©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Monocytic Differentiation of Acute Promyelocytic Leukemia Cells in Response to 1,25-Dihydroxyvitamin D Is Independent of Nuclear Receptor Binding (*)

Mickie Bhatia , James B. Kirkland , Kelly A. Meckling-Gill (§)

From the (1)Department of Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1

ABSTRACT
INTRODUCTION
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have shown that 1,25-dihydroxyvitamin D (1,25(OH)D) primes NB4 cells, the only available acute promyelocytic leukemia cell line, for 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiation. Here, we have used isomers of 1,25(OH)D to investigate the role of 1,25(OH)D and its putative nuclear receptor (VDR) in NB4 cell monocytic differentiation. 1,25-dihydroxyvitamin D (HL), a specific antagonist of only the nongenomic signals of 1,25(OH)D, attenuated the priming effect of 1,25(OH)D. The 6-cis conformer of 1,25(OH)D (HF), which is unable to bind to VDR, was 20 times more potent than 1,25(OH)D as a priming agent for monocytic differentiation. This response was also blocked by the HL antagonist. Unlike myelocytic HL-60 cells, which respond to 1,25(OH)D with increases in VDR expression and monocytic differentiation, neither HF nor 1,25(OH)D regulated VDR expression in NB4 cells. In the monocytic differentiation of acute promyelocytic leukemia cells, 1,25(OH)D appears to signal through a pathway independent of VDR/VDRE action.


INTRODUCTION

1,25-Dihydroxyvitamin D (1,25(OH)D)()is responsible for the classic biological activities of vitamin D, such as regulation of calcium homeostasis(1, 2) . In addition, it plays important roles in cell growth and differentiation(3, 4) . Cultured leukemia cells are the most widely used in vitro model system for studying the differentiation effects of 1,25(OH)D(5) . Several laboratories have shown that the phenotype of 1,25(OH)D-treated cells resembles that of circulating monocytes(6, 7) . Cultured HL-60 myelocytic leukemia cells have been the model of choice in identifying the pathways and genes that are regulated during cellular differentiation(8) . The biological responses seen in HL-60 cells can primarily be attributed to the genomic actions of 1,25(OH)D mediated through the nuclear vitamin D receptor, VDR, which is necessary for transcriptional regulation of targeted genes(9, 10) . Treatment of HL-60 cells with 1,25(OH)D results in increased expression of VDR at both transcriptional and post-transcriptional levels(11, 12) .

Recent reports have suggested that some of the activities of 1,25(OH)D may be membrane-mediated through pathways independent of the classic VDR(13, 14) . This is consistent with other steroid responsive systems, such as those for progesterone, glucocorticoids, and estradiol, for which evidence of nongenomic effects have been presented(15, 16) . 1,25(OH)D has been shown to cause changes in phospholipid metabolism in myoblasts causing activation of phospholipase C and has also been shown to generate diacylglycerol and activate protein kinase C in HL-60 cells within seconds of initial 1,25(OH)D exposure(17, 18) . Although rapid effects of 1,25(OH)D can be observed in HL-60 and U-937 cells, it is unknown whether these events have any effect on the differentiation program(18) . Current evidence suggests that different forms of 1,25(OH)D receptors are involved in transducing signals which are associated with these genomic and nongenomic responses(19) .

NB4 cells are genetically different from HL-60 cells in that they possess the diagnostic translocation characteristic of APL t(15;17) which causes the disruption of the retinoic acid receptor gene and expression of the PML/RAR fusion protein(20) . This aberration is thought to be the cause of oncogenesis in both this cell line and in APL(20) . HL-60 cells not only lack the t(15;17) translocation but have activating mutations in the Ni-ras and c-myc oncogenes, events thought to promote the leukemic phenotype in this cell type(21, 22) . NB4 cells respond to 1,25(OH)D and TPA differently than HL-60 cells. Either TPA or 1,25(OH)D alone will differentiate HL-60 cells along the monocytic pathway. We have recently shown that neither agent alone is able to induce monocytic differentiation in NB4 cells while combining the two induces elicits a very potent induction of monocytic differentiation(23) . 1,25(OH)D is required during the priming phase of NB4 cell differentiation which involves both protein kinase C and unidentified tyrosine kinases.()This led us to hypothesize that 1,25(OH)D was priming NB4 cells for monocytic differentiation via nongenomic pathways or what we have called VDR/VDRE-independent pathways.

Authentic 1,25(OH)D can undergo rotation about the 6-7 carbon-carbon bond to yield 6-transand 6-cis conformers, displaying extreme conformational flexibility (Fig. 1A). This enables 1,25(OH)D and its isoforms to assume a wide variety of shapes including conformational flexibility of the side chain, B ring, and A ring chair-chair interconversion. Analogues of 1,25(OH)D with restricted conformations have been developed to evaluate the biological roles of genomic and nongenomic pathways for 1,25(OH)D action. The 6-cis conformer of 1,25(OH)D (HF, Fig. 1B) has been shown by Norman et al. (25) to be a selective activator of the nongenomic functions of 1,25(OH)D. The HF analogue is virtually unable to bind and activate VDR, and attempts to induce monocytic differentiation of HL-60 cells have been unsuccessful(25) . Norman's group has also developed a potent antagonist of the nongenomic effects of 1,25(OH)D. This isomer, 1,25-dihydroxyvitamin D (HL, Fig. 1C), blocks all known nongenomic 1,25(OH)D responses (transcaltachia, Ca uptake by voltage-gated channels) but is unable to block genomic activities and the associated monocytic differentiation in HL-60 cells(26) . NB4 cells were treated with these isomers to elucidate the signaling mechanism of 1,25(OH)D that induces monocytic differentiation.


Figure 1: Structure of 1,25(OH)D isomers and analogues. The 6-s-cis conformer is similar to pre-D except in the position of double bonds at carbons 6 and 7. This allows for rotation around the 6- and 7-carbon and the resulting 6-s-cis conformation which can easily rotate back into the 6-s-trans conformation. The HF analogue is maintained in the cis conformation due to proton substitution with deuterium which makes the trans conformation energetically unfavorable.




RESULTS AND DISCUSSION

NB4 cells were treated with 1,25(OH)D, vehicle (ethanol), or the HL and HF analogues alone or in combination for 8 h, washed 3 times in PBS, and treated with 200 nM TPA for up to 72 h. They were then assessed for expression of differentiation markers, including adherence, CD14, and esterase expression and phagocytic capacity (Fig. 2, ). The percentage of cells expressing features of monocyte/macrophage differentiation was maximal at 200 nM 1,25(OH)D, and results were similar to those we have reported previously(23) . Adding the nongenomic antagonist, HL, at either 100 or 200 nM attenuated the 200 nM 1,25(OH)D response, although 200 nM HL was more effective than 100 nM. Maximal inhibition of the differentiation response was achieved at equimolar concentrations of HL and 1,25(OH)D whether 200 nM or 10 nM 1,25(OH)D was used. 200 nM HL, followed by TPA treatment, produced only a few percent more esterase-positive cells than ethanol followed by TPA. There was no increase in phagocytic activity, although increases in CD14 expression and adherence were demonstrated. 10 nM HL produced no significant changes in monocytic markers compared to ethanol alone (data not shown). Given that esterase expression and phagocytic ability represent functional activity of cells in the monocytic lineage, HL does not appear capable of inducing mature cell function even if some changes in phenotype are apparent. Since HL is capable of weak binding to VDR, it is possible that changes in CD14 expression and adherence properties are mediated through VDRE.


Figure 2: Expression of monocytic differentiation markers in NB4 cells exposed to 1,25(OH)D isomers. NB4 cells (2.5 10 cells/ml) were grown in Iscove's modified Dulbecco's medium + 10% fetal bovine serum and exposed to various agents for 8 h. Cells were washed and resuspended in media containing 200 nM TPA for up to 72 h and assessed for differentiation markers. All cultures had greater than 83% viability as assessed by trypan blue dye exclusion. Stocks of 1,25-(OH)-9,14,19,19,19-pentadeuterio-pre-D compound, an analogue of the 6-cis conformer (analogue HF) and the antagonist 1,25-dihydroxyvitamin D (analogue HL) were a generous gift from Dr. A. Norman (University of California, Riverside). -Napthol acetate esterase (Sigma) was assessed according to manufacturers' instructions in 12-well dishes as described previously (Bhatia et al. (23)) from a population of at least 200 cells. Adherence was assessed as a percentage of total cells in each well following a gentle PBS wash and removal of adherent cells with cold PBS-trypsin-10 mM EDTA. For assessment of phagocytosis, magnetic polystrenylene beads coated (4.5 µm) with primary monoclonal antibody IgG specific for the CD14 membrane antigen expressed on human monocyte/macrophage were used according to manufacturers' instructions (Dynabeads, Lake Success, NY). Positive cells were expressed as a percentage of total cells in each culture condition. Experimental data (n = 4) were analyzed by analysis of variance followed by Newman-Keuls test for differences between means using the general linear modelling procedure (Instat GraphPad Software, San Diego, CA). Bars with different superscripts are statistically different (p < 0.05).



The 6-cis conformer of 1,25(OH)D, HF, which does not bind VDR and is incapable of inducing monocytic differentiation in HL-60 cells, was used in NB4 cells to verify that 1,25(OH)D was indeed priming NB4 cells for monocytic differentiation without VDR complex formation. In fact, the HF analogue was a more potent priming agent than 1,25(OH)D; a near-maximal response was achieved at 10 nM, 20-fold lower than the concentration of 1,25(OH)D required for a complete response (Fig. 2, ). The HF priming effect could also be attenuated by the HL antagonist at concentrations similar to those used for inhibition of 1,25(OH)D priming (Fig. 2, ). The observations that HF mediates its effects through nongenomic pathways, and HL effectively inhibits the differentiation response, support the idea that both 1,25(OH)D and HF signal through pathways independent of VDR.

During 1,25(OH)D-induced differentiation in HL-60 cells, the increase in VDR expression is considered to be the primary function of 1,25(OH)D(11) . There is a direct relationship between high sensitivity to 1,25(OH)D-induced differentiation and the degree of up-regulation of VDR in sublines of HL-60 which vary in their responsiveness to 1,25(OH)D(27) . Moreover, HL-60 cells resistant to 1,25(OH)D were unable to up-regulate VDR(27) . Therefore, it was important to determine whether NB4 cells expressed VDR and, if so, to characterize its regulation when cells were exposed to 1,25(OH)D and the HF analogue. Western blot analysis (Fig. 3) demonstrated that although VDR is expressed in control cultures of both NB4 and HL-60 cells, its expression was only modulated in HL-60 cells. Neither ethanol, 1,25(OH)D, nor HF had any affect on VDR expression in NB4 cells. However, all three agents stimulated VDR expression in HL-60 cells even at the early 4-h time point. HL-60 cells up-regulate VDR by greater than 3-fold in response to either 1,25(OH)D or HF when compared to untreated cells at 24 h of exposure (Fig. 3). Surprisingly, VDR levels were also increased 2-fold in response to ethanol alone (vehicle). Ethanol is not an inert molecule and has the ability to modulate cellular signaling cascades including adenylyl cyclase (28) and phospholipase C(29) . Whether either of these pathways is involved in modulating VDR expression in ethanol-treated HL-60 cells remains to be determined. Previous studies investigating changes in VDR expression in response to 1,25(OH)D may not have considered control experiments in which cultures were exposed to vehicle alone and thus never reported any changes in VDR expression in response to ethanol.


Figure 3: Regulation of VDR in NB4 and HL-60 cells exposed to 1,25(OH)D and the HF analogue. NB4 and HL-60 cells were exposed to 200 nM 1,25(OH)D or ethanol (A) or 100 nM HF (B) for various lengths of time. Lysates were prepared from equal numbers of NB4 and HL-60 cells for SDS-PAGE at various time points, and proteins were separated on 15% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes using a semidry blotting apparatus for 2 h at 100 mA. Membranes were blocked with 5% milk powder in Tris-buffered saline (TBS), washed in TBS with 0.05% Tween 20, and incubated with rat monoclonal antibody (IgG) directed against the C-terminal end of the DNA binding domain of human VDR (Affinity Bioreagents, Neshanic Station, NJ). Horseradish peroxidase-conjugated anti-rat IgG was used as the secondary antibody with the ECL detection system (Amersham) and quantified by autoradiography and densitometry. Statistical differences between time 0 and all other time points were found for EtOH, 1,25(OH)D, and HF in HL-60 cells. No differences were found between any time points in NB4 cells regardless of the compound used. Shown is a representative example of at least four separate experiments.



Only a few genes that appear to be regulated by 1,25(OH)D have been shown to contain VDREs in their promoter regions and, thus, to be under direct control of VDR (30). The mechanism of up-regulation of nuclear receptor in HL-60 cells has been proposed to occur through an increase in protein stability rather than an increase in VDR transcription rate. HF treatment increases the level of VDR in HL-60 cells but is incapable of efficiently binding to VDR. This provides further credence to the idea that 1,25(OH)D need not bind VDR in order to modulate functional gene expression. Although increased VDR expression may be required for monocytic differentiation of HL-60 cells, increased VDR expression in response to HF in the absence of 1,25(OH)D does not lead to monocytic differentiation.

Since HF does not bind VDR and does not lead to HL-60 differentiation, the hypothesis still holds that VDR binding ability is a determinant of the potency of 1,25(OH)D analogues as monocytic differentiation inducers in the HL-60 model. Consistent with our observations, a recent evaluation of 1,25(OH)D analogues by Holick and co-workers (31) demonstrated that low affinity binding to VDR was correlated with a poor differentiation response in HL-60 cells. In contrast, NB4 cells neither regulate VDR expression in response to 1,25(OH)D nor require an increase in VDR expression to differentiate. This finding is consistent with the premise that monocytic differentiation in this cell type does not require the genomic actions of 1,25(OH)D.

HL-60 cell lines which are resistant to 1,25(OH)D continue to differentiate in response to TPA and, similarly, TPA-resistant HL-60 cells will differentiate in response to 1,25(OH)D(32) . These observations distinguish between two signaling pathways to induce monocytic differentiation. As NB4 cells are unable to completely differentiate in response to 1,25(OH)D or TPA alone and do not regulate VDR in response to 1,25(OH)D treatment, it appears that 1,25(OH)D and TPA signal through at least partially independent pathways. The responses to 1,25(OH)D are very different in NB4 cells as compared to HL-60 cells. This study suggest that 1,25(OH)D seems to initiate the monocytic differentiation program by more than one mechanism. HL-60 cells respond only to isoforms of 1,25(OH)D which are capable of VDR binding, and antagonists of the nongenomic effects of 1,25(OH)D are unable to block monocytic differentiation. NB4 cells, however, respond to the nongenomic signals of 1,25(OH)D. Differences in the mechanism of 1,25(OH)D action may be due to differences in the origin of these two cells types, likely involving differential gene expression and developmental arrest at different points during hematopoiesis. Potential heterodimerization between RAR and VDR(33) , along with the mutation in the RAR receptor in the NB4 cells and its ability to respond selectively to the nongenomic actions of 1,25(OH)D, raise many questions involving the potential evolution of different receptors to bind differently shaped 1,25(OH)D ligands(34) .

Since increases in VDR levels can be induced without ligand-nuclear receptor complex formation (ethanol alone stimulated VDR expression), a mechanism other than the VDR/VDRE-mediated pathway is functioning(35) . VDR may be being directly activated through a dephosphorylation of serine 51(36) , which could be responsible for the increased VDR expression in response to either 1,25(OH)D or the HF analogue in HL-60 cells.

The observations that NB4 cells do not differentiate in response to TPA or 1,25(OH)D alone(23) , but that 1,25(OH)D primes cells via VDR/VDRE independent pathways for TPA induced-differentiation and that all-trans retinoic acid is a potent priming agent in ATRA-resistant NB4 cells for cAMP-dependent differentiation (37) makes NB4 cells an excellent model for studying two-stage differentiation processes. The HL-60 cell model has been routinely used to test analogues of 1,25(OH)D that lack calcemic effects, for potential use as alternative differentiation agents to 1,25(OH)D(38, 39) . In theory, analogues with these characteristics could be used in differentiation therapy to treat patients with acute myelocytic leukemias. These analogues are consistently designed to achieve high affinity binding to VDR, a characteristic which also allows for binding to vitamin D binding protein. Bouillon et al.(24) have shown that vitamin D binding protein counteracts the effects of 1,25(OH)D and its isomers, an effect that is directly proportional to the binding affinity of the analogue for VDR. This suggests that isoforms which bind VDR with high affinity may be of less clinical use in vivo.

NB4 cells are derived from human APL blasts; therefore, analogues studied in this model may have greater utility in the treatment of APL than studies done in other leukemic cell lines including HL-60 cells. Since NB4 cells undergo monocytic differentiation independent of 1,25(OH)D nuclear receptor binding, we suggest that this model be considered when designing 1,25(OH)D analogues for use as differentiation agents. Moreover, this cell line provides an excellent model for characterization of 1,25(OH)D nongenomic signals and identification of the putative ``membrane recognition element'' for 1,25(OH)D.

  
Table: Phagocytic capacity of treated NB4 cells

Shown is the population distribution of phagocytic cells and the number of beads/cell engulfed when cultures were treated with 1,25(OH)D or related isomers in phase I treatment (8 h) followed by 200 nM TPA as phase II (24 h). Phagocytic activity was assessed 72 h after initial treatment. Latex beads (3 µm, Sigma) were added to cultures during the last 5 h of treatment, plates were washed, and cells were fixed and counterstained with Wright's stain. The number of cells containing beads and beads/cell (for at least 200 cells/treatment) were assessed by light microscopy (150 magnification). Values with different superscripts are statistically different from each other by analysis of variance followed by Newman-Keuls test for differences between means using the general linear modelling procedure (Instat GraphPad, Software). Differences were considered significant at p < 0.05 (n = 4).



FOOTNOTES

*
This work was supported by the American Institute for Cancer Research and The Natural Sciences Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 519-824-4120 (Ext. 3742); Fax: 519-763-5902; E-mail: kmeckling.ns@aps.uoguelph.ca.

The abbreviations used are: 1,25(OH)D, 1,25-dihydroxyvitamin D; PBS, phosphate-buffered saline; TPA, 12-O-tetradecanoylphorbol-13-acetate; VDR, vitamin D receptor; VDRE, vitamin D response element.

M. Bhatia, J. B. Kirkland, and K. A. Meckling-Gill, submitted for publication.


ACKNOWLEDGEMENTS

We would like to thank Dr. Anthony Norman for very generously providing the vitamin D analogues used in these studies. We also thank Dr. W. Woodward for helpful discussions during the progression of this work.


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