurs in chronic inflammatory diseases such as rheumatoid arthritis and periodontal disease.     

Anabolic effect of phosphogenistein and phosphodaidzein on bone components in rat femoral-metaphyseal tissues in vitro

The effect of phosphogenistein and phosphodaidzein, which are phosphorylated for the hydroxyl group (OH) at the 7-position of genistein and daidzein, on bone components was investigated. Femoral-metaphyseal tissues obtained from male rats (4 weeks old) were cultured for 24–72 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The presence of phosphogenistein (10⁻⁵ M) caused a significant increase in calcium content, alkaline phosphatase activity, and deoxyribonucleic acid (DNA) content in bone tissues cultured for 24 h. Phosphodaidzein (10⁻⁵ M) significantly elevated bone calcium and DNA content. These effects were completely prevented by the presence of cycloheximide (10⁻⁶ M), an inhibitor of protein synthesis. When femoral-metaphyseal tissues were cultured for 48 h in the presence of parathyroid hormone (1–34) (PTH; 10⁻⁸ M) or prostaglandin E₂ (PGE₂; 10⁻⁶ M), bone calcium content was significantly decreased. This decrease was significantly blocked by the presence of phosphogenistein (10⁻⁶ and 10⁻⁵ M) or phosphodaidzein (10⁻⁶ and 10⁻⁵ M). The presence of PTH (10⁻⁸ M) or PGE₂ (10⁻⁶ M) caused a significant increase in glucose consumption and lactic acid production by bone tissues. These increases were significantly inhibited by the presence of phosphogenistein (10⁻⁵ M) or phosphodaidzein (10⁻⁵ M), indicating their inhibitory effect on bone resorption. The present study has demonstrated that both phosphogenistein and phosphodaidzein have an anabolic effect on bone metabolism in rat femoral-metaphyseal tissues in vitro. Received: June 13, 2001 / Accepted: November 22, 2001     

1,25-dihydroxyvitamin D3 causes an increase in the number of osteoclastlike cells in cat bone marrow cultures

Summary The direct effect of 1,25(OH)₂D₃ upon osteoclast formation from precursor cells is still unknown. In the present experiments we have tested the effects of 1,25(OH)₂D₃ on the generation of osteoclastlike cells in cat bone marrow cultures. These cultures contain proliferating nonattached mononuclear cells and precursor cells that subsequently attach to the culture flask surface and then fuse to form multinucleated osteoclastlike cells. After 7 days of culture we separated the nonattached precursor cells from the attached cells and studied the effects of 1,25(OH)₂D₃ (10⁻¹⁰ M–10⁻⁸ M) on multinucleated cell formation in these two cell populations. In cultures derived from the non-attached precursor cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) resulted in a 180% increase in the number of attached mononuclear cells and a 90% increase in the number of nuclei contained within multinucleated cells. These effects were dose-dependent. 1,25(OH)₂D₃ did not have a consistent effect on the number of nonattached precursor cells. In cultures derived from attached cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) induced a 50% increase in the number of mononuclear attached cells and a 40% increase in the number of nuclei within polykaryons. The most likely explanation for these results is that 1,25(OH)₂D₃ promotes the differentiation and subsequent adhesion of nonattached precursor cells, stimulates proliferation of attached mononuclear precursor cells, and possibly stimulates fusion of these attached precursor cells.     

Effect of Vitamin D deficiency on macrophage and lymphocyte function in the rat

Summary Vitamin D deficiency has pronounced growth retardation effects on the skeletal system. Because the immune system has been implicated in the regulation of bone metabolism, we examined the effect of vitamin D deficiency on the functional development of immune function in a rachitic rat model. Rats deprived of vitamin D₃ bothin utero and in postnatal life (−/−) had significantly reduced thymocyte or splenocyte [³H]-thymidine incorporation to mitogens and decreased macrophage chemotaxis when compared with vitamin D₃-sufficient rats (+/+). Rats that were deficient in vitamin D₃ only duringin utero development (−/+) or during postnatal life (+/−) tended to have [³H]-thymidine incorporation levels that were intermediate to those of the −/− and +/+ group. Similarly, the chemotactic response of macrophages from the +/− and −/+ groups was intermediate to that of the −/− and +/+ group, except at high concentrations of C5a in which there was an overlap with the +/+ group. Interestingly, secretion of soluble mediators, including interleukin 2 by lymphocytes and interleukin 1 and PGE₂ by macrophages, was unaffected by vitamin D deficiency. These results suggest that vitamin D₃ is essential for the normal development of certain biological responses of lymphocytes and macrophages. Moreover, this rachitic rat model system will enable further evaluation of the role of vitamin D in the functional development of the cells of the immune system and their relationship to skeletal growth.     

Increase in femoral bone mass by ipriflavone alone and in combination with 1α-hydroxyvitamin D3 in growing rats with skeletal unloading

We assessed the possibility that ipriflavone treatment might result in bone restoration in immobilized rats. We also investigated the effect of combined treatment with ipriflavone and vitamin D₃ on the bone. Male Sprague-Dawley rats, 6 weeks of age, were subjected to unilateral sciatic neurectomy. Three weeks after the operation, ipriflavone (100 mg/kg), 1α-hydroxyvitamin D₃ [1α(OH)D₃, 25 ng/kg], or both ipriflavone and 1α(OH)D₃ were orally administered every day for 12 or 24 weeks. After 12 weeks of treatment, only the group receiving combined treatment with ipriflavone and 1α(OH)D₃ showed increases in total femur calcium content (+16.4%, compared with the control). After 24 weeks, both animals treated with ipriflavone alone and those that had received the combination of ipriflavone and 1α(OH)D₃ showed significant increases in femur calcium content (+18.0% and +23.8%, respectively). In these treatment groups, X-ray analysis revealed an increase in bone mineral density over the entire length of the femur, and an increase in cortical diameter at the midshaft without affecting medullary width. Administration of 1α(OH)D₃ (25 ng/kg) alone had no effect. Body weight, femur length, and serum markers of calcium and bone metabolism were not affected in any group. We evaluated the relationship between ipriflavone and vitamin D₃ in bone cells in a culture system using rat bone marrow stromal cells in which the cells subsequently form mineralized bone-like tissue. Continuous treatment with ipriflavone (10⁻⁵ M) for 21 days resulted in an increase in osteocalcin secretion, and enhanced its response to 1α,25-dihydroxyvitamin D₃ (10⁻¹¹ M-10⁻⁸ M). These findings indicate that ipriflavone treatment increases the femoral bone mass in immobilized rats. In addition, a low dose of 1α(OH)D₃, which did not induce hypercalcemia, in combination with ipriflavone, augmented the stimulatory effect of ipriflavone alone on the bone mass, possibly due to a direct effect of each agent on osteoblastic cells.     

Vitamin D3 and avian bonein vitro: Specificity of effect on Japanese quail calvaria

Summary Calcitriol (1,25(OH)₂D₃) has been shown, under certain conditions, to elicit anin vitro response in adult avian calvarium which may be interpreted as calcium uptake by the bone. The present investigation was undertaken to study the specificity of this response. Calvaria were removed from 6-week-old female Japanese quail and cultured for periods of up to 96 hours at 37°C in 5% CO₂/95% air. 1,25(OH)₂D₃ induced a fall in the medium total and ionic calcium concentrations at both 48 hours and 96 hours of incubation; these responses were not blocked by the presence of 10⁻⁴ M acetazolamide. Bovine parathyroid hormone (bPTH (1–34)) at 10⁻⁷ M, and dibutyryl cyclic AMP (DBcAMP) at 10⁻⁴M, had no effect on the medium calcium. In contrast, forskolin at 10⁻⁴ M induced a marked fall in medium calcium concentrations, particularly at 48 hours. The specificity was also studied with respect to vitamin D₃ and its two major metabolites. 1,25(OH)₂D₃ exhibited a bellshaped dose-response relationship with the maximal effect at 10⁻⁷ M. In contrast, the other two compounds elicited no effects at 10⁻⁷ M or 10⁻⁶ M; significant responses were observed at 10⁻⁵ M with both agents. In general, 25-dihydroxyvitamin D₃ (25OHD₃) was more potent than vitamin D₃. These findings suggest that the medium calcium response to 1,25(OH)₂D₃, interpreted as calcium uptake by the cultured adult avian bone, is relatively specific among calcemic agents; the response was elicited by forskolin but not by bPTH(1–34) or DBcAMP. The potency ratio exhibited by the vitamin D₃ analogs (1,25(OH)₂D₃>25OHD₃>vitamin D₃) reinforces the specificity claim.     

Comparison of the mechanisms of bone resorption induced by 1α,25-dihydroxyvitamin D3 and lipopolysaccharides

Summary The mechanisms of increase in bone resorption induced by 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] and bacterial lipopolysaccharides (LPS) were compared in anin vitro dead bone assay and a living bone assay. 1α,25(OH)₂D₃ at concentrations of 0.05–5 ng/ml dose-dependently enhanced the ability of alveolar macrophages to release⁴⁵Ca from prelabeled dead bone particles (dead bone assay). In addition, the vitamin promoted fusion of the macrophages to form multinucleated cells and also enhanced glucose consumption, a marker of activation of macrophages. LPS at 0.05–5 μg/ml similarly enhanced the release of⁴⁵Ca from the dead bone particles and glucose consumption by alveolar macrophages, but it did not induce fusion of the cells at any concentration. Both 1α,25(OH)₂D₃ and LPS dose-dependently stimulated the release of⁴⁵Ca from fetal mouse calvaria prelabeled with⁴⁵Ca (living bone assay). Compared to control bone, there were several times as many osteoclasts per given length of trabecular bone surface in calvaria treated for 5 days with either 5 ng/ml of 1α,25(OH)₂D₃ or 5 μg/ml of LPS. Indomethacin (10⁻⁵ M) completely inhibited the LPS-induced increase of osteoclasts, but not the 1α,25(OH)₂D₃-induced increase. These results suggest that 1α,25(OH)₂D₃ and LPS similarly stimulate bone resorption by activating macrophages as well as by promoting fusion of precursor cells to form multinucleated cells. 1α,25(OH)₂D₃ induced formation of multinucleated cells with bone-resorbing activity directly, whereas LPS appeared to induce multinucleated cells through prostaglandin synthesis by some other types of cells present in living bone tissues.