From the
A tetracycline-sensitive inducible expression system was used to
regulate the expression of neurotransmitter receptor genes in two
mammalian cell lines. The dopamine D
The ability to tightly control the transcription of heterologous
genes in transfected cells, or in complex systems such as transgenic
mice, is advantageous for a variety of studies. For example, inducible
expression systems provide a powerful tool for kinetic studies on the
synthesis and the processing of particular gene products. Inducible
expression systems are also useful for studies that seek to correlate
the level of gene expression with specific functional outcomes or for
investigations on the role of genes at defined developmental stages.
Furthermore, the ability to induce substantial and short-term increases
in gene expression may be essential for studies of stably transfected
cells that carry a heterologous gene encoding a protein whose
expression or activation is toxic to the cell.
A limitation of many
inducible eukaryotic promotor systems that respond to either metal ions
(Mayo et al., 1982), heat shock (Nouer, 1991), or hormones
(Lee et al., 1988) is their incomplete suppression of
transcription in the inactive state. Recently, however, a sensitive
inducible system has been described that is based on the
tetracycline-responsive transcriptional regulatory element of
Escherichia coli (tetO) and a bacterial
tet-repressor protein that is fused to the carboxyl-terminal
domain of the virion protein VP16 of the herpes simplex virus (Gossen
and Bujard, 1992). This hybrid protein acts as a transactivator to
stimulate transcription from minimal promotor sequences that are
positioned downstream of the tet-operator sequence. In
transfected HeLa cells, transcription from such a promotor was almost
completely suppressed when low concentrations of tetracycline were
present in the culture medium. In turn, very high levels of expression
of a reporter gene could be induced by the removal of tetracycline,
resulting in an induction of up to 5 orders of magnitude (Gossen and
Bujard, 1992).
It is often useful for structure-function studies of
neurotransmitter receptors to express these receptors in mammalian
cells. In some cases, however, obtaining high level stable expression
has proven difficult. For example, previous studies have failed to
obtain high levels of constitutive dopamine D
In this paper, we describe results from clonal mammalian cells that
stably express either the dopamine D
A second plasmid,
pTET-Spl (kindly provided by David Schatz, Yale University, New Haven,
CT), was used to clone the human cDNA encoding the dopamine D
HEK 293 cells were grown in
Eagle's minimum essential medium with Earle's salts that
was supplemented with 10% fetal bovine serum. Cells were transfected
using LipofectACE (Life Technologies, Inc.). Briefly, serum-free
Eagle's minimum essential medium with Earle's salts
containing 1% LipofectACE, pTET-GluR6, and pTA-N was added for 3 h to
100 mm plates of HEK 293 cells (
After
electrophoretic separation, proteins were transferred onto Immobilon
polyvinylidene difluoride membranes (Millipore) by electroblotting.
Blots were blocked in 10% newborn calf serum (Life Technologies, Inc.),
1
Cells were voltage-clamped at -80 mV, and inward currents were
evoked by local perifusion of the cells (via a wide mouthed glass
pipette) with extracellular solution containing 10 µM
kainate (a concentration about 25-fold higher than the apparent
EC
Currents were recorded with an
EPC9 amplifier that was controlled by software (Pulse, Instrutech.) run
on a Macintosh Quadra 800 computer. Analog signals were low
pass-filtered at 10 kHz (Bessel-type, 3 decibel) and were stored on
videotape with a VR 10b digital data recorder (Instrutech) and a VCR at
a sampling rate of 94 kHz. To measure kainate-evoked currents, signals
were replayed from tape and digitized at 10 kHz. The steady-state
amplitude of kainate-evoked currents was obtained from the digitized
records. To account for differences in cell size, the results were
expressed as current densities (in pA/pF) by dividing the amplitude of
the whole cell current by the cell capacitance. The cell capacitance
was taken as the C
To compare our GluR6 results
in HEK 293 cells more directly with the D
We have tested the efficacy of a tetracycline-responsive
promotor in regulating the transcription of the dopamine D
It seems likely that the noted
differences are related to differences between the two cell lines.
There were clear cell-specific differences in the expression of the
hybrid transactivator in the induced state; this expression was very
high in HEK 293 cells and low in GH3 cells. The results also indicate
that the high levels of GluR6 expression in noninduced HEK 293 cells
are not solely a function of the cis-acting elements of the
tetO/minimum promoter, because expression of the hybrid
transactivator in HEK 293 cells was tightly regulated by tetracycline.
One plausible explanation for the different levels of constitutive
expression in the two cell lines is that HEK 293 cells, but not GH3
cells, express trans-acting factors that can stimulate
transcription from the pTET-Spl plasmid in a tetracycline-insensitive
manner. Although such potential transactivators could be endogenous to
HEK 293 cells, it is also possible that they were introduced upon
transformation. In contrast to GH3 cells, the HEK 293 cell line was
established via adenovirus type 5 transformation (Graham et
al., 1977). The transforming region of adenovirus type 5 encodes
two proteins, E1A and E1B, which are necessary for complete
transformation (Van der Elsen et al., 1982, 1983). Northern
blot analysis of RNA extracted from nontransfected HEK 293 cells, as
well as from our noninduced or induced GluR6-transfected cells,
revealed expression of mRNA encoding the E1A (a 13 S mRNA species) and
E1B (the 22 S alternatively spliced mRNA) polypeptides (not shown). As
expected, we did not detect these mRNAs in GH3 cells. Although little
is known about the role of E1B, it is known that the adenoviral E1A
protein can act as a promiscuous transcriptional activator of many
cellular genes with no apparent cis-acting DNA sequence
requirement (Liu and Green, 1990, 1994; Lee et al., 1991;
Geisberg et al., 1994; Liu and Green, 1994)). However, if E1A
(or another transactivator) indeed overrides the tetracycline
responsiveness of the TATA box-containing tetO/minimum
promotor to stimulate transcription of the GluR6 cDNA, it is unclear
why it does not stimulate transcription of the hybrid transactivator,
which is also transcribed from the tetO/minimum promotor.
Therefore, additional experiments are required to determine whether E1A
can indeed selectively act as a promiscuous transactivator on the
tetO/minimum promotor of pTET-Spl.
The inducibility of
expression was much greater in GH3 cells than in HEK 293 cells. The
modest inducibility of GluR6 expression that we have found might simply
be related to the high level of constitutive expression in noninduced
HEK 293 cells. Whereas the levels of D
In
conclusion, this study examined the activity of tetracycline-responsive
promotors in GH3 and HEK 293 cells. Levels of D
We thank Steve Heinemann (Salk Institute, La Jolla)
for providing us with the cDNA encoding GluR6, Richard Huganir (The
Johns Hopkins University, Baltimore) for his generous gift of the
polyclonal GluR6/7 antibody, and David Schatz and Gary Rudnick (Yale
University, New Haven) for providing us with the plasmids pTET-Spl and
pTA-N, respectively. John Marshall (Yale University) subcloned the
GluR6 cDNA into the plasmid pTET-Spl.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-receptor was stably
expressed in GH3 cells, and GluR6 (a glutamate receptor subunit) was
stably expressed in human embryonic kidney (HEK 293) cells. Three
striking differences were found. 1) In the inactive state,
virtually no D
-receptor expression was found in GH3 cells,
whereas substantial levels of GluR6 expression were found in HEK 293
cells. 2) The induction of expression obtained upon removal of
tetracycline was robust in GH3 cells but only modest in HEK 293 cells.
3) Whereas in each clonal cell line, the expression of a
co-transfected hybrid transactivator is clearly regulated in a
tetracycline-responsive manner, in the induced state, its mRNA levels
were found to be very low in GH3 cells and very high in HEK 293 cells.
The results indicate that, in contrast to GH3 cells, HEK 293 cells do
not provide a cellular environment in which the expression of a
heterologous gene can be tightly controlled in a
tetracycline-responsive manner.
receptor
expression and significant amounts of functional G-protein-coupled
receptor complexes in transfected cell lines (Sokoloff et al.,
1990; 1992). Human embryonic kidney cells (HEK 293)
(
)
have been widely used for patch-clamp studies of
recombinant glutamate receptor (GluR) ion channels (for example, see
Keinänen et al. (1990), Partin et al.(1993), and
Raymond et al.(1993)). Because these studies are performed on
single cells, the generation of stably expressing cell lines offers
clear practical advantages over transient transfection protocols for
detailed structure-function studies. However, obtaining high level
constitutive expression of some GluR channels is likely to be
difficult, because the activation of these channels (by glutamate
released from cells in culture) is known to be cytotoxic (Choi, 1992).
receptor protein or
homomeric GluR ion channels formed from a kainate-type GluR subunit
(GluR6) (Egebjerg et al., 1991). An inducible expression
system based on that described by Gossen and Bujard(1992) was used in
which the transcription of both the hybrid transactivator and
the cDNA encoding the receptor protein are under the transcriptional
control of a tetracyline-responsive promotor. D
receptors
were expressed in clonal cells (GH3) derived from the rat pituitary
because GH3 cells are known to express several subtypes of G
and G
proteins (Hille, 1992) which are necessary for
functional D
receptor coupling. GluR6 was stably expressed
in HEK 293 cells because they are often used for studies of GluR ion
channels and because the transient transfection of HEK 293 cells with
cDNA encoding GluR6 leads to the expression of functional channels
(Herb et al., 1992). We were able to generate stably
transfected GH3 cells in which the expression of the human
D
-type receptor could be tightly regulated in a
tetracycline-responsive manner. We were also able to generate HEK 293
cell lines that stably express functional GluR6 ion channels; however,
the suppression of GluR6 expression by tetracycline was incomplete, and
the induction we obtained was only modest. The results indicate that a
tetracycline-responsive transactivator can efficiently regulate the
transcription of heterologous genes in GH3 cells but not in HEK 293
cells.
Plasmid Constructs and DNA Preparation
The
plasmid pTA-N (kindly provided by Gary Rudnick, Yale University, New
Haven, CT) encodes the hybrid transactivator described by Gossen and
Bujard (1992). This transactivator is under the transcriptional control
of the Tn10 tetracycline-resistance operator of E.
coli, which is linked to a TATA box-containing minimum promotor
sequence. Thus, in the presence of tetracycline, the transcription of
the transactivator gene is turned off. The plasmid pTA-N also encodes
the neomycin resistance gene, which is under the transcriptional
control of the SV40 early promotor region.
receptor into the HindIII restriction site found in the
polylinker region to generate the plasmid pTET-D
. The rat
cDNA encoding the fully edited version of the GluR6 receptor (kindly
provided by Steve Heinemann, Salk Institute, La Jolla, CA) was cloned
into the SalI/SpeI restriction sites in pTET-Spl to
generate pTET-GluR6. The polylinker is situated such that both
receptor-encoded cDNAs are under the transcriptional control of
identical tetO/minimum promotors. A 1.7-kb SV40-derived splice
poly(A) sequence is located downstream of the inserted cDNAs.
Generation of Stable Cell Lines
GH3 cells were
grown in 50% Ham's F-10 and 50% Dulbecco's modified Eagle
medium supplemented with 15% horse serum (Life Technologies, Inc.).
Cells were co-transfected using the lipofection-based method DOTAP
(Boehringer Mannheim) with the pTA-N and pTET-D plasmids.
48 h after transfection, the culture medium was supplemented with 0.3
mg/ml of G418 (Life Technologies, Inc.) until single transfected cell
colonies were isolated by ring cloning. Stably transfected cells were
expanded to cell lines in the presence of 0.1 mg/ml of G418, and 1
µg/ml of tetracycline (Sigma).
20% confluent). The cells were
then washed twice, harvested, and replated in Eagle's minimum
essential medium with Earle's salts (with 10% fetal bovine serum)
containing 0.8 mg/ml G418 and 2 µg/ml tetracycline.
Antibiotic-resistant cell clones were isolated by ring cloning and
expanded into cell lines. Stable cell lines were maintained in medium
containing 0.5 mg/ml of G418 and 2 µg/ml tetracycline.
RNA Extraction and Northern Blot Analysis
Total
cytoplasmic RNA was extracted from transfected cells using the
guanidine/cesium chloride ultracentrifugation method (Chirgwin et
al., 1979). RNA was separated on 1.2% formaldehyde/agarose gels
and transferred to Zeta Probe blotting membrane (Bio-Rad) by pressure
blotting. Blots were probed with P-radiolabeled random
primed DNAs encoding either the human D
receptor (a 1.2-kb
HindIII restriction fragment of the plasmid
pRc/CMV/D
; Schmauss et al.(1993)), the rat GluR6
receptor (a 3.3-kb HindIII restriction fragment; Egebjerg
et al. (1991)), the hybrid transactivator (a 1-kb
HindIII/SpeI restriction fragment of the plasmid
pTA-N), or the plasmid p3WT18 (adenovirus type 5 vector, map units
0-15.5, kindly provided by Selina Chen-Kiang, Mount Sinai School
of Medicine, New York; see Ruether et al.(1986)).
Protein Extraction and Immunoblot Analysis
GH3
cell pellets were homogenized in buffer containing 1% SDS, 0.1
M Tris (pH 7.5), 20 mM NaCl, 10 mM EGTA, and
10 mM EDTA in the presence of the protease inhibitors Pefabloc
(1 mg/ml), leupeptine (10 µg/ml), pepstatin (10 µg/ml), and
aprotinin (1 µg/ml) (Boehringer Mannheim) and boiled for 10 min.
The soluble protein fraction was separated from the insoluble fraction
by centrifugation, and 5 µg of soluble protein was dissolved in 1
Laemmli buffer (3% SDS, 0.1 M Tris, 10 mM
EDTA, 5%
-mercaptoethanol). HEK 293 cell pellets were resuspended
in 60 mM Tris (pH 6.8) and 2% SDS. Bromphenol blue and
-mercaptoethanol were added to 10-µg samples of each HEK 293
cell lysate to yield a final concentration of 0.01 and 5%,
respectively, and the samples were boiled prior to loading. Protein
samples (5 and 10 µg of protein for GH3 and HEK 293 cells,
respectively) were loaded onto 10% SDS-polyacrylamide gels. Protein
concentrations were determined using the Bradford assay.
Tris-buffered saline, 0.05% Tween 20, and 0.01% sodium azide
for 3 h. The primary antibodies used were either a rabbit polyclonal
anti-peptide antibody (dilution 1:12,500) that was raised against the
human D
receptor (Cambio; Cambridge, United Kingdom; see
Liu et al.(1994)) or a rabbit polyclonal antibody (dilution
1:300) that was raised against the carboxyl-terminal peptide sequence
of GluR6 (kindly provided by Richard Huganir, The Johns Hopkins
University, Baltimore, MD; see Raymond et al.(1993)). Blots
were incubated with primary antibody for 1 or 2 h in the same
incubation buffer, washed (1
Tris-buffered saline, 0.1% Tween
20) 6
10 min, and incubated for 1 h with secondary antibody
(peroxidase-conjugated goat anti-rabbit IgG; Kirkegaard and Perry
Laboratories, Inc, Gaithersburg, MD) in the same incubation buffer
without sodium azide. Bound antigens were visualized by enhanced
chemiluminescence using the ECL kit (Amersham Corp.).
Patch Clamp Recordings
Conventional patch-clamp
methods (Hamill et al., 1981) were used to record whole-cell
currents from GluR6-transfected HEK 293 cells. Patch pipettes were
pulled from capillary-filled borosilicate glass; they were coated to
within 100 µM of the tip with Sylgard (Dow Corning) and
were fire polished. The pipettes had initial resistances of 4-8
M when filled with the internal solution used for recording. HEK
293 cells were plated at low density on glass coverslips and allowed to
recover for at least 2 days before any experimental manipulations were
made. For electrophysiological recording, the coverslips of cells were
mounted in a recording chamber attached to the stage of an Axioskop
microscope (Zeiss) and were viewed with Nomarski optics at a total
magnification of 640
. The pipette solution (pH 7.2) contained
150 mM CsCl, 0.5 mM CaCl
, 5 mM
Na-EGTA, and 10 mM K-HEPES. The normal extracellular solution
(pH 7.2) contained 150 mM NaCl, 2.5 mM KCl, 1
mM CaCl
, and 10 mM Na-HEPES. All
recordings were performed at room temperature (20-22 °C).
for kainate activation of homomeric GluR6
channels; Marshall and Howe(1994)). Homomeric GluR6 channels show rapid
and complete desensitization, which can be removed by pretreatment of
the cells with concanavalin A (Egebjerg et al., 1991; Partin
et al., 1993; Wang et al., 1993). Therefore, in all
patch-clamp experiments, HEK 293 cells were preexposed to concanavalin
A (about 25 µM for 15-20 min). The effect of
concanavalin A to remove desensitization was complete and irreversible
over the course of the experiment.
value required to cancel the
capacitive current transients evoked by a 10-mV depolarizing voltage
step. Capacitive transient cancellation was complete for the cells
studied. The mean capacity of the cells was 20 pF, and current
densities were multiplied by 20 to give values indicative of the
absolute size of the whole-cell currents recorded.
Tetracycline Responsiveness of D
GH3 cells were transfected with the plasmid DNAs
pTA-N and pTET-DReceptor mRNA and Protein Expression in Stably Transfected GH3
Cells
as described under ``Materials and
Methods.'' G418-resistant cell clones were either grown in the
presence of tetracycline (1 µg/ml; noninduced) or without
tetracycline in the culture medium (induced). Clones were prescreened
for the expression of D
-encoded mRNA using reverse
transcriptase polymerase chain reaction amplification. Even under
saturating polymerase chain reaction conditions with 30 cycles of
amplification, we found significant differences in the amounts of
polymerase chain reaction products obtained from RNA templates of
noninduced cells (low amounts) and from induced cells (high amounts)
for all G418-resistant cell clones analyzed (not shown). One clone,
which Northern blot analysis indicated expressed the highest amount of
D
-encoded mRNA in the induced state, was selected for
further analysis. The time course of the induction (absence of
tetracycline) and the inhibition (1 µg/ml of tetracycline) of
D
-encoded mRNA expression in this clonal line is shown in
Fig. 1
. A low level of D
-encoded mRNA was detected 5
h after withdrawal of tetracycline from the culture medium
(Fig. 1, Induction). The amount of mRNA was found to be
further increased after 9 h of induction. Steady-state mRNA levels are
reached 24 h after induction (cf. lanes 3 and
4, Induction). There is very little fluctuation in
the levels of D
-encoded mRNA during a 2-9-day
induction period (Fig. 1, steady state). After
steady-state levels were reached, tetracycline (1 µg/ml) was added
to the culture medium (inhibition of induction). 3 days later,
D
mRNA was barely detectable on Northern blots, and the
mRNA levels remain undetectable at least up to 9 days of tetracycline
treatment (Fig. 1, inhibition).
Figure 1:
Northern blot
of RNA extracted from a stably D-expressing GH3 cell line.
The blot on the left shows the time-dependent increase in the
expression of D
-encoded mRNA between 5 to 24 h after
induction. Steady-state D
mRNA levels were monitored up to
9 days of induction and showed little fluctuation (middle). 3
days after inhibition of this induction (lane marked -3;
1 µg/ml tetracycline) no expression of D
-encoded mRNA
was detected. The same result was obtained with prolonged inhibition (6
and 9 days; marked -6 and -9) as shown on
the blot at the right. 20 µg of total RNA was loaded onto
each lane. The blots were probed with a
P-radiolabeled
cDNA encoding the human D
receptor. Blots were washed at 70
°C in 0.1
SSC and 0.1% SDS. Whereas the blot on the right
(6 and 9 days of inhibition) was exposed to film for 3 days, the
remaining blots were exposed for only 12 h. The length of the
D
-encoded mRNA (2.9 kb) results from the cloning of the
1.2-kb D
-encoded cDNA upstream of the SV40-derived splice
poly(A) sequence (1.7 kb). Size markers are derived from an RNA ladder
(Life Technologies, Inc.).
The kinetics of
induction and inhibition of D receptor expression were also
examined at the level of protein. To analyze the time course of the
synthesis of the D
receptor protein, cells were collected
5, 9, 24, and 48 h after withdrawal of tetracycline from the culture
medium (induction). The expression of the D
protein was
analyzed by immunoblotting using an antipeptide antibody that was
raised against the amino-terminal peptide sequence of the human D
receptor (see ``Materials and Methods''; Liu et
al.(1994)). The results are shown in Fig. 2. While no
specific immunoreactive signals were obtained on immunoblots of
proteins of nontransfected GH3 cells (lane marked
nt), three weak immunoreactive proteins with molecular sizes
of 35, 38, and 50 kDa are detected on immunoblots of proteins from
D
-transfected cells that were induced for 5 and 9 h. It is
likely that the 35- and 38-kDa proteins represent the D
receptor core protein (38 kDa) and a proteolytic fragment of the
D
protein (35 kDa), and that the 50-kDa protein results
from posttranslational modification (perhaps glycosylation) of the core
protein. 24 and 48 h after induction (Fig. 2), the major
immunoreactive protein is the 50-kDa protein, and two larger molecular
size bands are also visible (
70 and 80 kDa; representing perhaps
further post-translational modifications of a small percentage of the
D
proteins). The amounts of the 35- and 38-kDa proteins are
also increased significantly over those seen at 9 h. Continued
induction for 4 and 6 days results in high level and steady-state
D
receptor protein expression (Fig. 2, steadystate). Tetracycline was then reintroduced into the
medium, and proteins were collected from GH3 transfectants after 1, 2,
or 3 days of exposure (Fig. 2, inhibition). A
significant decrease in the signal intensity of the 35 and 38 kDa
immunoreactive bands, and a moderate decrease in the intensity of the
50 kDa immunoreactive band, are apparent after 24 and 48 h of
inhibition. 3 days of tetracycline treatment results in only weak
immunoreactivity, indicating that only spurious amounts of the D
receptor protein remain present in the cells at this time.
Figure 2:
Immunoblots of proteins extracted from
stably D-expressing GH3 cells. Transfected cells were
collected at the time points indicated at the top of the
lanes. The lane marked nt contains proteins
that were extracted from nontransfected GH3 cells. 5 µg of total
cellular proteins was loaded onto each lane. Blots were probed with a
rabbit polyclonal antiserum raised against the amino-terminal peptide
sequence of the human D
receptor (see ``Materials and
Methods''). Bound antigens were visualized using the ECL detection
reagents (Amersham Corp.). Size markers are derived from prestained
molecular size markers (Sigma).
In
summary, the expression of D receptor mRNA and protein in
our clonal GH3 cells is suppressed by tetracycline and is markedly
induced by removing tetracycline from the culture medium. The kinetics
of D
receptor mRNA and protein expression are convincingly
correlated for both induction and inhibition, and the kinetics are
similar to the corresponding kinetics reported by Gossen and Bujard
(1992) for the expression of the luciferase gene in transfected HeLa
cells with active tetracycline-responsive promotors. Steady-state mRNA
and protein levels were obtained 24 h after induction, and an
approximately 50% decrease in the amount of the D
receptor
protein is observed 24 h after inhibition of induction. This suggests
that tetracycline is rapidly taken up by GH3 cells and that the mean
lifetime of the recombinant D
receptor protein expressed in
these cells is between 1 and 2 days. Residual expression in the
inactive state (3 days of tetracycline treatment) was almost
undetectable on Northern blots (D
mRNA), and only spurious
levels of protein were detected on immunoblots (D
protein).
Tetracycline Responsiveness of GluR6 Expression in HEK
293 Cells
HEK 293 cells were co-transfected with the plasmids
pTA-N and pTET-GluR6 as described under ``Materials and
Methods'' and stable G418-resistant clones were selected.
Individual G418-resistant clones were grown in the absence of
tetracycline for 3-4 days and were screened for GluR6 expression
in patch-clamp experiments. Five GluR6-expressing clones were
identified that displayed similar levels of GluR6 expression in the
absence of tetracycline. Fig. 3A shows an example of a
whole cell current evoked by the application of 10 µM
kainate (a GluR agonist) in an induced cell from one of the clonal
lines.
Figure 3:
Functional expression of GluR6 homomeric
channels in HEK 293 cells. A, whole-cell current evoked by
kainate. An example of a whole-cell current recorded in response to the
application of 10 µM kainate in a stably transfected
GluR6-expressing HEK 293 cell. The cell was voltage clamped at a
membrane potential of -80 mV, and kainate was applied by local
perifusion during the time indicated by the bar above the
current trace. B, time course of induction and inhibition of
GluR6 channel expression. Stably transfected HEK 293 cells were grown
continuously in 2 µg/ml tetracycline. The tetracycline was removed
from the medium, and the steady-state amplitude of kainate-evoked
currents was measured from 6 h to 6 days later (induction,
-tet). The histogram on the left shows the mean
amplitude of the steady-state currents at each postinduction time. In
another set of cells, tetracycline (2 µg/ml) was reintroduced into
the medium after 5 or 6 days of induction, and currents were measured 1
or 2 days later (inhibition, +tet). To account
for differences in cell size, the currents were expressed as current
densities (pA/pF) and normalized to the average cell capacitance of 20
pF. For example, a value of 192 pA/20 pF was obtained for the cell in
panelA, which had a capacitance of 15 pF and gave a
mean inward current of 145 pA. All cells were voltage clamped at
-80 mV, and the currents were evoked by a saturating
concentration of kainate (10 µM). Measurements were made
from 7-15 cells at each time point. Bars indicate
S.E.
One of the clones
(24) was selected for
characterization of the time course of induction and inhibition of
GluR6 expression. Tetracycline was removed from the medium, and
functional GluR6 expression was measured from 6 h to 6 days later. The
results are shown in Fig. 3B (Induction -tet), where the mean amplitude of whole cell
currents recorded under identical conditions from several cells is
plotted as a function of time in the absence of tetracycline. As can be
seen, GluR6 expression reaches steady-state levels within 2-3
days. After induction for 4 or 5 days, tetracycline (2 µg/ml) was
reintroduced, and currents were measured from the cells 1 and 2 days
later (Fig. 3B, Inhibition+tet).
Exposure of the cells to tetracycline for 1 day significantly inhibited
GluR6 expression, and expression was further inhibited after 2 days of
exposure. The kinetics of the induction and inhibition of GluR6
expression are similar to the corresponding results obtained for
D receptor expression in our stable GH3 clone (
Fig. 1
and Fig. 2) and suggest that the mean lifetime of a
homomeric GluR6 channel is about 1 day in our transfected HEK 293
cells. Unlike the D
receptor results, however, maintaining
the HEK 293 cells in tetracycline for times longer than 2 days did not
eliminate GluR6 expression; functional expression remained constant at
about 50 pA/20 pF. Increasing the tetracycline concentration to 10 or
100 µg/ml also did not further inhibit expression, and 24 h
exposure to 100 µg/ml tetracycline killed a substantial proportion
of the cells. Thus, considerable tetracycline-insensitive expression of
GluR6 occurs in the HEK 293 clone examined (a whole-cell current of 50
pA corresponds to about 2500 homomeric GluR6 channels open
simultaneously), and only about a 4-fold induction of expression is
obtained upon removal of tetracycline.
results shown in
Fig. 1
and Fig. 2, we examined the levels of GluR6 mRNA and
protein in our stable HEK 293 clones. Fig. 4A shows a
Northern blot of GluR6 mRNA isolated from cells (clone 24) that were
either maintained continuously in tetracycline-containing (2 µg/ml)
media (noninduced) or that were kept in the absence of tetracycline for
3 days (induced). Similar amounts of GluR6 mRNA were found in the
induced and noninduced cells, indicating that significant transcription
of the GluR6 cDNA occurs in the inactive state. An immunoblot (using
antiserum raised against a GluR6-specific peptide sequence; see Raymond
et al.(1993)) of cell lysates from three stable
GluR6-expressing cell lines is shown in Fig. 4B. No
immunoreactive proteins were detected in nontransfected HEK 293 cells
(nt). Immunoreactive signals of the expected size for GluR6
(Raymond et al., 1993; Stern-Bach et al., 1994) were
clearly detected, however, in each sample isolated from cells of the
three different stable clones (24, 38, 39). A doublet band of
immunoreactive proteins that migrate at
120 kDa is clearly
detectable in each stable clone in both the noninduced (+
tetracycline) and induced state (without tetracycline for 3 days). The
increased amounts of GluR6 protein seen after 3 days induction are
consistent with the modest induction seen in the patch-clamp
experiments (Fig. 3B). Similar immunoblot results were
obtained in two additional clones (not shown).
Figure 4:
Expression of GluR6 mRNA and protein in
induced and noninduced HEK 293 cells. A, Northern blot of mRNA
extracted from nontransfected and stably GluR6-expressing cells. 20
µg of total RNA was loaded onto each lane. Whereas no mRNA encoding
the GluR6 receptor subunit was detected in nontransfected cells
(nt), both noninduced (+, cells grown in media containing
2 µg/ml tetracycline) and induced cells (-, cells grown for 3
days in the absence of tetracycline) express two mRNA species
(indicated by arrowheads) that hybridize to a
P-radiolabeled 3-kb HindIII restriction fragment
of the rat GluR6-encoded cDNA (see ``Materials and
Methods''). These two mRNA species result from the use of two
different polyadenylation signals in the plasmid pTET-Spl/GluR6. One is
located at the 3` end of the cloned GluR6 cDNA, and the other is
located 1.7 kb further downstream and is the SV40-derived
polyadenylation signal. A significant amount of transcription through
the most 5` located polyadenylation signal results in the smear between
both bands. The blot was exposed to film for 7 h. Under these
nonsaturating exposure conditions, no significant difference between
the GluR6-encoded mRNA levels in induced and noninduced HEK 293 cells
is apparent. Size markers are derived from an RNA ladder (Life
Technologies, Inc.). B, expression of GluR6 receptor protein
in three independent transfected HEK 293 cell clones. Blots were probed
with a rabbit polyclonal antibody raised against a carboxyl-terminal
peptide sequence of GluR6. No immunoreactive signals were detected when
proteins were isolated from nontransfected cells (nt).
Immunoreactive signals of the expected size for the GluR6 protein
(
120 kDa) are detected for each clone in both noninduced (+)
and induced (-) cells. For the induced cells, tetracycline was
removed from the medium for 3 days. A modest induction of GluR6 protein
expression is apparent for each clone. Ten µg of protein was loaded
in each lane. Size markers are derived from prestained molecular size
markers (Bio-Rad).
mRNA Expression of the Hybrid Transactivator in GH3 and
HEK 293 Cells
In order to begin to identify the reasons
underlying the tetracycline-insensitive expression in our
GluR6-expressing clones, we compared the expression of mRNA encoding
the hybrid transactivator in our transfected GH3 and HEK 293 cells. The
results are shown in Fig. 5. Interestingly, whereas GluR6 mRNA
expression was substantial in the inactive state
(Fig. 4A), the expression of the transactivator in the
stably transfected HEK 293 cells is clearly tetracycline responsive.
Furthermore, very high levels of its encoded mRNA are found in the
induced state. In contrast, in induced GH3 cells with an active
tetracycline-responsive promotor ( Fig. 1and 2), the expression
of the transactivator is much lower and its detection required 3 times
longer exposure of the Northern blot shown in Fig. 5. The latter
result is similar to the finding of Gossen and Bujard(1992) in
transfected HeLa cells with active tetracycline-responsive promotors
where the expression of the transactivator was barely detectable on
immunoblots.
Figure 5:
Expression of mRNA encoding the hybrid
transactivator in stably D-expressing GH3 cells and in
stably GluR6-expressing HEK 293 cells. The blot was probed with a
P-radiolabeled DNA encoding the hybrid transactivator (see
``Materials and Methods''). 20 µg of total RNA extracted
from noninduced (+) or induced (-) cells was loaded onto
each lane. Overnight exposure of the blot to film revealed a high level
of expression of the transactivator in induced, but not in noninduced,
HEK 293 cells. Only 3 times longer exposure of the blot showed the
presence of the transactivator-encoded mRNA in induced GH3 cells with
active tetracycline-responsive promotors. Size markers are derived from
an RNA ladder (Life Technologies, Inc.).
In summary, in contrast to the results obtained with
transfected GH3 cells, we could not increase the expression of the
GluR6 receptor in HEK 293 cells much above noninduced levels.
Substantial levels of tetracycline-insensitive expression were found in
patch-clamp experiments, and Northern blots and immunoblots showed high
levels of GluR6 expression in the inactive state. Similar immunoblot
results were obtained from five independent clones. The high level of
constitutive expression and the minimal responsiveness to tetracycline
appeared to be restricted to GluR6, since transcription of the hybrid
transactivator was strongly tetracycline-responsive.
receptor-encoded cDNA in GH3 cells and of the GluR6
receptor-encoded cDNA in HEK 293 cells. Strikingly different results
were obtained. On the one hand, the expression of the D
receptor in GH3 cells was found to be tightly controlled by the
tetracycline-responsive promotor. Expression was clearly suppressed by
tetracycline, and levels of the D
-encoded mRNA and protein
were found in the induced state that were substantially higher than
those we obtained previously in either transiently transfected cells or
in stably and constitutively D
-expressing GH3 cells (see
Schmauss et al., 1993). On the other hand, the GluR6 receptor
was expressed in HEK 293 cells in the noninduced state, and only a
modest induction of expression was obtained upon withdrawal of
tetracycline from the culture medium.
receptor expression
we have obtained here are much higher than those we have obtained in
GH3 cells with other expression strategies, the levels of functional
GluR6 expression we see in our stable transfectants after induction are
similar to the levels we have obtained transiently in HEK 293 cells,
either after transfection with plasmids employing cytomegalovirus
promoters or after infection of the cells with GluR6-containing viral
constructs. It is therefore possible that the levels of
tetracycline-insensitive GluR6 expression we report here may closely
approach the maximum levels of GluR6 expression that can be obtained in
these cells. It is also possible, however, that the high levels of the
hybrid transactivator expressed in induced HEK 293 cells might
contribute to the modest induction we have seen. It has been previously
reported that transactivators, at high concentrations, can inhibit
rather than stimulate transcription (Gill and Ptashne, 1988). This
phenomenon, called squelching, depends on increased concentrations of
the transactivator and on the strength of the activating region. In
this respect it is of interest to note that in our GH3 cells, where
transcription from the pTET-Spl template is clearly
tetracycline-responsive, the expression of the transactivator-encoded
mRNA was barely detectable on Northern blots (Fig. 5). This
finding is similar to the results of Gossen and Bujard (1992) who found
barely detectable levels of the transactivator protein on immunoblots
of proteins extracted from HeLa cells in which the
tetO/minimum promoter was very responsive to tetracycline.
They reasoned that this may have resulted because cells in which
squelching effects (caused by high concentrations of the
VP16-containing activating domain of the hybrid transactivator) were
significant had been eliminated by their selection protocol.
protein
were found in induced GH3 cells that are much higher than those we
obtained previously in cells constitutively expressing the D
receptor. The establishment of a cell line in which D
receptor expression can be tightly regulated will greatly aid
studies on the kinetics of the synthesis and processing, as well as on
the function, of this protein. In contrast, only minimal responsiveness
to tetracycline of GluR6 expression was observed in HEK 293 cells. We
have not identified conclusively the reasons for this lack of
tetracycline responsiveness. However, it is plausible that it reflects
the presence in these cells of promiscuous transactivators. Thus,
although the system studied here can clearly be used to obtain stable,
high level, and inducible expression of neurotransmitter receptors in
some cell lines, our results also indicate that it does not work
optimally in HEK 293 cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.