From the
We have shown that 1,25-dihydroxyvitamin D
1,25-Dihydroxyvitamin D
Recent reports have suggested that
some of the activities of 1,25(OH)
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)
Authentic
1,25(OH)
NB4 cells were treated with
1,25(OH)
During 1,25(OH)
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)
HL-60 cell lines which are resistant to 1,25(OH)
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)
The observations that NB4 cells do not differentiate in response to
TPA or 1,25(OH)
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)
Shown is the population distribution of phagocytic cells and the
number of beads/cell engulfed when cultures were treated with
1,25(OH)D
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.
(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.
(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) .
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) .
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.
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.
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.
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.
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
.
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) .
D
or the HF analogue in HL-60 cells.
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.
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
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).
D
, 1,25-dihydroxyvitamin D
;
PBS, phosphate-buffered saline; TPA,
12-O-tetradecanoylphorbol-13-acetate; VDR, vitamin D receptor;
VDRE, vitamin D response element.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.