Regulation of osteoblast growth by interactions between transforming growth factor-beta and 1alpha,25-dihydroxyvitamin D3

Osteoblast growth and differentiation encompass a series of events including proliferation, changes in cell shape, and expression of the markers specific for osteoblast phenotype. Both transforming growth factor-beta (TGF-beta) and 1alpha,25-dihydroxyvitamin D3 (1alpha,25[OH]2D3) are effective in regulating osteoblast proliferation, differentiation, bone matrix maturation and cell-specific gene expression. Although there is some degree of controversy regarding the influences on osteoblasts in vitro, it is generally agreed that TGF-beta stimulates osteoblast proliferation and growth, and inhibits the expression of the markers characteristic of the osteoblast phenotype such as osteocalcin. In contrast, 1alpha,25(OH)2D3 causes inhibition of the proliferation of osteoblasts, arrests their growth, and stimulates expression of specific markers. In many studies, complex interactions have been demonstrated between TGF-beta and 1alpha,25(OH)2D3 modulating their receptor expression, synthesis, and effects on osteoblast-specific gene expression. The cooperative actions of TGF-beta and 1alpha,25(OH)2D3 can be synergistic or antagonistic. It has recently been established that Smad proteins that transduce signals downstream the TGF-beta stimulation may mediate the crosstalk between TGF-beta and 1alpha,25(OH)2D3 signaling. Future studies should focus on the explanation of the molecular basis of these interactions and the in vivo consequences of the regulation of osteoblast growth and differentiation by TGF-beta and 1alpha,25(OH)2D3.     

Integrin beta1 silencing in osteoblasts alters substrate-dependent responses to 1,25-dihydroxy vitamin D3

Surface microroughness increases osteoblast differentiation and enhances responses of osteoblasts to 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. The observations that beta1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and that it is regulated by 1alpha,25(OH)2D3 in a surface-dependent manner, suggest that beta1 may play a role in mediating osteoblast response. To test this hypothesis, we silenced beta1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA) and examined the responses of the beta1-silenced osteoblasts to surface microtopography and 1alpha,25(OH)2D3. To better understand the role of beta1, MG63 cells were also treated with two different monoclonal antibodies to human beta1 to block ligand binding. beta1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, beta1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)2D3. Anti beta1 antibody AIIB2 had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z caused dose-dependent decreases in cell number and alkaline phosphatase and an increase in osteocalcin. Effects of 1alpha,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)2D3. The results also show that beta1 mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)2D3.     

Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1 alpha,25-dihydroxyvitamin D3 have ruffled borders and resorb dentine

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1 alpha,25(OH)2D3 were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that 1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1 alpha,25(OH)2D3 exhibit non-functional basic features of osteoclast morphology, and 2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.     

Steroid hormone action in musculoskeletal cells involves membrane receptor and nuclear receptor mechanisms

Steroid hormones regulate target cells through traditional nuclear mechanisms as well as by membrane mechanisms. 1alpha,25(OH)2D3 and 24R,25(OH)2D3 bind membrane receptors (mVDR) and mediate their effects on the physiological responses of musculoskeletal cells via protein kinase C (PKC). In cultures of costochondral growth plate chondrocytes, 1alpha,25(OH)2D3 binds the 1,25-mVDR in growth zone cells, activating phospholipase C (PLC), leading to diacylglycerol (DAG) production and PKC translocation to the plasma membrane. It also activates PLA2, increasing arachidonic acid release and prostaglandin synthesis. 24R,25(OH)2D3 binds its membrane receptor in resting zone chondrocytes, activating phospholipase D (PLD), and increasing DAG and PKC activity, but translocation does not occur. PLA2 activity is decreased, reducing arachidonic acid and prostaglandin production. 17Beta-estradiol (E2) activates PKC in both cartilage cells, but DAG is not involved. 1alpha,25(OH)2D3 and 24R,25(OH)2D3 also increase PKC in osteoblasts in a cell-specific manner. Antibodies to the 1,25-mVDR block PKC activation. Membrane-mediated events influence gene expression via signaling cascades, including the ERK1/2 MAP kinases. The ability of steroid hormones to initiate events nongenomically is important for regulation of matrix vesicle (MV) function in the extracellular matrix. MVs have mVDRs, but ligand binding inhibits PKC-zeta (PKCzeta) via a mechanism that differs from PKCalpha activation in the plasma membranes. Treatment of MVs from growth zone chondrocyte cultures with 1alpha,25(OH)2D3 releases stromelysin-1 (MMP-3) and increases TGF-beta activation. MMP-3 is also involved in proteoglycan degradation, facilitating calcification. 24R,25(OH)2D3 inhibits PKCzeta in MV from resting zone cell cultures and inhibits MMP-3 release. Chondrocytes and osteoblasts produce 1,25(OH)2D3, 24,25(OH)2D3, and E2; thus, locally produced steroids may function as autocrine regulators of matrix events, including matrix vesicle enzyme activity and matrix protein remodelling during longitudinal growth, calcification, and growth factor activation.     

Nonclassical effects of 1alpha,25-dihydroxyvitamin D(3) and its analogs

The activated form of vitamin D(3), 1alpha,25(OH)(2)D(3), not only plays a central role in bone and calcium metabolism but has also potent antiproliferative and prodifferentiating effects. Moreover, the combined presence of 25(OH)D(3)-1alpha-hydroxylase as well as the vitamin D receptor in several tissues introduced the idea of a paracrine role for 1alpha,25(OH)(2)D(3). By introducing chemical modifications into the flexible parent molecule 1alpha,25(OH)(2)D(3), a whole generation of vitamin D analogs was created. Due to a clear dissociation of the antiproliferative and prodifferentiating effects from calcemic effects, these analogs can be used not only for the treatment of bone disorders but also for non-classical applications.     

1alpha,25-Dihydroxyvitamin D3 modulates the murine antibody response to pneumococcal capsular polysaccharide serotype 3 through IL-12

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is a steroid hormone that regulates calcium metabolism. Besides, 1alpha,25(OH)(2)D(3 )also has pronounced immunomodulatory effects: it strongly inhibits dendritic cell (DC) maturation and impairs IL-12 production. We studied the effect of 1alpha,25(OH)(2)D(3 )on the antibody response to pneumococcal capsular polysaccharide (caps-PS) serotype 3. 1alpha,25(OH)(2)D(3) inhibited the IgG2a antibody response to caps-PS serotype 3. Besides, 1alpha,25(OH)(2)D(3) also inhibited IL-12 production and maturation of DC. Anti-IL-12 and exogenous IL-12, respectively, inhibited and stimulated the IgG2a antibody response to caps-PS serotype 3. Exogenous IL-12 abrogated the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3, indicating that the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3 was mediated through IL-12. In conclusion, we demonstrate that 1alpha,25(OH)(2)D(3) has an inhibitory effect on the IgG2a antibody response to caps-PS serotype 3, and that this effect was mediated trough IL-12.     

Osteoblastic NF-κB pathway is involved in 1α, 25(OH)2D3-induced osteoclast-like cells formation in vitro

1α, 25-dihydroxyvitamin D₃ (1α, 25(OH)₂D₃) acts on the osteoblasts to enhance the expressions of receptor activator of nuclear factor κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) and induce the formation of osteoclasts. However, the mechanism in osteoblasts by which 1α, 25(OH)₂D₃ promotes osteoclastogenesis has not yet been completely understood. This study aimed to select the first generation of murine osteoblasts to explore the underlying mechanism of 1α, 25(OH)₂D₃-induced osteoclastic formation from bone marrow mononuclear cells (BMMNCs). We discovered the activation of osteoblastic NF-κB pathway under 10^-8 mol/L 1α, 25(OH)₂D₃ treatment, as evidenced by the transfer of NF-κB p65 from cytoplasm to nuclei. Then, the NF-κB p65-siRNA was designed, constructed, and transfected into osteoblastic cells. Immunofluorescence assay confirmed the successfully silenced NF-κB p65 gene in osteoblasts. In the co-culture system of osteoblasts and BMMNCs with 1α, 25(OH)₂D₃ added, the multinucleated osteoclast-like cells containing 2-3 nuclei were observed in BMMNCs co-cultured with non-transfection osteoblasts, conversely, silencing osteoblastic NF-κB p65 resulted in failed differentiation of BMMNCs along with substantial vacuolar degeneration in cytoplasm. In addition, the expressions of RANKL and M-CSF were notably decreased in NF-κB p65-silenced osteoblasts. Taken together, our data indicated that osteoblastic NF-κB pathway was involved in 1α, 25(OH)₂D₃-induced osteoclast-like cells formation from BMMNCs through regulating the expression of RANKL and M-CSF. Therefore, our findings further identified the mechanism of 1α, 25(OH)₂D₃-induced osteoclastogenesis on the basis of prior studies.     

Distribution of metabotropic glutamate receptors in the parabrachial and Kölliker-Fuse nuclei of the rat

In the present study, we analysed the distribution and cellular localization of metabotropic glutamate receptors (1alpha, 2/3, 5) in parabrachial and K?lliker-Fuse nuclei using subtype-specific antisera. Immunolabelling revealed that different nuclei express different sets of metabotropic glutamate receptors. Metabotropic glutamate receptor la immunoreactivity was found in the K?lliker-Fuse nucleus and in several parabrachial nuclei, including the waist area, lateral crescent, medial, external medial and ventral lateral nuclei. The external lateral and internal lateral parabrachial nuclei were devoid of metabotropic glutamate receptor 1alpha immunoreactivity. Metabotropic glutamate receptor 5 immunoreactivity was observed in the K?lliker-Fuse and in the medial parabrachial nuclei, while in the remaining nuclei the staining was very weak. Again, the external lateral nucleus was devoid of metabotropic glutamate receptor 5 immunoreactivity. The metabotropic glutamate receptor 2/3 antisera stained all lateral parabrachial nuclei as well as the K?lliker-Fuse nucleus, while staining in the medial parabrachial nucleus was weak. Metabotropic glutamate receptor 1alpha immunoreactivity was observed on presumed dendritic profiles, while metabotropic glutamate receptor 5 immunoreactivity was found predominantly on neuronal cell bodies. Metabotropic glutamate receptor 2/3 immunoreactivity was present as a fine, punctate immunostaining in the neuropil. Our data suggest that glutamate release in the parabrachial and K?lliker-Fuse nuclei might induce a variety of second messenger cascades, as indicated by the presence or absence of certain types of metabotropic glutamate receptors.     

Osteoblast response to titanium surface roughness and 1alpha,25-(OH)(2)D(3) is mediated through the mitogen-activated protein kinase (MAPK) pathway

When osteoblasts are cultured on surfaces of increasing microroughness, they exhibit decreases in proliferation, increases in differentiation and local factor production, and enhanced response to 1alpha,25(OH)(2)D(3). The cells interact with surfaces through integrins, which signal by the same pathways used by 1alpha,25(OH)(2)D(3), including protein kinase C via phospholipase C and protein kinase A via phospholipase A(2). This provides opportunities for crosstalk that may contribute to the synergistic effects of surface roughness and the vitamin D metabolite. Because these pathways converge at mitogen-activated protein kinase (MAPK), we tested the hypothesis that the extracellular signal-regulated kinase (ERK1/2) subclass of MAPKs mediates the effects of surface roughness and 1alpha,25(OH)(2)D(3). MG63 osteoblast-like osteosarcoma cells were cultured on commercially pure Ti disks with various surface roughnesses: pretreatment (PT; 0.6 microm average roughness [Ra]), coarse grit-blasted and acid-etched (SLA; 4 microm RA), and titanium plasma-sprayed (TPS; 5.2-microm R(a)). At confluence, cells were treated for 24 h with control media or media containing 10(-7) M 1alpha,25(OH)(2)D(3). One-half of the cultures received 1 microm or 10 microm PD98059, a specific inhibitor of the ERK family of MAPKs. PD98059 alone did not affect proliferation, osteocalcin production, or production of transforming growth factor-beta1 or nitric oxide, regardless of the surface roughness. Alkaline phosphatase was reduced by the inhibition of the ERK family kinases on all surfaces to a comparable extent. However, when PD98059 was added to the cultures with 1alpha,25(OH)(2)D(3), the effects of the seco-steroid were blocked, including the synergistic increases seen in MG63 cells cultured on SLA or TPS. These results indicate that ERK1/2 MAPK is required for the maintenance of alkaline phosphatase at control levels and that the effects of 1alpha,25(OH)(2)D(3) are mediated by ERK1/2. However, the effects of surface roughness are not due to the ERK family of MAPKs. This suggests that alternative pathways may be used, including those mediated by other MAPK subclasses.     

Interactions of 1 alpha,25-dihydroxyvitamin D3 with IL-12 and IL-4 on cytokine expression of human T lymphocytes

BACKGROUND: 1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25[OH](2)D(3)) exerts its effects on the immune system, particularly through suppression of T helper/cytotoxic cell 1 (T(H)/T(C)1)-mediated reactions, although direct actions of 1 alpha,25(OH)(2)D(3) on human T lymphocytes have not yet been studied in detail. OBJECTIVE: We evaluated the effect of 1 alpha,25(OH)(2)D(3) on basal and cytokine-driven T-cell functions at the single-cell level. METHODS: We used 4-color flow cytometry for simultaneous detection of intracellular cytokines in CD4(+) and CD8(+) human PBMCs that had been cultured in the presence of 1 alpha,25(OH)(2)D(3) singly or in combination with either IL-12 or IL-4. According to the exploratory nature of these investigations, the Bonferroni correction was not applied for data analysis and presentation. RESULTS: 1 alpha,25(OH)(2)D(3) had little effect on T(H)/T(C)1 cytokines but significantly inhibited IL-12-induced IFN-gamma production. Constitutive synthesis of T(H)/T(C)2-related cytokines was also only modestly affected by 1 alpha,25(OH)(2)D(3) alone. When T(H)/T(C)2 differentiation was induced by IL-4, 1 alpha,25(OH)(2)D(3) significantly expanded the percentages of IL-4(+) and IL-13(+) cells. However, the predominant effect of 1 alpha,25(OH)(2)D(3) on T lymphocytes, particularly in the presence of IL-4, was the induction of separate CD4(+) and CD8(+) subpopulations with almost exclusive expression of IL-6. This might be an important facet of the immunomodulatory action of 1 alpha,25(OH)(2)D(3) because IL-6 might act in parallel with 1 alpha,25(OH)(2)D(3) in modulation of T(H)/T(C) effector cell functions. CONCLUSIONS: Our data imply that the specific actions of 1 alpha,25(OH)(2)D(3) on cytokine-stimulated T-cell functions could play a role in the prevention of T(H)/T(C)1-related autoimmune diseases but also predispose toward T(H)/T(C)2-mediated allergic reactions.     

1Alpha,25-dihydroxyvitamin D3 restores thymocyte apoptosis sensitivity in non-obese diabetic (NOD) mice through dendritic cells

AIMS/HYPOTHESIS: Resistance of NOD thymocytes to apoptosis-inducing signals is restored by 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3), a therapy preventing diabetes in NOD mice. We studied whether modulation of thymocyte apoptosis is due to direct effects on thymic T lymphocytes or indirect effects via thymic dendritic cells, since both cell types constitute known targets for 1alpha,25OH2D3. METHODS AND RESULTS: Female NOD mice were treated with 1alpha,25OH2D3 (5microg/kg/2d) from 21 to 70 days. Vehicle-treated NOD and NOR mice served as controls. Analysis of thymic T lymphocytes from 1alpha,25OH2D3)-treated mice revealed a decrease in number of apoptosis-resistant CD4+CD8+ and CD4+CD8-HSA(high) T lymphocyte subsets, higher pro-apoptotic IL-2 and FasL, and lower anti-apoptotic Bclx-L mRNA expression levels. Thymic dendritic cells from 1alpha,25OH2D3-treated NOD mice had increased CD8alpha+FasL+ and CD80+/86+ expression compared to control NOD mice. In a syngeneic co-culture system of thymocytes and thymic dendritic cells, apoptosis levels were 20% higher only in co-cultures where both T cell- and dendritic cell-compartments originated from 1alpha,25OH2D3-treated mice. Activation-induced cell death-sensitivity in peripheral T lymphocytes was comparable to levels present in NOR mice, confirming better thymic selection in 1alpha,25OH2D3-treated mice. CONCLUSION/INTERPRETATION: We conclude that 1alpha,25OH2D3 needs both thymic T cell- and dendritic cell-compartments to exert its apoptosis-restorative effects in NOD thymocytes.     

Steroid Hormone Action in Musculoskeletal Cells Involves Membrane Receptor and Nuclear Receptor Mechanisms

Steroid hormones regulate target cells through traditional nuclear mechanisms as well as by membrane mechanisms. 1 &#102 ,25(OH) 2 D 3 and 24R,25(OH) 2 D 3 bind membrane receptors (mVDR) and mediate their effects on the physiological responses of musculoskeletal cells via protein kinase C (PKC). In cultures of costochondral growth plate chondrocytes, 1 &#102 ,25(OH) 2 D 3 binds the 1,25-mVDR in growth zone cells, activating phospholipase C (PLC), leading to diacylglycerol (DAG) production and PKC translocation to the plasma membrane. It also activates PLA 2 , increasing arachidonic acid release and prostaglandin synthesis. 24R,25(OH) 2 D 3 binds its membrane receptor in resting zone chondrocytes, activating phospholipase D (PLD), and increasing DAG and PKC activity, but translocation does not occur. PLA 2 activity is decreased, reducing arachidonic acid and prostaglandin production. 17 &#103 -Estradiol (E 2 ) activates PKC in both cartilage cells, but DAG is not involved. 1 &#102 ,25(OH) 2 D 3 and 24R,25(OH) 2 D 3 also increase PKC in osteoblasts in a cell-specific manner. Antibodies to the 1,25-mVDR block PKC activation. Membrane-mediated events influence gene expression via signaling cascades, including the ERK1/2 MAP kinases. The ability of steroid hormones to initiate events nongenomically is important for regulation of matrix vesicle (MV) function in the extracellular matrix. MVs have mVDRs, but ligand binding inhibits PKC-zeta (PKC &#145 ) via a mechanism that differs from PKC &#102 activation in the plasma membranes. Treatment of MVs from growth zone chondrocyte cultures with 1 &#102 ,25(OH) 2 D 3 releases stromelysin-1 (MMP-3) and increases TGF- &#103 activation. MMP-3 is also involved in proteoglycan degradation, facilitating calcification. 24R,25(OH) 2 D 3 inhibits PKC &#145 in MV from resting zone cell cultures and inhibits MMP-3 release. Chondrocytes and osteoblasts produce 1,25(OH) 2 D 3 , 24,25(OH) 2 D 3 , and E 2 ; thus, locally produced steroids may function as autocrine regulators of matrix events, including matrix vesicle enzyme activity and matrix protein remodelling during longitudinal growth, calcification, and growth factor activation.     

Effect of Clostridium difficile enterotoxin A on ultrastructure of Chinese hamster ovary cells.

Electron microscopical immunocytochemistry and light microscopy were used to study the effect of Clostridium difficile enterotoxin A (EA) on cultured Chinese hamster ovary (CHO) cells. At 4 degrees C, immunocytochemically detected EA was randomly distributed along the plasma membrane; when cells were subsequently transferred to 37 degrees C, the EA moved into coated pits and coated vesicles within 2 min. Within 2 h of incubation at 37 degrees C with EA, the perinuclear cytoplasm of the CHO cells became highly refractile, and in 4 h, most of the cells were round; however, the majority of rounded cells excluded erythrosin B while remaining firmly attached to the culture dish plastic. When EA was removed from the cultured cells within 2 h, the cells returned to a normal morphology and formed a confluent monolayer. The nuclei of rounded cells were irregularly shaped; cytoplasmic intermediate filaments were collapsed toward the nucleus. Long bundles of parallel, 11-nm-diameter filaments appeared in the nuclei after 3 h of incubation with EA and disappeared by the fourth hour of incubation. Intoxicated cells did not undergo mitosis. Thus, EA was internalized, at least in part, by receptor-mediated endocytosis and subsequently affected the nuclei of CHO cells.     

1alpha,25(OH)2D3 regulation of integrin expression is substrate dependent

Osteoblasts are attachment-dependent cells that interact with their surface through integrin-mediated mechanisms. Their differentiation is regulated by 1,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D(3)] and is affected by substrate chemistry and microtopography, suggesting that 1alpha,25(OH)(2)D(3) may regulate integrin expression in a surface-specific manner. To test this hypothesis, osteoblast-like human MG63 cells were grown on tissue culture plastic and on grit-blasted and acid-etched titanium disks with a complex microtopography to induce osteoblast differentiation. Expression of alpha(2), alpha(5), alpha(v), beta(1), and beta(3) integrins were quantified by real-time polymerase chain reaction (PCR) as a function of time in culture and treatment with 1alpha,25(OH)(2)D(3). Results were correlated with expression of osteocalcin, a marker of a differentiated osteoblast. Osteocalcin mRNA increased with time and 1alpha,25(OH)(2)D(3) treatment and these changes were greater in cultures on the titanium disks. Integrin expression varied with time in culture and this was also surface dependent. At each time point, beta(1) and alpha(2) mRNAs were greater on titanium than on plastic, whereas alpha(5) expression was reduced and alpha(v),beta(3) expression was unaffected. 1alpha,25(OH)(2)D(3) increased beta(1) mRNA on both surfaces at all time points, but it increased alpha(2) expression only in 8-d cultures. 1alpha,25(OH)(2)D(3) caused reduced alpha(5) expression only in cultures grown on plastic for 8 d, and had no effect on either alpha(v) or beta(3) expression regardless of surface. These results show that integrin expression in human osteoblast-like cells is differentially modulated by 1alpha,25(OH)(2)D(3) in a time-dependent manner that is sensitive to the surface on which the cells are grown.     

Age-dependent stimulation or inhibition of calcium release from bone cultures by 1 alpha, 25-dihydroxyvitamin D3

The effect of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3) on calcium release from neonatal and young adult mouse calvaria in tissue culture was studied. At concentrations from 2.6 X 10(-6) M to 2.6 X 10(-9) M, 1 alpha 25-(OH)2D3 enhanced calcium release from 5-day-old calvaria. At the same concentrations, 1 alpha, 25-(OH)2D3 either inhibited or failed to enhance calcium release from 75-day-old calvaria. Parathyroid hormone-enhanced calcium release from 75-day-old calvaria was inhibited considerably by 1 alpha, 25-(OH)2D3 at 2.6 X 10(-6) M and 2.6 X 10(-7) M. These findings suggest that cells involved in the bone resorption process may respond differently to the same stimulus at different stages in the development of the organism. This direct inhibitory effect of 1 alpha, 25-(OH)2D3 on bone resorption in cultured calvaria of older animals may explain, in part, its beneficial effect in the treatment of osteoporosis.     

Influence of estrogen on renal vitamin D hydroxylases and serum 1alpha,25-(OH)2D3 in chicks.

The influence of estrogen on the metabolism of 25-hydroxyvitamin D3 was studied in 2- to 5-wk-old chicks. Single injections of at least 500 microgram diethylstibestrol (DES) increased the conversion of 25-hydroxyvitamin D3 to 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) and suppressed the production of 24,25-dihydroxyvitamin D3 in chick kidney homogenates. Acute (one 5-mg) injections of testosterone or progesterone did not enhance the 25-hydroxyvitamin D3-1alpha-hydroxylase, indicating specificity. However, chronic pretreatment with DES appeared to allow the potentiation of previously unstimulatory steroids such as progesterone and testosterone. In addition, the hormonal metabolite of vitamin D3, 1alpha,25-(OH)2D3, was measured in 4- to 6-wk chick plasma after steroid treatment. Greater than 1 mg DES per day for 5 days was necessary to enhance the circulating level of 1alpha,25-(OH)2D3; testosterone alone had no effect. This elevation was rapid, occurring within 12--24 h after injection. These data suggest that estrogen (as evidenced by DES treatment) is a modulator of vitamin D metabolism along with other known regulators such as parathyroid hormone, phosphate, and 1alpha,25-(OH)2D3. The mechanism of the regulation is as yet unknown.