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,25-Dihydroxyvitamin D3 in Promyelocytic NB4 Leukemia Cells: A Structure-Function Study1
Department of Biochemistry (X.S., J.E.B., A.W.N.), Division of Biomedical Sciences (A.W.N.), and the Department of Chemistry (W.H.O.), University of California, Riverside, California 92521
Address all correspondence and requests for reprints to: Prof. Anthony W. Norman, Department of Biochemistry, University of California, Riverside, California 92521. E-mail: Norman{at}ucrac1.ucr.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3
[1,25-(OH)2D3] actions in cell growth and
differentiation are mediated by both its nuclear receptor
(VDRnuc) and its rapid membrane-related effects. In the
present study, we investigated the effect of
1,25-(OH)2D3 on p42mapk
phosphorylation using human acute promyelocytic leukemia cells (NB4).
1,25-(OH)2D3 (10-8
M) significantly increased p42mapk
phosphorylation in a time- and dose-dependent manner, with the earliest
response detectable at 30 sec. Because
1,25-(OH)2D3 is a conformationally flexible
molecule, we have used a series of conformationally locked
(6-s-cis vs. 6-s-trans) analogs to
evaluate which shape is optimal for activation. Four
6-s-cis-locked analogs (HF, JM, JN, and JP) and two
6-s-trans-locked analog (JB and JD) were studied. HF,
JM, JN, and JP all increased p42mapk phosphorylation at 1
and 5 min (10-8 M), but JB and JD had little
effect. Analog HL [1ß,25-(OH)2D3], a
specific antagonist for only the rapid effects of
1,25-(OH)2D3, attenuated
1,25-(OH)2D3-induced p42mapk
phosphorylation 65–90%. To assess the potential involvement of the
VDRnuc in mediating the analog’s action, the relative
abilities of the analogs to compete with
[3H]1,25-(OH)2D3 for binding
in vitro to the VDRnuc of NB4 cells was
measured. All 6-s-cis analogs bound poorly to
VDRnuc (relative competitive index, 0.5–2%) compared with
1,25-(OH)2D3 (relative competitive index,
100%). The present studies demonstrate for the first time that in NB4
cells 1,25-(OH)2D3 rapidly activates the
p42mapk pathway, and that this effect can be selectively
mediated by analogs that can assume a 6-s-cis
conformation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3
[1,25-(OH)2D3] is derived from its parent
vitamin D3 by sequential hydroxylation in the liver and
kidney. It is active in regulating mineral homeostasis, cell
differentiation, and proliferation (1). Many biological functions of
1,25-(OH)2D3 are mediated by its nuclear
receptor (VDRnuc). However, recent studies in our
laboratory and others have identified a series of rapid nongenomic
effects of 1,25-(OH)2D3 that occur within
seconds to minutes of exposure of cells to this steroid hormone (2). We
and others have presented evidence supporting the existence of a
membrane receptor for 1,25-(OH)2D3
(VDRmem) that mediates the initiation of rapid responses in
some cells (3, 4). These rapid effects of
1,25-(OH)2D3 have been demonstrated in a
variety of systems, including the following: a rapid stimulation of
intestinal calcium transport in the perfused chick intestine (termed
transcaltachia) (2), a rapid increase in intracellular Ca2+
in human keratinocytes and skeletal muscle (5, 6), a rapid opening of
voltage-gated Ca2+ (7) and chloride channels (8) in the ROS
17/2.8 cell line, a rapid stimulation of sodium proton exchange in
opossum kidney cells (9), a rapid action on phospholipid metabolism in
several tissues and cell lines (3, 10, 11), a rapid activation of
protein kinase C (PKC) in rat epithelium cells (12), and a rapid
activation of mitogen-activated protein kinase (MAP kinase or
p42mapk and p44mapk) in hepatic Ito cells (13)
and human keratinocytes (14). These rapid actions of
1,25-(OH)2D3 on the cell membrane are
postulated to regulate cell biological function and potentially to
interact with other membrane-mediated kinase cascades or to engage in
cross-talk with the cell nucleus to modify genomic responses of cell
differentiation and proliferation (15).
MAP kinase belongs to the family of serine/threonine protein kinases
and can be activated by phosphorylation of a tyrosine residue induced
by mitogens or cell-differentiating agents (16, 17). MAP kinase
integrates multiple intracellular signals transmitted by various second
messengers (13, 17) and regulates many cellular functions by
phosphorylation of several cytoplasmic kinases and nuclear
transcription factors, including the epidermal growth factor receptor,
c-Myc, and c-Jun (18). However, it is unknown whether the
differentiation of NB4 cells by 1,25-(OH)2D3
involves the activation of MAP kinase.
The human acute promyelocytic leukemia cell line (NB4) is a model for
study of the rapid membrane actions of
1,25-(OH)2D3.
1,25-(OH)2D3 is required during the priming
phase of NB4 cell differentiation (19), and tyrosine phosphorylation
was reported to be involved in this priming phase process (20). It is
not clear which tyrosine phosphorylation cascade plays a role in this
priming phase.
1,25-(OH)2D3 is a highly flexible
molecule (21) that is capable of rotating around its 6,7 carbon-carbon
bond to generate a continuum of shapes/structures bounded by the
so-called 6-s-trans and the 6-s-cis
conformers (Fig. 1A
). Since
1,25-(OH)2D3 as a natural ligand can induce
both genomic and rapid cellular responses, it was suggested that
different shapes or conformers of the seco steroid hormone might
activate the genomic and rapid response signal transduction pathways
(15, 22, 23). Thus, we have reported that although the
6-s-cis-locked conformers, but not the
6-s-trans-locked analogs, can mimic the rapid membrane
effect of 1,25-(OH)2D3, the
6-s-cis-locked analogs are only a weak agonist for the
VDRnuc and stimulation of gene transcription (2).
|
,25-(OH)2D3, several analogs of
1,25-(OH)2D3 were employed that had
previously been shown to discriminate the rapid effects from the
VDRnuc-mediated genomic effects (15). In this report, we
studied MAP kinase phosphorylation regulated by
1,25-(OH)2D3 as well as
6-s-cis- and 6-s-trans-locked analogs of
1,25-(OH)2D3 in NB4 cells. Our results show
that 1,25-(OH)2D3 rapidly activated MAP
kinase in NB4 cells and that 6-s-cis-locked, but not
6-s-trans-locked, analogs increased p42mapk
phosphorylation in the same manner as
1,25-(OH)2D3. This is the first report that
1,25-(OH)2D3 is able to activate the
p42mapk pathway in NB4 cells and that this MAP kinase
activation can discriminate among various shapes of the
conformationally flexible 1,25-(OH)2D3. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1,25-(OH)2D3 and
analogs
1,25-(OH)2D3 was a gift from Dr.
M. R. Uskokovic (Hoffmann-La Roche, Nutley, NJ). Analogs HF, JM,
JN, JP, JB, JD, and HL were provided by Dr. W. H. Okamura
(University of California, Riverside, CA).
1,25-Dihydroxy[23,24-N-3H]cholecalciferol was
purchased from Amersham. 1,25-(OH)2D3 and
its analogs were initially dissolved in ethanol at a concentration of
10-4 M. The final concentration of ethanol in
all experiments was less than 0.1%. An equivalent amount of ethanol as
vehicle was included in the control. All solutions of
1,25-(OH)2D3 and analogs were stored in the
dark at -20 C until used.
Cell culture
NB4 cells were obtained from Dr. K. A. Meckling-Gill
(Guelph, Canada) and were originally isolated from a human patient with
acute promyelocytic leukemia by Dr. Michel Lanotte at the Hospital
Saint-Louis (INSERM U-301, Paris, France) (24). The cell line is
characterized by a translocation involving chromosomes 15 and 17, which
is typical of the classical form of acute promyelocytic leukemia-M3 in
the French-American-British classification. NB4 cells were cultured in
DMEM-Ham’s F-12 medium with 10% FCS at 5% CO2 balanced
air and were routinely passaged as suspension cultures; only passages
8–20 were used for each assay. Cell growth and viability were assessed
using the trypan blue dye exclusion method, and 95% of the cells
showed viability in the experimental culture conditions.
Immunoprecipitation of tyrosine-phosphorylated proteins
NB4 cells were cultured in 60-mm diameter dishes and
treated with 1,25-(OH)2D3 or analogs in 4 ml
DMEM-Ham’s F-12 containing 10% charcoal-stripped FCS. At the end of
the incubation period, cells were washed once in cold PBS containing
sodium vanadate at a concentration of 100 µM and further
extracted with RIPA buffer containing 50 mM Tris-HCl (pH
7.4), 150 mM NaCl, 0.2 mM
Na3VO4, 2 mM EGTA, 25
mM NaF, 1 mM phenylmethylsulfonylfluoride,
0.25% sodium deoxycholate, 1% Nonidet P-40, 2 µg/ml leupeptin, 2
µg/ml aprotinin, and 2 µg/ml pepstatin. Insoluble material was
removed in a microcentrifuge at 14,000 rpm for 10 min. The protein
concentration was determined with a protein assay kit (Bio-Rad
Laboratories, Hercules, CA). For immunoprecipitation, the supernatant
was incubated with bead-conjugated monoclonal antiphosphotyrosine
antibody overnight at 4 C. The immunoprecipitates containing the
tyrosine-phosphorylated proteins were washed four times with freshly
prepared RIPA buffer and further eluted with 2 x Laemmli gel
buffer. At this point, the samples were either stored at -20 C for
further use or processed via Western blots. Equal loading of MAP kinase
protein was determined by running the Western blots using polyclonal
anti-p42mapk antibody. For this purpose, samples were
aliquoted from each cell extract before immunoprecipitation.
SDS-PAGE and Western blot
Antiphosphotyrosine immunoprecipitates of cell extract were
resolved on 7.5% SDS-PAGE and transferred to polyvinylidene difluoride
(PVDF) membranes according to the manufacturer’s instructions
(Amersham). The membrane was further immunoblotted using a rabbit
anti-p42mapk polyclonal antibody overnight at 4 C, followed
by incubation with secondary horseradish peroxidase-conjugated mouse
antirabbit antibody for 1 h at 25 C. The phosphorylated MAP kinase
bands were then visualized by enhanced chemiluminescence. A
densitometer with a CCD camera (Eagleeye II, Stratagene, La Jolla, CA)
was used to scan the density of the immunophosphoprotein bands. The
results were normalized for protein loading and further plotted as a
percentage of the density of the corresponding band in the control
lane. The specificity of p42mapk phosphorylation was
determined by resolving the tyrosine-phosphorylated proteins in
SDS-PAGE, transferring the proteins to PVDF membrane, and then
incubating the membrane with anti-p42mapk polyclonal
antibody that had or had not been preexposed to MAP kinase peptide for
2 h.
Ligand receptor competition assay
The relative affinity of nonradioactive
1,25-(OH)2D3 and each analog to compete with
[3H]1,25-(OH)2D3 for binding
to the VDRnuc of NB4 cells and to the plasma D-binding
protein (DBP) was carried out in vitro according to a
previously described procedure (25). Briefly, the NB4 cells were
collected from a fast growing stage, and the cellular
VDRnuc of 1,25-(OH)2D3 were
extracted with KTED buffer containing 10 mM Tris-HCl (pH
7.4), 300 mM KCl, 1 mM EDTA, and 5
mM dithiothreitol. After sonication, the cell extract was
further centrifuged at 500 x g for 10 min. The
supernatant was collected for use in a ligand receptor binding assay.
In this assay, increasing concentrations of nonradioactive
1,25-(OH)2D3 or the tested analogs were
incubated with NB4 cell extracts or dilutions of human serum (as a
source of DBP) in the presence of a fixed saturating amount of
[3H]1,25-(OH)2D3. The
reciprocal of the percentage of maximal binding of
[3H]1,25-(OH)2D3 was then
calculated and plotted as a function of the relative analog
concentration vs.
[3H]1,25-(OH)2D3. Each analog
showed a linear plot, and the slope of each curve represents the
analog’s competitive index value. The competitive index value for each
analog is then normalized to the competitive index value of the
nonradioactive 1,25-(OH)2D3, thereby
generative the value of the relative competitive index (RCI), where the
RCI for 1,25-(OH)2D3 is defined as
100%.
Statistics
All results are expressed as the mean ± SEM. A
Newman-Keuls two-way ANOVA for multiple comparisons was used for
statistical analysis. A difference between experimental groups was
considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-(OH)2D3 that were
employed in this study are shown in Fig. 1
. HF (kinetically suppressed
in its 6-s-cis orientation) and JM, JN, and JP (three
permanent 6-s-cis-locked analogs) are the most potent known
agonists to mimic the 1,25-(OH)2D3-induced
rapid responses (Fig. 1B), but all have a poor binding affinity for the
VDRnuc and little ability to mediate genomic actions in
chick and other cell systems (15, 19, 22). Figure 1C shows the
structures of the 6-s-trans-locked analogs, JB and JD.
Functionally, JB and JD are not active in either genomic or rapid
response assays of several in vitro and in vivo
systems (15). Analog HL [1ß,25-(OH)2D3],
which is an epimer of 1,25-(OH)2D3 at
carbon-1, has been demonstrated to block all known rapid effects of
1,25-(OH)2D3, but is unable to block its
genomic activities, including monocytic differentiation (26) (Fig. 1D).
1,25-(OH)2D3
rapidly increased phosphorylation of p42mapk in
NB4 cells
To determine whether MAP kinase phosphorylation is altered
by 1,25-(OH)2D3, we first examined the
time-dependent effects of 1,25-(OH)2D3 on
p42mapk phosphorylation. The NB4 cells, cultured in 10%
charcoal-stripped FCS medium, were treated with
1,25-(OH)2D3 at 10-8
M for various times. Cells were extracted, and the
phosphorylated MAP kinase was immunoprecipitated with
antiphosphotyrosine antibody and further analyzed by Western blot using
the antibodies against p42mapk. The specificity of the
immunodetected MAP kinase was confirmed by preblocking the primary
anti-MAP kinase antibody with purified MAP kinase peptide in a Western
blot step (Fig. 2). Figure 3A shows that
1,25-(OH)2D3 at 10-8
M increased p42mapk phosphorylation. Increased
p42mapk phosphorylation was observed from 30 sec to 15 min.
The quantitative results from six separate experiment indicate that the
mean elevated p42mapk phosphorylation was 2.6-, 2.2-, and
2.2-fold over the control level at 1, 5, and 15 min, respectively (Fig. 3C). Equal MAP kinase protein loading in each lane was verified as
shown in Fig. 3B. Thus, our results suggest that MAP kinase activation
is one of the rapid cell membrane-mediated events that can be initiated
by 1,25-(OH)2D3.
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,25-(OH)2D3,25-(OH)2D3 on p42mapk
phosphorylation was dose dependent, NB4 cells were treated with
1,25-(OH)2D3 at
10-10-10-8 M at 1 and 5 min.
Figure 4A shows that the
p42mapk phosphorylation of NB4 cells induced by
1,25-(OH)2D3 was dose dependent. The highest
p42mapk phosphorylation occurred at 10-8
M at both 1 and 5 min, when the increased levels of
phosphorylation were 2.8- and 4.0-fold above the control value,
respectively (Fig. 4C). Both 10-9 and 10-10
M 1,25-(OH)2D3 increased
p42mapk phosphorylation to a lesser extent at 1 or 5 min.
Furthermore, the p44mapk phosphorylation bands were
observed with a higher concentration (10-8 M)
of 1,25-(OH)2D3 treatment. Equal loading of
total MAP kinase proteins is shown in Fig. 4B. Therefore, our data show
that p42mapk activation in NB4 cells by
1,25-(OH)2D3 is dose dependent, with a
maximal effect achieved at 10-8 M.
|
,25-(OH)2D3, have been shown to mediate
rapid effects on cell membrane-mediated biological events, such as
activation of transcaltachia and opening of calcium channel (8, 15, 22). To assess whether 1,25-(OH)2D3-induced
p42mapk activation is related to the 6-s-cis
conformation, we determined the efficacy of HF, JM, JN, and JP on MAP
kinase phosphorylation in NB4 cells. In this experiment, the cells were
treated with HF, JM, JN, and JP at 10-8 M for
1 and 5 min. Vehicle and 1,25-(OH)2D3 at
10-8 M were used as negative and positive
controls. The results show that all HF, JM, JN, and JP increased
p42mapk phosphorylation with the same efficacy as
1,25-(OH)2D3 (Fig. 5). The increased p42mapk
phosphorylation by the 6-s-cis analogs was approximately 2-
to 3-fold above the control level.
|
,25-(OH)2D3 on
p42mapk phosphorylation. NB4 cells were exposed to vehicle,
1,25-(OH)2D3, JB, or JD (Fig. 6), all at 10-8
M, for 1 and 5 min. Compared with the control value,
1,25-(OH)2D3 increased p42mapk
phosphorylation 2- to 4-fold at 1 and 5 min, respectively. JB and JD
did not activate p42mapk phosphorylation at either 1 or 5
min. This result suggests that the 6-s-trans conformer of
1,25-(OH)2D3 is not an acceptable shape to
initiate rapid membrane response, possibly through a putative membrane
receptor (4).
|
,25-(OH)2D3-induced
p42mapk phosphorylation in NB4 cells,25-(OH)2D3 in stimulating
both transcaltachia and the opening of chloride channels (23, 26). In
this experiment, NB4 cells were treated with vehicle, either analog HL
at 10-9 M,
1,25-(OH)2D3 alone, or the combination of HL
with 1,25-(OH)2D3 for 5 min. The analog HL
alone had little effect on MAP kinase phosphorylation.
1,25-(OH)2D3 induced p42mapk
phosphorylation in a dose-dependent manner, with a maximum effect at
10-8 M. The simultaneous presence of HL
(10-9 M) greatly attenuated the
1,25-(OH)2D3-induced p42mapk
phosphorylation (Fig. 7) and achieved an
inhibition of 92%, 64%, and 86% at
1,25-(OH)2D3 concentrations of
10-10, 10-9, and 10-8
M.
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,25-(OH)2D3 to
compete under in vitro conditions with
[3H]1,25-(OH)2D3 for binding
to the classical VDRnuc present in nuclear and cytosolic
extracts obtained from NB4 cells or to the DBP present in serum. The
RCI is defined as a relative measure of an analog’s ability to compete
with [3H]1,25-(OH)2D3 for binding
to the VDRnuc; the RCI of
1,25-(OH)2D3 is by definition set at 100%
(15, 27, 28). Table 1 tabulates the RCI
for 1,25-(OH)2D3 and all of the analogs
evaluated in this report. With respect to the NB4 cells, neither the
6-s-cis analogs (HF, JM, JN, and JP) nor the
6-s-trans analog (JB and JD) interact effectively with the
VDRnuc; collectively, these RCI values ranged from
0.4–4.5%. Therefore, our results indicates that NB4 cells possess a
VDRnuc for 1,25-(OH)2D3 that has
a structural specificity similar to those of receptors in chick and
other cell types. Furthermore, our data indicate that the action of the
6-s-cis analogs, HF, JM, JN, and JP, are probably
independent of classical VDRnuc in NB4 cells. Table 1 also
reports the RCI for 1,25-(OH)2D3 and its
analogs with respect to binding to the plasma transport protein, DBP.
It is clear that neither the 6-s-cis analogs nor the
6-s-trans analogs bind at all effectively to the plasma
transport protein; thus, it is unlikely that DBP is making a
significant contribution to the delivery of the analogs to the target
cell or is participating in some way to initiate the stimulation of MAP
kinase.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-(OH)2D3 increased the
p42mapk phosphorylation of NB4 cells in a dose- and
time-dependent manner and that this effect can be mimicked by its
6-s-cis analogs (HF, JM, JN, and JP), but not the
6-s-trans-locked analog (JB and JD). Elevated MAP kinase
phosphorylation can be seen as early as 30 sec, with higher
phosphorylation bands occurring 1–5 min after exposure to the steroid
hormone and analogs. Although the fold p42mapk
phosphorylation induced by 1,25-(OH)2D3 is
variable between experiments, an increased level of p42mapk
phosphorylation of 2- to 5-fold was observed consistently. Furthermore,
p44mapk phosphorylation was detected in some experiments
with a higher concentration of 1,25-(OH)2D3.
The reason for this may be that the anti-MAP kinase antibody was raised
against peptide fragments of the ERK2 (p42mapk), is known
to have broad interspecies reactivity (human, chick, and murine), and
cross-reacts with p44mapk protein to a lesser extent. This
communication is the first demonstration that
1,25-(OH)2D3 activates MAP kinase pathway in
NB4 cells.
HF is a synthetic, kinetically locked, 6-s-cis previtamin
analog, whereas analogs JM, JN, and JP are 6-s-cis
permanently locked analogs of 1,25-(OH)2D3.
Our data demonstrate that all four 6-s-cis analogs bind
poorly to the VDRnuc of NB4 cells (0.1–2%); the RCI
values are the same as those reported in the well characterized chick
intestine (15). As the 6-s-cis analogs activated MAP kinase
in NB4 cells, this result supports the concept that the
6-s-cis shape/structure of
1,25-(OH)2D3 is preferred for cell
membrane-initiated actions, and its function is less dependent on
involvement of VDRnuc. It has been reported that analogs
HF, JM, JN, and JP are active in eliciting
1,25-(OH)2D3-induced rapid membrane effects,
such as stimulating rapid calcium channel opening without promoting
gene transcription (15, 22). Furthermore, the lack of a genomic
response for JN at a physiological concentration has been observed in
several in vitro and in vivo systems, such as in
a vitamin D-deficient chick, HL60 cells, MCF-7 cells, or ROS 17/2.8
cells (15). The present studies demonstrate that the 6-s-cis
analogs were able to induce MAP kinase phosphorylation in NB4 cells and
support the view that 1,25-(OH)2D3-induced
MAP kinase phosphorylation is a cell membrane-mediated event.
In the present studies, we further confirmed that the
6-s-trans structure of
1,25-(OH)2D3 is not a preferred conformer to
elicit the rapid phosphorylation of p42mapk in NB4 cells.
In NB4 cells, the 6-s-trans-locked analogs, JB and JD,
neither bind to VDRnuc nor affect p42mapk
phosphorylation, which is consistent with results from other systems
showing that JB and JD were not active in either the genomic assays or
the rapid response assays (15).
Analog HL or 1ß,25-(OH)2D3 is known to be an
antagonist capable of blocking
1,25-(OH)2D3-induced rapid membrane effects,
but it is unable to block genomic activities induced by
1,25-(OH)2D3 (23, 26). In the present
experiments, analog HL at 10-9 M maximally
blocked the p42mapk phosphorylation induced by
1,25-(OH)2D3 at equimolar or lower
concentration. This blocking effect of HL on the rapid membrane action
of 1,25-(OH)2D3 has also been reported in
NB4 cell differentiation (19). It is conceivable that there exists a
membrane protein that can interact with
1,25-(OH)2D3 to initiate the MAP kinase
pathway, and this interaction can be blocked by the isomer
1ß,25-(OH)2D3.
The structure-function results presented in this communication with
regard to 1,25-(OH)2D3-mediated activation
of MAP kinases represent the first chapter of a more complete study.
The present results focus on only three families of analogs and present
results for the 6-s-cis (active) and 6-s-trans
(inactive) relationships and 1ß,25-(OH)2D3
(antagonist). It is to be anticipated, as is the case with
VDRnuc, that there are components of the parent
1,25-(OH)2D3 molecule (e.g.
20-epi vs. normal side-chain; modification of the C and D
rings) that may also be structural components necessary for full
agonist activity for activation of MAP kinase.
MAP kinases belong to a family of dual specificity serine/threonine
protein kinases (29) in which the activation process is achieved by the
phosphorylation of both specific tyrosine and threonine residues (30).
MAP kinases are known to be able to integrate multiple intracellular
signals transmitted by various second messengers initiated by mitogens
or cell differentiation agents. It is clear that the MAP kinase pathway
sequentially links cell surface receptor-mediated signals to the
nucleus and can regulate the expression of a specific pattern of gene
(17). 1,25-(OH)2D3 as a steroid hormone is
known to control cell differentiation and proliferation by the
VDRnuc-mediated genomic pathway (1). Recently, evidence has
accumulated to show that many rapid cell membrane-related biological
events, such as Ca2+ channel opening, activation of PKC,
and priming of cell differentiation, can be initiated by
1,25-(OH)2D3 (2). The existence of a
putative VDRmem has been suggested (3, 31), and a report
describing a partial purification of a VDRmem from chick
intestine has been reported (4). Although many gaps remain to be filled
in this concept, one of the questions that needs to be answered is
whether there is a signaling pathway transducing the
1,25-(OH)2D3 membrane-initiated signals to
the nucleus. As the MAP kinase pathway has been shown in other systems
to play a role in transducing the ligand signal from the outer cell
membrane to the nucleus, we postulate that p42mapk
activation by 1,25-(OH)2D3 in NB4 cells may
represent one form of cross-talk between the membrane and nucleus,
which could modulate the genomic pathways of
1,25-(OH)2D3.
The activation of p42mapk in NB4 cells was clearly
dependent on the exposure of cells to
1,25-(OH)2D3. Because the
p42mapk phosphorylation by
1,25-(OH)2D3 in NB4 cells was rapid, it
virtually rules out the possibility that
1,25-(OH)2D3 induces the synthesis of any
endogenous factors that further activate cell surface receptors.
1,25-(OH)2D3-induced MAP kinase activation
was also previously reported in rat liver Ito cells and human
keratinocytes by Beno and Gniadecki, respectively (13, 14). The
mechanism for 1,25-(OH)2D3-induced MAP
kinase phosphorylation was reported to be dependent on PKC pathway
activation in Ito cells (13) or involved in the tyrosine
phosphorylation of Shc protein and a complex formation between
VDRnuc and Shc protein, which are preceded by MAP kinase
activation (14).
Taken together, our results provide evidence that MAP kinase activation
by 1,25-(OH)2D3 is a cell membrane-mediated
process. Recent studies have suggested that the level and duration of
activation of MAP kinase are important factors in determining which
specific cellular events will be initiated (17, 32). Furthermore,
besides the classical MAP kinase pathway that is dependent on Ras, Raf,
and MAP kinase activation, PKC activation was linked to the activation
of MAP kinase (33). 1,25-(OH)2D3 and analog
HF have been reported to activate and stimulate both PKC and PKC
translocation into the nucleus of NB4 cells (34). It is still unknown
whether the PKC activation and p42mapk phosphorylation
induced by 1,25-(OH)2D3 in NB4 cells are
connected or separate events in
1,25-(OH)2D3-signaling pathways. Also,
1,25-(OH)2D3 has been shown to be able to
directly activate PKC, which is incorporated into a liposome; this
indicates the possibility of a ligand-binding domain on PKC for
1,25-(OH)2D3 (35).
In summary, the current studies are the first to demonstrate the
following: 1) the MAP kinase pathway in NB4 cells can be activated by
1,25-(OH)2D3 and its
6-s-cis-locked analogs, but not by its
6-s-trans-locked analogs; 2) HL, an antagonist only for the
rapid membrane actions of 1,25-(OH)2D3, can
attenuate the MAP kinase phosphorylation induced by
1,25-(OH)2D3; and 3) both
6-s-cis-and 6-s-trans-locked analogs are poor
binders to the VDRnuc of NB4 cells. This latter observation
could rule out a direct involvement of the VDRnuc in these
analog actions. Collectively, the results suggest that the
6-s-cis conformer of 1,25-(OH)2D3
can play a major role in this rapid event of MAP kinase activation.
Further studies will focus on the upstream pathway of
p42mapk by using tyrosine kinase inhibitors or the mutant
kinases to investigate whether
1,25-(OH)2D3-induced MAP kinase pathway has
classical upstream MAP kinase activators, such as MAP kinase kinase
(MEK), MEK kinase (MEKK), and receptor-related tyrosine kinase.
Finally, our findings provide new insights into the study of the
VDRmem and will shed light on further studies of the
cross-talk between the cell membrane and VDRnuc-mediated
genomic pathway of 1,25-(OH)2D3.
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Received July 7, 1997.
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