From the
We have studied the role of intracellular calcium sequestration
on human immunodeficiency virus (HIV) production by latently infected
T-lymphocytic cells. Inhibition of the sarco-endoplasmic reticulum-type
calcium transport ATPases by thapsigargin or cyclopiazonic acid induced
activation of HIV production in the CEM-derived ACH-2 cells. An
approximately 50% depletion of the thapsigargin-sensitive calcium pools
as measured fluorimetrically of Indo-loaded cells fully activated virus
production. Viral activation was manifest by increases in soluble viral
core p24 production, increases in cellular immunofluorescent staining
for viral antigens, and increased viral transcription as measured by
HIV long terminal repeat-directed expression of the chloramphenicol
acetyltransferase reporter gene. Virus induction could be blocked in a
dose-dependent manner by the calcium channel blocker econazole. Virus
production by the Jurkat-derived HIV-1-inducible J1.1 cells was not
significantly stimulated by thapsigargin. These data indicate that
intracellular calcium pool function is involved in the control of the
transcription of proviral HIV in a cell type-specific manner within the
T-lymphoid lineage and that ACH-2 cells represent a useful model for
the study of calcium dependent activation of the transcription of
proviral HIV.
Intracellular calcium storage organelles play a key role in
calcium signaling. These organelles sequester calcium ions from the
cytoplasm through the action of the sarco-endoplasmic reticulum-type
calcium transport ATPases (SERCA)
Calcium homeostasis plays a
key role in lymphocyte activation, and the resulting modulation of
transcription factor activities could contribute to the regulation of
HIV gene expression
(26, 27, 28, 29, 30, 31, 32) .
Recent studies of HIV pathogenesis have shown
(33, 34, 35, 36, 37) that even
during the asymptomatic phase of HIV disease, a significant fraction of
lymphocytes found in lymph nodes carries proviral HIV, whereas only a
small subfraction of these cells actively expresses virus at any given
time. Thus the study of signaling systems involved in the regulation of
HIV expression in latently infected cells may contribute to our
understanding of the natural course of HIV-induced disease.
In the
present work the effect of inhibition of the SERCA-type calcium pumps
on the expression of proviral HIV in latently infected lymphocytic
cells was studied. CEM-derived ACH-2 cells were treated with inhibitors
of the SERCA-type calcium pumps and virus production was measured by
supernatant viral core p24 antigen production and by immunofluorescent
staining for HIV antigens. HIV LTR-driven transcription was studied
using an LTR-CAT-transfected CEM-derived cell line. In parallel,
intracellular calcium pool function was assessed by fluorimetry in
Indo-loaded cells. Our results indicate that intracellular calcium
pools associated with SERCA-type calcium pumps can exert an important
control of the transcription of proviral HIV in latently infected
cells.
Cell counts and
viabilities were determined using a hemocytometer and the trypan blue
exclusion test and by an MTT conversion assay as described previously
(40, 41) . In several experiments these two techniques
were performed simultaneously on the same cells and gave closely
comparable results.
Data presented in this work represent the
results of at least three independent experiments.
HIV-specific transcription was assessed in a CEM-derived cell line
expressing the chloramphenicol acetyltransferase gene driven by the
HIV-1 LTR. The pU3R-III CAT plasmid was modified by insertion of the
hygromycin-B-phosphotransferase gene
(44) . Stable introduction
of this expression vector into CEM-SS cells was accomplished by
electroporation
(44) . Selection of stable transfectants was
performed by culturing the cells for 5 weeks in the above medium
supplemented with 400 units/ml hygromycin-B (Calbiochem). The resulting
line, BWV-899, showed low basal levels of CAT expression. CAT assays
following thapsigargin treatment of the cells were performed by
standard methods
(45) . Chloramphenicol acetylation was
quantitated using an ImageQuant PhosphorImager (Molecular Dynamics,
Sunnyvale, CA).
As shown in this report, the inhibition of intracellular
calcium sequestration by thapsigargin, cyclopiazonic acid, or tBHQ,
three structurally unrelated inhibitors of the SERCA-type calcium
pumps, fully activated HIV production by the latently infected ACH-2
T-cell line. Over a 5-day period of treatment more than 95% of the
cells became positive for HIV antigen production as opposed to less
than 5% prior to treatment. This stimulation occurred in the range of
inhibitor concentrations previously shown to inhibit the SERCA calcium
pumps in situ in membrane preparations made from uninfected
cells
(50) . Thapsigargin induction of HIV synthesis could be
blocked by the calcium channel blocker econazole. This indicates that
calcium influx into the cells through econazole-sensitive calcium
channels, present in lymphoid cells
(51, 52) , opened by
the depletion of thapsigargin-sensitive calcium pools is involved in
virus induction in ACH-2 cells.
Intracellular calcium pump inhibitor
treatment induced the synthesis and release of soluble viral antigen
into the culture supernatant and increased virus antigen expression
within the cell, as measured by p24 ELISA and immunofluorescent
staining, respectively, indicating de novo virus synthesis. On
the transcriptional level, assuming that thapsigargin treatment did not
modify CAT mRNA or protein turnover, it can be concluded that
thapsigargin treatment of the CEM-SS-derived BWV-899 cells increased
the transcription of the chloramphenicol acetyltransferase reporter
gene directed by an HIV LTR. A 50% depletion of the
thapsigargin-mobilizable calcium pool was sufficient for the maximal
stimulation of HIV production. Such a treatment, similar to that seen
on uninfected cells
(3) , resulted in an enhanced calcium influx
across plasma membrane calcium channels opened as a consequence of pool
depletion.
Thapsigargin action appeared to be specific to ACH-2
T-cells. Virus production of the Jurkat-derived latently infected
T-cell line J1.1 did not change significantly following exposure to
this drug, even though growth arrest was observed. This is in agreement
with other reported observations, indicating that HIV production by
J1.1 cells is controlled by distinct mechanisms from those acting in
ACH-2 cells
(53) . Uninduced ACH-2 cells contain significantly
higher levels of the viral rev protein than J1.1 cells
(53) . As this protein is involved in the transition from latent
to productive infection, it can be hypothesized that intracellular
calcium pool depletion by SERCA inhibitors sets in motion-activating
mechanisms that require a certain level of preexisting rev.
Moreover, HIV synthesis is regulated by a complex set of cellular
transcription factors
(26, 27, 28, 29, 30, 31, 32) .
Differences in the expression of transcription factors that are
activated upon calcium pool depletion may also account for the
different responsiveness of ACH-2 and J1.1 cells. Thapsigargin may be
useful in future studies to elucidate the differences between the HIV
control mechanisms of these two model T-cell types.
The mobilization
of calcium from intracellular storage pools is a key component of
lymphocyte activation. Since the activation of latently HIV-infected
lymphocytes leads to virus production and cytopathy, the study of the
calcium-dependent signaling systems involved in the regulation of HIV
expression may contribute to our understanding of the natural course of
HIV-induced disease.
SERCA inhibitors offer a unique tool to study
the mechanisms by which intracellular calcium pools and calcium entry
from the extracellular medium interact in regulating HIV expression,
as, unlike the action of calcium ionophores, the selective depletion of
intracellular calcium pools activates the cells' own calcium
entry mechanisms. In this work this process was exemplified by the
inhibition of thapsigargin-induced HIV synthesis by the imidazole-type
calcium channel blocker econazole. Therefore the data and the new
experimental model presented in this paper may be useful in elucidating
the effects of calcium homeostasis on the virus production of latently
infected cells.
HIV is known to induce several perturbations of the
calcium-dependent cellular signal transduction system in the infected
cell. Elevated resting cytoplasmic calcium concentrations and increased
inositol trisphosphate levels have been observed
(54, 55) . Such changes may activate calcium release
from intracellular pools
(1, 2, 22, 56, 57, 58, 59, 60, 61, 62, 63) and, as shown in our work, could thereby stimulate virus
production. The activation of calcium signaling by HIV therefore may
represent a positive feedback mechanism in the regulation of virus
replication and may represent a new target for the pharmacological
modulation of HIV expression.
This work is
dedicated to the memory of Lugosi Antalné.
We are indebted to Drs. J. E. Groopman, D. Zhang, J.
Enouf, . Enyedi, T. Kovcs, and B. Sarkadi for helpful discussions.
ACH-2 and J1.1 cells were obtained through the AIDS Research and
Reference Program (Division of AIDS, NIAID, National Institutes of
Health) contributed by Dr. T. Folks. The excellent technical assistance
of H. Chhay and C. Falcione is greatly appreciated.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
and release
calcium into the cytoplasm via receptor operated calcium channels (see
Refs. 1-5 and references therein). The depletion of intracellular
calcium pools can be specifically achieved by inhibition of the
SERCA-type calcium pumps using agents such as thapsigargin (Tg),
cyclopiazonic acid, or di- tert-butyl benzohydroquinone (tBHQ,
Refs. 6-13). These agents induce leakage of stored calcium into
the cytosol, mimicking second messenger-induced calcium mobilization.
The initial calcium release event sets in motion several distinct
cellular signaling systems. Signals coupled to intracellular calcium
pool depletion result in calcium influx into the cell
(2, 14, 15, 16, 17, 18, 19, 20, 21) ,
and the changes in cytosolic calcium levels modulate calcium and
calmodulin-regulated processes
(2, 19, 20, 21, 22) . The use of
SERCA inhibitors has already led to the observation that mobilization
of intracellular calcium pools can generate signals that modulate cell
activation and differentiation
(3, 6, 23) ,
adaptation to altered metabolic conditions
(24) , and expression
of the c- fos, c- jun, and c- myb transcription
factor genes
(6, 25) .
Chemicals
Cadmium chloride, caffeine, acetyl
coenzyme-A, phorbol myristate acetate, ionomycin, fetal calf serum,
hydrocortisone hemisuccinate, econazole, and Indo 1-AM were obtained
from Sigma. TNF was obtained from Genzyme, Cambridge, MA.
Thapsigargin, cyclopiazonic acid, and
2,5-di- tert-butyl-1,4-benzohydroquinone were obtained from
Biomol Inc., Plymouth Meeting, PA.
Cell Culture and Inhibitor Treatments
The
T-lymphoblastoid CEM-SS cells, as well as the chronically HIV-1
infected ACH-2 and J1.1 cells
(38, 39) , were obtained
from the AIDS Research and Reference Reagent Program, National
Institutes of Health and were grown in RPMI 1640 medium (Mediatech
Inc., Washington, D. C.) supplemented with 10% heat-inactivated fetal
calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2
mM glutamine (Mediatech). The cells were harvested in the
exponential phase of growth, then centrifuged at 1000 g for 10 min. Cell pellets were washed twice with 45 ml of complete
medium by centrifugation, as above, and resuspended in complete medium
at a density of 1
10
cells/ml. Thapsigargin,
cyclopiazonic acid, or tBHQ were added to the cells from stock
solutions of 5 mM, 10 mM, and 0.5 M in
Me
SO, or ethanol, respectively, and the cells were further
cultured in a humidified incubator at 37 °C and 5% CO
.
The concentration of the Me
SO and ethanol diluents was less
than 0.1% in the experiments, was included in appropriate control
experiments, and did not interfere with the assays.
Calcium Flux Measurements
Cells were harvested by
centrifugation and resuspended in complete medium at a density of 2
10
cells/ml and incubated with 1 µM Indo 1-AM for 30 min at 37 °C. The cells were then
centrifuged, and the cell pellet was resuspended in 45 ml of complete
medium and centrifuged as above. The cells were thereafter resuspended
in complete medium at a density of 2
10
cells/ml
and stored at room temperature. 3-ml cell suspensions were mixed with
10 ml of Tyrode-HEPES solution
(3) , centrifuged, and the cells
were resuspended in 3 ml of Tyrode-HEPES. Cytoplasmic calcium
concentrations were recorded as described previously
(3) using
a Perkin-Elmer LS-5B spectrofluorimeter equipped with a
thermostatically controlled (37 °C) cuvette holder and stirring
capability (C. N. Wood & Co, Longhorn, PA). Free cytoplasmic
calcium concentration was calculated as described previously
(3, 42) .
Virus Quantitation
HIV production was assessed by
quantitating core p24 concentration in culture supernatants using the
DuPont HIV-1 antigen capture ELISA system (DuPont), according to the
manufacturer's instructions. Samples were assayed in duplicate.
In this work we tested several lots of ACH-2 cells of different passage
history and obtained essentially similar results. A slight variation in
the basal level of core p24 production (ranging from 150 to 700
pg/10cells/day), however, was observed. Indirect
immunofluorescence staining of methanol fixed cells was performed using
human HIV-positive serum and anti-human IgG-fluorescein isothiocyanate
conjugate (Tago, Burlingame, CA) as described previously
(43) .
Concentration-dependent Stimulation of HIV Production
by Calcium Pump Inhibitors
Thapsigargin (Fig. 1 A)
as well as cyclopiazonic acid (Fig. 1 B), two
structurally unrelated inhibitors of the SERCA-type calcium pumps,
induced a concentration-dependent stimulation of virus production of
ACH-2 cells. The effect occurred in the low nanomolar range for
thapsigargin and in the low micromolar range for cyclopiazonic acid.
The proliferation of the cells was also inhibited by these agents, as
detected by MTT conversion. Similar results on cell growth have been
observed previously with uninfected cells
(46, 47, 48) . The magnitude of the stimulation
of HIV production by the cells was comparable with that obtained using
maximally effective TNF or phorbol myristate acetate treatment
(Fig. 1, C and D).
Figure 1:
Concentration-dependent activation by
calcium pump inhibitors of HIV production of ACH-2 T-cells. 10cells/ml were incubated with various concentrations of
thapsigargin ( A), cyclopiazonic acid ( B), TNF
( C), and phorbol myristate acetate ( D). The
supernatant viral p24 antigen concentrations ( squares) and the
viable cell densities ( circles) were determined on day 5 of
culture using ELISA and MTT conversion,
respectively.
In the cases above,
growth arrest accompanied virus induction. Similar effects ( i.e. growth arrest and augmentation of HIV production) were obtained
with tBHQ and the calcium ionophore ionomycin in the concentration
ranges of 6.25-50 and 0.03-2 µM, respectively.
On the other hand, hydrocortisone (0.156-10 µM),
caffeine (0.15-5 mM), and cadmium ions (4-250
µM) blocked the proliferation of ACH-2 cells in a
concentration-dependent manner without stimulating HIV production
(results not shown).
Time Course of HIV Production
As shown on
Fig. 2A, thapsigargin treatment of ACH-2 cells induced
virus production within 48 h and sustained stimulation of virus
production was observed during a 5-day period. This resulted in an
approximately 70-fold enhancement of HIV p24 production in treated
cultures, as compared with controls.
Figure 2:
Time course of thapsigargin action on HIV
production and growth of ACH-2 cells. A, the cells were
exposed to 12.5 nM thapsigargin ( circles) or diluent
( squares) at initial cell densities of 10cells/ml, and supernatant core p24 levels were measured daily for
5 days. B, growth of ACH-2 cells in the absence
( squares) and in the presence ( circles) of 12.5
nM thapsigargin. Thapsigargin treatment resulted in growth
arrest. C, cell growth and viability of control
( squares) and 12.5 nM thapsigargin-treated
( circles) ACH-2 cells, measured by MTT conversion. D,
viability of control ( squares) and thapsigargin-treated
( circles) ACH-2 cells, as determined by trypan blue exclusion.
E, inhibition of thapsigargin-induced HIV synthesis by
econazole. Cells were exposed to various concentrations of econazole
and treated with 2.7 nM thapsigargin ( squares). As a
control, cells were exposed to econazole in the absence of thapsigargin
( triangles). Relative virus production is expressed as the
ratio of supernatant p24 concentrations (pg/ml) and the MTT
metabolizing activity (milli-optical density) of the cells at day 4
relative to control ( i.e. with thapsigargin without
econazole).
Thapsigargin treatment of these
cells resulted in an arrest of cell growth, as measured by cell counts
(Fig. 2 B) and by MTT conversion
(Fig. 2 C), without significant loss of viability
(Fig. 2 D). When ACH-2 cells were treated with 2.7 nM thapsigargin in the presence of various concentrations of the
calcium channel blocker econazole (Fig. 2 E) for 4 days,
a dose-dependent inhibition of thapsigargin-induced HIV production
could be obtained in the low micromolar econazole concentration range.
Econazole alone did not modify viability or virus production of ACH-2
cells.
Immunofluorescence Analysis of HIV Expression
When
HIV production by ACH-2 cells was evaluated by indirect
immunofluorescence, less than 5% of the cells stained positive at base
line (Fig. 3 A). After 12.5 nM thapsigargin
treatment for 5 days, more than 95% of the cells became positive for
HIV antigens (Fig. 3 B). TNF treatment (1000
units/ml, 5 days) served as a positive control in these studies
(39) and fully activated HIV production by ACH-2 cells
(Fig. 3 C).
Figure 3:
Immunofluorescent staining of HIV antigen
production of latently infected cells. A, nonstimulated ACH-2
cells; B, ACH-2 cells stimulated for 5 days with 12.5 nM thapsigargin. C, ACH-2 cells after 5 days treatment with
10units/ml TNF
. D, chloramphenicol
acetyltransferase activity of CEM-SS cells carrying an HIV LTR-CAT
construct treated by thapsigargin for 4 days. CAT activity is
stimulated by thapsigargin in a concentration-dependent manner in the
nanomolar range.
CAT Assays
In order to study the effect of
inhibition of SERCA-type calcium pumps on HIV transcription in a
cellular context similar to ACH-2 cells, CEM-SS were transfected with a
plasmid carrying an HIV-LTR CAT construct. As shown on
Fig. 3D, 4 days of treatment with thapsigargin in the
nanomolar range resulted in a concentration-dependent enhancement of
CAT activity. This enhancement, up to 8-fold, indicates that inhibition
of SERCA-type calcium pumps modulates the transcription of HIV-1
directed by the viral LTR.
Calcium Flux Determinations
In order to correlate
the extent of intracellular calcium pump inhibition with the degree of
stimulation of HIV production, ACH-2 cells were exposed to thapsigargin
and studied fluorimetrically using Indo-1. The inhibitor concentration
of 12.5 nM used in these experiments induced a complete
activation of HIV production (see Fig. 1). Although thapsigargin
treatment of ACH-2 cells induced an acute rise in cytoplasmic calcium
levels, the resting cytoplasmic calcium concentration of the cells
returned to that of nontreated cells after an overnight incubation in
the presence of the inhibitor (Fig. 4 A). Similar results
have been reported previously for uninfected lymphoid cells
(46, 48, 49) .
Figure 4:
Calcium flux measurements in control
( Tg) and previously thapsigargin-treated
( +Tg) ACH-2 cells. A, at day 1 following 12.5
nM thapsigargin treatment the cells were treated with a
second, 4 µM dose of thapsigargin in the absence of
extracellular calcium. After the completion of the calcium release from
internal pools, 0.5 mM calcium was added to the medium, and
calcium influx was observed. B, thapsigargin-treated cells
were loaded by Indo in calcium-free medium, and by consecutive
additions of calcium and EGTA, the cells were placed in 0.5 mM calcium-containing and calcium-free medium. The
thapsigargin-treated cells repeatedly exhibited an initial rise in the
cytoplasmic calcium concentration when placed in calcium containing
medium.
The addition of a second,
higher dose of thapsigargin (4 µM) revealed that the
internal calcium pools were partially depleted, as the second
thapsigargin treatment resulted in an approximate 50% diminished
calcium mobilization, as compared with untreated cells
(Fig. 4 A). The initial thapsigargin treatment resulted
in transient rises of the cytosolic calcium concentration when the
cells were placed in calcium containing medium
(Fig. 4 B), a phenomenon not seen in control cells.
Similar results were obtained after 5 days of continuous thapsigargin
treatment (results not shown). A partial depletion of the
thapsigargin-sensitive calcium pools was therefore sufficient to fully
activate HIV production.
, tumor
necrosis factor-
; CAT, chloramphenicol acetyltransferase; MTT,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;
ELISA, enzyme-linked immunosorbent assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.