Augmentation of monocyte chemotaxis by 1 alpha,25-dihydroxyvitamin D3. Stimulation of defective migration of AIDS patients

Preincubation for 20 h with 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) markedly augmented the chemotactic responsiveness of human blood monocytes to the classical chemoattractant, FMLP. A modest enhancement of monocyte spontaneous locomotion in the absence of chemoattractants was also observed. Maximal increase of monocyte migration was observed after pretreatment with 10(-9) M 1,25(OH)2D3 and was detectable at FMLP concentrations ranging from 10(-10) to 10(-7) M. Pretreatment with 1,25(OH)2D3 augmented the number of monocyte high affinity FMLP receptors (1500 +/- 220 and 3800 +/- 300 sites per cell for untreated and 1,25(OH)2D3-pretreated cells, respectively) with no significant changes in Kd values (2 +/- 0.5 nM and 4 +/- 1 nM, for untreated and 1,25(OH)2D3-pretreated monocytes). Enhanced chemotaxis was not restricted to FMLP, because 1,25(OH)2D3-treated monocytes showed enhanced migration also in response to activated C components and chemotactic cytokines. In agreement with previous observations, monocytes from AIDS patients showed defective migration capacity. In vitro exposure to 1,25(OH)2D3 stimulated monocyte migration in all 10 patients examined with considerable quantitative differences among individuals. Regulating the responsiveness of mature monocytes to chemo-attractants, 1,25(OH)2D3, produced systemically or in situ by immunocompetent cells, could play a role in the regulation of the recruitment of monocytes at sites of inflammation, cell-mediated immunity, or bone resorption. The potential of 1,25(OH)2D3 as a restorative agent under conditions of defective phagocyte recruitment deserves further exploration.     

Regulation of RANKL promoter activity is associated with histone remodeling in murine bone stromal cells

Receptor activator of NFkappa-B ligand (RANKL) is essential for osteoclast formation, function, and survival. Although RANKL mRNA and protein levels are modulated by 1,25(OH)2D3 and other osteoactive factors, regulatory mechanisms remain unclear. In this study, we show that 2 kb or 2 kb plus exon 1 of a RANKL promoter sequence conferred neither 1,25(OH)2D3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), however, strongly increased RANKL promoter activity. A series of 5'-deleted RANKL promoter constructs from 2,020 to 110 bp showed fourfold increased activity after TSA treatment. TSA also dose dependently enhanced endogenous RANKL mRNA expression with 50 microM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)2D3. Using a chromatin immunoprecipitation (ChIP) assay we showed that TSA significantly enhanced association of both acetylated histone H3 and H4 on the RANKL promoter, with H4 > H3. A similar increase in acetylated histone H4 on the RANKL gene locus was seen after 1,25(OH)2D3 treatment, but ChIP assay did not reveal localization of VDR/RXR heterodimers on the putative VDRE of the RANKL promoter. To explore the role of H4 acetylation of 1,25(OH)2D3 stimulated RANKL, we added both TSA and 1,25(OH)2D3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)2D3-induced RANKL mRNA expression by 70% at all doses of 1 ,25(OH)2D3 studied. These results suggest that TSA increases of endogenous expression of RANKL involve enhanced acetylation of histones on the proximal RANKL promoter. Preventing deacetylation, however, blocks 1,25(OH)2D3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression.     

Vitamin D(3) and analogues modulate the expression of CSF-1 and its receptor in human dendritic cells

The active vitamin D(3)-metabolite 1,25(OH)(2)D(3) inhibits the interleukin 4/granulocyte-macrophage colony-stimulating factor (IL-4/GM-CSF)-induced differentiation of human monocytes into dendritic cells without altering survival. Colony-stimulating factor 1 (CSF-1) is an important survival factor for cells of the monocytic lineage. We therefore investigated whether the inhibitory activity of 1,25(OH)(2)D(3) is paralleled by a regulation of CSF-1 and its receptor. Purified human monocytes were cultured together with IL-4/GM-CSF in the presence of 1,25(OH)(2)D(3), its analogue tacalcitol, the low-affinity vitamin D receptor ligand 24,25(OH)(2)D(3), or the solvent ethanol for up to 5 days. Expression of CSF-1, CSF-1R, and GM-CSF mRNA was measured by RT-PCR. Protein secretion for CSF-1 was measured by ELISA, expression of CSF-1R by flow cytometry. The results showed that 1,25(OH)(2)D(3) and tacalcitol significantly up-regulated CSF-1 mRNA-expression and protein secretion in a dose-dependent manner. The effect of 1,25(OH)(2)D(3) occurred already after 1h of pre-treatment. In contrast, CSF-1R mRNA- and cell surface-expression was down-regulated simultaneously. The solvent ethanol and 24,25(OH)(2)D(3) were without effect. GM-CSF mRNA expression was not modulated in 1,25(OH)(2)D(3)-treated cells. These data point towards a distinct and specific regulation of CSF-1 and its receptor by 1,25(OH)(2)D(3) and its analogue tacalcitol in human monocytes which parallels the inhibition of differentiation into dendritic cells without altering survival.     

Role of c-Myc in nitric oxide-mediated suppression of cytochrome P450 3A4

Cytochrome P450 (CYP) 3A4, which is abundant in human liver and small intestine and participates in the metabolism of various drugs and xenochemicals, is known to be induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the colon carcinoma cell line Caco-2 cells. Nitric oxide (NO) is able to inhibit CYP3A4 expression and catalytic activity. In this study, we investigated the mechanism of suppression by NO of 1,25(OH)2D3-induced CYP3A4 expression in Caco-2 cells. Caco-2 cells were exposed for 36 h to 400 nM 1,25(OH)2D3, and the induction of CYP3A4 mRNA expression was detected by real-time PCR. Because c-Myc regulates the expression of several genes, we examined its effect on the CYP3A4 expression induced by 1,25(OH)2D3. The expression of c-myc mRNA was increased in the early stage but decreased 36 h after the treatment of Caco-2 cells with 1,25(OH)2D3. The NO donor NOR-4 suppressed CYP3A4 expression induced by 1,25(OH)2D3 in Caco-2 cells in contrast, it significantly induced c-myc gene expression. Treatment of Caco-2 cells with the c-myc antisense oligonucleotide reversed the inhibitory effect of NOR-4 on CYP3A4 expression induced by 1,25(OH)2D3. These results suggest that the suppression of 1,25(OH)2D3-induced CYP3A4 expression by NO is due to c-myc expression.     

Increased level of calcitonin mRNA after 1,25-dihydroxyvitamin D3 injection in the rat

Vitamin D metabolites are able to change plasma calcitonin (CT) levels, but nothing is known about a possible effect at the CT gene level. Here we have investigated the acute effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on the CT biosynthetic activity of thyroid glands from adult rats. Plasma CT levels were significantly increased (X2) 1 and 2 h after 1,25-(OH)2D3 injection in the face of unchanged plasma calcium values. The thyroidal CT content also was unchanged. A 2-fold increase in CT mRNA level measured by dot-blot hybridization occurred 1 and 2 h after 1,25-(OH)2D3 administration. Expression of CT gene products was examined in the rabbit reticulocyte lysate cell-free translation assay. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A single precursor of Mr approximately equal to 15 000 could be specifically immunoprecipitated with CT antisera. A 3-4-fold rise in translatable CT mRNA activity was observed 1 and 2 h after 1,25-(OH)2D3 injection. Thus, parallel changes in CT mRNA level, CT mRNA activity and plasma CT levels were observed in adult female rats after administration of 1,25-(OH)2D3. These findings demonstrate for the first time that 1,25-(OH)2D3 enhanced CT gene expression in the face of unchanged plasma calcium levels.     

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.     

1,25-Dihydroxyvitamin D3 and the regulation of the differentiation and function of macrophages and granulocytes

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) exerts a differential inhibitory effect on the formation of granulocyte, granulocyte/macrophage, and macrophage colonies grown from mouse bone marrow precursor cells; 50% inhibition was attained at 1.1, 2.3, and 23 nM 1,25(OH)2D3, respectively. The inhibition of colony formation, as well as phagocyte proliferation in liquid cultures, requires the presence of 1,25(OH)2D3 in the early stages of culture (up to 72 h after culture initiation). 1,25(OH)2D3 induces a dose- and time-dependent augmentation of the phagocytic capability of mononuclear phagocytes (up to 100%) towards both heat-killed yeast cells and IgG-coated sheep red blood cells. The augmentation of the phagocytic capability of the mononuclear phagocytes depends critically on when 1,25(OH)2D3 is added. It is effective when added up to 72 h after culture initiation, while at later stages (greater than or equal to 96 h) the cells are no longer induced to express enhanced phagocytic capability. We suggest that these phenomena may be relevant to hemopoietic processes.     

1 alpha,25-dihydroxyvitamin D3 inhibits productive infection of human monocytes by HIV-1.

Pretreatment of freshly isolated human peripheral blood monocytes with the steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)D), markedly reduced (by 95%) productive infection of human monocytes by HIV-1. Equivalent concentrations (10nM) of 25-hydroxyvitamin D3 (25(OH)D), the biologic precursor of 1,25(OH)D, were ineffective at reducing either CD4 expression or HIV-1 production. Pretreatment was required for modulation of HIV-1 infection by 1,25(OH)D. Interestingly, 1,25(OH)D-mediated decreases in p24 antigen production were observed prior to any observed reduction in CD4 expression, suggesting that 1,25(OH)D treatment may modulate HIV-1 infection of monocytes through additional factors besides decreased HIV-1 binding. These data raise the possibility that 1,25(OH)D compounds may be important in host resistance to HIV-1.     

Differential effects of 1,25-dihydroxyvitamin D3 on human lymphocytes and monocyte/macrophages: inhibition of interleukin-2 and augmentation of interleukin-1 production

Human peripheral blood monocytes and activated, but not resting, lymphocytes possess specific intracellular receptors for the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The effects of 1,25-(OH)2D3 on the function of these cells was therefore examined. The addition of physiologic concentrations of the hormone (0.001-0.1 nM) to lectin- or antigen-activated lymphocytes resulted in inhibition of lymphocyte proliferation. Supernatants from lectin-activated lymphocytes incubated with 1,25-(OH)2D3 had reduced interleukin-2 (IL-2) activity. The immediate biological precursor of 1,25-(OH)2D3, 25-hydroxyvitamin D3, did not affect function of lymphocytes or monocytes. The ability of exogenous recombinant IL-2 to reverse the inhibitory effects of the hormone on lymphocyte proliferation suggest that 1,25-(OH)2D3 does not alter the generation of IL-2 receptors. In contrast to its effects on IL-2 production, 1,25-(OH)2D3 caused a dose-dependent increase in the production of interleukin-1 (IL-1) by monocyte/macrophages. These results suggest that immune cells and their products can be regulated in a specific but diverse fashion by the vitamin D3-endocrine system.     

Divergent regulation of 1,25-dihydroxyvitamin D3 on human bone marrow osteoclastogenesis and myelopoiesis

The physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has influence over osteoclastogenesis and myelopoiesis, but the regulational mechanism is not well-defined. In this report, formation of osteoclast-like (OCL) cells from primitive myeloid colony-forming cells (PM-CFC) as mediated by 1,25(OH)2D3 was examined. Our results present in this report clearly show that 1,25(OH)2D3 dose-dependently stimulated OCL cell formation when added to suspension cultures of individually replated PM-CFC colonies. Marrow cells were plated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or the human bladder carcinoma cell line 5637 conditioned medium (5637 CM) as the source of colony-stimulating activity. The 1,25(OH)2D3 effect of osteoclast differentiation was associated with a concomitant decrease in clonogenic growth of myelopoietic progenitors in response to colony-stimulating activity. Secondly, the effect of adding the known stimulator of hematopoiesis, interleukin-1beta (IL-1beta) and/or 1,25(OH)2D3 on human myeloid colony growth was assessed. IL-1beta enhanced the formation of primitive myeloid colonies in response to GM-CSF by 160%. On the other hand, 1,25(OH)2D3 dose-dependently inhibited both GM-CSF- and 5637 CM-driven myeloid colony formation by as much as 90% at 100 nM. Addition of IL-1beta to GM-CSF-stimulated cultures dampened the inhibitory effect of 1,25(OH)2D3. The inhibition of myeloid clonogenic growth by 1,25(OH)2D3 was almost abolished (89%) by simultaneously adding anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNF-alpha MoAb) to the culture medium. These results collectively suggest divergent roles for 1,25(OH)2D3 in osteoclastogenesis and myelopoiesis, promoting the differentiation of OCL cells from primitive myeloid cells but inhibiting the proliferation of later myeloid progenitor cells. This inhibition of myeloid progenitors may be mediated by TNF-alpha.     

Modulation by cAMP of 1alpha,25-dihydroxyvitamin D3 sensitivity of murine erythroleukemia cells.

As we previously reported, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently inhibited not only proliferation of undifferentiated murine erythroleukemia (MEL) cells but also activin A-induced erythroid differentiation of MEL cells. However, the effect of 1,25(OH)2D3 on MEL cell proliferation was significantly greater by one order of magnitude than that on differentiation (IC(50): 9.2 vs 0.8 nM, respectively). The response of activin A-treated mature MEL cells to 1,25(OH)2D3 in the induction of 1,25(OH)2D3-24-hydroxylase (24-OHase) activity, a rapid effect of 1,25(OH)2D3, was enhanced to the same degree as in untreated immature cells, suggesting that differences in capacity of cells to inactivate 1,25(OH)2D3 did not contribute to augmentation of 1,25(OH)2D3 effect in activin A-treated mature cells. Furthermore, neither the number nor the affinity of vitamin D receptors (VDR) differed significantly between activin A-treated cells and untreated immature cells. The intracellular cAMP level, which affects 1,25(OH)2D3-mediated induction of 24-OHase activity, was significantly less in activin A-treated mature cells than in immature MEL cells. The addition of dibutyryl cAMP (dbc AMP) to activin A-treated MEL cells dose-dependently attenuated 1,25(OH)2D3-mediated induction of 24-OHase activity, finally to a level comparable to that of the untreated cells at the final concentration of 100 nM dbcAMP, while dbcAMP itself by 100 nM did not affect MEL cell differentiation by 24 h. In summary, we have shown for the first time that 1,25(OH)2D3 exerted its effect on leukemia cells at physiological concentration and that the magnitude of this effect depended on the changes in intracellular cAMP level through stages of differentiation, suggesting that the cAMP-protein kinase A system may be useful as a target for clinical application of vitamin D analogs by improving the sensitivity of leukemic cells to 1,25(OH)2D3.     

Effect of 1,25-dihydroxyvitamin D3 on human cancer cells in vitro

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dependent growth and differentiation of 6 tumor cell lines has been determined by the use of the monolayer proliferation assay. Cell lines of 4 gastro-intestinal carcinomas, 1 malignant schwannoma, and 1 malignant histiocytoma have been established and characterized. Cells were incubated for 4, 7, and 11 days in the presence of 0.8 or 8 nM 1,25(OH)2D3 and for control without addition of the hormone. Proliferation rates of 1,25(OH)2D3 treated cells were compared with cell growth in the untreated controls. Five out of 6 cell lines showed a 1,25(OH)2D3 dependent growth pattern. With 8 nM 1,25(OH)2D3 they were all inhibited. With 0.8 nM, 3 of them were inhibited at any time of the test period, whereas 1 was stimulated at day 4 and inhibited at days 7 and 11. One cell line was stimulated at days 4, 7, and 11 when incubated with 0.8 nM 1,25(OH)2D3. No striking morphological changes could be observed in the presence of 1,25(OH)2D3. We conclude that 1,25(OH)2D3 dependent cells in vitro are not necessarily growth-inhibited by this compound. Thus, 1,25(OH)2D3 is not an exclusively proliferation inhibiting agent.     

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.     

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

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.