Induction Of Monocytic Differentiation Research Articles

A specific type of ganglioside as a modulator of insulin-dependent cell growth and insulin receptor tyrosine kinase activity: Possible association of ganglioside-induced inhibition of insulin receptor function and monocytic differentiation induction in HL-60 cells

Insulin-dependent cell growth has been correlated with insulin receptor function, particularly receptor-associated kinase activity, in in vitro studies. The insulin-dependent phosphorylation of the 95-kDa receptor subunit was clearly inhibited, in a concentration-dependent manner, by the presence of unbranched neolacto series gangliosides having a NeuAc2----3Gal terminus, particularly 2----3-sialosylparagloboside (2----3SPG; IV3NeuAc-nLc4), but not by other gangliosides with a NeuAc2----6Gal terminus or by branched neolacto series gangliosides (e.g. G10). Such inhibition of phosphorylation was minimal with ganglio series gangliosides and negligible with sphingosine, neutral glycolipids, or sulfatide. 2----3SPG did not affect insulin binding to the insulin receptor. Insulin-dependent cell growth and its inhibition by 2----3SPG were observed in three human cell lines so far tested: lymphoid cell line IM9, promyelocytic leukemia cell line HL-60, and erythroleukemia cell line K562. Since IM9 cells contain a much higher quantity of insulin receptor than do HL-60 or K562 cells, insulin-dependent receptor phosphorylation and its inhibition by 2----3SPG in intact cells were clearly observed with IM9 cells. Receptor phosphorylation in intact cells was inhibited when cells were preincubated in the presence of 2----3SPG. Insulin-dependent growth of HL-60 and K562 cells was also inhibited by prolonged culture (96-144 h) with exogenous 2----3SPG. Subsequent to the inhibition of insulin-dependent HL-60 cell growth, a remarkable phenotypic transformation was observed, i.e. changes in morphology, enzymes, and cell-surface markers to those characteristic of monocytes. The level of 2----3SPG in HL-60 cells increased when cells were cultured with 1 alpha,25-dihydroxyvitamin D3 to the same degree seen in cells cultured with 5 microM 2----3SPG. Both these treatments led to inhibition of insulin-dependent cell growth, followed by induction of monocytic differentiation. Thus, the cellular level of 2----3SPG may modulate insulin-dependent cell growth and define the lineage specificity of differentiation through modulation of receptor-associated kinase activity.

Induction of monocytic differentiation and modulation of the expression of c-fos, c-fms and c-myc protooncogenes in human monoblasts by cytokines and phorbolester

Growing evidence suggests that proto-oncogenes regulate central aspects of cellular physiology such as cell proliferation and differentiation. The proto-oncogenes c-fos, c-fms and c-myc are thought to be involved in these processes. In this study the human myelomonoblast line THP-1 has been used to study monocytic differentiation in response to various cytokines and the phorbolester TPA. After treatment of THP-1 cells with Tumor Necrosis Factor (TNF)-alpha, Interleukin (IL-6) and TPA the cells became adherent, lost their division potential and expressed new surface structures associated with monocytic differentiation. The expression of c-fos and c-fms transcripts was rapidly induced within 45 min by these agents and declined to undectable levels within 24 h. Exposure of THP-1 to TNF-alpha, IL-6 and TPA was associated with a rapid downregulation of c-myc expression, that returned to starting levels within 36 h. However, treatment of THP-1 with other cytokines including Granulocyte (G)-, Macrophage (M)-, Granulocyte/Macrophage (GM)-Colony Stimulating Factor (CSF), Interleukin (IL)-3 and Interleukin (IL)-4 failed to result in monocytic differentiation. These data suggest that changes in c-fms, c-myc and c-fos expression may be related to induction of monocytic differentiation and that their appearance or downregulation can be induced by certain cytokines.

Induction of Monocytic Differentiation by Tumor Necrosis Factor in Phorbol Ester-resistant KG-1a Cells

Tumor necrosis factor (TNF) is a regulatory cytokine that has pleiotropic effects on hematopoietic cell growth and differentiation. The present studies have examined the effects of TNF on the differentiation of phorbol-ester resistant human KG-la leukemia cells. Treatment with 100 U/mL of TNF or 33 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) had no detectable effect on the growth of KG-1a cells. In contrast, TNF, but not TPA, induced cellular aggregation and expression of the ICAM-1 adhesion molecule in KG-1a cells. Furthermore, KG-1a cells responded to TNF, but not to TPA, with a partial down-regulation of c-myc mRNA levels and induction of M-CSF gene transcription. Previous work suggested that TNF induces M-CSF gene expression through activation of phospholipase A2 and eicosanoid production. The present studies also demonstrate that TNF stimulated phospholipase A2 activity. In contrast, there was no detectable increase in phospholipase A2 activity following TPA treatment. These results indicate that: 1) certain characteristics of the differentiated monocytic phenotype were induced by TNF in the phorbol ester-resistant KG-1a line, and 2) treatment with TNF and not TPA was associated with activation of phospholipase A2 during induction of monocytic differentiation in these cells.

Inhibition of phorbol ester-induced monocytic differentiation and c-fms gene expression by dexamethasone: potential involvement of arachidonic acid metabolites

The treatment of human U-937 leukemia cells with 12-O- tetradecanoylphorbol-13-acetate (TPA) is associated with induction of monocytic differentiation. However, the signaling pathways responsible for induction of the differentiated monocytic phenotype remain unclear. The present studies demonstrate that dexamethasone blocks TPA-induced U- 937 cell growth inhibition, adherence, and alpha-naphthyl acetate esterase staining. The results also demonstrate that dexamethasone inhibits the appearance of c-fms transcripts associated with TPA treatment. Run-on transcription assays demonstrated that the c-fms gene is transcriptionally active in uninduced U-937 cells and that the rate of transcription is unchanged after dexamethasone and/or TPA treatment. These findings indicated that TPA increases c-fms expression by a dexamethasone-sensitive posttranscriptional mechanism. Treatment of U- 937 cells with TPA was also associated with stimulation of arachidonic acid metabolism. Furthermore, dexamethasone, an inhibitor of phospholipase A2 activity, blocked TPA-induced increases in arachidonic acid release. These findings suggested that TPA may regulate certain features of monocytic differentiation, such as c-fms gene expression, through the formation of arachidonic acid metabolites. Indomethacin, an inhibitor of cyclooxygenase, had no detectable effect on c-fms gene expression. However, the cyclooxygenase metabolite, prostaglandin E2, inhibited the TPA-induced increases in c-fms mRNA levels. Taken together, the results indicate that TPA regulates c-fms gene expression by a dexamethasone-sensitive mechanism and that c-fms mRNA levels are controlled by metabolites of the arachidonic acid pathway.

Inhibition of Phorbol Ester-Induced Monocytic Differentiation and c-fms Gene Expression by Dexamethasone: Potential Involvement of Arachidonic Acid Metabolites

The treatment of human U-937 leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of monocytic differentiation. However, the signaling pathways responsible for induction of the differentiated monocytic phenotype remain unclear. The present studies demonstrate that dexamethasone blocks TPA-induced U-937 cell growth inhibition, adherence, and alpha-naphthyl acetate esterase staining. The results also demonstrate that dexamethasone inhibits the appearance of c-fms transcripts associated with TPA treatment. Run-on transcription assays demonstrated that the c-fms gene is transcriptionally active in uninduced U-937 cells and that the rate of transcription is unchanged after dexamethasone and/or TPA treatment. These findings indicated that TPA increases c-fms expression by a dexamethasone-sensitive posttranscriptional mechanism. Treatment of U-937 cells with TPA was also associated with stimulation of arachidonic acid metabolism. Furthermore, dexamethasone, an inhibitor of phospholipase A2 activity, blocked TPA-induced increases in arachidonic acid release. These findings suggested that TPA may regulate certain features of monocytic differentiation, such as c-fms gene expression, through the formation of arachidonic acid metabolites. Indomethacin, an inhibitor of cyclooxygenase, had no detectable effect on c-fms gene expression. However, the cyclooxygenase metabolite, prostaglandin E2, inhibited the TPA-induced increases in c-fms mRNA levels. Taken together, the results indicate that TPA regulates c-fms gene expression by a dexamethasone-sensitive mechanism and that c-fms mRNA levels are controlled by metabolites of the arachidonic acid pathway.

Post-transcriptional regulation of thymidine kinase gene expression during monocytic differentiation of HL60 promyelocytes

The regulatory mechanism of human thymidine kinase (TK) gene expression was investigated in HL-60 promyelocytes during induction of monocytic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA). The steady-state levels of TK mRNA diminished gradually as cells were treated with TPA. The nuclear run-on experiments were performed and revealed that TPA treatment did not change TK gene activity in HL-60 cells. These findings suggested that the expression of TK mRNA was controlled by a post-transcriptional mechanism. The half-life of mature TK mRNA transcript was found to be more than 8 hours in both proliferating and differentiated HL-60 cells, which indicated that the stability of mature TK mRNA does not play a role in regulating TK gene expression. Analysis of poly(A-) TK mRNAs showed the high molecular weight precursors of TK mRNA which appeared in proliferating cells were not detectable in TPA-treated cells. This finding suggested that the TK mRNA processing event is implicated in the regulation of human TK gene expression in HL-60 cells during monocytic terminal differentiation.

Effects of dexamethasone on induction of monocytic differentiation in human U‐937 cells by dimethylsulfoxide

The present studies demonstrate that dimethylsulfoxide (DMSO) treatment of human U-937 myelomonocytic leukemia cells is associated with induction of monocytic differentiation. The DMSO-induced U-937 monocytic phenotype was associated with 1) growth inhibition, 2) loss of clonogenic survival, 3) increases in alpha-naphthyl acetate esterase (NSE) staining, and 4) increases in cell surface expression of the monocyte marker Mac-1. DMSO treatment of U-937 cells was also associated with down-regulation of c-myc and c-myb gene expression as well as with increases in tumor necrosis factor (TNF) mRNA levels. The results further demonstrate that induction of U-937 monocytic differentiation by DMSO is accompanied by increases in phospholipase A2 activity. Moreover, this stimulation of phospholipase A2 was sensitive to dexamethasone. We therefore studied the effects of dexamethasone on DMSO-induced differentiation of U-937 cells. Although dexamethasone had no effect on growth inhibition or loss of clonogenic survival by DMSO, this glucocorticoid blocked increases in NSE staining and cell surface Mac-1 expression. Dexamethasone also had no effect on the down-regulation of c-myc and c-myb expression but blocked the reappearance of c-myb transcripts after 6 hr of DMSO treatment. Finally, dexamethasone inhibited DMSO-induced increases in TNF gene expression. Taken together, the results demonstrate that dexamethasone inhibits multiple characteristics, including the stimulation of phospholipase A2 activity, associated with DMSO-induced monocytic differentiation of U-937 cells.

M-CSF and M-CSF-receptor gene expression in acute myelomonocytic leukemias

The role of hematopoietic growth factors in the pathogenesis of human leukemias is still obscure. In this study, RNA from 24 human acute myelomonocytic leukemias (AML) was used to analyze the expression of the macrophage colony stimulating factor (M-CSF) and its corresponding receptor (c- fms). Fifty percent of AML cells exhibited c- fms transcripts of regular length but at a lower level than in normal monocytes/macrophages. In most cases the reduced c- fms expression of AML cells was not associated with autostimulatory M-CSF expression. Only a few cases of AML showed co-expression of M-CSF and c- fms, which by contrast was regularly observed in cultivated blood monocytes and some tissue macrophage subsets. Higher levels of c- fms expression could be found in AMLs with a more mature monocytic immunophenotype. Permanent myelomonocytic cell lines expressed c- fms only after induction of monocytic differentiation. Neither the M-CSF gene nor the c- fms gene were rearranged in AML cells. In AML cells the homozygote genotype of the c- fms gene predominated. Our results do not provide evidence for the involvement of M-CSF and c- fms genes in human myeloid leukemogenesis. c- fms expression appears to indicate monocytic differentiation within the myelomonocytic lineage. We found autostimulatory M-CSF expression to be a physiologic feature of some tissue macrophages and hence not necessarily associated with neoplastic proliferation.

Growth, differentiation and the β-adrenergic signal system of HL-60 cells: Characterization in a medium with insulin as the only added protein

The purpose of the present investigation was to define experimental conditions for studies on growth, differentiation and the β-adrenergic signal system of HL-60 cells. The cell medium was completely devoid of added proteins and hormones other than insulin. The HL-60 cell was able to grow and differentiate in this medium. The spontaneous differentiation along the granulocytic pathway after 72 hr, as assessed by the Nitro Blue tetrazolium test, increased by 400% compared to the serum supplemented medium, but the response to 1 μM retinoic acid was equal in the two media. Induction of monocytic differentiation by 0.16 μM phorbol-13-acetate-12-myristate, as determined by surface adherence after 24 hr, was lower in the absence than in the presence of serum. cAMP levels were elevated in response to (−)-isoproterenol. The ec 50 was 0.36 ± 0.01 μM (mean ± SE, N = 3). The β-adrenergic ligand 3H-CGP 12177 was specifically bound to 1 single class of binding sites ( K d : 0.15 ± 0.04 nM, B max: 2220 ± 150, mean ± SE, N = 3). These data are comparable to our previously reported findings in serum supplemented medium. The present data show that HL-60 cells are able to grow and differentiate in the absence of serum proteins and hormones other than insulin. Under the present experimental conditions, these cells possessed functional β-adrenergic receptors.

Phospholipase C activates protein kinase C and induces monocytic differentiation of HL-60 cells

Phospholipase C (PLC)-mediated hydrolysis of membrane phospholipids results in the production of diacylglycerol, inositol phosphates, and choline metabolites. Inositol triphosphate increases calcium levels, while diacylglycerol activates protein kinase C. The present studies demonstrate that exogenous PLC generates inositol phosphates, releases choline metabolites, and activates protein kinase C in human HL-60 promyelocytic leukemia cells. PLC also induced monocytic differentiation of HL-60 cells as manifested by adherence, growth inhibition, and appearance of monocytic cell surface antigens. Furthermore, PLC treatment decreased c-myc mRNA levels and induced c- fos, c-fms, and tumor necrosis factor transcripts. The changes in gene expression induced by PLC are similar to those previously shown to be associated with phorbol ester-induced monocytic differentiation of HL- 60 cells. Our results thus demonstrate that exogenous PLC activates HL- 60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation. PLC may therefore play a role in transducing signals from physiological inducers of monocytic differentiation.

Phospholipase C Activates Protein Kinase C and Induces Monocytic Differentiation of HL-60 Cells

Phospholipase C (PLC)-mediated hydrolysis of membrane phospholipids results in the production of diacylglycerol, inositol phosphates, and choline metabolites. Inositol triphosphate increases calcium levels, while diacylglycerol activates protein kinase C. The present studies demonstrate that exogenous PLC generates inositol phosphates, releases choline metabolites, and activates protein kinase C in human HL-60 promyelocytic leukemia cells. PLC also induced monocytic differentiation of HL-60 cells as manifested by adherence, growth inhibition, and appearance of monocytic cell surface antigens. Furthermore, PLC treatment decreased c-myc mRNA levels and induced c-fos, c-fms, and tumor necrosis factor transcripts. The changes in gene expression induced by PLC are similar to those previously shown to be associated with phorbol ester-induced monocytic differentiation of HL-60 cells. Our results thus demonstrate that exogenous PLC activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation. PLC may therefore play a role in transducing signals from physiological inducers of monocytic differentiation.

Bryostatin 1 activates protein kinase C and induces monocytic differentiation of HL-60 cells

Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL- 60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation.

Bryostatin 1 Activates Protein Kinase C and Induces Monocytic Differentiation of HL-60 Cells

Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL-60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation.

Induction of monocytic differentiation by calcitriol (1,25-dihydroxyvitamin D 3) in the human promyelocytic leukemic cell line (HL-60) in serum-free medium

The effect of calcitriol on the induction of differentiation in human promyelocytic leukemic cell line (HL-60) cultured in serum-free chemically defined medium (SFM) was investigated. The utilization of SFM containing RPMI-1640 basal medium supplemented with insulin (5 μg/ml), transferrin (5 μg/ml), sodium selenite (5 ng/ml), and bovine serum albumin (0.5 mg/ml) allowed a more precise examination of the cellular/molecular mechanism of calcitriol's action in HL-60 cell differentiation without interference of components present in serum. HL-60 cells grown in SFM were induced to differentiate into monocytes/macrophages by calcitriol as indicated by induction of differentiation-associated biological and biochemical parameters: chemiluminescent (CL) responsiveness, lysozyme activity, nonspecific esterase, expression of cell surface antigens, and reduced proliferation. The exposure of HL-60 cells in SFM to calcitriol (from 10 −10 to 10 −8 M) resulted in dose-dependent induction of these parameters, which was similar to those obtained with cells grown in 10% fetal calf serum containing medium (10% SCM). However, calcitriol was 5-fold more potent for HL-60 cells cultured in SFM than those cultured in 10% SCM as indicated by shifts in dose-response curves for induction of CL responsiveness and lysozyme activity. The effect of calcitriol on the proliferation and acquisition of several monocyte-associated cell surface antigens was also more sensitive for HL-60 cells cultured in SFM than for cells grown in 10% SCM. We characterized and quantitated calcitriol receptors in HL-60 cells cultured in SFM in comparison to those in 10% SCM after exposing intact cells to radiolabeled calcitriol. Cells cultured in either SFM or 10% SCM exhibited calcitriol receptors that migrated at 3.4S as a single peak on sucrose gradients and elicited inherent DNA binding ability. There was essentially no difference in the apparent dissociation constants ( K d ) nor in the number of calcitriol binding sites per HL-60 cell, that is ~6.0 × 10 −11 M and ~3000 binding sites/cell respectively. It is concluded that culturing HL-60 cells in SFM results in full expression of calcitriol-induced phenotypic changes excluding the possibility that such changes result from the indirect effect of calcitriol mediated by identified and/ or unidentified components present in serum.

Disparate effects of activators of protein kinase C on HL-60 promyelocytic leukemia cell differentiation.

In previously published studies (Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260, 5979-5984), we demonstrated that the activation of the calcium- and phospholipid-dependent protein kinase C by phorbol esters was dissociable from the induction of monocytic differentiation by these agents in HL-60 promyelocytic leukemia cells. We have now compared the effects of two related diterpenes (mezerein and 12-O-tetradecanoylphorbol-13-acetate) and two cell-permeable diacylglycerols (1-oleoyl-2-acetoylglycerol and 1,2-dioctanoylglycerol) on the induction of differentiation in HL-60 cells. Each of these agents activated protein kinase C in vitro and stimulated the phosphorylation of a number of identical proteins in intact HL-60 cells. Exposure to either of the diterpenes at nanomolar concentrations resulted in an inhibition of cell growth and the induction of qualitatively distinct types of monocytic maturation in HL-60 cells. Conversely, neither of the two diacylglycerols was found to be a potent or efficacious inducer of differentiation, as measured by increases in cell adhesion, nonspecific esterase activity, or phagocytosis, even at growth-inhibitory concentrations. However, concurrent exposure of HL-60 cells to both 1,2-dioctanoylglycerol and the calcium ionophore A23187, at concentrations which were without maturational activity when used separately, resulted in measurable increases in both protein phosphorylation and in the fraction of cells expressing a differentiated phenotype. Taken together, these results suggest that specific biochemical effects associated with 12-O-tetradecanoylphorbol-13-acetate, in addition to the activation of protein kinase C, may be important determinants for the induction of leukemia cell differentiation.

Expression of the human c-fms proto-oncogene product (colony-stimulating factor-1 receptor) on peripheral blood mononuclear cells and choriocarcinoma cell lines.

The c-fms gene product is related, and possibly identical, to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. Using antisera to a recombinant v-fms--coded polypeptide, glycoproteins encoded by the human c-fms locus were detected in mononuclear cells from normal peripheral blood and in promyelocytic HL-60 cells 24 h after induction of monocytic differentiation with phorbol ester. The 150-kD human c-fms--coded glycoprotein was expressed at the cell surface, was active as a tyrosine-specific protein kinase in vitro, and shared primary structural features with the product of the feline retroviral v-fms oncogene. A biochemically indistinguishable glycoprotein was detected in human choriocarcinoma cell lines. Like peripheral blood mononuclear cells and phorbol ester-treated HL-60 cells, the choriocarcinoma cells expressed high affinity binding sites for human CSF-1. In addition to serving as a lineage specific growth factor in hematopoiesis, CSF-1 may play a role in normal trophoblast development.

Induction of monocytic differentiation and bone resorption by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulates bone resorption in man and other vertebrates, in part, by increasing the number of osteoclasts, the principal resorbing cells of bone. Because osteoclasts are very likely derived from a member(s) of the mononuclear phagocyte family, we determined if 1,25(OH)2D3 promotes maturation of these cells by studying its effects on the human promyelocytic leukemia cell line HL-60. Of the vitamin D3 metabolites tested, only 1,25(OH)2D3, at 10(-10) to 10(-7) M, induces the differentiation of HL60 into mono- and multinucleated macrophage-like cells. Phenotypic change is evident within 24 hr and reaches a plateau between 72 and 96 hr of incubation. The changes are metabolite-specific and include (i) adherence to substrate, (ii) acquisition of the morphological features of mature monocytes, (iii) a 4- to 6-fold enhancement in lysozyme synthesis and secretion, (iv) increase in the fraction of alpha-naphthyl acetate esterase-positive cells from approximately 2% to 100% of the population, and (v) the acquisition of several monocyte-associated cell surface antigens. More importantly, treated HL-60 cells acquire the capacity to bind and degrade bone matrix, two of the essential, functional characteristics of osteoclasts and related bone-resorbing cells. These results, considered together with the reported action of 1,25(OH)2D3 on nontransformed mononuclear cells, are consistent with the view that vitamin D3 enhances bone resorption and osteoclastogenesis in vivo by promoting the differentiation of precursor cells.