Role of ceramide as a lipid mediator of 1 alpha,25-dihydroxyvitamin D3-induced HL-60 cell differentiation

The treatment of HL-60 myelocytic leukemia cells with 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) resulted in the activation of a neutral sphingomyelinase and in sphingomyelin turnover (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). In this paper, the effects of 1,25-(OH)2D3 on the product of sphingomyelin hydrolysis, ceramide, and the possible function of ceramide as a lipid mediator of the effects of 1,25-(OH)2D3 on HL-60 cell differentiation were investigated. Treatment of HL-60 cells with 1,25-(OH)2D3 resulted in a time- and dose-dependent increase in ceramide mass levels. Ceramide levels peaked at 2 h following treatment of HL-60 cells with 100 nM 1,25-(OH)2D3 with an increase of 41% over base line. The mass of generated ceramide (13 +/- 2 pmol/nmol of phospholipid) agreed with the mass of hydrolyzed sphingomyelin (17 +/- 4 pmol/nmol of phospholipid). Cell-permeable ceramides with shorter N-acyl chains induced HL-60 cell differentiation at subthreshold concentrations of 1,25-(OH)2D3. Higher concentrations of cell-permeable ceramides potently induced HL-60 cell differentiation independent of 1,25-(OH)2D3. A 2-h exposure of HL-60 cells to N-acetyl-sphingosine was sufficient to cause differentiation. Morphologically, N-acetylsphingosine caused a similar monocytic differentiation of HL-60 cells as did 1,25-(OH)2D3. Exogenous ceramide was further metabolized to sphingomyelin and other sphingolipids, but no conversion to sphingosine was detected. Moreover, sphingosine and its analogs failed to affect monocytic differentiation of HL-60 cells in response to subthreshold 1,25-(OH)2D3, indicating that the effect of ceramide was independent of sphingosine generation. These studies demonstrate that ceramide is a lipid mediator that may transduce the action of 1,25-(OH)2D3 on HL-60 cell differentiation.     

Evidence for the functional involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 in bone.

In the present study the involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast-like cells and in the stimulation of in vitro bone resorption by 1,25(OH)2D3 was examined. Incubation for 24 h with 1,25(OH)2D3 potently stimulated osteocalcin synthesis by ROS 17/2.8 cells. This stimulation was inhibited (30-70% inhibition) by 25 microM of the protein kinase C (PKC) inhibitors 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG) and sphingosine without affecting basal osteocalcin synthesis. 1,25(OH)2D3-stimulated osteocalcin secretion by nontransformed isolated fetal rat osteoblasts was also inhibited (30-55%) by AMG. Also, AMG inhibited 10(-9) M 1,25(OH)2D3-induced up-regulation of vitamin D receptor in ROS 17/2.8 cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) did not cause an increase in osteocalcin secretion, while only a small increase in cellular content of osteocalcin in ROS 17/2.8 cells was observed. Addition of PMA together with 1,25(OH)2D3 did not change the response to 1,25(OH)2D3. The PKC inhibitors were not toxic for the cells. 1,25(OH)2D3 did not stimulate diacylglycerol production in ROS 17/2.8 cells up to 5 min after administration. However, 4- and 24-h incubation with 10 nM 1,25(OH)2D3 increased phorbol ester binding in ROS 17/2.8 cells. 1,25(OH)2D3 potently stimulated bone resorption after 3 and 6 days of culture in fetal mouse long bones and calvaria. Both the PKC inhibitors AMG (25 microM) and staurosporine (50 nM) strongly inhibited (60-86% inhibition) 1,25(OH)2D3-stimulated bone resorption without affecting basal 45Ca release. These effects were not due to a cytotoxic effect of both PKC inhibitors. Nor is it likely that the effects of AMG and staurosporine are due to inhibition of cell proliferation as hydroxyurea did not affect 1,25(OH)2D3-stimulated bone resorption. The inhibition of 1,25(OH)2D3-stimulated bone resorption by PKC inhibitors suggests that besides osteocalcin synthesis PKC is also involved in other responses of 1,25(OH)2D3 in bone. 1,25(OH)2D3 does not directly activate PKC via an increase in diacylglycerol production but more likely via an increase in PKC. Together, the present study demonstrates a functional involvement of PKC in the action of 1,25(OH)2D3 in bone and bone cells which may have consequences for the development of 1,25(OH)2D3 analogs, e.g. with less hypercalcemic and relatively more antiproliferative activity.     

Magnesium deprivation inhibits the expression of differentiation-related phenotypes in human promyelocytic leukemia HL-60 cells

The role of magnesium ions in the differentiation of human promyelocytic leukemia HL-60 cells was investigated. When HL-60 extracellular magnesium was deficient (less than 0.01 mM), the total intracellular magnesium content and [3H] leucine incorporation rates decreased to 61 and 28%, respectively, on day 3. When the cells were treated with various inducers (100 nM 1 alpha, 25 dihydroxyitamine D3 (1,25(OH)2D3), 100 nM beta-all-trans retinoic acid (RA), 20 nM 12-o-tetradecanoyl phorbol-13-acetate (TPA), 1.25% dimethylsulfoxide (DMSO) and 30 nM aclacinomycin (AcM] in magnesium-deficient medium, the expression of differentiation-related phenotypes (nitroblue tetrazolium (NBT) reducing ability, nonspecific esterase (NSE) activity and monoclonal antibody, OKM1 binding activity) was almost completely inhibited. After a 2-day treatment with 100 nM 1,25(OH)2D3 in magnesium-deficient medium, the expression of differentiation-related phenotypes was restored by further incubation in the absence of inducer in standard magnesium medium (0.4 mM). These results suggested that magnesium deprivation inhibited the expression of HL-60 differentiation-related phenotypes but not their commitment to differentiation. These phenotypes were expressed without inducer in standard magnesium medium after a 2-day simultaneous treatment with 1,25(OH)2D3 and cyclohexamide (protein synthesis inhibitor) in magnesium-deficient medium, but not after simultaneous pretreatment with 1,25(OH)2D3 and alpha-amanitin (RNA synthesis inhibitor). Thus, it was suggested that the magnesium-requiring step in HL-60 cell differentiation is in protein but not mRNA synthesis. This conclusion is supported by the findings that changes in c-myc and c-fms mRNA levels in HL-60 cells treated with 100 nM 1,25(OH)2D3 in magnesium-deficient medium and those in standard magnesium medium were the same. In addition, dibutyryl cyclic adenosine monophosphate (dbc AMP) could restore expression of differentiation-related phenotypes inhibited by magnesium deprivation but not those inhibited by cyclohexamide, even though magnesium deprivation inhibited protein synthesis as much as did cyclohexamide. This suggests that magnesium-requiring step in HL-60 cell differentiation is different from that inhibited by cyclohexamide.     

Enhancing effects of ceramide derivatives on 1,25-dihydroxyvitamin D(3)-induced differentiation of human HL-60 leukemia cells

Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] represents a useful approach for the treatment for cancer, including acute myeloid leukemia (AML). Recent studies demonstrated that the combined administration of 1,25-(OH)(2)D(3) and differentiation-enhancing agents could alleviate the side effects of 1,25-(OH)(2)D(3) and improve the rate of long term survival. In this study, we determined the enhancing activities of ceramide derivatives on 1,25-(OH)(2)D(3)-induced differentiation of human myeloid leukemia HL-60 cells. Importantly, some of these derivatives -- namely, A2, B3, and H9 -- enhanced the 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells in a concentration-dependent manner. In addition, the morphologic studies using Giemsa staining and flow cytometric analysis demonstrated that the combined treatment of 1,25-(OH)(2)D(3) with one of the three analogues, A2, B3, and H9, directed the HL-60 cells into monocytic lineage, but not into granulocytic lineage. The inhibition studies demonstrated that A2, B3, and H9, enhanced 1,25-(OH)(2)D(3)-induced differentiation of HL-60 cells via the PI3-K/PKC/JNK/ERK pathways. The ability of ceramide derivatives to enhance the differentiation-inducing potential of 1,25-(OH)(2)D(3) may contribute to an effective therapy for AML.     

1,25-Dihydroxyvitamin D3 regulation of phorbol ester receptors in HL-60 leukemia cells

In this study the relationship between cell binding of phorbol 12,13-dibutyrate (PDBu) and induction of differentiation by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was examined. Binding of [3H]PDBu increased within 12 h of 1,25-(OH)2D3 treatment, and a 60-130% increase in [3H]PDBu receptor levels was observed within 24 h. By 48 h, however, [3H]PDBu binding was not different from control. Scatchard analysis of [3H]PDBu binding showed no statistical differences in Kd value (Kd approximately equal to 30 nM) between 1,25-(OH)2D3-treated and control cells 22 h post-treatment; however, a 2-fold increase in Bmax was observed in treated (338 +/- 24 pmol/10(9) cells) compared to control cultures (170 +/- 14 pmol/10(9) cells). Stimulation of [3H]PDBu binding was dependent on 1,25-(OH)2D3 concentrations over a range of 1-100 nM. Homogenates from 1,25-(OH)2D3-treated HL-60 cells also demonstrated an increase (70%) in [3H]PDBu binding to the Ca2+/phospholipid-dependent enzyme protein kinase C as assessed by incubation of cell homogenates with [3H]PDBu in the presence of saturating phosphatidylserine and calcium concentrations. This suggests that the increase in [3H]PDBu binding cannot be entirely explained by modulation of the latter two agents. Cycloheximide (5 microM), an inhibitor of protein synthesis, ablated the 1,25-(OH)2D3-stimulated increase in [3H]PDBu binding to intact HL-60 cells. These data demonstrate that an increase in [3H]PDBu binding occurs early in the course of 1,25-(OH)2D3-induced differentiation, results from an increased number of [3H]PDBu-binding site, and is dependent on protein synthesis.     

pp75: A novel tyrosine-phosphorylated protein that heralds differentiation of HL-60 cells

The human promyeloid cell line HL-60 differentiates toward monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or dibutyryl cAMP, respectively. When nondifferentiated cells were incubated for 20 min with 2 mM H2O2 and 0.1 mM sodium orthovanadate to inhibit their protein-tyrosine-phosphatase activity (Heffetz, D., Bushkin, I., Dror, R., and Zick, Y. (1990) J. Biol. Chem. 265, 2896-2902), we found marked tyrosine phosphorylation of a single major protein of 53 kDa. Induction of differentiation of HL-60 cells was accompanied by the appearance of an additional major cytosolic tyrosine-phosphorylated protein of 75 kDa (pp75). In dibutyryl cAMP-treated cells, tyrosine phosphorylation of pp75 peaked after 24 h and then declined rapidly. In 1,25(OH)2D3-treated cells, increased tyrosine phosphorylation was detected as early as 2 h and peaked after 3 days, whereas the presence of differentiated phenotypes, assessed by the capacity of the cells to reduce nitro blue tetrazolium, was detected no earlier than 24 h. Doses of 1,25(OH)2D3 as low as 1 nM induced the appearance of pp75 at a stage where almost no differentiation measured by nitro blue tetrazolium reduction was detected. Phosphorylation of pp75 was not stimulated by adriamycin, which induced growth arrest without initiation of differentiation. pp75 could also be detected in U-937, a monocytic cell line that is more advanced in its differentiation state, and also in terminally differentiated circulating human monocytes treated with H2O2/vanadate. pp75 underwent in vitro tyrosine phosphorylation in cytosolic extracts derived from 1,25(OH)2D3-induced HL-60 cells, but not in extracts derived from uninduced cells. Our results raise the possibility that tyrosine phosphorylation of pp75 may be a common early event that heralds the differentiation of HL-60 cells into both the monocytic and granulocytic pathways.     

Ca2+ priming during vitamin D-induced monocytic differentiation of a human leukemia cell line

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces monocytic differentiation of the human promyelocytic leukemia line, HL-60, and enhances Ca2+ transport in target cells of the mineral metabolism system. Hence, we determined whether the steroid's maturational effect on HL-60 involves alterations of intracellular calcium [( Ca2+]i). We found that, as detected by indo-1 fluorescence, [Ca2+]i increases in a slow tonic manner from 99 +/- 11 nM in virgin HL-60 to 182 +/- 19 nM (p less than 0.001) in those treated with 1,25-(OH)2D3 for 24 h. The first apparent rise in [Ca2+]i occurs at between 6 and 12 h and parallels expression of alpha-thrombin and N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptors. This increase in [Ca2+]i is derived from extracellular calcium as its reduction abolishes the effect. The increase in [Ca2+]i is associated with an increase in inositol trisphosphate-stimulated Ca2+ flux from intracellular stores. Interestingly, 1,25-(OH)2D3-mediated HL-60 differentiation as manifest by expression of the macrophage-specific antigen, 63D3, is not blocked by low extracellular calcium. In contrast, the fMLP-induced superoxide ion generation is diminished if the increase in [Ca2+]i is prevented. Furthermore, fMLP-stimulated signal transduction is also reduced by limiting the stimulation of [Ca2+]i during 1,25-(OH)2D3 treatment. Thus, although differentiation of HL-60 to the monocytic phenotype by 1,25-(OH)2D3 is Ca2+-independent, expression of response to regulatory stimuli requires priming of cellular Ca2+ stores. The latter appears to be induced by 1,25-(OH)2D3 via stimulated Ca2+ entry through the plasma membrane.     

Dibutyryl cAMP enhances the effect of 1,25-dihydroxyvitamin D3 on a human promyelocytic leukemia cell, HL-60, at both the receptor and the postreceptor steps.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces differentiation of a human promyelocytic leukemia cell line, HL-60, into monocytes/macrophages, and 25-hydroxyvitamin D3- and 1,25-(OH)2D3-24-hydroxylase activities in HL-60 mitochondria via a steroid-hormone receptor mechanism. Dibutyryl cyclic adenosine monophosphate (dbcAMP), a granulocyte inducer, significantly augmented the differentiation-inducing effect of 1,25-(OH)2D3 along the monocyte/macrophage pathway. Furthermore, dbcAMP significantly potentiated the effect of 1,25-(OH)2D3 on HL-60 cells to hydroxylate 1,25-(OH)2[26,27-3H]D3 to form 1,24,25-(OH)3[26,27-3H]D3. DbcAMP seemed to augment the effect of 1,25-(OH)2D3 in part through upregulation of the 1,25-(OH)2D3 receptor, because 10(-7) M dbcAMP increased 1,25-(OH)2D3 receptor levels approximately 2.3-fold, which was similar to a 1.9-fold augmentation by the same concentrations of dbcAMP of 1,25-(OH)2D3-induced cell characteristics to hydroxylate C-24 of 1,25-(OH)2[26,27-3H]D3. However, dbcAMP is also known to enhance HL-60 cell differentiation caused by other differentiation inducers. We have established another HL-60 clone which acquires resistance to 1,25-(OH)2D3 in the induction of cell differentiation by a defect at the postreceptor step, as reflected by resistance to other differentiation inducers, such as retinoic acid and dimethyl sulfoxide. Even in this resistant clone, dbcAMP significantly enhanced the differentiation-inducing effect of 1,25-(OH)2D3. Of interest, this clone showed resistance to dbcAMP in the induction of cell differentiation. Furthermore, we have demonstrated that intracellular cAMP levels were significantly lower in uremic serum-treated cells than in cells treated with normal human serum and that a significant positive correlation was found between intracellular cAMP levels and 1,25-(OH)2D3-induced cell differentiation. These data indicated that the intracellular cAMP level is one of the major determinants of 1,25-(OH)2D3-induced HL-60 cell differentiation and that dbcAMP could enhance the effects of 1,25-(OH)2D3 on HL-60 cells not only by increasing 1,25-(OH)2D3 receptor levels but also at the postreceptor step.     

1,25(OH)2D3 increases membrane associated protein kinase C in MDBK cells.

To determine whether 1,25-dihydroxycholecalciferol [1,25(OH)2D3] affects protein kinase C (PKC) activity in kidney, as has been demonstrated in HL-60 cells we measured 1,25(OH)2D3 binding, PKC activity and PKC immunoreactivity in Madin Darby bovine kidney (MDBK) cells, a normal renal epithelial cell line derived from bovine kidney. Our data demonstrate that MDBK cells exhibit specific high affinity binding for 1,25(OH)2D3, indicating the presence of the vitamin D receptor (VDR). Treatment of MDBK cells with 1,25(OH)2D3 for 24 h increased membrane PKC activity and immunoreactivity. The effect of 1,25(OH)2D3 was dose-dependent, with a peak effect observed at 10(-7)M 1,25(OH)2D3. The 1,25(OH)2D3 induced increase in membrane PKC was paralleled by a comparable decrease in cytosolic PKC activity and amount. Although time course studies were consistent with a VDR mediated effect of 1,25(OH)2D3 on PKC protein synthesis, total PKC activity was not increased by 1,25(OH)2D3, suggesting an effect on PKC translocation or localization. These results suggest that 1,25(OH)2D3 modulates PKC mediated events in kidney, a classic target for this steroid hormone.     

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

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

Development of receptors for leukotriene B4 on HL-60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3

The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL-60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH-LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12-dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl-methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL.     

Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60

Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.     

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts

The rapid, non-genomic actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(-/-) osteoblasts isolated from wild-type and VDR null mice to study the increase in intracellular calcium ([Ca(2+)](i)) and activation of protein kinase C (PKC) induced by 1,25(OH)(2)D(3). Within 1 min of 1,25(OH)(2)D(3) (100 nM) treatment, an increase of 58 and 53 nM in [Ca(2+)](i) (n = 3) was detected in VDR(+/+) and VDR(-/-) cells, respectively. By 5 min, 1,25(OH)(2)D(3) caused a 2.1- and 1.9-fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated-MBP(4-14) in VDR(+/+) and VDR(-/-) osteoblasts. The 1,25(OH)(2)D(3)-induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)(2)D(3) treatment resulted in the same degree of translocation of PKC-alpha and PKC-delta, but not of PKC-zeta, from cytosol to plasma membrane in both VDR(+/+) and VDR(-/-) cells. These experiments demonstrate that the 1,25(OH)(2)D(3)-induced rapid increases in [Ca(2+)](i) and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)(2)D(3) in osteoblasts.     

Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts

A fluorescent Ca2+ indicator, acetoxymethyl Quin2, was used to quantify changes in the cytosolic free calcium concentration ([Ca2+]i) of confluent mouse osteoblasts. 1,25 - Dihydroxycholecalciferol (1,25 - (OH)2D3, 10-100 pM), 25-hydroxycholecalciferol (25-(OH)D3, 10-100 nM), parathyroid hormone (PTH(1-84), 0.1-10 nM), and prostaglandin E2 (PGE2, 10-1000 nM) all induced immediate (t less than 15 s) transient increases in [Ca2+]i, from a basal level of 135 +/- 8 nM to levels of 179-224 nM. These increases rapidly returned to a plateau approximately 10% higher than the basal level. 24,25-Dihydroxycholecalciferol (24,25-(OH)2D2, 0.1-10 nM) induced a rapid increase in [Ca2+]i which remained elevated for 5 min before decreasing. The 1,25-(OH)2D3- and PTH-induced spikes were abolished by the prior addition of EGTA and Ca2+ entry blockers (verapamil, nifedipine, 1 microM) while the responses to 25-(OH)D3, 24,25-(OH)2D3, and PGE2 were unaffected. Addition of 1,25-(OH)2D3 + EGTA or PTH + EGTA caused enhanced Ca efflux. Addition of drugs which interfere with calcium sequestration by the endoplasmic reticulum (ER) (caffeine, 4 mM; 8-(diethyl-amino)-octyl 3,4,5-trimethoxybenzoate HCl, 0.5 mM) or mitochondria (antimycin, 10 microM; oligomycin, 5 microM) showed that 25-(OH)D3 and PGE2 mainly mobilized Ca2+ from ER. 1,25-(OH)2D3 and bovine PTH caused a transient increase in [Ca2+]i, 70% of which resulted from Ca2+ influx from outside the cells and 30% by release from the ER. The [Ca2+]i increase induced by 24,25-(OH)2D3 included a 30% contribution from the ER and 70% from the mitochondria.     

Regulation of cellular adhesion and fibronectin synthesis by 1 alpha,25-dihydroxyvitamin D3

We recently reported that the steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) can inhibit growth, alter morphology, and increase cell associated and medium concentrations of fibronectin (FN) in MG-63 human osteosarcoma cells (Franceschi, R. T., James, W., and Zerlauth, G. (1985) J. Cell. Physiol. 123, 401-409). In the present study, we have tested the hypothesis that 1,25-(OH)2D3 increases cellular adhesion by stimulating FN synthesis. Hormone treatment altered cell morphology and increased cell/substratum adhesion in MG-63 cells, effects which could be mimicked by exogenously added FN. 1,25-(OH)2D3-dependent increases in FN production were due to a rapid (within 12 h) increase in FN synthesis. Maximal (2 to 5-fold) stimulation was observed after 48 h. Hormone treatment did not alter apparent FN stability or distribution during this time. The FN response was specific to 1,25-(OH)2D3 when compared with other vitamin D metabolites. In contrast, triamcinolone acetonide, another known inducer of FN synthesis in certain cells, was only slightly stimulatory up to a concentration of 1 microM. FN mRNA, as measured by Northern blot hybridization, increased within 6 h of 1,25-(OH)2D3 addition with maximal (5-fold) induction seen at 24 h. 1,25-(OH)2D3 also stimulated FN synthesis in several other transformed cell lines (TE-85 human osteosarcomas, SW-480 human colon carcinomas, and HL-60 myeloid leukemia cells). These results may be related to known actions of 1,25-(OH)2D3 on cell differentiation and tumor metastasis.     

Disassociation of the macrophage-maturational effects of vitamin D from respiratory burst priming.

During the process of enhancing monocytic differentiation of the human leukemia line HL-60, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) also "primes" the cell for respiratory burst by increasing the uptake of Ca2+ across the plasma membrane (Hruska, K.A., Bar-Shavit, Z., Malone, J.D., and Teitelbaum, S.L. (1988) J. Biol. Chem. 263, 16039-16044). The present study asked if the maturational effect of vitamin D is dependent upon this "priming" phenomenon. To this end, we exposed HL-60 to either 1,25(OH)2D3 or its synthetic analogue (1 alpha, 3 beta, 5Z, 7E)-9-10-Secocholesta-5,7,10(19)-triene-1, 3, 25-triol (22-oxa). We found that 22-oxa induced HL-60 maturation as effectively as does the natural steroid. As expected, 48 h of 1,25(OH)2D3 exposure more than doubles (p less than 0.005) HL-60 basal cytosolic Ca2+ and increases inositol triphosphate-sensitive Ca2+ stores approximately 4-fold (p less than 0.01). 22-oxa in contrast alters neither Ca(2+)- nor inositol triphosphate-mobilizable deposits. Moreover, 1,25(OH)2D3 treatment prompts a transient Ca2+ "spike" in response to formyl-methionyl-leucyl-phenylalanine (fMLP) and a marked increase in superoxide (O-2) generation when exposed to the chemotactic peptide (p less than 0.01) or phorbol ester (p less than 0.02). Treatment with 22-oxa does not enable HL-60 to respond to fMLP with a Ca2+ spike or prime the cell for respiratory burst unless it is co-incubated with the Ca2+ ionophore, ionomycin. Similarly, phorbol ester impacts more profoundly on O-2 generation by 1,25(OH)2D3 than 22-oxa preincubated cells (p less than 0.02), unless the latter is added with ionomycin. Our findings indicate that the maturational effects of vitamin D sterols are independent of their capacity to prime cells for respiratory burst and that the Ca2+ ionophoretic effects of 1,25(OH)2D3 play a major role in such priming.