1,25-Dihydroxyvitamin D3 enhances cytosolic free calcium in HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) caused a rise in the concentration of intracellular free calcium ions ([Ca2+]i) in HL-60 cells. This effect of 1,25(OH)2D3 parallels its suppression of cell proliferation and its induction of cell differentiation into monocyte-like cells. The changes in [Ca2+]i are dose and time dependent. The concentration of 1,25(OH)2D3 (10(-7) M) that induced maximal differentiation also caused the maximal increase in intracellular Ca2+. The rise in cytoplasmic free Ca2+ concentration was not immediate and reached statistical significance only after 24 h. The [Ca2+]i reached its peak at 48 h (134 +/- 4 nM vs 101 +/- 3 nM in controls) and remained stable at this level. The increase in intracellular Ca2+ was found to be related to new protein synthesis, because it was inhibited in the presence of specific RNA and protein synthesis inhibitors. The rise in [Ca2+]i was not observed during incubation of HL-60 cells with 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), a vitamin D metabolite that does not induce the differentiation of HL-60 cells. In contrast, 25-hydroxyvitamin D3 (25-OH-D3) and phorbol 12-myristate 13-acetate (TPA), both of which induce differentiation in this cell line, also increase [Ca2+]i. In conclusion, the present study emphasizes that a significant increase in intracellular free Ca2+ occurs in the effect of 1,25(OH)2D3 on HL-60 cells.     

The involvement of calcium ions in the effect of 1,25-dihydroxyvitamin D3 on HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) was found to suppress growth of human leukemic cells (HL-60), and to induce the differentiation of these cells to monocyte-like cells. The purpose of the present study was to examine the role of calcium ions in the effects of 1,25(OH)2D3 on HL-60 cells. Incubation of the HL-60 cells with 1,25(OH)2D3 (10(-7) M) for 4 days caused a significant inhibition of 50% of cell growth. The number of differentiated cells increased simultaneously from 24 x 10(3) +/- 2 x 10(3) in the controls to 658 x 10(3) +/- 32 x 10(3) in the 1,25(OH)2D3 (10(-7) M)-treated cells. The role of calcium ions in the effects of 1,25(OH)2D3 on HL-60 cells was first studied by changing the available calcium in the medium and by measuring the effect of 1,25(OH)2D3 on intracellular Ca2+ levels. Limitation of the available Ca2+ by means of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or verapamil enhanced the inhibitory effect on proliferation and decreased the number of differentiated cells obtained by 1,25(OH)2D3 alone. These effects could be abolished by restoring the Ca2+ levels. The role of the intracellular free Ca2+ ions in the effect of 1,25(OH)2D3 was further illustrated by measuring the intracellular Ca2+ levels. The intracellular free Ca2+ concentration in 1,25(OH)2D3 (10(-7) M)-treated HL-60 cells rose significantly from 117.0 +/- 6.3 nM in the untreated HL-60 cells to 145.0 +/- 7.5 nM in the treated cells (p less than 0.02). Addition of verapamil moderated the increase in intracellular free Ca2+ (125.0 +/- 5.2 nM) obtained by 1,25(OH)2D3 alone. Thus the elevation of intracellular free Ca2+ caused by 1,25(OH)2D3 treatment may be involved in the effect of the hormone on the HL-60 cells.     

1 alpha,25-dihydroxyvitamin D3-induced upregulation of calcineurin during leukemic HL-60 cell differentiation

Cyclosporin A and FK506, at concentrations that inhibited phosphatase activity of calcineurin in HL-60 cellular lysates, augmented the proliferation of leukemic HL-60 cells. These immunosuppressants did not affect 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced monocytic differentiation of HL-60 cells, but did abrogate the 1,25(OH)2D3- induced inhibition of HL-60 cell growth. Treatment with 20 nmol/L 1,25(OH)2D3 led to a progressive increase in calcineurin phosphatase activity in subcellular fractions from HL-60 cell extracts, the increase in this activity appeared to parallel the phenotypic and functional changes of HL-60 cells during monocytic differentiation induced by 1,25(OH)2D3. Immunoblot analysis indicated that increase in calcineurin activity was concordant with the increased expressions of calcineurin catalytic subunit isozymes, calcineurin A alpha (CNA alpha), and calcineurin A beta(CNA beta), and a regulatory calcineurin B subunit (CNB) proteins, which were preceded by a coordinate increase in the levels of CNA alpha, CNA beta and CNB mRNAs. The expression of calmodulin remained unaltered throughout 1,25(OH)2D3-induced monocytic differentiation. These results suggest that calcineurin activation has a net negative effect on HL-60 cell proliferation, and that the increased expression of calcineurin may be involved in 1,25(OH)2D3- induced inhibition of HL-60 cell proliferation.     

Combinations of interferon-gamma and retinoic acid or 1 alpha, 25-dihydroxycholecalciferol induce differentiation of the human monoblast leukemia cell line U-937

The monoblastlike leukemia cell line, U-937, is induced to differentiate into monocytelike cells by incubation with 200-500 U/ml of recombinant human immune interferon (IFN-gamma) judging from capacity to reduce nitroblue tetrazolium. At least an additive differentiation-inducing effect was found between IFN-gamma and 1-100 nM retinoic acid (RA). A marked synergistic differentiation-inducing effect was found between IFN-gamma and 0.1-1.0 nM 1 alpha,25-dihydroxycholecalciferol (1,25[OH]2D3). It is also shown that U-937 can be primed for differentiation by treatment for approximately one day with 1,25(OH)2D3 followed by exposure to IFN-gamma. Priming of these cells does not depend on the normal rate of RNA synthesis, as it occurs even better in the presence of cordycepin, suggesting that a decrease in RNA synthesis favors IFN-induced differentiation. Actually, the addition of cordycepin during initial incubation with IFN increased the subsequent response to IFN-gamma (and also to RA and 1,25[OH]2D3). These results, indicating that combinations of IFN-gamma and either RA or 1,25(OH)2D3 induce differentiation of U-937, may be of importance in combination biotherapy of leukemia.     

Development of a novel 1,25(OH)2-vitamin D3 analog with potent ability to induce HL-60 cell differentiation without modulating calcium metabolism.

We describe several novel analogs of the seco-steroid 1,25(OH)2-vitamin D3[1,25(OH)2D3] and their effects on differentiation and proliferation of HL-60 human myeloid leukemic cells in vitro as well as their effects on calcium metabolism in vivo. The 1 alpha-25(OH)2-16ene-23yne-26,27F6-vitamin D3 is the most potent analog reported to date, having about 80-fold more activity than the reference 1,25(OH)2D3 for inhibition of proliferation and induction of differentiation of HL-60 cells. Also, this analog decreased RNA expression of MYC oncogene in HL-60 by 90% at 5 x 10(-10) mol/L. Intriguingly, intestinal calcium absorption and bone calcium mobilization mediated in vivo by 1 alpha-25(OH)2-16ene-23yne-26,27F6-D3 was found to be markedly (15-fold) less than that of 1,25(OH)2D3. In addition, 1 alpha-25(OH)2D3 bound to 1,25(OH)2D3 receptors of both HL-60 and intestine more avidly than did 1 alpha-25(OH)2-16ene-23yne-26,27F6-D3. This novel analog may open up new therapeutic strategies for several hematopoietic, skin, and bone abnormalities and may provide a new tool to understand how vitamin D3 seco-steroids induce cellular differentiation.     

1,25-Dihydroxyvitamin D3 enhances phorbol ester-stimulated differentiation and protein kinase C-dependent substrate phosphorylation activity in the U937 human monoblastoid cell

In the U937 human monoblastoid cell line, 1,25-dihydroxyvitamin D3[1,25(OH)2D3] through a specific interaction with the 1,25(OH)2D3 receptor promotes differentiation toward a more mature phenotype. In addition to this direct effect, 1,25(OH)2D3 also potentiates differentiation in response to lymphokines and (Bu)2cAMP. We examined the effect of 1,25(OH)2D3 on phorbol ester-stimulated differentiation. Either preincubation with or simultaneous exposure to 1,25(OH)2D3 enhanced phorbol ester-stimulated differentiation. Over a 72-h period, the increase in phorbol ester responsiveness was dependent on the duration of 1,25(OH)2D3 exposure. Enhancement of phorbol ester-induced differentiation was observed with 1,25(OH)2D3 concentrations ranging from 0.1-10 nM. The 1,25(OH)2D3 vitamin D metabolite was more potent than the 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 metabolites in potentiating phorbol ester-induced differentiation. Phorbol esters can exert at least a portion of their effects on cellular function by activating protein kinase C. Thus, one mechanism by which 1,25(OH)2D3 could amplify signal transduction leading to potentiation of phorbol ester-stimulated differentiation would be by enhancing phorbol ester-stimulated phosphorylation. To examine this possibility, we measured protein kinase C-dependent substrate phosphorylation in extracts derived from cells pretreated with 1,25(OH)2D3. In extracts derived from cells treated with 1,25(OH)2D3, the protein kinase C-dependent phosphorylation of endogenous U937 substrates stimulated by calcium, phosphatidyl serine, and diolein was increased compared to that observed in vehicle-treated cells. The conditions required for 1,25(OH)2D3 to increase protein kinase C-dependent phosphorylation of endogenous substrates (concentration, duration of exposure, and metabolite specificity) were similar to those required to enhance phorbol ester-stimulated differentiation. Possibly mediating this enhanced phosphorylation was an increase in protein kinase C activity observed in extracts derived from 1,25(OH)2D3-treated cells.     

Induced differentiation of HL-60 promyelocytic leukemia cells to monocyte/macrophages is inhibited by hydroquinone, a hematotoxic metabolite of benzene.

Chronic exposure of humans to benzene has been shown to have a cytotoxic effect on hematopoietic progenitor cells in intermediate stages of differentiation, which can lead to aplastic anemia and acute myelogenous leukemia. We studied the effect of hydroquinone (HQ), a toxic metabolite of benzene found in the bone marrow, on the human promyelocytic leukemia cell line (HL-60), which can be induced to differentiate to both monocyte and myeloid cells, and thus has been used as a surrogate for a granulocyte/macrophage progenitor cell. Exposure of HL-60 cells to noncytotoxic concentrations of HQ for 3 hours before induction with phorbol myristate acetate (TPA) caused a dose-dependent inhibition of the acquisition of characteristics of monocytic differentiation, such as adherence, nonspecific esterase (NSE) activity, and phagocytosis, but had no effect on cell proliferation. HQ appeared to be affecting maturation beyond the monoblast/promonocyte stages. HQ also prevented differentiation induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]; however, the block occurred after the acquisition of adherence. HQ at concentrations that inhibited monocytic differentiation had no effect on differentiation to granulocytes, suggesting that the block in the differentiation of these bipotential cells is a step unique to the monocytic pathway. HQ was unable to prevent differentiation induced by the macrophage-derived cytokine, interleukin (IL)-1, a differentiation factor for cells of the monocytic lineage.     

1,25-Dihydroxycholecalciferol and macrophage differentiation with aging

Aging is attended by both decreased levels of circulating 1,25-dihydroxy-vitamin D (1,25(OH)2D) and alterations of immune function. We have explored the relationship of these events via the effects of the steroid hormone on macrophage differentiation, using both the human leukemic cell line HL-60, which has the capacity to differentiate along a monocytic or granulocytic pathway, and authentic bone-marrow-derived macrophage precursors. When treated with 1,25(OH)2D, HL-60 cells undergo monocytic differentiation, as documented by the appearance of macrophage-specific membrane antigens and esterase activity. Also, 1,25(OH)2D increases [Ca2+]i in a slow tonic manner, an event that parallels f-Met-Leu-Phe (fMLP) receptor expression. The rise of [Ca2+]i is derived from influx of extracellular Ca2+ and is associated with increased inositol trisphosphate (IP3)-stimulated Ca2+ release from intracellular stores. On the other hand, while prevention of the 1,25(OH)2D-generated increase in [Ca2+]i leads to reduced superoxide generation, it does not block monocytic differentiation. 1,25(OH)2D also targets to authentic bone-marrow-derived macrophage precursors at all stages of differentiation. In CSF-1-dependent cells, the steroid produces doubling of expression of the mannose receptor, a macrophage-specific membrane protein, which is also expressed by differentiated osteoclasts. The macrophage-maturing effect of 1,25(OH)2D was further explored by analyzing its effect on fMLP signal transduction in HL-60 cells. While virgin HL-60 cells are unresponsive to fMLP, cells incubated for 24 h with 1,25(OH)2D respond to fMLP stimulation with a 60% increase in [Ca2+]i, and possess greater IP3-sensitive calcium stores than virgin cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 and its sensitivity to ketoconazole in 1 alpha,25-dihydroxyvitamin D3-differentiated HL60 cells

Regulation of the metabolism of [3H]25-hydroxyvitamin D3 ([ 3H]25-(OH)D3) in vitro to material with the characteristics of [3H]24,25-dihydroxyvitamin D3 ([3H]24,25-(OH)2D3) has been studied in the human promyelocytic cell line HL60. Synthesis of 24,25-(OH)2D3 was induced in a dose-dependent manner in cells pretreated with 0.1-100 nM 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) for 4 days. This treatment also inhibited cell proliferation and stimulated differentiation to a macrophage phenotype that was characterized by staining for non-specific esterase (NSE) activity. The ability to synthesize [3H]24,25-(OH)2D3 from [3H]25-(OH)D3 and the expression of NSE activity both responded to changes in concentration of 1 alpha,25-(OH)2D3 in the culture medium in a parallel manner. Synthesis of [3H]24,25-(OH)2D3 was linear when the incubation time was between 1 and 8 h and the cell number between 1 and 12 x 10(6) cells/incubation. The optimum substrate concentration for its synthesis was 125 nM, giving an apparent Michaelis constant of 360 nM. The identity of the [3H]24,25-(OH)2D3 synthesized by these cells was confirmed by co-chromatography with authentic 24,25-(OH)2D3 on normal-phase and reverse-phase high-performance liquid chromatography systems and by its reaction to sodium-m-periodate. Cells that had been exposed to 100 nM 1 alpha,25-(OH)2D3 for 4 days synthesized 2.17 +/- 0.07 (S.E.M.) pmol 24,25-(OH)2D3/10(6) cells per h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Staurosporine, a novel protein kinase inhibitor, enhances HL-60-cell differentiation induced by various compounds

The effect of staurosporine, a novel calcium/phospholipid-dependent protein kinase (protein kinase C) inhibitor, on differentiation of human promyelocytic leukemic HL-60 cells, was investigated. Staurosporine inhibited HL-60-cell proliferation in a concentration-dependent manner, but did not induce HL-60-cell differentiation by itself. When staurosporine was added to HL-60 cells treated with a suboptimal concentration (1 nM) of 1 alpha,25 dihydroxyvitamin D3 (1,25(OH)2D3), cell differentiation was enhanced in a concentration-dependent manner and the percentages of nitro blue tetrazolium reducing ability and nonspecific esterase activity-positive cells increased from 6% to 51% and from 8% to 54%, respectively, on day 4 at a concentration of 5 nM. Staurosporine (5 nM) achieved almost the same enhancement effect in cultures treated with suboptimal concentrations of 1 nM all-trans-beta-retinoic acid (RA), 3 ng/ml actinomycin D (Act D), 100 microM dibutyryl cyclic adenosine 3':5'-monophosphate (dbc AMP), and 50 microM prostaglandin E1 (PG E1). These results suggest that the inhibition of protein kinase C activity by staurosporine exerts an important role in HL-60-cell differentiation induced by various compounds. Moreover, staurosporine (5 nM) completely inhibited optimal concentrations (50 nM) of [12-o-tetradecanoyl phorbol-13-acetate (TPA)]-induced cell differentiation, but enhanced optimal concentrations of dbc AMP (1 mM)-induced cell differentiation. On the other hand, 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine, which has been reported to inhibit cyclic adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) as much as protein kinase C, completely inhibited both cell differentiations induced by optimal concentrations of TPA (50 nM) and induced by optimal concentrations of dbc AMP (1 mM), and did not significantly enhance HL-60-cell differentiation induced by suboptimal concentrations of 1,25(OH)2D3, RA, and dbc AMP. Therefore, these results suggest that the inhibition of protein kinase C, which is not accompanied by that of protein kinase A, is concerned with the induction of HL-60-cell differentiation.     

Novel vitamin D analogs that modulate leukemic cell growth and differentiation with little effect on either intestinal calcium absorption or bone mobilization

Induction of terminal differentiation of leukemic and preleukemic cells is a therapeutic approach to leukemia and preleukemia. The 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active form of vitamin D3, can induce differentiation and inhibit proliferation of leukemia cells, but concentrations required to achieve these effects cause life-threatening hypercalcemia. Seven new analogs of 1,25(OH)2D3 were discovered to be either equivalent or more potent than 1,25(OH)2D3 as assessed by: (a) inhibition of clonal proliferation of HL-60, EM-2, U937, and patients' myeloid leukemic cells: and (b) induction of differentiation of HL-60 promyelocytes. Furthermore, these analogs stimulated clonal growth of normal human myeloid stem cells. The most potent analog, 1,25-dihydroxy-16ene-23yne-vitamin D3, was about fourfold more potent than 1,25(OH)2D3. This analog decreased clonal growth and expression of c-myc oncogene in HL-60 cells by 50% within ten hours of exposure. Effects on calcium metabolism of these novel analogs in vivo was assessed by intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Each of the analogs mediated markedly less (10 to 200-fold) ICA and BCM as compared with 1,25(OH)2D3. To gain insight into the possible mechanism of action of these new analogs, receptor binding studies were done with 1,25(OH)2-16ene-23yne-D3 and showed that it competed only about 60% as effectively as 1,25(OH)2D3 for 1,25(OH)2D3 receptors present in HL-60 cells and 98% as effective as 1,25(OH)2D3 for receptors present in chick intestinal cells. In summary, we have discovered seven novel vitamin D analogs that are more potent than the physiologic 1,25(OH)2D3 as measured by a variety of hematopoietic assays. In contrast, these compounds appear to have the potential to be markedly less toxic (induction of hypercalcemia). These novel vitamin D compounds may be superior to 1,25(OH)2D3 in a number of clinical situations including leukemia/preleukemia; they will provide a tool to dissect the mechanism of action of vitamin D seco-steroids in promoting cellular differentiation.     

1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.     

The metabolite 1 alpha,25-dihydroxyvitamin D3 enhances lymphokine- mediated activation of the oxidative metabolism of the U937 cell line and phosphorylation of a 48-kD respiratory burst-associated protein

U937 cells respond to a variety of stimuli with increased differentiation as manifested by reduced growth, increased adherence, increased expression of several surface receptors, and increased capacity for phagocytosis and formation of reactive oxygen intermediates. In the present study the effects of lymphocyte conditioned media, recombinant interferon-gamma (IFN-gamma), and 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the ability to form reactive oxygen intermediates by U937 cells were measured by using the luminol-dependent luminescence (LDL) assay. Neither 1,25(OH)2D3 alone nor IFN-gamma alone enhanced competence for phorbol myristate acetate- stimulated LDL. Cells were capable of moderate LDL after exposure to lymphocyte conditioned media, and this was enhanced by 1,25(OH)2D3 (10(- 8) mol/L) and other vitamin D metabolites at higher concentrations. This effect was not secondary to accelerated production of myeloperoxidase, which is important in the LDL assay. Enhanced phorbol myristate acetate-stimulated phosphorylation of a 48-kd substrate was observed in 32P-labeled intact cells treated with 1,25(OH)2D3 alone or in combination with IFN-gamma. Treatment of cells with IFN-gamma or lymphocyte conditioned media did not alter phosphorylation. These results support the concept that 1,25(OH)2D3 plays a role in phagocyte differentiation and activation beyond the effects of lymphokines. Protein kinase C-mediated phosphorylation reactions may be necessary for the ability of U937 cells to reduce O2 and required for maximal activity under some conditions of incubation.     

Synergistic enhancement by 12-O-tetradecanoylphorbol-13-acetate and dibutyryl cAMP of 1alpha,25-dihydroxyvitamin D3 action in human promyelocytic leukemic HL-60 cells.

We have reported that dibutyryl cAMP (dbcAMP), an activator of cAMP-dependent protein kinase (PKA), potentiated the effects of 1alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3)-induced 24-hydroxylation activity in HL-60 cells by increasing 1,25-(OH)2D3 receptor (VDR). The present study demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent phorbol ester, also potentiated the effect of 1,25-(OH)2D3 on HL-60 cells and that TPA and dbcAMP acted in a synergistic manner to enhance the effect of 1,25-(OH)2D3. It is interesting that TPA induced 24-hydroxylation activity far more efficiently than dbcAMP, in addition to their effects in increasing VDR. TPA increased basal levels of c-fos mRNA to the maximum by 1 h after the treatment, whereas dbcAMP failed to affect c-fos gene expression. Together with the previous data indicating the presence of AP-1-like sequence in the promoter of 24-hydroxylase gene, it was suggested that TPA potentiated the effect of 1,25-(OH)2D3 through an activation of c-fos gene expression. This notion was further supported by the data showing that TPA and dbcAMP also acted in a synergistic manner to activate c-fos gene expression. Neither TPA nor dbcAMP affected c-jun early response gene in the HL-60 clone used in the present study. The present study suggested that the activation of early c-fos response gene by TPA might be another mechanism to enhance the effect of 1,25-(OH)2D3, besides up-regulation of VDR.     

1 alpha,25-Dihydroxyvitamin D3 inhibits gamma-interferon synthesis by normal human peripheral blood lymphocytes.

1 alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active metabolite of vitamin D3, inhibited synthesis of gamma-interferon (IFN-gamma) by phytohemagglutinin-activated peripheral blood lymphocytes (PBLs). A significant reduction of IFN-gamma protein levels in PBL culture medium was achieved with a physiologic 1,25-(OH)2D3 concentration (0.1 nM). 1,25-(OH)2D3 also inhibited accumulation of IFN-gamma mRNA in activated PBLs in a dose-dependent fashion. The ability of 1,25-(OH)2D3 to modulate IFN-gamma protein synthesis was unaltered in the presence of high concentrations of recombinant human interleukin 2. The suppression of IFN-gamma synthesis by PBLs was specific for 1,25-(OH)2D3; the potencies of other vitamin D3 metabolites were correlated with their affinities for the cellular 1,25-(OH)2D3 receptor. The time course of 1,25-(OH)2D3 receptor expression in phytohemagglutinin-activated PBLs was correlated with the time course of 1,25-(OH)2D3-mediated inhibition of IFN-gamma synthesis. In selected experiments, T-lymphocyte-enriched cell preparations were utilized. In these experiments, 1,25-(OH)2D3 was equally active as in PBL preparations. Finally, we examined the effects of 1,25-(OH)2D3 on the constitutive IFN-gamma production by two human T-lymphocyte lines transformed by human T-lymphotropic virus type I. The cell lines were established from a normal donor (cell line S-LB1) and from a patient with vitamin D-dependent rickets type 2 (cell line Ab-VDR). IFN-gamma synthesis by S-LB1 cells was inhibited in a dose-dependent fashion by 1,25-(OH)2D3, whereas IFN-gamma synthesis by Ab-VDR cells was not altered by 1,25-(OH)2D3. The data presented in this study provide further evidence for a role of 1,25-(OH)2D3 in immunoregulation.     

Receptor-mediated monocytoid differentiation of human promyelocytic cells by tumor necrosis factor: synergistic actions with interferon-gamma and 1,25-dihydroxyvitamin D3

Human myeloid leukemia cells respond to various signals by differentiating to more mature cells. This study was designed to evaluate the effects of a mononuclear phagocyte-derived factor, tumor necrosis factor/cachectin (TNF), on the proliferation and differentiation of the human cell lines HL-60 (promyelocytic) and U937 (monoblastic), and to characterize TNF receptors on these cells. TNF had no effect on HL-60 cell growth or thymidine incorporation, but it markedly inhibited that of U937 cells. HL-60 cells treated with TNF formed osteoclast-like polykaryons and developed nonspecific esterase positivity. In a dose-dependent fashion, TNF enhanced HL-60 cell nonspecific esterase activity, H2O2 production, NBT reduction, and acid phosphatase content. Together, TNF and interferon-gamma (IFN-gamma) additively and synergistically caused increases in these activities as well as the expression of HLA-DR and the monocyte antigens LeuM3 (CDw14) and OKM1 (CD11). TNF also synergistically enhanced the differentiating effects of 1,25-dihydroxyvitamin D3. The potentiating actions of D3 of IFN-gamma on the TNF effect were maximal when the two agents were present together throughout the incubation, and pretreatment with TNF augmented the subsequent response to D3, but not IFN-gamma. HL-60 and U937 cells bound 125I-labeled TNF specifically, rapidly, and reversibly with binding constants of 227 and 333 pmol/L and receptors per cell of 4,435 and 6,806 for HL-60 and U937, respectively. Scatchard plots were linear, which suggested single classes of receptors. HL-60 TNF receptors were not changed by a three-day treatment with IFN-gamma or D3. U937 and HL-60 cells internalized and degraded 125I-labeled TNF to comparable degrees. TNF has differing effects on HL-60 and U937 cells that are apparently mediated through comparable high-affinity TNF receptors. The unique responses of different cell types to TNF may be due to postreceptor factors.