From the Department of Pharmacology and ** Department
of Periodontics/Prevention/Geriatrics, the University of Michigan,
Ann Arbor, Michigan 48109-0632
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ABSTRACT |
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It is now recognized that protein kinase C (PKC)
plays a critical role in 1,25-dihydroxyvitamin D3
(1,25-(OH)2D3) promotion of HL-60 cell
differentiation. In this study, the effects of phosphorothioate antisense oligonucleotides directed against PKC, PKC
, PKC
I, and PKC
II on HL-60 promyelocyte cell differentiation and
proliferation were examined. Cellular differentiation was determined by
nonspecific esterase activity, nitro blue tetrazolium reduction, and
CD14 surface antigen expression. Differentiation promoted by
1,25-(OH)2D3 (20 nM for 48 h)
was inhibited similarly in cells treated with PKC
antisense (30 µM) 24 h prior to or at the same time as hormone treatment (86 ± 9% inhibition; n = 4 versus 82 ± 8% inhibition; n = 4 (mean ± S.E.), respectively). In contrast, cells treated with
PKC
antisense 24 h after 1,25-(OH)2D3
were unaffected and fully differentiated. PKC
antisense did not
block 1,25-(OH)2D3 promotion of HL-60 cell
differentiation. Next, the ability of PKC
I- and PKC
II-specific
antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation
was examined. PKC
II antisense (30 µM) completely
blocked CD14 expression induced by 1,25-(OH)2D3, whereas PKC
I antisense had
little effect. Interestingly, PKC
II antisense blocked
differentiation by 87 ± 7% (n = 2, mean ± S.D.) but had no effect on 1,25-(OH)2D3
inhibition of cellular proliferation. These results indicate that the
effects of 1,25-(OH)2D3 on HL-60 cell
differentiation and proliferation can be dissociated by blocking
PKC
II expression.
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INTRODUCTION |
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The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)1 regulates the growth and maturation of numerous organs and cell types. 1,25-(OH)2D3 is involved in the control of calcium and phosphorus homeostasis, muscle function, immunity, endocrine secretions, and neurotransmission (1). It is accepted that, in part, this hormone alters cell function by enhancing or repressing expression of specific genes (2, 3). Other studies have revealed that 1,25-(OH)2D3 regulates cellular processes without altering gene expression (4). These observations suggest that nongenomic effects of the hormone occur and result in the rapid alteration of cell membrane phospholipid metabolism and intracellular calcium concentrations (5, 6). Although the exact mechanism by which 1,25-(OH)2D3 promotes HL-60 cell differentiation is not fully understood, a number of studies from our laboratory and others have implicated protein kinase C (PKC) as a critical component of this process (7-9).
PKC is a family of serine-threonine protein kinases, which play major
roles in regulation of many cellular processes. To date, 11 PKC
isoenzymes have been characterized and classified into three groups
based on their structure and activation requirements (10, 11). The
classical PKCs, PKC, PKC
I, PKC
II, and PKC
, require calcium
for activation. A second class of PKCs has been termed the novel PKCs
and consist of PKC
, PKC
, PKC
, and PKC
(11). These novel
PKCs do not have a calcium binding motif, and therefore calcium is not
required for activation. The third class of PKCs are called the
atypical PKCs and include PKC
, PKCµ, and PKC
. These PKCs differ
significantly in structure to the other PKCs. Furthermore, atypical
PKCs do not respond to phorbol ester activation.
The importance of PKC in 1,25-(OH)2D3 promotion
of HL-60 cells along the monocyte/macrophage pathway is now
appreciated. Our laboratory reported that
1,25-(OH)2D3 increases PKC levels in HL-60
cells (7). Additionally, we found that classical inhibitors of PKC, H-7
and staturosporine, block the ability of
1,25-(OH)2D3 to promote HL-60 cell
differentiation (12, 13). Using similar PKC inhibitors, PKC activation
by 1,25-(OH)2D3 has been shown to be involved
in skin, heart, skeletal muscle, and renal cell gene expression and
function (14-17). Unfortunately, such chemical inhibitors are of
little use in determining isoenzyme specificity for a cellular
transduction mechanism. Recent studies have used overexpression and
antisense techniques to provide evidence that PKC is, to some
extent, the isoenzyme involved in 1,25-(OH)2D3 promotion of HL-60 cell differentiation (18, 19). In this study, we
showed that increased PKC
II levels by
1,25-(OH)2D3 is required to promote HL-60 cell
differentiation. Interestingly, PKC
II antisense had no effect on
1,25-(OH)2D3 inhibition of HL-60 cell
proliferation. Our report shows that increases in PKC
II levels and
activation are important events in 1,25-(OH)2D3
promotion of cell differentiation. Moreover, we suggest that the events leading to cellular differentiation most likely require protein phosphorylation.
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EXPERIMENTAL PROCEDURES |
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Chemicals-- 1,25-(OH)2D3 was purchased from Tetrionics Inc. (Madison, WI). Vitamin D3 metabolite purity and structural integrity were confirmed by high performance liquid chromatography and UV spectroscopy. All other reagents were reagent grade or better.
Cell Culture-- HL-60 promyelocytic leukemia cells were obtained from American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 medium supplemented with 10% horse serum, 1000 units/ml penicillin G, and 0.5 mg/ml streptomycin. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Cells used in this study were from passages 21-45. All experiments were initiated with cells in log phase growth at 2 × 105 cells/ml and then allowed to equilibrate in growth medium for 24 h prior to any treatment. Cellular differentiation was assessed by nonspecific esterase activity, nitro blue tetrazolium dye reduction, and CD14 surface antigen expression (7, 12, 36).
Oligonucleotides--
Phosphorothioate oligonucleotides were
synthesized by the DNA Synthesis Center at the University of Michigan.
PKC antisense was designed to interact with bases +4 to +18 of the
PKC
mRNA. PKC
sense had the complementary sequence of the
same region. PKC
oligonucleotides used were: PKC
sense, 5'-GCT
GAC CCG GCT GCG-3'; PKC
antisense, 5'-CGC AGC CGG GTC AGC-3'. PKC
antisense was designed to interact with bases +6 to +20 of the PKC
mRNA. PKC
sense had the complementary sequence over the same
region. PKC
oligonucleotides used were: PKC
sense, 5'-TCG GGG GGG
ACC ATG-3'; PKC
antisense, 5'-CAT GGT CCC CCC CGA-3'. PKC
I and
PKC
II specific antisenses were designed to interact with bases +1942 to +1956 that exist 3' from the splice site. PKC
I sense and PKC
II sense had the complementary sequences to its respectful antisense pair.
PKC
I antisense was: 5'-GTT TTA AGC ATT TCG-3'; PKC
II antisense was: 5'-GTT GGA GGT GTC TCT-3'.
Western Blot Analysis of PKC and PKC
Protein
Levels--
Cells were washed two times with phosphate-buffered saline
then resuspended in lysis buffer (0.2 M Tris, 0.5 mM EGTA, 0.5 mM EDTA, 0.5% Triton X-100, 100 mM leupeptin, 0.4 mM phenylmethylsulfonyl fluoride, pH 7.5) and homogenized using a Dounce homogenizer. Protein
content of total cell homogenates was determined by the Bradford
protein assay (37). Equal amounts of protein from each condition were
run on a 10% polyacrylamide gel, and proteins were subsequently
transferred to Immobilon paper (Millipore, Bedford, MA). The blot was
blocked with buffer containing 1% bovine serum albumin (10 mM Tris, 0.1% Tween 20, and 1% bovine serum albumin, pH
7.4). It was then probed for 2 h with primary antibodies (PKC
, PKC
I, PKC
II, and PKC
antibodies; Life Technologies Inc.), then washed three times with blocking buffer and incubated for 1.5 h
with a secondary antibody conjugated with horseradish peroxidase (Sigma). The blot was then washed five times with Tween-TBS (10 mM Tris and 0.2% Tween 20, pH 7.4). Finally, it was
developed using enhanced chemiluminescence (Amersham Pharmacia Biotech) and exposed to x-ray film.
Cell Proliferation Assay-- Cell number was determined using a Coulter (Coulter Electronics, Hialeah, FL) model Zf cell counter. Cell viability was determined using trypan blue dye exclusion.
Statistical Analysis of Data-- Differences between 1,25-(OH)2D3 treated cells and untreated cells for all assays were evaluated by unpaired Student's t test.
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RESULTS |
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Effect of PKC Antisense on 1,25-(OH)2D3
Induction of PKC
Levels--
HL-60 cells were treated with control
(0.1% ethanol) or 20 nM
1,25-(OH)2D3 and PKC
sense or antisense for
48 h. PKC
protein levels were determined by Western blot
analysis (Fig. 1). Cells treated with
1,25-(OH)2D3 in the presence of PKC
sense
oligonucleotide showed similar increases in PKC
levels as compared
with cells exposed to 1,25-(OH)2D3 alone.
Importantly, cells treated with PKC
antisense (lane 4)
exhibited a mark inhibition in 1,25-(OH)2D3 induction of PKC
levels. PKC
levels were decreased by 81 ± 9% (mean ± S.E.) relative to sense or oligonucleotide free
cultures. The antibody used to detect PKC
in this experiment was not
specific for the splice isoenzymes
I or
II. As seen in Fig. 1,
untreated (control) cells routinely exhibited minimal levels of PKC
.
Furthermore, PKC
levels remain unchanged in uninduced (control)
cells even after 48 h of PKC
antisense treatment
(n = 13, lanes 1 and 2, Fig. 1).
This observation is expected, because PKC
has a half-life of greater
than 70 h. Therefore, blocking translation with PKC
antisense
would not greatly influence existing levels of PKC
. As shown in Fig.
2, PKC
levels were increased within
24 h of 1,25-(OH)2D3 treatment.
Furthermore, PKC
antisense significantly blocked
1,25-(OH)2D3 induction of PKC
levels at 24 and 48 h of hormone treatment. Therefore, these results
demonstrate that the PKC
antisense oligonucleotide is able to block
the induction of PKC
levels by
1,25-(OH)2D3.
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Specificity of PKC Antisense to Inhibit
1,25-(OH)2D3 Induction of PKC
Protein
Levels--
HL-60 cells were treated with 20 nM
1,25-(OH)2D3 alone (C) or with
PKC
sense, PKC
antisense, PKC
sense, or PKC
antisense (30 µM amount of either oligonucleotide) for 48 h.
PKC
levels were determined by Western blot analysis (Fig.
3A). PKC
levels in cells
treated with 1,25-(OH)2D3 and PKC
sense,
PKC
antisense, or PKC
sense were not significantly different from
cells treated with 1,25-(OH)2D3 alone. As
expected, PKC
antisense was able to inhibit the induction of PKC
by 1,25-(OH)2D3 (lane 5). Moreover, as shown in Fig. 3B, PKC
antisense was able to
specifically block 1,25-(OH)2D3 enhancement of
PKC
levels. Importantly, PKC
antisense had no effect on
1,25-(OH)2D3 induction of PKC
levels. These results demonstrate that PKC
antisense has specificity in blocking 1,25-(OH)2D3-induced increases of PKC
.
Western blot analysis of PKC
I and PKC
II splice isoenzymes was
performed using specific antibodies. PKC
II protein levels were
detectable using these specific antibodies, whereas PKC
I levels were
not detectible. This observation in HL-60 cells is similar to ones
reported previously (20, 21). Also, the PKC
antisense
oligonucleotide at concentrations up to 60 µM had no
effect on 1,25-(OH)2D3 promotion of cell
differentiation (data not shown).
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Dose Response of PKC Antisense to Inhibit
1,25-(OH)2D3 Induction of PKC
Levels and
Cell Differentiation--
HL-60 cells were treated with 20 nM 1,25-(OH)2D3 and either 30 µM PKC
sense or 1, 10, or 30 µM PKC
antisense for 48 h. PKC
levels were determined by Western blot
analysis (Fig. 4A). As shown
in Fig. 4A, a dose-dependent decrease in PKC
levels was observed with increasing concentrations of PKC
antisense.
In this experiment an 85% decrease, as determined by scanning
densitometry, in PKC
levels was observed with 30 µM
PKC
antisense. Next, the effects of antisense constructs on HL-60
cell differentiation and the importance of the time of PKC
antisense
addition, relative to 1,25-(OH)2D3 treatment,
were also examined. HL-60 cells were treated with PKC
sense or
PKC
antisense 24 h prior to (open symbols) or at the
same time (closed symbols) as 20 nM
1,25-(OH)2D3. Cell differentiation was
determined by nitro blue tetrazolium dye reduction (circles)
and nonspecific esterase activity (squares) (Fig.
4B). HL-60 cells treated with
1,25-(OH)2D3 in the absence of oligonucleotide
treatment were induced to differentiate to the same extent as cells
pretreated or co-treated with PKC
sense (data not shown).
Differentiation promoted by 1,25-(OH)2D3 was inhibited by 86 ± 9% in cells pretreated with PKC
antisense
(30 µM) and 82 ± 8% in cells co-treated with
PKC
antisense (Fig. 4B). Therefore, it is likely that the
action of the antisense construct is not to lower existing PKC
levels but to block 1,25-(OH)2D3-induced increases in PKC
synthesis. However, if cells were first treated with 1,25-(OH)2D3 for 24 h prior to PKC
antisense, antisense treatment was ineffective in blocking
1,25-(OH)2D3 promotion of cell differentiation
(hatched circles and squares; Fig.
4B). This observation suggests that
1,25-(OH)2D3 has induced sufficient de
novo synthesis of PKC
within 24 h to render the antisense PKC
construct impotent. Thus, these experiments reveal that a relevant and required action of 1,25-(OH)2D3 in
promoting HL-60 cell differentiation is to up-regulate PKC
levels by
increasing the synthesis of the enzyme.
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Effects of PKCI and PKC
II Antisense on HL-60 Cell
Differentiation--
HL-60 cells were treated with vehicle, 20 nM 1,25-(OH)2D3, or 20 nM 1,25-(OH)2D3 and PKC
sense,
PKC
antisense, PKC
I sense, PKC
I antisense, PKC
II sense, or
PKC
II antisense (30 µM) for 72 h, and cell
differentiation was determined by CD14 surface antigen expression using
flow cytometry (Fig. 5). CD14 is a cell surface marker of mature monocytes/macrophages. Treatment with 1,25-(OH)2D3 significantly increased cell
differentiation as shown by the substantial increase in CD14 expression
(Fig. 5A). PKC
sense did not affect
1,25-(OH)2D3-induced expression of CD14, whereas PKC
antisense completely blocked the ability of
1,25-(OH)2D3 to increase CD14 expression. This
is consistent with all previous observations and again demonstrates
that PKC
is essential for 1,25-(OH)2D3-induced differentiation of HL-60
cells. In Fig. 5, C and D, the potency of
antisense constructs designed to hybridize specifically with PKC
I
and PKC
II was examined. A complete block of
1,25-(OH)2D3-induced CD14 expression was
observed with the PKC
II-specific antisense oligonucleotide (Fig.
5C). In contrast, PKC
I-specific antisense failed to
reverse the enhanced expression of CD14 by
1,25-(OH)2D3 (Fig. 5D).
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Effects of PKCII Antisense on Cell
Proliferation--
Interestingly, PKC
II antisense did not block
1,25-(OH)2D3 inhibition of HL-60 cell
proliferation (Fig. 6). Thus, these data show that blocking 1,25-(OH)2D3-stimulated
increase in PKC
II decreased the induction of cell differentiation by
80% but had no effect on 1,25-(OH)2D3
inhibition of cell proliferation. Similar results were obtained with
the less specific PKC
antisense construct (data not shown).
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DISCUSSION |
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1,25-(OH)2D3 affects the growth and
differentiation of numerous cell types (22-26). Relevant to this
report HL-60 cells have been shown to differentiate into
monocytes-macrophages (24) and osteoclast-like cells (23) upon exposure
to 1,25-(OH)2D3. Expression of several genes
including c-myc, c-fos, and PKC, PKC
, and
PKC
are regulated prior to the appearance of the mature monocytic-macrophage phenotype (7-9, 28). c-myc gene
expression is decreased, c-fos gene expression is
transiently increased, and PKC levels are increased in HL-60 cells
during the process of cellular differentiation (25, 29, 30).
Considering the nature of these early events and the accepted
importance of these gene products in cell signaling and growth, it is
likely that regulation of these genes is critical for induced HL-60
cell differentiation.
Recent reports revealed that the 1,25-(OH)2D3
receptor (vitamin D receptor) is a substrate for PKC and that
phosphorylation of vitamin D receptor is important for controlling
osteocalcin expression (3, 27). Such studies support and extend the
possible roles PKCs have in modulating
1,25-(OH)2D3's actions. Transcriptional response elements for 1,25-(OH)2D3 have also
been identified. Interestingly, the response element for
1,25-(OH)2D3 in the osteocalcin gene contains a
phorbol ester response element (31, 32). One factor that interacts with
this AP-1 sequence is a heterodimer made up of c-fos and
c-jun. PKC-directed phosphorylation of c-fos and
c-jun regulates their AP-1 binding activity (33). However, the precise molecular nature of the interaction between the
1,25-(OH)2D3 signal transduction pathway and
PKC for regulation of gene expression is still not clear. Several
nuclear proteins have been shown to be phosphorylated by PKC during the
course of myeloid cell differentiation (34). Our laboratory reported
that 10 nuclear proteins undergo phosphorylation state changes within
6-40 h of 1,25-(OH)2D3 treatment (34). We
identified several of these proteins as nuclear matrix or DNA packaging
proteins, including several histones and lamin B. Therefore, PKCs act
as regulators of nuclear events and may be intimately involved in the
transduction of the 1,25-(OH)2D3 signal
ultimately regulating gene expression and HL-60 cell
differentiation.
Increasing evidence exists to indicate that PKC plays an important
role in 1,25-(OH)2D3 promotion of HL-60 cell
differentiation. A variant HL-60 cell line (HL-525) lacking basal
levels of PKC
is resistant to phorbol ester-induced differentiation
(18). However, susceptibility to phorbol ester differentiation was
restored if HL-525 cells were transfected to overexpress PKC
.
Additionally, phorbol 12-myristate 13-acetate resistance of HL-525
cells was reversed by pretreating with
1,25-(OH)2D3, which increased PKC
levels
(18). Also, it was shown that a 25-mer PKC
antisense construct
different from the one used here was capable of partially blocking
(averaging
30%) 1,25-(OH)2D3's induction
of cell differentiation (19). Although a partial inhibition of
1,25-(OH)2D3-promoted cell differentiation was
observed using their antisense construct, it had little effect on
1,25-(OH)2D3 inhibition of cell proliferation. In our study, novel 15-mer PKC
and PKC
II antisense constructs were found to inhibit 1,25-(OH)2D3 promotion of
cell differentiation by 80-90%. However, these antisense
oligonucleotides had no effect on
1,25-(OH)2D3's ability to inhibit cell
proliferation. Moreover, reduction of basal levels of PKC
was not
required for PKC
antisense to inhibit
1,25-(OH)2D3 promotion of cell differentiation.
This result suggests that blocking de novo synthesis of
PKC
is the mechanism of action for the antisense construct. We
demonstrated that PKC
II is uniquely responsible for
1,25-(OH)2D3 promotion of cell differentiation.
There is controversy as to whether PKC
I is expressed in HL-60 cells.
In all reports, PKC
I levels in unstimulated HL-60 cells is
significantly lower than PKC
II levels (19-21, 35). In this study,
we failed to detect measurable levels of PKC
I. This finding is in
agreement with several reports (20, 21). However, others have shown,
using different antibodies or Northern blot analysis, that
1,25-(OH)2D3 increased PKC
I protein levels or mRNA levels (19, 35).
The findings reported here indicate that PKCII specifically
participates in the signal transduction mechanisms employed by 1,25-(OH)2D3 to promote HL-60 cell
differentiation. Interestingly, we found a direct correlation between
the quantitative lowering of PKC
II protein levels and the degree of
induced differentiation. The correlation between the increased levels
of PKC
induced by 1,25-(OH)2D3 and the
extent of cellular differentiation (7-9) suggest that there are not
spare PKC
s in these cells. Thus, we suggest that the levels of
PKC
are stoichometrically related to promotion of differentiation.
This study provides clear and convincing evidence that promotion of
cell differentiation and inhibition of cell proliferation are two
distinct processes by 1,25-(OH)2D3 that can be
disassociated by blocking the expression of a single gene, PKC
II. To
date, several analogs of 1,25-(OH)2D3 have been
developed that are selective at affecting calcium mobilization and
promoting terminal cellular differentiation. Our study suggests that it
may be possible to further separate the actions of
1,25-(OH)2D3 into its capacity to promote
cellular differentiation versus its capacity to inhibit cell
proliferation.
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ACKNOWLEDGEMENT |
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We thank Li Huang for her technical efforts and expertise.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DE 10337 (to R. U. S. and M. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Pharmacology, 2301D MSRB III, University of Michigan, Ann Arbor, MI 48109. Tel.: 734-763-3255; Fax: 734-763-4450; E-mail: robsim{at}umich.edu.
¶ Current address: Cardiovascular Institute and Division of Cardiology, San Francisco Veterans Hospital, San Francisco, CA 94105.
Newton-Loeb Fund/University of Michigan Cancer Center
Predoctoral Fellow.
1 The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; PKC, protein kinase C.
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REFERENCES |
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