Download Inhibition of Phorbol Ester-Induced Monocytic

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Inhibition of Phorbol Ester-Induced Monocytic Differentiation and c-fms Gene
Expression by Dexamethasone: Potential Involvement of Arachidonic Acid
Metabolites
By Richard M. Stone, Kyoko Imamura, Rakesh Datta, Matthew L. Sherman, and Donald W. Kufe
The treatment of human U-937 leukemia cells with 12-0tetradecanoylphorbol-13-acetate (TPA) is associated with
induction of monocytic differentiation. However, the signaling pathways responsible for induction of the differentiated
monocytic phenotype remain unclear. The present studies
demonstrate that dexamethasone blocks TPA-induced
U-937 cell growth inhibition, adherence, and alphanaphthyl acetate esterase staining. The results also demonstrate that dexamethasone inhibits the appearance of
c-fms transcripts associated with TPA treatment. Run-on
transcription assays demonstrated that the c-fms gene is
transcriptionally active in uninduced U-937 cells and that
the rate of transcription is unchanged after dexamethasone and/or TPA treatment. These findings indicated that
TPA increases c-fms expression by a dexamethasonesensitive posttranscriptional mechanism. Treatment of
U-937 cells with TPA was also associated with stimulation
of arachidonic acid metabolism. Furthermore, dexamethasone, an inhibitor of phospholipase A, activity, blocked
TPA-induced increases in arachidonic acid release. These
findings suggested that TPA may regulate certain features
of monocytic differentiation, such as c-fms gene expression, through the formation of arachidonic acid metabolites. Indomethacin, an inhibitor of cyclooxygenase, had no
detectable effect on c-fms gene expression. However, the
cyclooxygenase metabolite, prostaglandin E, inhibited the
TPA-induced increases in c-fms mRNA levels. Taken together, the results indicate that TPA regulates c-fms gene
expression by a dexamethasone-sensitive mechanism and
that c-fms mRNA levels are controlled by metabolites of
the arachidonic acid pathway.
0 7990 by The American Society o f Hematology.
T
demonstrated that both the CSF-1 and c-fms genes are
expressed during monocytic differentiati~n.~.’The c-fms
gene shares homology with other tyrosine kinase-encoding
protooncogenes.” Moreover, the c-fms product is identical to
the CSF-1 receptor.’ These findings have suggested that
CSF-1 is capable of regulating monocyte functions by an
autocrine mechanism. The c-fms transcripts become detectable a t 12 to 24 hours during TPA-induced monocytic
differentiation, while the CSF-1 and T N F genes are expressed as earlier events.3s6Recent studies have demonstrated
that c-fms gene expression is regulated a t least in part by a
labile protein that stabilizes the c-fms transcript.21However,
little is known about the intracellular signal transduction
mechanisms associated with the induction of c-fms expression.
The results reported here suggest that arachidonic acid
metabolites may be involved in TPA-induced signaling
events that result in the appearance of a differentiated
monocytic phenotype and the regulation of c-fms gene
expression.
PA (12-0-tetradecanoy1phorbo1-13-acetate)and other
tumor-promoting phorbol diesters induce human myeloid leukemic cell lines to differentiate along the monocytic
lineage. This induction of a monocytic phenotype includes
inhibition of growth, adherence, an increase in monocytic
surface markers, and induction of alpha-naphthyl acetate
esterase (NSE) staining.’.’ Furthermore, in addition to the
down-regulation of c-myc. TPA-induced monocytic differentiation of HL-60 promyelocytic leukemia cells is associated
with the appearance of c-fos, c-fms, colony-stimulating
factor- 1 (CSF- l ) , platelet-derived growth factor- 1 ( PDGFl), platelet-derived growth factor-2 (PDGF-2), and T N F
transcript^.^.^ The TNF, CSF-1, and c-fms (CSF-1 receptor)
genes code for proteins involved in monocyte proliferation
and a~tivation.’.~
However, the signal transduction pathways
responsible for these changes in gene expression during
induction of monocytic differentiation by TPA remain unclear.
TPA-induced activation of the calcium- and phospholipiddependent protein kinase C’’.” is presumed to represent a
critical step in the induction of monocytic differentiation by
phorbol diesters. For example, other structurally unrelated
agents, such as bryostatin 112 and tele~cidin,’~
also activate
protein kinase C and induce monocytic differentiation. Moreover, sphingoid basesL4 and palmitoyl carnitineL5 inhibit
activation of protein kinase C as well as phorbol esterinduced monocytic differentiation. However, it is not clear
whether the multiple events associated with TPA-induced
monocytic differentiation of myeloid leukemic cell lines are a
direct consequence of protein kinase C activation. In this
regard, TPA may mediate additional biochemical eventsL6
Furthermore, agents that activate protein kinase C are
capable of stimulating arachidonic acid metabolism via the
activation of phospholipase A2.10,’7,’8
Indeed, recent findings
have suggested that TPA induces T N F gene expression
through the arachidonic acid cascade and that T N F mRNA
levels are controlled by metabolites of this pathway.”
CSF-1 is required for the proliferation and differentiation
of cells along the monocytic lineage.’ Previous work has
Blood, Vol 76, No 6 (September 15). 1990: pp 1225-1232
From the Laboratory of Clinical Pharmacology, Dana-Farber
Cancer Institute, Hantard Medical School, Boston, MA.
Submitted August 3,1989;accepted May 22,1990.
Supported in part by PHS grant no. CA 34183 awarded by the
National Cancer Institute, DHHS, by an American Cancer Society
Clinical Career Development Award (R.M.S.),and by a Burroughs
Wellcome Award in Clinical Pharmacology ( 0 .W.K.).
Presented in part at the American Association for Cancer
Research, May 1989, San Francisco, CA.
Address reprint requests to Richard M. Stone, MD. Laboratory
of Clinical Pharmacology. Dana-Farber Cancer Institute. 44 Binney
St. Boston, M A 021 15.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7606-0012$3.00/0
1225
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1226
STONE ET AL
MATERIALS AND METHODS
Cell culture. U-937 cells were grown in RPMI 1640 medium
(Hazelton Laboratories, Vienna, VA) containing 100 U/mL penicillin, 100 pg/mL streptomycin, and 2 mmol/L L-glutamine in 10%
heat-inactivated fetal bovine serum (Sigma Chemical Co, St Louis,
MO) at a density of 2 x lO’/mL in a 5% CO, humidified atmosphere
at 37OC. Viable cells were determined by trypan blue exclusion. TPA
was dissolved in 1% acetone at a stock concentration of 3.3 pmol/L.
Dexamethasone, nordihydroguaiaretic acid (NDGA), esculetin (BIOMOL Research Laboratories, Plymouth Meeting, PA), ketoconazole, caffeic acid, 5(S)-hydroperoxyeicosatetraenoic acid (5HPETE) (BIOMOL), indomethacin, and prostaglandin E, (PGE,)
were dissolved in 100% ethanol. The final concentration of ethanol
did not exceed 0.1%. All chemicals were obtained from Sigma,
unless otherwise noted.
Induction of monocytic differentiation. Cell adhesion was determined by calculating the percentage of nonadherent cells compared
with total cells (after adherent cells were scraped from plastic
culture flasks) after 1 day of treatment. Cytocentrifuge smears of
cells were examined for NSE stainingz2after 2 days of treatment.
RNA extraction and hybridization. Total cellular RNA was
purified by the guanidine isothiocyanate-cesium chloride method,23
analyzed by gel electrophoresis through 1% agarose-formaldehyde
gels and transferred to nitrocellulose filters.24Hybridization conditions were as described.’ Autoradiograms were scanned by laser
densitometry.
Probes included the 1.6-kilobase (Kb) ClaI/EcoRI fragment of
the human c-myc 3’ exon purified from the pM C41-3 RC plasmid,25
the 1.8-Kb PstI fragment of the chicken @-actingene purified from
the pAI plasmid,z6the 4.0-Kb EcoRI fragment of the c-fms gene
isolated from the pclfms 102 plasmid,” and the 1.1-kb PstI fragment
of the pE4 plasmid containing tumor necrosis factor (TNF) cDNA.6
Run-on transcription assay. Labeled nuclear RNA was
prepared2’ and hybridized to plasmid DNA (equivalent to 2 pg of
insert) that had been run in a 1% agarose gel and transferred to a
nitrocellulose filter. The prehybridization and hybridization conditions were as previously described” except that the hybridizations
were performed for 72 hours at 55OC and the concentration of
RNase in the wash was 500 ng/mL. The ”P-labeled RNA was
hybridized to PstI-digested chicken @-actinpAl plasmid, resulting
in a 1.8-Kb insert,26 and EcoRI-digested pclfms 102 plasmid,
resulting in a 4.0-Kb fragment containing the human clfms cDNA.”
Measurement of arachidonic acid release. Arachidonic acid
release was monitored as described.” Cells in logarithmic growth
phase were incubated for 16 hours in 0.1 pCi/mL [5,6,8,9,
11,12,14,15-3H]arachidonic
acid (87 Ci/mmol; Amersham Corp,
Arlington Heights, IL). The cells were then washed in phosphatebuffered saline, resuspended in medium with or without dexamethasone for 30 minutes, then exposed to 8 nmol/L TPA. Release of
tritium into cell-free supernatants was then determined by scintillation counting.
Measurement of protein kinase C activity. U-937 cells were
suspended in serum-free RPMI 1640 medium and treated with 8
nmol/L TPA for 5 minutes at 37OC. In certain experiments, the cells
were pretreated with 1 pmol/L dexamethasone or 50 pmol/L
NDGA for 30 minutes. After pelleting and washing in phosphatebuffered saline (Ca++- and Mg++-free), cells were lysed by 10
passages through a 25-gauge needle and reconstituted in buffer A
(20 mmol/L Tris-HCI, pH 7.5, 100 pg/mL aprotinin, 0.25 mmol/L
leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride). The cytosolic fraction was obtained after microcentrifugation. The particulate fraction was solubilized with buffer A containing 1% Triton
X-100for 15 minutes on ice. Protein kinase C was partially purified
by passage through 0.5 mL diethylaminethyl-cellulose columns and
eluted with 80 mmol/L NaCI. Aliquots of column eluate were
assayed in the presence of 10 mmol/L MgCI,, 20 pg of histone HI,
lo6 cpm [y-32P]adenosine triphosphate (3,000 Ci/mmol, Amersham; final specific activity, approximately 400 cpm/pmol), and
11.7 nmol/L TPA with or without 8 pg/mL phosphatidylserine and
1 mmol/L CaCI,. After incubation for 10 minutes at 3OoC, bound
radioactivity was determined by precipitation with 25% trichloroacetic acid and collection onto filters under vacuum. Protein kinase C
activity was determined by subtracting the amount of ”P (per
milligram protein) incorporation into histone noted in the absence of
added phospholipids and calcium from the amount of ”P incorporation in their presence.
RESULTS
The treatment of U-937 cells with 8 nmol/L TPA resulted
in the appearance of a differentiated monocytic phenotype.
For example, U-937 cell proliferation was completely inhibited by TPA exposure (Fig 1). In contrast, while dexamethasone alone had no detectable effect on proliferation of U-937
cells, this agent partially blocked the growth inhibitory
effects of TPA (Fig 1). Dexamethasone also inhibited the
effects of TPA on other phenotypic markers. In this regard,
56% of the U-937 cells were adherent after a 24-hour
exposure to TPA (Table 1). However, only 9% of these cells
adhered to the flask following treatment with both dexamethasone and TPA. Dexamethasone also blocked TPA-induced
increases in NSE staining (Table 1). These findings suggested that dexamethasone inhibits multiple phenotypic
changes associated with the induction of monocytic differentiation.
Further studies were performed to determine whether
dexamethasone also blocks changes in gene expression during TPA-induced monocytic differentiation. We previously
demonstrated that c-fms transcripts are induced in HL-60
I
E
v)
0
r
l o 0 r - l
I
I
Days
Fig 1. Effects of TPA and dexamethasone (DEX) on U-937 cell
growth. Viable cell counts were determined by trypan blue
exclusion for untreated U-937 cells (0).cells treated with 8 nmol/L
TPA (W)* 1 pmol/L dexamethasone (01, or 1 pmol/L dexamethasone for 30 minutes before adding 8 nmol/L TPA ( 0 ) .Values
represent the mean f SE for three separate experiments.
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DEXAMETHASONE INHIBITS C-FMS INDUCTION
1227
a
a
Table 1. Effects of TPA and Dexamethasoneon Monocytic
Differentiation in U-937Cells
U-937 cells (untreated)
TPA (8 nmol/L)
Dexamethasone ( 1 pmol/L)
Dexamethasone (1 pmol/L), 30 min
prior to TPA (8 nmol/L)
% Adherence.
% NSE Positivet
0
5 6 + 11
12 f 1
4+2
29 + 8
7 + 1
9+8
11 + 6
In each case, data was obtained from three separate experiments.
*Mean + SE percent adherent cells after 1 day of treatment.
tMean + SE percent of strongly positive cells after 2 days of
treatment.
cells by 24 hours of TPA exposure.’ Similar findings were
obtained in the present study (Fig 2). In contrast, T N F
mRNA was detectable at 3 to 6 hours and was downregulated by 24 hours of TPA treatment (Fig 2). The level of
c-myc transcripts was also down-regulated by 24 hours of
exposure to this agent (Fig 2). The effects of dexamethasone
on changes in c-fms and c-myc expression were therefore
monitored at 24 hours of TPA treatment, while similar
studies were performed at 3 hours for changes in T N F
mRNA levels. Dexamethasone alone had no detectable effect
on c-fms expression (Fig 3). However, this agent completely
inhibited the induction of c-fms transcripts by TPA (Fig 3).
28s -
c
i
28s - 1
18s -
28s -
18s Fig 3. Effects of TPA and dexamethasone (DEX) on c-fms,
c-myc, and actin expression. U-937 cells were treated with 8
pmol/L TPA, 1 pmol/L dexamethasone, or 1 pmol/L dexamethasone for 30 minutes before adding TPA. Total cellular RNA (20 p g )
was isolated at 24 hours for Northern analysis with the ’*P-labeled
c-fms, c-myc, and actin probes.
c-fms
18s -
28s -
TNF
18s -
28s -
c-myc
18s Fig 2. Effects of TPA on c-fms. TNF, and c-myc mRNA levels in
US37 cells. U-937cells were treated with 8 nmol/L TPA for the
indicated times. Total cellular RNA (20 pg) was analyzed as
described6and hybridized t o the 32P-labeledc-fms, TNF, and c-myc
cDNA probes.
Dexamethasone also blocked TPA-induced T N F expression
(data not shown), as previously shown in HL-60 cells,” but
had no effect on the down-regulation of c-myc expression
(Fig 3). Moreover, dexamethasone alone or in combination
with TPA had no detectable effect on actin mRNA levels
(Fig 3). These findings indicated that dexamethasone inhibits pathways involved in the induction of c-fms and T N F
expression, but not in the down-regulation of c-myc expression.
To further analyze the effects of dexamethasone on c-fms
expression, we measured the rate of c-fms gene transcription
in isolated nuclei by the run-on transcription assay procedure. Although c-fms transcripts were undetectable by
Northern analysis (Fig 2), the c-fms gene was transcriptionally active in uninduced U-937 cells (Fig 4). Furthermore,
there was no detectable increase in c-fms gene transcription
associated with the appearance of c-fms transcripts after 24
hours of TPA treatment. These findings suggested that c-fms
expression is controlled posttranscriptionally in these cells.
Treatment with dexamethasone alone or in combination with
TPA also had no effect on rates of c-fms transcription (Fig
4). These results further implied that dexamethasone inhibits
the posttranscriptional regulation of c-fms expression by
TPA.
The finding that dexamethasone, an inhibitor of phospholi-
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I228
Fb4.
STONEETM
Hhen d TPA and dounw(h..arw (WX) on rotom d e - . l k r g . r w t . blg..nd~andolkrplomld
DMAmm e run
on0 1% .gwoaooI(. trwa(.rrdto nhrocr*ub.. p . 9 by
~ Bovmwn bhtlng. wd hybrldh.d ro "Uu.mountrd " P - b b d d nodwr RNA
(4 x le cpmlmL) W r d from control (U-937). T P A - n w d ah.d o r o n w t h w - t r w t d cob. and cell8 rrwnd with d o x o m m
for
*ut@# b.tor. Odd- TPA. -Id [email protected] 24 hour8 . h W drug r r ~ r ~Kbg.
t .kibb@wp h 8 .
m
paw A? activity.?' blocked certain features associated with
TPA-induced monocytic differentiation prompted further
studies monitoring the etTects of this agent on arachidonic
acid release. In this context. phorbol aten that activate
protein kinase C also stimulate arachidonic acid metab
olism."" Similar findings were obtained in TPA-treated
W-937 cells. For example. TPA increased tritium release
from ['H ]arachidonic acid-labeled cells by approximately
4 0 7 over that in uninduced cells (Fig SA). In contrast. while
dexamethasone alone had nodetectable effect on arachidonic
acid release. thisagent blocked the increases induced by TPA
(Fig 3R). These findings suppated that the effects of
dexamethasone may be. at least in part. related to inhibition
of arachidonic acid release.
After its release from membranebound phaopholipids.
arachidonic acid is further metabolized to leukotriena by
5-lipoxygenase and to pmtaglandins by cyclooxygenase.'Y
Consequently. further experiments were performed in the
pmencc of N X J A . an inhibitor of 5-lipoxygenase."' N f f i A
(30pmollL) alone had no detectable etTm on levelsof c-/mJ
mRWA (Fig 6).However. treatment with this 5-lipoxygenasc inhibitor and TPA multed in nearly complete inhibition
of TPA-induced c - 1 . 5 expration (fig 6). ND(;A also
blocked the induction of T S F expression by TPA. but had no
detectable effect on actin mRXA levels (Fig 6). However.
given the potential of this concentration of N D S A to affecl
other pathways." additional inhibitors of 5-lipoxygenase
were tested." I' Neither I O to 100 pmol/L caffeic acid. 20 to
F8g 6. Hhen d W A and od
I W X ) on "hMo& odd rdowo (rom U-W7 cdn. (AI UW7OrOr worm pnhbdodrrkh
pt)."k
addfor 18 bar.. Wnh.d. and expawd to8 nmd/L WA. R.k...d
nhkm Imo lha rupwn" W n m0nhor.d n rho
Miutdr h o 8 for the u n t r n t d (0)
ond T P A - t r w d (0)cob. (E) U-937 & were p o b b d d whh ['H)u.dJdmkodd. mrh.d. and
k u b t d with 1 Lmo(lL dononmhauww. A b r 30 mlnucw. 8 nmo(/L TPA rm .bd.d for n .ddtrrorul2 houn and the cJh*e
w p u r u t m s hard(a rrirkm reloow. V o k m reprosent rho myn (R for *wee s o p r o t e erprknmtr. CO. u w r w d &.
-
~
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1229
DEXAMETHASONE INHlBrrS C - n S INDUCTION
Q
Q
c
I
28s -
1
I
18s 28s 18s -
TNF
(3h)
N D G A in these studies may have blocked the increases in
c-fms expression by altering TPA-induced activation of
protein kinase C. We therefore monitored the effects of these
agents on the translocation of protein kinase C activity from
the cytosol to membrane fractions. Treatment of U-937cells
with TPA was associated with an increase in membranebound protein kinase C activity and a decline in that found in
the cytosol (Fig 8). Dexamethasone or NDGA alone had
little. if any. effect on the distribution of protein kinase C
activity in u n i n d u d U-937 cells (data not shown). Moreover, TPA-induced translocation of protein kinase C activity
was similar in the presence of these agents to that obtained
with TPA alone (Fig 8). These results suggested that the
effects of dexamethasone and NDGA on TPA-induced c-fms
expression are not associated with inhibition of protein kinase C.
I
G
O cu
W 6 W
0 (3 0 (3
Z
- a. Z a.
28s y
Fig 6. EChcn of TPA and NodA on c-*nr. TNF. and actin
mRNA lovole. U-937 d l r w u a treated with 8 mndlL TPA. 60
#tmol/L NDGA, or 60 #tmdlL N W A for 30 minutee bofora adding
TPA. Total d l u b r RNA was iwbted at 3 and 24 hours for
hybridization to the mP-labaledDNA prok..
100 pmol/L ketoconazole. nor 5 to 50 pmol/L esculetin
inhibited the induction of c-fms expression observed after 24
hours of TPA treatment (data not shown). Furthermore.
treatment of U-937cells for 24 hours with lOor 100 nmol/L
5-HPETE. the primary metabolite of arachidonic acid via
the 5-lipoxygenase pathway. failed to induce c-Jms mRNA
(data not shown). These findings suggested that the formation of eicosanoidsother than through the activity of S-lipoxygenase may be involved in the regulation of c-fms gene
expression.
Studies were also performed with indomethacin, an inhibitor of cyclooxygenase.” and PGE,. a prostaglandin metabolite of the cyclooxygenase pathway. Treatment of U-937cells
with indomethacin or PGE,alone had nodetectable effect on
c-fms mRNA levels (Fig 7). Furthermore. indomethacin had
no effect on the induction of c-fms expression by TPA (Fig
7). However. exposure of U-937cells to PGE, was associated
with a 53% decrease in levels of c-fms transcripts induced
during TPA treatment (Fig 7). These agents had similar
effects on TNFexpression. although PGE,completely blocked
the induction of T N F transcripts by TPA (Fig 7). In
contrast. indomethacin and PGE, had little. if any, erect on
changes in c-myc expression or actin mRNA levels (Fig 7).
Taken together. these results suggested that the induction
of c-/” expression by TPA is mediated through the arachidonic acid cascade. However. the use of dexamethasone and
28s-
-
-
9
0
;+
W
18s -
28s
18s
-
7
-1
1
I
28s 18s -
Fig 7. EChcn of TPA, hrd0nnt)UdnImoO),and WE, on gono
o x p r . u k n in U-937 colts. U-937 edlr ware treated with 8 d l L
TPA. 10 p d l L indomethacin. 1 pmol/L PGE,. 10 pmotlL Momothadn for 30 minutoe before adding TPA. or 1 #tmd/L PGE, for 30
minutoe befor0 adding TPA. Total cellular RNA (20 pg) war
ieolated at 3 and 24 hours for hybridization to the indicated
=P-labd.d prob...
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1230
STONE ET AL
arachidonic acid release. Furthermore, inhibitors of phospholipase A, block both TPA-induced increases in arachidonic
acid metabolism and T N F gene expression.” These findings
suggested that arachidonic acid metabolites are involved in
the regulation of T N F expression during monocytic differentiation. The studies reported here in U-937 cells have
examined whether the arachidonic acid cascade is similarly
involved in the regulation of other events associated with
TPA-induced monocytic differentiation. TPA treatment of
U-937 cells was associated with increases in arachidonic acid
.E 0
@ E
release that were comparable to that reported for HL-60
- P
o w
cells.” Moreover, dexamethasone inhibited the stimulation
L
0.0
n
of arachidonic acid metabolism by TPA. The hydrolysis of
TPA
DEX
NDGA
co
membrane phospholipids by phospholipase A, is ratelimiting
in leukotriene and prostaglandin ~ynthesis.~’
FurtherTPA
TPA
more, glucocorticoids, such as dexamethasone, have been
Fig 8. Effects of TPA, dexamethasone (DEXI, and NDGA on
shown to inhibit phospholipase A,, presumably through the
protein kinase C activity. U-937 cells were treated with 8 nmol/L
induction of l i p ~ c o r t i n .Treatment
~~
of U-937 cells with
TPA for 5 minutes. Cells were also pretreated with 1 pmol/L
dexamethasone
also
blocked
certain
phenotypic
characterisdexamethasone or 50 pmol/L NDGA for 30 minutes before adding
tics of TPA-induced monocytic differentiation, such as growth
the TPA. Protein kinase C activity was determined for the
inhibition, adherence, and NSE staining. While other work
cytosolic ( 0 )and membrane (H) fractions. The results are expressed as the mean ? SD for three determinations. CO, untreated
has demonstrated that dexamethasone blocks the appearance
cells.
of a differentiated murine erythroleukemia cell phen~type,~’
previous studies have not demonstrated an inhibitory effect
DISCUSSION
of this agent on induction of human myeloid differentiation.
The possible role of arachidonic acid metabolites in
Human myeloid leukemia cell lines differentiate along the
TPA-induced monocytic differentiation of U-937 cells was
monocytic lineage following treatment with certain phorbol
esters.’ This induction of monocytic differentiation is associexamined further in terms of the regulation of c-fms gene
expression. The c-fms gene codes for the CSF-1 receptor’
ated with changes in both phenotypic characteristics and
While these findings have demonstrated
and is expressed during induction of monocytic, but not
gene
granulocytic, differentiati~n.~
While c-fms transcripts are
that the block in differentiation associated with leukemogenalso detectable in trophoblasts:’ expression of this gene
esis is reversible, little is known about the intracellular
appears to be specific for the monocytic lineage in hematopoisignaling mechanisms responsible for inducing the differentietic cells. Recent studies have demonstrated that c-fms
ated phenotype. Phorbol esters, such as TPA, that activate
transcripts are stabilized by a labile protein during induction
protein kinase C also induce differentiation. However, the
of monocytic differentiation.” The present studies similarly
relationship between this activation and induction of differendemonstrate posttranscriptional regulation of c-fms mRNA
tiation has remained unclear.
levels in TPA-treated U-937 cells. Furthermore, the results
Previous studies have demonstrated that induction of
indicate that dexamethasone inhibits this effect of TPA on
monocytic differentiation is associated with the downc-fms expression. While these findings suggested that arachiregulation of c-myc expression and appearance of c-fos
donic acid metabolites are involved in the posttranscriptional
transcript^.^ The decrease in c-myc mRNA levels may be
regulation of this gene, dexamethasone may have blocked
sufficient to induce myeloid d i f f e r e n t i a t i ~ nIn
. ~ contrast,
~
the
this effect of TPA by modulating other signaling event^.^'
induction of c-fos expression is neither sufficient nor required
for induction of the differentiated monocytic phen~type.’~ However, there was no detectable inhibitory effect of dexamethasone on TPA-induced activation of protein kinase C in
Other studies have demonstrated that TPA-induced monothese cells.
cytic differentiation is associated with expression of the
Previous studies have examined arachidonic acid metaboTNF, c-fms, CSF-1, PDGF-1, and PDGF-2 genes.’”*’’
lism during induction of monocytic differentiation. For
These genes code for products involved in the regulation and
example, treatment of U-937 cells with TPA is associated
function of cells differentiated along the monocytic lineage.
with an increase in phospholipase A, activity and secretion of
While certain insights are available regarding the downcyclooxygenase metabolites, including PGE,, thromboxane
regulation of c-myc transcripts during monocytic
A,, and thromboxane B,.4,s4’ Other studies have demondifferentiati~n,~’
signaling events involved in the regulation
strated that TPA-induced monocytic differentiation of HL-60
of genes coding for monocyte membrane or secreted products
cells is associated with increases in the activities of prostaglanremain poorly understood.
din H synthetase and thromboxane synthase.44 In contrast,
The induction of T N F transcripts is one of the earliest
there is no available evidence that leukotriene synthesis is
detectable changes in gene expression associated with TPAincreased during differentiation of these cells along the
induced monocytic differentiation.” Treatment of certain
monocytic lineage.42.44In the present studies, NDGA, an
myeloid cells with TPA is associated with increases in
+
+
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1231
DEXAMETHASONE INHIBITS C-FMSINDUCTION
inhibitor of 5-lipoxygenase and leukotriene synthesis, blocked
TPA-induced c-fms gene expression. These findings suggested that 5-lipoxygenase metabolites might be involved in
the regulation of this gene. However, other known inhibitors
of 5-lipoxygenase had no detectable effect on the induction of
c-fms transcripts by TPA. Furthermore, there was no detectable effect of adding 5-HPETE on c-fms expression. Taken
together with the previous findings in TPA-treated U-937
and HL-60 cells, the present results would suggest that
leukotrienes may have little, if any, involvement in regulating
c-fms mRNA levels during monocytic differentiation.
The involvement of eicosanoids in the regulation of c-fms
gene expreSsion is, however, supported by the findings with
PGE,. In the present work, PGE, partially down-regulated
TPA-induced increases in c-fms m R N A levels. Previous
studies have also demonstrated that PGE, inhibits macrophage colony formation.45The basis for these effects remains
unclear, although PGE, has been shown to increase cyclic
adenosine monophosphate (CAMP) levels in myeloid cells46
and, thus, certain monocyte functions may be inhibited
through CAMP-dependent protein kinase activity. On the
basis of the present results, indomethacin, an inhibitor of
cyclooxygenase activity, might have been expected to increase levels of c-fms transcripts, unless PGE, synthesis was
already partly inhibited during induction of differentiation.
Indeed, the increase in PGE, secretion from TPA-treated
U-937 cells was relatively low in the absence of exogeneous
arachidonic acid.42
In summary, the finding that dexamethasone blocks TPAinduced increases in c-fms gene expression has suggested
involvement of the arachidonic acid cascade. The present
results and previous studies4, have demonstrated that T P A
treatment of U-937 cells is associated with increases in
phospholipase A, activity and that this effect is sensitive to
dexamethasone. While there is little evidence to support
leukotrienes in the regulation of c-fms mRNA levels, the
results suggest that the cyclooxygenase metabolite, PGE,,
down-regulates expression of this gene. Further studies are
required to define which arachidonic acid metabolites play a
role in the induction of c-fms expression.
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1990 76: 1225-1232
Inhibition of phorbol ester-induced monocytic differentiation and
c-fms gene expression by dexamethasone: potential involvement of
arachidonic acid metabolites
RM Stone, K Imamura, R Datta, ML Sherman and DW Kufe
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