Vol. 266, No. 21, Issue of July 25, pp. 13485-13488,1991
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
Na'/H' Antiporter Gene
Expression during Monocytic
Differentiation of HL60 Cells*
(Received for publication, March 20, 1991)
Gadiparthi N. RaoSB, Nicolas de RouxS,
Claude Sardetq, Jacques Pouyssegurl, and
Bradford C. BerkS
From the $Cardiology Division, Emory University School
of Medicine, Atlanta, Georgia 30322 and the ([Centre de
Biochimie, Centre National de la Recherche Scientijique,
Universite de Nice, Parc Valrose, 06034 Nice, France
During differentiation of human
HL60 cells into monocytes there are sustained in-
creases in intracellular pH and Na+/H+ antiporter ac-
tivity. Here we show that increased transcription and
expression of the gene for the Na+/H+ antiporter pre-
cedes phorbol 12-myristate 13-acetate (PMA)-induced
HL60 cell differentiation. PMA increased steady-state
Na+/H+ antiporter mRNA levels -50-fold within 8 h
(at which time <15% of cells had differentiated). This
increase was due to an increased transcriptional rate
as determined by nuclear run on. Immunoprecipitation
of [3BS]methionine-labeled Na+/H+ antiporter using an
antiporter fusion protein antibody (RPl-cZ8) showed
an equivalent increase in Na+/H+ antiporter protein
synthesis. The synthetic diacylglycerol, l-oleolyl-2-
acetylglycerol, an activator of protein kinase C, which
unlike PMA did not cause differentiation, failed to
induce Na+/H+ antiporter mRNA. Furthermore, inhi-
bition of PMA-induced differentiation by either sphin-
gosine or cycloheximide prevented accumulation of
Na+/H+ antiporter mRNA. Together, these findings
strongly suggest a close association of Na+/H+ anti-
porter induction with HL60 cell differentiation. The
HL60 cell system is a promising model to study the
mechanisms of Na+/H+ antiporter gene regulation and
its function in differentiation.
An increase in activity of the Na+/H+ antiporter is one of
the earliest events stimulated by growth factors (1-4). Because
this stimulation results in an intracellular alkalinization in
the absence of HCO;, it was proposed that the function of
the antiporter was to cause intracellular alkalinization (1-4).
However, experiments performed in the presence of HCO;
suggest that Na+/H' antiporter-mediatedpHi changes are not
involved in the early mitogenic response. Specifically, in the
presence of HCO;, growth factors cause no change in pHi or
even an acidification (5,6), while Na+/H+ antiporter-deficient
mutants are able to grow well at pH > 7.4 (7, 8). These
findings led us to investigate the possibility that the role of
the Na'/H+ antiporter may be more important in other long
* The costs of publication of this article were defrayed in part by
the payment of page charges. This 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: Division of Car-
diology, Emory University School of Medicine, P. 0. Drawer LL,
Atlanta, GA 30322. Tel.: 404-727-8147; Fax: 404-727-3330.
term cellular responses. One model system in which long term
alterations in Na+/H' antiporter activity and pHi have been
shown to be essential is the differentiation of the HL60
promyelocytic leukemia cell line into granulocytes (in re-
sponse to retinoic acid or dimethyl sulfoxide (9, 10)) or into
monocyte/macrophages (in response to phorbol 12-myristate
13-acetate (PMA)' or vitamin DS (11, 12)). Differentiation
into either cell type is associated with a sustained increase in
pHi and antiporter activity (9-12). Furthermore, alkaliniza-
tion with NH4C1 is sufficient to trigger differentiation (11).
Therefore, we have examined Na'/H+ antiporter expression
at the mRNA and protein levels in HL60 cells during differ-
entiation to determine whether regulation of the antiporter
gene plays a role in these long term cellular responses.
MATERIALS AND METHODS
Cell Culture-HL6O cells were grown in RPMI 1640 medium sup-
plemented with 10% (v/v) heat-inactivated fetal bovine serum, 100
units/ml penicillin, and 100 pg/ml streptomycin. The cultures were
maintained in humidified 95% air, 5% CO, at 37 "C by passage of 1-
2 X lo5 cells/ml every other day. Cell viability was tested by trypan
blue exclusion assay. HL60 cells were differentiated by exposure to
PMA (20 ng/ml) for various lengths of time, and the extent
differentiation was measured by attachment of cells to the substra-
RNA Blot Analysis-HLGO cells were treated with 20 ng/ml PMA,
and total cellular RNA was isolated from all cells (approximately 8
X lo7 cells for each time point) at the indicated times by the guani-
dinium isothiocyanate-cesium chloride protocol (13). Poly(A+) RNA
was selected by passing total cellular RNA in high salt containing 20
mM Tris-HC1 buffer (pH 7.5) through an oligo(dT)-cellulose column
and eluting it with salt-free 20 mM Tris-HC1 buffer (pH 7.5) as
described earlier (14). Poly(A+) RNA (10 fig) from each time point
was size-fractionated by electrophoresis on 1% agarose, 2% formal-
dehyde gel. After transfer to Nytran membrane (15), the RNA was
cross-linked to the membrane using
Stratagene, La Jolla, CA). After 4 h of prehybridization in 50% (v/v)
formamide, 5 X Ssc (1 X SSC = 0.15 M NaC1, 0.015 M sodium
citrate), 5 X Denhardt's (1 X Denhardt's = 0.02% (w/v) each of Ficoll,
polyvinylpyrrolidone, and bovine serum albumin), 50 mM sodium
phosphate (pH 6.5), and 250 pg/ml sheared salmon sperm DNA at
42 "C, the Nytran membrane was hybridized in the above solution
containing 10% (w/v) dextran sulfate and 1 X lo6 cpm/ml cDNA
probe for 16 h at 42 "C. The cDNA probes Na+/H+ antiporter cDNA
(BamHI fragment, base pairs 731-2646 of human cDNA, clone c28
(16)) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
(a full-length rat cDNA, clone pRGAPD 13 (17)) were radiolabeled
using a GIBCO/BRL random primers labeling kit as per the manu-
facturer'sprotocol with [a-"PIdCTP (specific activity, 3000 Ci/mmol,
Du Pont-New England Nuclear). After hybridization, the Nytran
membrane was washed 3 times in 2 X SSC, 0.2% SDS (15 min, 25 "C)
and twice in 0.1 X SSC, 0.1% SDS (15 min, 60 "C). The membrane
was then exposed to Kodak X-Omat AR x-ray film with an intensi-
fying screen at -70 "C for 16 h. Densitometric analysis of the auto-
radiograms exposed in the linear range of film density was made on
a Pharmacia Ultroscan XL laser densitometer.
Nuclear Run-on Assay-Nuclei from uninduced and induced HL60
cells (approximately 2 X 10' cells) were prepared by the technique of
Groudine et al. (18). Run-on transcription was carried out at 30 "C
for 30 min in a reaction mixture consisting
suspension (3-5 X 10' nuclei in 40% (v/v) glycerol, 50 mM Tris-HC1,
pH 8.3, 5 mM MgCI,, and 0.1 M EDTA), 60 pl of 5 X nuclear run-on
UV irradiation (Stratalinker,
p1 of nuclear
The abbreviations used are: PMA, phorbol 12-myristate 13-ace-
tate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SDS, so-
dium dodecyl sulfate; TES, N-tris(hydroxymethyl)methyl-2-amino-
ethanesulfonic acid PAGE, polyacrylamide gel electrophoresis; PKC,
protein kinase C; OAG, 1-oleoyl-2-acetylglycerol.
Na+/H+ Antiporter Expression
in HL60 Cells
buffer (12.5 mM MgCI,, 750 mM KCI, 1.25 mM each ATP, GTP, and
CTP, 25 mM Tris-HCI (pH
8.0)), and 50 pl of [n-:"P]UTP (500 pCi,
specific activity, 3000 Ci/mmol, Du Pont-New
simultaneous reaction with n-amanitin (2 pg/ml) served as a control.
Reactions were terminated by the addition of 8 pl of 11 mg/ml RNase-
free DNase I and incubation at 30 "C for 5 min. "'P-Labeled RNA
was purified as described (19). Plasmids containing cDNAs for the
Na'/H+ antiporter and GAPDH were linearized, denatured (0.2 M
NaOH for 30 min at 25 "C), neutralized with 6 X SSC (10 volumes),
and applied to a Nytran membrane
apparatus. After prehybridization for 3 h in 100 mM TES-HCI buffer
(pH 7.4). 0.2% SDS, 10 mM EDTA, 0.3 M NaCI, 1 X Denhardt's, and
250 pg/ml Escherichia coli tRNA, the membrane was hybridized in
the above solution containing 1
X 10' cpm/ml "'P-labeled nuclear
RNA transcripts for 48 h at 65 "C. Membranes were washed twice in
2 X SSC, 0.1% SDS (15 min, 25 "C) and twice in 0.1 X SSC, 0.1%
SDS (60 min, 60 "C).
Immunoprecipitation Analvsis"HL60 cells were metabolically la-
heled for 24 h using [:"S]methionine
and membranes were isolated as described (1). Equal amounts of
membrane proteins from uninduced and
immunoprecipitated using affinity-purified anti-Na+/H+ antiporter
antibodies (RPl-c28), and the immunoprecipitated proteins
separated on SDS, '7.5% PAGE under
jected to autoradiography
England Nuclear). A
(10 pg/slot) using a slot blot
f 20 ng/ml PMA,
induced HL60 cells were
reducing conditions and sub-
FIG. 2. Steady-state levels of Na+/H+ antiPorter and
GAPDH mRNA during PMA-induced
tion of human promyelocytic HL60 cells. Equal amounts of
poly(A+) RNA (10 pgllane) from PMA-treated and untreated HL60
cells were analyzed by Northern blotting for steady-state levels of
Na+/H+ antiporter and GAPDH mRNA
Initially, we compared the time course for PMA-stimulated
HL60 cell differentiation to the steady-state levels of Na+/H+
antiporter mRNA. Differentiated cells were identified by at-
tachment to the substratum and change in
shown in Fig. 1, HL60 cells differentiated
manner into monocytic macrophage-like
20 ng/ml PMA. After 8 h, 12% of HL60 cells were differen-
tiated, and by 24 h 50% were differentiated. The viability, as
tested by trypan blue exclusion assay, was >90% both in
control and PMA-treated cells. Steady-state levels of Na+/H+
antiporter mRNA were then analyzed
a "'P-labeled human Na+/H+ antiporter cDNA (16). As is
evident from Figs. 1 and 2. little change was observed in the
steady-state levels of Na+jH+ antiporter mRNA within 4 h.
However, after 8 h of PMA treatment, Na+/H+ antiporter
mRNA levels increased by 42-fold as compared with levels in
untreated HL60 cells. Increases in the steady-state levels of
Na+/H+ antiporter mRNA persisted, reaching
50 f 10-fold by 24 h. To demonstrate that these increases in
the steady-state Na+/H+ antiporter mRNA levels in PMA-
treated HL60 cells reflected a specific induction by PMA and
using the respective '"p-
'% ' $
8 Q $ * 8% 8 Q $ *
RESULTS AND DISCUSSION
cell shape. As
in response to
by Northern blot using
r. r'y- C GAPDH
. + Na/H
a maximum of
0 12 24
FIG. 1. Time course for PMA-induced monocytic differen-
tiation and induction of Na'/H+ antiporter mRNA in HL60
cells. HL60 cells were treated with 20 ng/ml PMA. Right axis, cell
differentiation was measured as the percentage of cells at.tached to
the substratum. Left axis, Na'/H+
determined by quantitative laser densitometry (Pharmacia Ultrascan)
of Northern blots prepared from poly(A') RNA obtained
indicated time points and hybridized to antiporter cDNA (see Fig. 2).
antiporter mRNA levels were
FIG. 3. Transcription rates of Na+/H+ antiporter and
GAPDH genes during PMA-induced differentiation of HL60
cells. Radiolabeled RNA transcripts were isolated from nuclei
PMA-treated and untreated HL6O cells and hybridized to filter-
immobilized Na+/H+ antiporter cDNA and GAPDH cDNA as de-
scribed under "Materials and Methods."
not a generalized increase in total RNA (or unequal loading
of poly(A+) RNA), the blots were reprobed with a '"P-labeled
cDNA for the glycolytic enzyme, GAPDH, which is constitu-
tively expressed in most cells (17). Steady-state mRNA levels
for GAPDH did not vary between PMA-treated and untreated
HL60 cells (Fig. 2, lower panel). These findings show that the
Na+/H+ antiporter is specifically induced by PMA. Compar-
ison of the time courses for antiporter mRNA accumulation
and cell differentiation (Fig. 1) indicated that the increase in
antiporter mRNA levels preceded differentiation.
To determine whether the increase in Na+/H' antiporter
mRNA in PMA-treated HL60 cells was due to a corresponding
increase in its transcription rate, nuclear run-on assays were
performed in nuclei prepared from PMA-treated and un-
treated HL60 cells (Fig. 3). The rate of Na+/H+ antiporter
gene transcription increased 17
HL60 cells. No significant change was observed in the rate of
GAPDH gene transcription, a result in agreement with its
steady-state mRNA levels. The 17-fold increase in the rate of
Na+/H+ antiporter gene transcription may not account com-
pletely for the 50-fold increase in its steady-state
levels, suggesting a role for post-transcriptional mechanisms
such as mRNA stabilization in PMA-treated HL60 cells.
k 4-fold in PMA-treated
in HL60 Cells
Earlier studies suggested that PMA-induced monocytic dif-
ferentiation of HL60 cells is mediat,ed by protein kinase C
(PKC) (21,22). To define the role of PKC in PMA-stimulated
Na+/H+ antiporter mRNA accumulation, two experiments
were performed. 1) HL60 cells were treated with l-oleoyl-2-
acetylglycerol (OAG), a synthetic diacylglycerol and specific
activator of PKC. OAG, which does not induce diffrrentiation
(23, 24), also failed to induce Na+/H+ antiporter mRNA (Fig.
4A). This result clearly indicates that PKC activation alone
is insufficient to induce Na+/H+ antiporter mRNA and dif-
ferentiation. 2) HL60 cells were treated with PMA for 8 h in
the presence or absence of the PKC inhibitor, sphingosine
(10 p ~ ) .
Sphingosine blocked PMA-induced monocytic dif-
ferentiation of HL60 cells by 78% (as determined by cell
adherence), in agreement with previous
Sphingosine also inhibited PMA-induced Na+/H+ antiporter
mRNA accumulation significantly (70% decrease) but had no
effect on GAPDH mRNA levels (Fig. 4R).
These findings present a paradox regarding the
OAG, although an activator of PKC, caused neither differ-
entiation nor Na+/H' antiporter mRNA accumulation. PMA,
also an activator of PKC, induced both differentiation and
Na+/H+ antiporter mRNA accumulation. Both
were significantly inhibited by sphingosine which is thought
to inhibit PKC (21, 22). However, in light of the failure of
OAG to induce either differentiation or antiporter expression,
activation of PKC alone is insufficient to mediate these
events. Thus, it appears that inhibition
HL60 cell differentiation and Na+/H+ antiporter expression
by sphingosine may be mediated by blockade of signal trans-
duction event(s) other than PKC. Recently, it was reported
that sphingosine affects a number
events at the plasma membrane level (25). These data suggest
that signals in addition to activation of PKC are required for
stimulation of antiporter expression and HL60 cell differen-
To further characterize the mechanisms underlying the
PMA-induced increases in the Na'/H+ antiporter mRNA
accumulation, we studied the effect of cycloheximide, a potent
inhibitor of protein synthesis, on steady-state Na'/H+ anti-
porter mRNA levels. Cycloheximide (20 wg/ml) significantly
inhibited the PMA-induced increase
studies (21, 22).
role of PKC.
of these events
of signal transduction
in Na+/H+ antiporter
FIG. 4. Effect of OAG, sphingosine, and cycloheximide on
Na+/H+ antiporter mRNA accumulation in HL60 cells. A, effect
of OAG. HLW cells were treated with 20 ng/ml PMA or 50 ng/ml
OAG for 24 h. H , effect of sphingosine. HL6O cells were treat.ed with
20 ng/ml PMA -C addition of sphingosine (10 PM every 3 h (21, 22)).
C, effect of cycloheximide. HLFO cells were treated with 20 ng/ml
PMA k 20 pg/ml cycloheximide for 8 h. In all experiments poly(A')
RNA was isolated and analyzed for Na+/H+ antiporter and GAPDH
mRNA as described under "Materials and Methods."
FIG. 5. Na'/H+ antiporter protein synthesis during PMA-
induced monocytic differentiation of HL60 cells.
size markers were phosphorylase
kI1a). Preimmune serum did not precipitate the 110-. 100-, or 42-kI)a
proteins from induced or uninduced HIAO cells as shown.
h (110 kDa) and ovalhumin
mRNA level by 80% (Fig. 4C). Cycloheximide also decreased
PMA-induced HL60 cell differentiation by 80-8596. These
findings show that continuous protein synthesis is required
for PMA induction of Na+/H+ antiporter mRNA levels and
differentiation of HL60 cells. In summary, agents that
differentiation (PMA) increase antiporter expression, while
agents which fail to cause differentiation (OAG) or inhibit
PMA-induced differentiation (sphingosine and
mide) do not. Thus there is a strong correlation
antiporter expression and differentiation.
To relate the increases in antiporter mRNA levels to anti-
porter protein expression, HL60 cells were metabolically la-
beled using [""Slmethionine (kPMA), and Na+/H+ antiporter
protein was isolated by immunoprecipitation using affinity-
purified Na+/H' antiporter antibodies (1). Following immu-
noprecipitation, proteins were separated by SDS-PAGE and
visualized by autoradiography. As shown in
protein bands of molecular sizes 110, 100, and 42 kDa were
immunoprecipitated. The 100-kDa protein was identified as
the Na+/H+ antiporter b?sed on its molecular size (1) and the
fact that it was the only band phosphorylated (data not
shown). As shown in Fig. 5, there was -30-fold increase in
newly synthesized antiporter protein, a finding in agreement
with the mRNA levels (Figs. 1 and 2). The 110- and 42-kDa
proteins were also induced to the same extent as the antiporter
during PMA-induced monocytic differentiation of HL60 cells.
However, the nature of these proteins remains to be deter-
mined, as they were not precipitated by preimmune rabbit
serum (Fig. 5) nor by antibodies lacking specificity for the
In cultured, growth-arrested smooth muscle cells we previ-
ously observed a relative increase
mRNA levels in response to calf serum (26). However, there
was only a 1.2-fold increase in V,,,,, (27), suggesting important
post-translational regulation. To determine whether similar
regulatory mechanisms were present in the
HL60 cells, Na+/H+. antiporter activity was measured by
amiloride-sensitive ""Na influx following acid loading (28).
There was an increase of -3-fold in apparent V,,,, (pHi = 5.5,
Na+(out) = 20 mM) in PMA-treated cells. In comparison with
the 30-fold increase in antiporter protein these results
Fig. 5, three
of -9-fold in antiporter
G. N. Rao and H. C. Herk, unpublished ohservations.
13488 Download full-text
Na+/H+ Antiporter Expression in HL60 Cells
that many of the antiporters are functionally inactive.
Our data suggest that increased expression of the Na'/H'
antiporter is closely associated with HL60 cell differentiation
because: 1) the increase in antiporter expression temporally
preceded differentiation; 2) inhibition of Na'/H'
mRNA accumulation by cycloheximide or sphingosine was
associated with inhibition of differentiation; 3) although OAG
stimulated PKC it failed to induce the antiporter or differ-
entiation; and 4) initiation of HL60 cell differentiation into
granulocytes by retinoic acid (1 /LM for 72 h) was also associ-
ated with an increase of -15-fold in antiporter mRNA levels
(preliminary data). Furthermore, Besterman et al. (12) have
reported that the Na+/H+ antiporter
blocks PMA-induced differentiation of HL60 cells.
The role for such massive increases in Na'/H'
gene expression during HL60 cell differentiation is unclear.
It may be essential for differentiation by virtue of its effects
on pHi (9-12). This appears plausible because during differ-
entiation into both monocytes and granulocytes there are
sustained increases in pHi (9-12). However, the magnitude of
the changes in pHi (0.25 pH units after 24 h (9-12)) and Vmsx
(-3-fold after 24 h of PMA treatment) suggests that the
relatively larger increase in Na'/H+ antiporter mRNA and
protein levels may be involved in other signal transduction
events at the plasma membrane such as Na+ transport (29).
Alternatively, antiporters synthesized during differentiation
may be functionally inactive due to concomitant induction of
regulatory proteins that are inhibitory (e.g. phosphatases) or
to insertion of antiporters into sites other than the plasma
membrane (although no antiporter activity could be detected
in phagosomal membranes of neutrophils (30)). It appears to
be prudent for a differentiating monocyte or granulocyte to
increase its Na'/H' antiporter levels to defend itself against
the cytoplasmic acidification which occurs upon activation by
a variety of agents (31). In summary, the present findings
suggest that increased expression of the Na'/H'
closely associated with HL60 cell differentiation, and this cell
system will be useful to study the transcriptional regulation
of antiporter gene expression.
Acknowledgments-We thank Drs. Jeffrey N. Davidson and R. W.
Alexander for critical reading of the manuscript.
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