From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Changes in Na,K-ATPase Gene Expression During Granulocytic Differentiation
of HL60 Cells
By Setsuko K. Chambers, Maureen Gilmore-Hebert, Barry M. Kacinski, and Edward J. Benz Jr
During granulocytic differentiation of HL60 cells, immediate
reduction of ouabain-sensitive potassium flux is observed
within the first 12 hours of addition of dimethyl sulfoxide
(DMSO). We show that gene expression of the a3 isoform of
Na+,K+-ATPase, which encodes an ouabain-inhibitable
Na+,K+-ATPaseactivity, significantly declines during the first
24 hours of granulocytic differentiation by DMSO of HL60
cells. The more common a1 isoform decreases, but more
gradually over 72 hours of DMSO induction. Loss of a3 and a1
messenger RNA (mRNA) are due to changes in mRNA decay;
their transcriptionis not altered. a3 mRNA half-life is 3 hours
in HL60 cells; upon induction by 16 hours of DMSO, it
decreases to approximately 2 hours. a1 transcripts are less
sensitive to DMSO induction, with their half-life being 3.5
hours in HL60 cells; upon induction, their half-life decreases
to 3 hours. Experiments measuring protein stability confirm
that a3 protein is more labile than al. In uninduced HL60
cells, a3 membrane protein comprises 30% of the total Q
isoforms, and is less stable than al, with a protein half-life of
only 9 hours. Upon DMSO induction, steady-state a3 protein
decreases markedly within 10 hours, whereas a1 protein
remains stable. These results show that posttranscriptional
changes during induction play a major role in the differential
regulation of a1 and a3 isoforms of Na+,K+-ATPase; regulation of the latter may be important for early granulocytic
differentiation, or for one of the differentiated functions of
mature granulocytes.
o 1992by TheAmerican Society of Hematology.
M
isoform showed that in uninduced HMO cells, d mRNA
comprised 20% to 30% of total Na+,K+-ATPasea isoform
mRNA, with a1 isoform mRNA comprising the rest.5 There
was considerably more a1 than d mRNA synthesis in
uninduced HL60 cells, and this difference persisted unchanged during induction of HL60 cells with DMSO:
These results implied that posttranscriptional mechanisms
may play an important role in the changes seen in a1 and,
more importantly, d mRNA expression after induction.
We now report studies that show the importance of mRNA
stability in the differential regulation of a1 and a3 isoform
mRNA during granulocytic differentiation of HL60 cells.
Moreover, we show that the human a3 isoform membrane
protein is significantly more labile than a1 in HL60 cells,
with its levels declining markedly by 10 hours of DMSO
induction. These findings suggest that the d isoform may
contribute to the timely changes in ion flux seen during
early granulocytic differentiation.
ORPHOLOGIC features of granulocytic differentiation of HL60 cells are evident within 2 to 3 days of
the addition of dimethyl sulfoxide (DMSO). One of the
immediate changes accompanying DMSO-induced granulocytic differentiation is a rapid reduction in bidirectional K+
flux, with the ouabain-sensitive component of IC+ influx
decreasing with a half-life of 11 hours and the rate constant
for K+ efflux decreasing with a half-life of 14 hours.’
However, the number of ouabain binding sites remains
stable for the first 24 hours of DMSO induction, after which
they decline with a half-life of approximately 3 days? The
functions of both the a1 and a3 isoforms of Na+,K+ATPase, a membrane-bound heterodimeric enzyme responsible for active maintenance of sodium and potassium
gradients across the plasma membrane, are ouabainDuring granulocytic differentiation of HMO
cells by DMSO, we have observed a dramatic decline of d
messenger RNA (mRNA) to undetectable levels, whereas
a1 mRNA declined by only 50%: The possibility that the
a3 isoform may participate in early granulocytic differentiation prompted us to further investigate the mechanism of
regulation of d gene expression in this setting.
The a1 and d subunits of Na+,K+-ATPase are two
members of the a subunit gene family:,’ the a genes code
for catalytic subunits of Na+,K+-ATPase. The three identified a subunit genes differ in their expression patterns in
different tissues and during development, both at mRNA
and protein levels.4.s-11a1 and d mRNA, but not a2,are
present in some human cells of hematopoietic origin, with
a3 mRNA distribution restricted to cells of granulocyte,
monocyte, and T-cell lineage.5 The d isoform mRNA and
protein are also found in tissues of neuromuscular ontogeny, such as brain and heart, and tend to decrease and/or
disappear during fetal rat development.4,sJ1 The a1 isoform, on the other hand, corresponds to the ubiquitous Na+
pump found in both adult and fetal rat tissues, although
more abundant in adult kidney, brain, and heart than its
fetal equivalent^.^,^ The prominence of the a3 isoform in
fetal or undifferentiated cells leads us to suspect that
changes in the d isoform participate in development
and/or early differentiation.
Our initial investigation into the regulation of the a3
Blood, Vol80, No 6 (September 15), 1992: pp 1559-1564
MATERIALS AND METHODS
Cell and cell culture. HL60 cells were grown in suspension in
RPMI 1640 medium with 10% fetal calf serum (FCS) and mainFrom the Departments of Obstetrics and Gynecology, Therapeutic
Radiology, and Intemal Medicine and Human Genetics, Yale University School of Medicine, New Haven, CT.
Submitted January 21,1992; accepted May 21, 1992.
Supported by the Reproductive Scientist Development Program
Grant No. 5-KI2-HD 00849 awarded by the National Instiiute of
Child Health and Human Development (NICHD) and American
College of Obstetrics and Gynecology, and the Yale University
Comprehensive Cancer Center Support Grant No. 5-P3O-CA16359-16
awarded by the National Cancer Institute (S.K C.),National Institutes
of Health (NIH) Grant No. CA-47292 and Bristol-Myers R100-063
(B.M.K.), and NIH Grant No. ROI-HL44430 (E.J.B.).
Address reprint requests to Setsuko K Chambers, MD, Departments
of Obstetricsand Gynecology, Yale University School of Medicine, Box
3333,333 Cedar St, Nav Haven, CT 06510.
The publication costs of this arzicle 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.
8 1992 by The American Society of Hematology.
0006-4971/92/8006-OOO1$3.00/0
1559
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
CHAMBERS ET AL
1560
tained at 37°C in a humidified atmosphere containing 5% CO2. All
media was supplemented with 2 mmol/L glutamine-1% penicillinstreptomycin. Inductions were performed by the addition of 1.25%
DMSO to cells at a concentration 1 x l@/mL, for the specified
time periods. When mRNA half-life was measured, actinomycin-D
(10 pg/mL) was added to cells at a concentration of 1 x 106/mL.
When protein half-life was measured, cycloheximide (10 pg/mL)
was added to cells at a concentration of 5 x l@/mL.
Isolation and analysis of total cellular RNA. HL60 cells were
harvested at various intervals after drug treatment. Total cellular
RNA was prepared from cells by the guanidium isothiocyanate/
CsCl gradient method.12 Total RNAs (30 pg/well) were separated
by 1% agarose/formaldehyde gel electrophoresis, transferred to
Gene Screen Plus (New England Nuclear, Boston, MA), and
hybridized to the following 3ZP-labeledcDNAs: the 2.2-kb EcoRI
fragment of human Na+,K+-ATPasea1 cDNAor the 2.0-kbEcoRI
fragment of human Na+,K+-ATPase a3 cDNA purified from M13
phage host? The same blots were then stripped and rehybridized to
the 1.0-kb Pst I-Xba fragment of human y-actin cDNA purified
from a pHF-1 plasmid host.13 Filters were then washed to high
stringency (final wash, 0 . 1 ~
SSC at 60°C twice each for 30
minutes) and dried, and transcripts were visualized by autoradiography. Relative signal intensity was determined by densitometric
analysis of all films, and all photographs of ethidium bromidestained gels by the Bio Image Visage 2000 system (Millipore,
Bedford, MA). Integrated optical intensity over the area of the
bands was calculated and then normalized to that of the 18s RNA
from the corresponding ethidium bromide-stained gel for Figs 1,3,
and 4, and to that of y-actin for Fig 2. Calculation of relative
mRNA half-life was derived from the ratio of transcript studied to
18sRNA or y-actin (relative transcript levels), at serial time points
during the actinomycin-D chase experiments. Our consistent
findings of a biphasic curve of (a1 and a3) transcripts during
actinomycin-D chase experiments have been described by othe r ~ .An
~ initial
~ - ~increase
~
during actinomycin-D chase followed by
terminal transcript decay of colony-stimulating factor-1 (CSF-1)
transcripts in fibroblasts exposed to tumor necrosis factor-a (TNFa),16 of granulocyte-CSF (G-CSF) transcripts in monocytes stimuof c-myc transcripts in fibroblasts
lated by lipop~lysaccharide,~~
exposed to cycl~heximide,’~
and of c-fos transcripts in monocytes
treated with 12-0-tetradecanoylphorbol-13-acetate
(TPA) or cycloheximide have been dem0n~trated.l~
It is possible that actinomycin-D may inhibit transcription of a labile mRNA regulatory
protein, resulting in a transient superinduction of the transcripts,
before terminal transcript decay. Each result was obtained from at
least three different experiments, and the results of mean transcript half-lives with standard errors are presented in Figs 3 and 4,
so as to provide validity to these difficult half-life measurements in
the face of ongoing differentiation and inhibition of new transcription by actinomycin-D. The ethidium bromide-stained gels are also
shown in Figs 1,3, and 4 to show equality of the load as well as the
lack of nonspecific effects, such as RNA cytotoxicity, for all time
points in Fig 1 (cytotoxicity from actinomycin-D was noted at the
8-hour time point in uninduced HL60 cells), and all time points
through 3 hours in Figs 3 and 4. The 4-hour time point in the latter
two figures shows signs of some degree of RNA breakdown of the
28s RNA from cytotoxicity of the combination of drugs. Ethidium
bromide and y-actin correlate with each other in Fig 2, as the
duration of actinomycin-D chase is 3.5 hours in uninduced HL60
cells. Thus, the results of hybridization with y-actin are shown
instead of the ethidium bromide-stained gel.
Isolation and analysis of membrane preparations of cell protein.
HL60 cells were harvested at various intervals after drug treatment. Membrane preparations were then performed on ice. Cell
pellets were suspended in reticulocyte standard buffer (RSB)
without Nonidet P-40 (10 mmol/L Tris, pH 7.4,lO mmol/L NaCI, 3
mmol/L MgCIz), with added proteinase inhibitors (1 pg/mL
aprotinin, 2 pg/mL leupeptin, 0.5 mmol/L phenyl-methyl-sulfonic
fluoride, and 10 pg/mL soybean trypsin inhibitor), then dounced
20 times. Nuclear pellets were isolated after centrifugation at 3,000
rpm for 5 minutes. Supernatants were then centrifuged at 14,000
rpm for 15 minutes to recover the membrane pellet. The final
supernatants comprised the cytoplasmic fraction. Samples were
taken up in 5 x sample buffer without dye (75 mmol/L Tris, pH 7,
10% vol/vol glycerol, 2% wtlvol sodium dodecyl sulfate [SDS], and
5% vol/vol 2-mercaptoethanol). Protein concentrations of each
sample was determined by the BioRad protein assay kit (BioRad,
Melville, NY). Solubilized membrane proteins were analyzed
either by direct application onto nitrocellulose filters using a slot
blot manifold (Schleicher and Schuell, Keene, NH) after dilution
of sample buffer SDS by phosphate-buffered saline (PBS), or by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (6.5% gel),
and electroblotted to Immobilon P. The filters were blocked by
immersion into “Blotto” blocking solution for a1 (15% wt/vol
nonfat dry milk and 5% wt/vol bovine serum albumin in PBS), or
into Tris-buffered saline blocking solution for a3 (30 mmol/L Tris,
pH 7.5, 150 mmol/L NaCI, 5% wt/vol nonfat dry milk, and 0.5%
Tween 20) at 4°C overnight. Afterwards, the filters were rocked in a
1:250 dilution of rabbit antirat a1 polyclonal IgG antibody 6408
(gift of R.W. Mercer, Washington University School of Medicine,
St Louis, MO) or a 1:lOO dilution of rabbit antirat a3 polyclonal
antibody (Upstate Biotechnology, Inc, Lake Placid, NY) in blocking solution overnight at 4°C. Unbound antibody was removed
from the filters by three 10-minute incubations in rinsing solution
(10 mmol/L Tris, pH 8,150 mmol/L NaCI, and 0.05% Tween 20).
This was followed by incubation of the filters in 12.5 pCi IlZ
Staph-protein A (ICN, Costa Mesa, CA) in blocking solution for 1
hour. Unbound Staph-protein A was removed using the same
washing technique. Autoradiography and densitometry were then
performed as described above.
RESULTS
mRNA stability in uninduced HL6Opromyelocytic leukemia
cells. a3 isoform mRNA of Na+,K+-ATPase comprised
20% to 30% of total a isoform mRNA in HL60 cells, with
a1 isoform mRNA comprising the rest? Baseline a1 mRNA
synthesis rate as measured by nuclear run-off analysis was
considerably greater than that of a3;these rates were not
found to change with DMSO induction? When we measured relative mRNA half-lives of a3 and a1 isoforms in
uninduced HL60 cells (Fig l), we found that a3 mRNA
(transcript half-life, 3 hours) was less stable than a1
(transcript half-life, 3.5 hours), resulting in a shift of the
mRNA decay curve during actinomycin-D chase, to the left.
y-Actin was a more stable transcript, with 57% remaining at
6 hours. When the actinomycin-D chase was extended to 8
hours, cytotoxicity precluded further measurements. Densitometric measurements of the results depicted in Fig 2
confirmed that the relative of a1 mRNA half-life was as
long as 3.5 hours in uninduced HL60 cells, with the finding
that 46% of a1 transcripts remained at this time point.
These findings suggest that the mRNA half-life of the a1
isoform was slightly greater than that of the a3 isoform in
uninduced HL60 cells. However, transcriptional, rather
than posttranscriptional, effects, remain largely responsible
for the baseline abundance of a1 over a3 isoform mRNAs.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1561
CHANGES IN NA,K-ATPASEGENE EXPRESSION
A
Y
0
0
II
It
II
(3m
II
II
c c c c c
Fig 1. mRNA half-lives of a1
and 03 W o r m s of Na+,K+-ATPose, and y-actln in uninduced
HL60 cells. (A) HL60 cells were
exposed to actinomycin-D (10
pglml.) over a &hour chase period. Total cellular RNA was analyzed as described in Materials
and Methods. (E) The levels of
a l , d,?actin, and the 18s RNA
of the ethidium bromidestained
gel were quantftated by densitometric scanning. Integrated intensity over the area of each
band was calculated and normalized to its 18s RNA control. Relative transcript levels were then
plotted versus time of actinomycin-D chase.
.
285-
-
0
-
.J
28s-
m
28s
18S-
i
P
Tiid-
mRNA srabiliry in DMSO-induced HL60 cells. We determined the mRNA half-lives of the a1 and a3 isoforms
during granulocytic differentiation. HL60 cells were treated
with DMSO for 16 hours, after which actinomycin-D was
added for various timc intervals. Longer exposures of HL60
cells to DMSO before measurement of mRNA half-lives
were not possible because of the cytotoxicity of the drug
combination. After 16 hours of DMSO induction, steadystate levels of a3 isoform mRNA declined to SO%, whereas
that of a1 still remained at greater than 90%: Relative a3
isoform mRNA half-life was shortened on DMSO induction to slightly more than 2 hours (Fig 3). At the 3-hour
time point, only 5% of a3 transcripts remained, whereas in
uninduced HL60 cells, 40% of a3 transcripts remained.
RNA cytotoxicity was not noted until 4 hours of actinomycin-D chase. Thus, the finding of a shortened a3 mRNA
half-life upon DMSO induction does not appear to be a
nonspecific effect. Moreover, Fig 3 represents the mean a3
o r ~ c u c H
r i m Y
C 9
H
C
I
C
n
0
H
II
II
C
C
C
I1
C
II
C
Fig 2 mRNA half-life of a1 hoformof Na+,K+ -ATPase in unlnducd
HL60 cdh. HUiO celh were exposed to actinomycin-D over a 3.5-hour
chase period. Serial samples for total cellular RNA were extracted
every 30 minutes after the first hour for analysis. The levels of 01
Isofom and Tactin were analyzed as in Materials and Methods.
Integrated intensity over each band was calculated and normalizedto
its y-actin control. Relative 01 transcript levels were then plotted
versus time of actinomycin-D chase.
mRNA half-life with standard error measurements of three
separate experiments. Thus, a3 isoform mRNA half-life
was shortened from 3 hours in uninduced HL60 cells to
little more than 2 hours after 16 hours of DMSO induction.
a1 isoform mRNA half-life was shortened to 3 hours
upon 16 hours of DMSO induction (Fig 4). Figure 4
represents the mean a1 mRNA half-life with standard error
measurements of three separate experiments. a1 transcript
levels at 4 hours of actinomycin-D chase in induced HL60
cells can be interpreted to have resulted from a combination of a1 mRNA decay and nonspecific effects due to RNA
cytotoxicity, first evident at 4 hours. Measurement of a1
mRNA decay at all time points before 4 hours of actinomycin-D chase appears to be valid and not secondary to
nonspecific effects. a1 transcripts decreased by 50% by 3
hours, while in uninduced HL60 cells,90% of a1 transcripts
remained at that time point of the actinomycin-D chase.
Thus, a1 isoform mRNA half-life was shortened from 3.5
hours in uninduced HL60 cells to 3 hours after 16 hours of
DMSO treatment.
a1 mRNA half-life remained longer than that of a3
mRNA both in uninduced and DMSO-treated HL60 cells,
with the difference between a1 and a3 isoform mRNA
half-lives 30 minutes in uninduced cells, and 1 hour in
induced cells. Both half-lives decreased after 16 hours of
DMSO induction: a3 by almost 1 hour, a1 by 30 minutes.
Taken together, these findings would explain why the
decrease in a1 steady-state levels after induction was more
gradual than that of a3, while always remaining greater
than that of a3. Presumably, although this is technically
impossible to show, a1 and a3 isoform mRNA become less
stable during continued differentiation. After 16 hours of
DMSO induction, steady-state levels of a1 remained at
greater than 90%. decreasing to 50% after 72 hours of
induction. On the other hand, after 16 hours of DMSO
induction, steady-state levels of a3 decreased to 50%. and
were undetectable by 24 hours of induction?
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
CHAMBERS ET AL
1562
A
205-
0
-
w
-
II
II
C C t : !
a3
Fig 3. mRNA half-llfe of a3 Mom of Na*,K*
-ATPase In DMSO-treated HLBO cells. (A) HLBO cells
were exposed t o 1.25% DMSO for 16 hours before
the addition of actinomycin-D (10 pg/mL) over a
Chour chase period. Total cellular RNA was analyzed
as described in Materials and Methods. (B) The levels
of 03 isoform mRNA were analyzed and quantitated
as described in Materials and Methods and in the
legend t o Fig 1. The level of 03 isoform mRNA was
normalized t o its respective 18s RNA control. Mean
(&EM) relative transcript levels were plotted versus
time of actinomycin-0 chase.
105-
Findings at the membrane pmtein level parallel that of
"A.
Studies were performed in uninduced HL60 cells
to determine a1 and a3 isoform levels in the uninduced
state. a3 protein was shown in the membrane fraction of
uninduced HL60 cells with minimal reactivity demonstrated to the nuclear fraction. The cytoplasmic fraction
and fetal kidney showed no a3 reactivity, as expected. Fetal
brain was the positive control for a3 protein (Western blot,
Fig 5A). a1 and a3 membrane proteins were shown in
human HL60 cells by cross-species reactivity of rabbit
antirat a1 or a3 antibodies to the human epitopes (slot blot,
Fig 5B). While the relative reactivity of the antisera makes
absolute quantification difficult, densitometric scanning of
baseline HMO cells (time = 0 in Fig 5B) showed that a3
isoform comprised 30% of the total a isoform found in
HL60 cells at the protein level, when corrected for protein
load, with a1 isoforms comprising the rest. This relative
difference of a1 and a3 isoforms at the protein level
reflected that seen at the steady-state mRNA level. Measurements of protein stability were then performed in the
presence of cycloheximide, an inhibitor of protein chain
elongation. Densitometric scanning showed that a3 isoform
protein half-life was approximately 9 hours, with no decay
of the a1 isoform membrane protein seen over 12 hours.
Flg 4. mRNA half-Me of a1 Mom of Na*,K*ATPase In DMSO-treated HLBO cells. (A) HUW) cella
were exposed t o 1.25% DMSO for 16 hours before
the addition of actinomycin-D (10 pg/mL) over a
4-hour chaw period. Total cellular RNA was analyzed
as described in Materials and Methods. (B) The levels
of a1 isoform mRNA were analyzed and quantitated
as described in Materials and Methods and in the
legend t o Fig 1. The level of ul isoform mRNA was
normalized to its respective 18s RNA control. Mean
(*SEM) relative transcript levels were plotted versus
time of actinomycin-D chase.
28s-
Although it may appear that there was a relative increase in
a1 protein expression by 9 and 12 hours of cycloheximide
exposure, densitometric analysis showed a 25% decrease in
peak optical density accompanied by only a 1.1-fold increase in integrated optical intensity over the whole band.
This can be explained by the samples spreading beyond the
confines of the slot blot manifold during loading of the
protein samplcs at 9 and 12 hours. There was no evidence of
cytotoxicity from cycloheximide, as the a1 isoform motility
and signal intensity did not change during the time course
on a Western blot (results not shown). This would suggest
that, in uninduced HL60 cells, membrane protein half-lives
of the a1 and a3 isofonns bear the same relationship to
each other that their mRNA half-lives do, and that they
appear to be equally translated, although direct measurements of translational efficiency were not performed.
During DMSO induction, a1 membrane protein remained stable over 70 hours, whereas a3 protein was
markedly decreased by the 10-hour time point (Fig 6).
While the time course of the protein findings was not
strictly in keeping with the time course of mRNA steadystate levels over a prolonged DMSO induction, it is clear
that a3 membrane protein is more labile than a1 on DMSO
induction.
al
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1583
CHANGES IN NA,K-ATPASEGENE EXPRESSION
HL60
HL60
2"-
a3
925lrDa-
B
a1
a3
T=O
T=3
T=6
T=9
T=l2
Fetal Kidney
Fetal Brain
Fig 5. Rotein half-lives of the a1 and a3 hoforms of Na+,K+ATPase In u n l n d d HL60 cells. (A) Westem blot of nuclear, membrane, and cytoplasmic proteins of uninduwd HUW) cells (150 pg/
well), incubatedwith rabbitantirator3poIyclonalantibody asdescribed
In Materials and Methods. Fetal kidney and brain membrane proteins
are included as controls. (E) Slot blot of membrane proteins of HL60
cells (100 pg/slot for a1 and 180 pglslot for a3). HL60 cells were
exposed to cycloheximide (10 pg/mL) over a 12-hour time period.
Cells were harvested at the indicated times, and membrane protein
isolated and analyzed as described in Materials and Methods.
DISCUSSION
mRNA decay is now recognized to be a major control
point in the regulation of gene expression.'* This study
confirms the importance of changes in mRNA stability for
regulation of Na+,K+-ATPasea1 and especially a3 isoform
mRNA expression during granulocytic differentiation of
HL60 cells. It has been reported that low-dose actinomycin-D treatment of HL60 cells inducesgranulocyticdifferentiation after 6 days.'" However, the actinomycin-D stock
solution loses its differentiative effect after several days,
despite proper storage.'" As all our experiments were
performed using the same concentrated stock solution, the
time period of the drug exposure was a maximum of 6
hours, and the data were reproducibly produced each time.
We do not believe our results were confounded by the
potential differentiative effectsof actinomycin-D. However,
measurement of mRNA half-lives during the process of
differentiation is challenging due to the cytotoxicity of the
combination of drugs. As we confined our analysis of
mRNA decay to time points before evidence of RNA
cytotoxicity, and the data are reproducible, we feel that the
results of this study are consistent with the hypothesis that
changes in mRNA half-life of both the a1 and a3 isoforms
of Na+,K+-ATPasedirectly govern the ultimate expression
of both genes during DMSO induction. Posttranscriptional
controls have been shown to be important in the regulation
of CSF-I and c-fos mRNA during macrophage differentiation of human monocytes by phorbol ester treatment,I4aM
and of c - w mRNA during monocytic differentiation of
HL60 cells.21A conserved AU-rich sequence from the 3'
untranslated region of several mRNAs, including that of
c-fos, has been implicated in conferring mRNA instability.14.2221This AU-rich signal has been proposed to function
as a recognition site for RNAses, or a gene product that
causes destabilization by interaction with the poly A tail
and its associated ribonucleoprotein complexes.'*Examination of the published human a3 isoform gene sequence
shows no such consensus sequence. However, a noncanonic
polyadenylation site AATATA is located IS to 17 nucleotides upstream from the poly A attachment site." This
sequence has been observed in a mutant with a markedly
decreased ability to polyadenylate mRNA."
Our finding that a1 and d membrane protein levels and
half-lives parallel our data at the mRNA level in uninduced
HL60 cells lends support to the notion that these isoforms
are equally translated under the conditions of our study. A
difference in translational efficiency has already been observed between @ and a isoform mRNAs,R while a difference in translational regulation of a3 and a1 mRNA had
been suspected? Rapid potassium fluxes have been shown
to occur during early granulocytic differentiation,' but it is
not known whether they have a causative role in differentiation. Were the a isoforms causally important to granulocytic differentiation, then the rapid ion fluxes that occur
within the first 12 hours of addition of DMSO'**would have
to be explained in light of the a1 and a3 mRNA half-lives
and the time course of a1 and a3 membrane protein
turnover after addition of DMSO. The transport capacity of
the a3 isoform relative to a1 is unknown. If the a3 isoform
had a higher transport capacity than a1 in HL60 cells, then
a1
a3
T=O
T=lO
T=70
Fetal Kidney
Fetal Brain
Fig 8. a1 and a3 h o f m s of Na+,K+ -ATPase In DMSO-lndd
HL60 cells. Slot blot of membrane proteins of HUW)cell5 (100 pg/slot
fora1 and 180 pg/slotford). Fetal kidney and fetal brain protoinsam
Included as COntrOlS. HL60 cells were exposed to 1.25% DMSOfor the
Indicated time periods.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
CHAMBERS ET AL
1564
the decrease in a3 protein expression that is observed in the
first 10 hours of DMSO induction may well account for the
changes in K+ influx that occur during early granulocytic
differentiation. If the activity of the a isoforms was roughly
equal, then alteration of posttranslational activity of the a3
isoform could lead to the early decline in Na+, K+-ATPase
activity observed during granulocytic differentiation. However, the number of ouabain binding sites remains stable
during the first 24 hours of DMSO induction and then
declines gradually with a half-life of approximately 3 days.*
Because a1 is the predominant ouabain-sensitive isoform,
the number of ouabain binding sites (as contrasted to
activity) may well correlate with the time course of decay of
the a1 isoform mRNA and protein. Although greater than
90% of steady-state a1 mRNA is present 16 hours after
initiation of DMSO induction, it declines to approximately
50% by 72 hours of induction. The a1 protein level still
remains stable at that time point. We theorize that upon
prolonged granulocytic differentiation by DMSO, the a1
protein levels begin to decrease such that altered gene
expression of a1 could well be responsible for the decline in
ouabain binding sites.
REFERENCES
1. Gargus JJ, Adelberg EA, Slayman CW: Rapid changes in
bidirectional K+ fluxes preceding DMSO-induced granulocytic
differentiation of HL-60 human leukemic cells. J Cell Physiol
120233,1984
2. Ladoux A, Geny B, Marrec N, Abita JP: [3H] Ouabain
binding and %rubidium uptake during the dimethyl sulfoxideinduced differentiation of human promyelocytic leukemia HL-60
cells. FEBS Lett 176467, 1984
3. Hara Y, Nikamoto A, Kojima T, Matsumoto A, Nakao M:
Expression of sodium pump activities in BALB/c3T3 cells transfected with cDNA encoding a3-subunits of rat brain Na+,K+ATPase. FEBS Lett 238:27,1988
4. Shyjan AW, Levenson R: Antisera specific for the a l , a2, a3
and b subunits of the Na,K-ATPase: Differential expression of a
and b subunits in rat tissue membranes. Biochemistry 28:4531,1989
5. Gilmore-Hebert M, Schneider JW,Greene AL, Berliner N,
Stolle CA, Lomax K, Mercer RW, Benz ET Jr: Expression of
multiple Na+,K+-adenosine triphosphatase isoform genes in human hematopoietic cells. J Clin Invest 84:387,1989
6. Shull MM, Lingrel JB: Multiple genes encode the human
Na+,K+-ATPase catalytic subunit. Proc Natl Acad Sci USA
84:4039,1987
7. Yang-Feng TL, Schneider JW, Lindgren V, Shull MM, Benz
El Jr, Lingrel JB, Francke U: Chromosomal localization of human
Na+,K+-ATPase a- and b-subunit genes. Genomics 2:128,1988
8. Schneider JW, Mercer RW, Gilmore-Herbert M, Utset MF,
Lai C, Greene A, Benz EJ Jr: Tissue specificity, localization in
brain, and cell-free translation of mRNA encoding the A3 isoform
of Na+-ATPase. Proc Natl Acad Sci USA 85:284,1988
9. Sweadner KJ: Isozymes of the Na+/K+/ATPase. Biochim
Biophys Acta 988:185,1989
10. Urayama 0, Shutt H, Sweadner KJ: Identification of three
isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat
brain. J Biol Chem 264:8271,1989
11. Young RM, Lingrel JB: Tissue distribution of mRNAs
encoding the a isoforms and b subunit of rat Na+,K+-ATPase.
Biochem Biophys Res Commun 14552,1987
12. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ:
Isolation of biologically active ribonucleic acid from sources
enriched in ribonuclease. Biochemistry 185294,1979
13. Gunning P, Ponte P, Okayama H, Engel J, Blau H, Kedes L:
Isolation and characterization of full-length cDNA clones for
human a, p, and y actin mRNAs: Skeletal but not cytoplasmic
actins have an amino-terminal cysteine that is subsequently removed. Mol Cell Biol3:787,1983
14. Sariban E, Luebbers R, Kufe D: Transcriptional and posttranscriptional control of c-fos gene expression in human monocytes. Mol Cell Biol8:340,1988
15. Koeffler H, Gasson J, Tobler A Transcriptional and posttranscriptional modulation of myeloid colony-stimulating factor expression by tumor necrosis factor and other agents. Mol Cell Biol
8:3432,1988
16. Mantovani L, Henschler R, Brach M, Mertelsmann R,
Hernnann F: Regulation of gene expression of macrophage-colony
stimulating factor in human fibroblasts by the acute phase response
mediators interleukin (IL)-lp, tumor necrosis factor-u and IL-6.
FEBS Lett 28097,1991
17. Ernst T, Ritchie A, Demetri G, Griffin J: Regulation of
granulocyte- and monocyte-colony stimulating factor mRNA levels
in human blood monocytes is mediated primarily at a posttranscriptional level. J Biol Chem 2645700,1989
18. Brawerman G: mRNA decay: Finding the right targets. Cell
57:9,1989
19. Collins S, Bodner A, Ting R, Gallo R: Induction of morphological and functional differentiation of human promyelocytic
leukemia cells (HL-60) by compounds which induce differentiation
of murine leukemia cells. Int J Cancer 25:213,1980
20. Horiguchi J, Sariban E, Kufe D: Transcriptional and posttranscriptional regulation of CSF-1 gene expression in human monocytes. Mol Cell Biol8:3951,1988
21. Shaw G, Kamen R: A conserved AU sequence from the 3‘
untranslated region of GM-CSF mRNA mediates selective mRNA
degradation. Cell 46:659,1986
22. Weber B, Horiguchi J, Luebbers R, Sherman M, Kufe D:
Posttranscriptional stabilization of c-fms mRNA by a labile protein
during human monocytic differentiation. Mol Cell Biol9:769,1989
23. Caput D, Beutler B, Hartog K, Thayer R, Brown-Shimer S,
Cerami A: Identification of a common nucleotide sequence in the
3”Itranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci USA 83:1670,1986
24. Ovchinnikov YA, Monastyrskaya GS, Broude NE, Ushkaryov YA, Melkow AM, Smirnow YV, Malychev IV, Allikmets
RL, Kostina MB, Dulubova IE, Kiyatkin NI, Grishin AV, Modyanov NN, Sverdlov ED: Family of human Na+, K+-ATPase genes.
FEBS Lett 233237,1988
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1992 80: 1559-1564
Changes in Na,K-ATPase gene expression during granulocytic
differentiation of HL60 cells
SK Chambers, M Gilmore-Hebert, BM Kacinski and EJ Jr Benz
Updated information and services can be found at:
http://www.bloodjournal.org/content/80/6/1559.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.