The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro

Effects of dexamethasone and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were studied in cultures of adult human marrow stromal cells. In primary culture, dexamethasone (10(-8) M) increased the number of fibroblast colonies formed but decreased their average size. The number of colonies expressing alkaline phosphatase activity was increased, consistent with the enhancement of osteogenic differentiation by this glucocorticoid. In secondary culture, osteogenic differentiation was assessed by measurement of the steady-state levels of particular mRNAs that are characteristic of cells of the osteoblast lineage. The mRNAs for alpha 1(I)-procollagen, alkaline phosphatase, osteopontin and bone sialoprotein were expressed under all culture conditions used. In contrast, osteocalcin mRNA expression was detectable only in cultures treated with 1,25(OH)2D3 (10(-8) M). Addition of 1,25(OH)2D3 to control increased the expression of the mRNAs for alkaline phosphatase and osteopontin but had no significant effect on bone sialoprotein expression. The highest levels of expression of the mRNAs for alkaline phosphatase, bone sialoprotein and osteocalcin were observed in dexamethasone-treated cultures to which 1,25(OH)2D3 had been added. These results demonstrate that, as earlier found in other species, dexamethasone and 1,25(OH)2D3 promote the osteogenic differentiation of human marrow stromal cells as measured by expression of these osteogenic markers.     

Stromal cell-mediated stimulation of osteoclastogenesis

In the bone marrow microenvironment, stromal cells or their products are known to regulate proliferation and differentiation of hematopoietic stem cells. The purpose of this investigation was to characterize stroma-mediated effects of differentiation-inducing factors on osteoclastogenesis in defined murine cultures. Hematopoietic progenitors (derived from long-term bone marrow cultures, LTBMCs) were cocultured with cloned stromal cell lines to demonstrate the indirect effects of various differentiation-inducing factors. Osteoclastogenesis was compared in three murine marrow systems (whole bone marrow, progenitors cultured alone, and cocultures of progenitors with stromal cell lines) by analysis of multinuclearity and tartrateresistant acid phosphatase (TRAP) activity. The cultures were treated for two weeks with murine recombinant GM-CSF (5 U/ml), 1,25-dihydroxyvitamin D3 (10(-8) M), or parathyroid hormone (PTH, 10(-8) M). In whole bone marrow cultures, osteoclast differentiation was stimulated by GM-CSF, PTH and 1,25-dihydroxyvitamin D3. With progenitors alone, only GM-CSF promoted osteoclastogenesis. Each agent stimulated osteoclastogenesis in cocultures of progenitors with a stromal cell line (GBLneo'). Thus, the coculture system is a partially defined model for whole bone marrow cultures. In contrast, progenitors that were cocultured with a stromal cell line derived from an osteopetrotic op/op mouse failed to differentiate in the presence of PTH or 1,25-dihydroxyvitamin D3. These results indicate that stimulation of osteoclastogenesis by PTH or 1,25-dihydroxyvitamin D3 is mediated indirectly through factors present in normal marrow stromal cells and that an osteopetrotic stromal cell line failed to support differentiation.     

Inhibition of cholesterol absorption by plant sterols for mass intervention

Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)2D3 and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)2D3, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS.     

Application of the extremum stack to neurological MRI

The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by increased marrow adipose tissue formation. Reversal of this process may provide a novel therapeutic approach for osteopenic disorders. We have shown that cells cultured from human trabecular bone are not only osteogenic, but are able also to undergo adipocyte differentiation under defined culture conditions. Osteoblast differentiation was induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and adipocyte differentiation by dexamethasone (dex) plus 3-isobutyl-1-methylxanthine (IBMX) treatment. Adipogenesis was characterized by lineage-specific enzyme and gene activities, alpha-glycerophosphate-3-dehydrogenase activity, fatty acid binding protein, aP2 and lipoprotein lipase expression. Osteoblastogenesis was assessed by osteoblast characteristic 1,25(OH)2D3 induction of alkaline phosphatase activity and osteoblast-specific 1,25(OH)2D3-induced osteocalcin synthesis and release. We provide evidence for a common pluripotent mesenchymal stem cell that is able either to undergo adipogenesis or osteoblastogenesis, using clonal cell lines derived from human trabecular bone cell cultures. Adipogenesis can be induced also by long chain fatty acids and the thiazolidinedione troglitazone. Dex plus IBMX-induced adipogenesis can be inhibited by interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta. Interestingly, and in contrast to extramedullary adipocyte differentiation as shown by mouse 3T3L-1 and a human liposarcoma SW872 cell line, trabecular bone adipogenesis was unaffected by insulin. Also, the formation of fully differentiated adipocytes from trabecular bone cells after troglitazone treatment and long chain fatty acids was dependent on increased expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma2 caused by dex plus IBMX. Specific inhibition of marrow adipogenesis and promotion of osteoblastogenesis of a common precursor cell may provide a novel therapeutic approach to the treatment of osteopenic disorders.     

The cellular actions of interleukin-11 on bone resorption in vitro

The pleiotropic cytokine interleukin-11 (IL-11) stimulates osteoclast formation in vitro, but it is not known whether it influences other steps in the bone-resorptive cascade. Using a variety of in vitro model systems for studying bone resorption we have investigated the effects of IL-11 on 1) osteoclast formation, fusion, migration, and activity; and 2) osteoblast-mediated osteoid degradation. The involvement of matrix metalloproteinases (MMPs) and products of arachidonic acid metabolism in IL-11-mediated resorption were also assessed. We first examined the bone-resorptive effects of IL-11 by assessing 45Ca release from neonatal mouse calvarial bones. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10(-10) M. The kinetics of IL-11-mediated 45Ca release demonstrated that it was without effect for the first 48 h of culture, but by 96 h, it stimulated 45Ca release to the same level as that produced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (a hormone that stimulates osteoclast formation and activity). IL-11 also produced a dose-dependent increase in osteoblast-mediated type I collagen degradation with a maximum of 58.0 +/- 6.2% at 5 x 10(-9) M; this effect of IL-11 was less than that produced by 1,25-(OH)2D3 (76.5 +/- 7.1%) and was prevented by an inhibitor of MMPs, but not those blocking arachidonic acid metabolism. We then tested the effects of IL-11 on isolated mouse osteoclasts cultured on ivory slices in the presence and absence of primary mouse osteoblasts. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts. We then examined the effects of IL-11 on the formation of osteoclast-like multinucleate cells in mouse bone marrow cultures and the resorptive activity of such cultures using ivory as a substrate. IL-11 dose-dependently increased 1) the number of tartrate-resistant acid phosphatase-positive osteoclast-like multinucleate cells and 2) the surface area of lacunar resorption, although the effects were less than that of 1,25-(OH)2D3. The effect of IL-11 on bone marrow lacunar resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. These results provide strong evidence that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating osteoclast migration and activity. Our data also suggest that the stimulatory effects of IL-11 involve both MMPs and products of arachidonic acid metabolism.     

Effects of macrophage colony stimulating factor and granulocyte-macrophage colony stimulating factor on osteoclastic differentiation of hematopoietic progenitor cells

Although the hematopoietic origin of the osteoclast is generally accepted, the precise phenotype of the progenitor and the regulation of its differentiation are unclear. This study compares proliferation and differentiation of progenitors in response to macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Nonadherent progenitor cells from murine long-term bone marrow cultures (LTBMC) (as a source of osteoclast progenitors) demonstrated a significant proliferative response to M-CSF. In addition, M-CSF increased the number of multinucleated cells, only a small percent of which (14-16%) were tartrate-resistant, acid phosphatase (TRAP)-positive. In contrast, cells cultured with GM-CSF generated more TRAP-positive multinucleated cells even at concentrations less stimulatory of proliferation than M-CSF. The osteoclast phenotype of these multinucleated cells was also assessed by ultrastructural characterization of ruffled borders in association with bone fragments. The bone-active hormone 1,25-dihydroxyvitamin D3 inhibited the proliferation of this subset of progenitor cells in the presence of M-CSF or GM-CSF. All of these results show effects on progenitors in the absence of the stromal cell microenvironment in this system. These results provide evidence for a divergence in the biological responsiveness of osteoclast progenitor cells to M-CSF compared with GM-CSF; they support the notion that M-CSF has a "priming" effect on osteoclast progenitors whose subsequent differentiation to osteoclastic multinucleated cells is promoted by GM-CSF.     

Induction of surface tumor necrosis factor (TNF) expression and possible facilitation of surface TNF release from human monocytic cells by granulocyte-macrophage colony-stimulating factor or gamma interferon in combination with 1,25-dihydroxyvitamin D3

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), gamma interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can regulate monocyte maturation and activation. Using the human monocytoid cell line U937, we have shown that these agents increase surface tumor necrosis factor (TNF) expression without directly affecting TNF release. GM-CSF and IFN-gamma combined with 1,25(OH)2D3 increased cellular TNF secretion to levels not seen with these agents alone. Ability to express and secrete TNF in part depended on degree of monocytic maturation. The combination of 1,25(OH)2D3 and GM-CSF, however, facilitated lipopolysaccharide (LPS)-mediated release of surface TNF from U937 cells, an effect that was temporally independent of maximal maturation. 1,25(OH)2D3 plus IFN-gamma was less effective than 1,25(OH)2D3 plus GM-CSF at facilitating TNF secretion. We postulate that 1,25(OH)2D3 and GM-CSF are required together to prime a specific mechanism, probably a protease, which cleaves TNF from the surface of monocytic cells. This protease, once primed, can be activated by a secondary stimulus such as LPS.     

1,25-Dihydroxy vitamin D3 inhibits adipocyte differentiation and gene expression in murine bone marrow stromal cell clones and primary cultures

Bone marrow stromal stem cells differentiate into adipocytes and osteoblasts. These two lineages are thought to be reciprocally related, in part due to the observation that the osteoblast-inducing factor, 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], inhibited adipogenesis of rat femoral-derived stromal cell cultures. However, the literature is divided concerning the adipogenic effects of this steroid hormone. This work examined the effect of 1,25(OH)2D3 (10(-12)-10(-8) M) on murine femoral-derived bone marrow stromal cell differentiation in response to adipogenic agonists employing two different classes of nuclear hormone receptors: the glucocorticoid receptor (hydrocortisone) or peroxisome proliferator-activated receptors (thiazolidinediones). Experiments used the multipotent murine bone marrow stromal cell line, BMS2, and its subclones, as well as primary-derived murine bone marrow stromal cell cultures. In all systems examined, 1,25(OH)2D3 blocked adipogenesis induced by hydrocortisone, methylisobutylxanthine, and indomethacin based on flow cytometric analysis of lipid accumulation. This correlated with reduced messenger RNA levels of the late adipocyte gene markers, aP2 and adipsin. In the BMS2 subclone no. 24, the 1,25(OH)2D3 actions were concentration dependent. Whereas 1,25(OH)2D3 partially inhibited thiazolidinedione-induced adipogenesis in the parental BMS2 cell line, it had minimal effect on the thiazolidinedione-induced differentiation of the BMS2 subclone and primary cultures. These findings indicate that 1,25(OH)2D3, at nanomolar concentrations, completely inhibits murine bone marrow stromal cell differentiation in response to glucocorticoid-based adipogenic agonists but is a less effective adipogenic antagonist following induction with thiazolidinediones. This work supports the conclusion that 1,25(OH)2D3 inhibits murine femoral-derived bone marrow stromal cell adipogenesis.     

Stimulation of osteoclast formation by 1,25-dihydroxyvitamin D requires its binding to vitamin D receptor (VDR) in osteoblastic cells: studies using VDR knockout mice

Previous studies have shown that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays important roles in the formation of osteoclasts through its actions on osteoblastic cells. We have generated mice lacking vitamin D receptor (VDR) by gene targeting (VDR-/-). These mice had tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and exhibited similar levels of parameters for bone resorption to those in wild type mice. The present studies were undertaken to clarify whether effects of 1,25(OH)2D on osteoclast formation require VDR in osteoblasts, and to examine mechanisms of the formation of osteoclasts without VDR-mediated actions using VDR-/- mice. When wild-type calvarial osteoblasts and spleen cells were co-cultured with 1,25(OH)2D, TRAP-positive osteoclasts were formed regardless of the genotypes of spleen cells. In contrast, when osteoblasts from VDR-/- mice were co-cultured, no osteoclasts could be formed even with wild-type spleen cells. Parathyroid hormone and interleukin-1alpha stimulated osteoclast formation by co-cultures from VDR-/- mice, and the generated osteoclasts showed resorbing activity. These results demonstrate that VDR-mediated actions of 1,25(OH)2D in osteoblasts are essential for osteoclast formation by 1,25(OH)2D, and that functionally intact osteoclasts can be formed without 1,25(OH)2D actions under stimulations by other agents. It is suggested that osteoclastic bone resorption can be maintained without 1,25(OH)2D actions by other stimulatory agents.     

Effects of Pasteurella multocida toxin on porcine bone marrow cell differentiation into osteoclasts and osteoblasts

The effect of Pasteurella multocida toxin (PMT) on porcine osteoclast and osteoblast differentiation was studied using in vitro cell culture systems. When grown in the presence of Vitamin D3, isolated porcine bone marrow cells formed multinucleated cells with features characteristic of osteoclasts. Exposure of bone marrow cells to Vitamin D3 and PMT during growth resulted in formation of increased numbers and earlier appearance of osteoclasts compared to controls. Ultrafiltered medium form PMT-treated cells likewise increased osteoclast numbers, suggesting that a soluble mediator may be involved in the action of PMT. When cell cultures were treated with fluorescein-labeled PMT, fluorescence was found within the cytoplasm of small, round cells that did not resemble either osteoclasts or osteoclastic precursor cells. Cultures of porcine bone marrow cells exposed to dexamethasone, ascorbic acid, and beta-glycerophosphate developed into osteoblastic cells that formed multilayered, mineralized nodules. Exposure of osteoblastic cultures to low concentration of PMT resulted in retarded cell growth, formation of decreased numbers of nodules and minimal to no mineralization in the nodules; higher concentration of PMT resulted in increased cellular debris and poor growth of cells, with no nodule formation. These findings suggest that PMT may induce turbinate atrophy in pigs by increasing osteoclast numbers and inhibiting osteoblastic bone formation. The effect of PMT on osteoclastic differentiation and growth may not be due to a direct effect on preosteoclastic cells, but rather due to alterations in the soluble mediator secretion by marrow stromal cells.     

Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption

Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Paget's disease, bone metastasis, and hypercalcemia of malignancy. However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited. Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity. A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined. This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide. Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein. Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures. In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types. These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption.     

Involvement of prostaglandin E2 and interleukin-1 alpha in the differentiation and survival of osteoclasts induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions. LPS from various periodontal pathogens is supposed to be a major virulence factor of periodontal diseases. In the present study, we demonstrated that LPS from periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (Y4 LPS) stimulated osteoclast formation in mouse bone marrow culture systems. Addition of anti-interleukin-1 alpha (IL-1 alpha) antibody or indomethacin in the marrow cultures resulted in the suppression of osteoclast differentiation. Quantitative analyses revealed that Y4 LPS stimulated the production of IL-1 alpha and prostaglandin E2 (PGE2) by bone marrow cells. Furthermore, an immunoblot analysis showed that Y4 LPS stimulated bone marrow cells to upregulate the expression of cyclooxygenase-2, a rate-limiting enzyme for the conversion of arachidonic acid to prostanoids. These findings suggest that both IL-1 alpha and PGE2 are involved in the LPS-mediated osteoclast differentiation. In addition, we found that Y4 LPS supported the survival of osteoclasts. Addition of anti-IL-1 alpha antibody in the osteoclast culture resulted in a reduction of osteoclast survival. Indomethacin, however, showed no effect on osteoclast survival. These findings suggest that the increased PGE2 and IL-1 alpha synthesis by bone marrow cells may play an important role in the differentiation and survival of osteoclasts induced by A. actinomycetemcomitans LPS.     

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.     

Interleukin 4, interferon-gamma, and prostaglandin E impact the osteoclastic cell-forming potential of murine bone marrow macrophages

Interleukin 4 (IL-4) is an immune cytokine that inhibits bone resorption in mice and suppresses osteoclastic cell formation in vitro through an undefined mechanism. In this report, we have established the cellular identity of the IL-4 target cell using a variety of bone marrow/stromal cell coculture methods. Initially, we found that the majority of IL-4's inhibition of osteoclastic cell formation was due to its effect on bone marrow cells, not stromal cells. Consequently, bone marrow macrophages were used as osteoclastic cell progenitors after they had been transiently exposed to IL-4 (48 h), before the addition of stromal cells, 1,25-dihydroxyvitamin D3, and dexamethasone. In this circumstance, IL-4 impaired subsequent osteoclastic cell formation, suggesting that the macrophage may be potentially targeted by many factors known to influence osteoclast formation. Consequently, we discovered that interferon-gamma (IFN gamma), prostaglandin E (PGE), and cell-permeant cAMP analogs also impacted osteoclastic cell formation when used to selectively treat bone marrow macrophages. IFN gamma suppressed osteoclastic cell formation, whereas PGE and cAMP analog treatment led to the formation of significantly enlarged osteoclastic cells. Importantly, PGE antagonized the inhibitory effects of both IL-4 and IFN gamma on the osteoclastic cell-forming potential of bone marrow macrophages. Collectively, these findings establish bone marrow macrophages as osteoclastic cell precursors with the degree of their commitment to the osteoclast pathway sensitive to the effects of soluble mediators, including IL-4, IFN gamma, and PGE.     

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.     

17Beta-estradiol antagonizes effects of 1alpha,25-dihydroxyvitamin D3 on interleukin-6 production and osteoclast-like cell formation in mouse bone marrow primary cultures

In mouse bone marrow primary cultures, the formation of osteoclast-like, i.e. tartrate-resistant acid phosphatase (TRAP)- and calcitonin receptor-positive multinucleated cells (MNC), when induced by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), can be suppressed by 17beta-estradiol (17beta-E2), whereas 17alpha-E2 is without any effect. 17beta-E2, above 10(-11) M, significantly reduced 1alpha,25(OH)2D3-mediated TRAP+ MNC formation in cultured bone marrow cells from both female and male mice. The estrogen at 10(-8) M suppressed the peak response to the vitamin D sterol by 50%. 17beta-E2 significantly suppressed basal and 1alpha,25(OH)2D3-stimulated cellular production of interleukin (IL)-6. IL-6 alone, although bone marrow cells in hormone-free culture produced appreciable amounts of the cytokine, did not induce any TRAP+ MNC. Therefore, the changes in IL-6 production induced by the hormones could not be the sole determinant for the extent of TRAP+ MNC formation. However, the stimulatory effect of 1alpha,25(OH)2D3 on osteoclastogenesis nevertheless can be significantly reduced by a neutralizing monoclonal anti-IL-6 antibody. In the presence of 10(-8) M 17beta-E2, the anti-IL-6 monoclonal antibody does not achieve any further suppression of 1alpha,25(OH)2D3-related osteoclast-like cell formation. Our data suggest that induction of osteoclastogenesis by 1alpha,25(OH)2D3 is partially dependent on IL-6 signaling and can be modulated by 17beta-E2 through interference with IL-6 receptor activation, in addition to inhibition of IL-6 production by marrow stromal cells.     

Triiodothyronine restricts myofibrillar growth and enhances beating frequency in cultured adult rat cardiomyocytes

Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.     

Effects of 1,25-dihydroxyvitamin D3 on tumor cell invasion to the extracellular matrix in human fibrosarcoma HT1080 cells and its correlation with laminin

We investigated the role of the active form of vitamin D, 1,25-dihydroxyvitamin D3[1,25(OH)2D3], in promoting tumor cell invasiveness through the extracellular matrix, and showed that 1,25(OH)2D3-induced reduction of laminin production by the cells was correlated with the inhibitory effect of the hormone on tumor cell invasiveness. 1,25(OH)2D3 significantly inhibited invasiveness through the matrix, type IV collagenolytic and migratory activity, but not cell attachment to the matrix in human fibrosarcoma HT1080 cells. The 1,25(OH)2D3-induced inhibition showed the same dose dependency and magnitude for invasiveness as for the effects on type IV collagenolysis and cell migration. 1,25(OH)2D3 inhibited laminin production from the cells in a dose-dependent manner. The inhibitory effects of 1,25(OH)2D3 on the invasiveness, type IV collagenolysis and cell migration appeared to parallel the hormone-induced reduction of laminin production. Antilaminin monoclonal antibody, blocking the activity of laminin in the culture medium, inhibited HT1080 cell invasiveness. In the presence of exogenous laminin, 1,25(OH)2D3-induced inhibition of invasion was not observed. These findings suggest that 1,25(OH)2D3 acts on HT1080 cells, inhibiting the expression of laminin from the cells, and that the reduced laminin expression leads to the inhibition in the type IV collagenolytic and migratory activity of the cells, and consequently, to the inhibition of invasiveness through the extracellular matrix.