©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Responsiveness of a Tetracycline-sensitive Expression System Differs in Different Cell Lines (*)

James R. Howe (1), Boris V. Skryabin (2), Scott M. Belcher (1), Cynthia A. Zerillo (1), Claudia Schmauss (2)(§)

From the (1) Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066 and the (2) Department of Psychiatry and Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A tetracycline-sensitive inducible expression system was used to regulate the expression of neurotransmitter receptor genes in two mammalian cell lines. The dopamine D-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.


INTRODUCTION

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 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).

In this paper, we describe results from clonal mammalian cells that stably express either the dopamine D 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.


MATERIALS AND METHODS

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.

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 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).

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 (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.

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 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).

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 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.

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 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.


RESULTS

Tetracycline Responsiveness of DReceptor mRNA and Protein Expression in Stably Transfected GH3 Cells

GH3 cells were transfected with the plasmid DNAs pTA-N and pTET-D 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.

To compare our GluR6 results in HEK 293 cells more directly with the D 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.


DISCUSSION

We have tested the efficacy of a tetracycline-responsive promotor in regulating the transcription of the dopamine D 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.

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 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.

In conclusion, this study examined the activity of tetracycline-responsive promotors in GH3 and HEK 293 cells. Levels of D 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.


FOOTNOTES

*
This work was supported by the National Alliance for Research on Schizophrenia and Depression (to C. S.), a Grant IBN-9409772 from the National Science Foundation (to C. S.), and Grant NS 30996 from the National Institutes of Health (to J. R. H.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Mount Sinai School of Medicine, Box 1229, One Gustave L. Levy Pl., New York, NY 10029. Tel.: 212-241-6085; Fax: 212-831-1947; E-mail: Schmauss@ smtplink.mssm.edu.

The abbreviations used are: HEK, human embryonic kidney cells; GluR, glutamate receptor; GluR6, kainate-type GluR subunit; kb, kilobase pair(s); pF, picofarad.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J.(1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  2. Choi, D. W.(1992) J. Neurobiol. 23, 1261-1274 [Medline] [Order article via Infotrieve]
  3. Egebjerg, J., Bettler, B., Hermans-Borgmeyer, I. & Heinemann, S.(1991) Nature 351, 745-748 [CrossRef][Medline] [Order article via Infotrieve]
  4. Geisberg, J. V., Lee, W. S., Berk, A. & Riccardi, R. P.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2488-2492 [Abstract]
  5. Gill, G. & Ptashne, M.(1988) Nature 334, 721-724 [CrossRef][Medline] [Order article via Infotrieve]
  6. Gossen, M. & Bujard, H.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547-5551 [Abstract]
  7. Graham, F. L., Smiley, J. S., Russell, W. C. & Nairn, R.(1977) J. Gen. Virol. 36, 59-72 [Abstract]
  8. Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981) Pflügers Arch. 391, 85-100
  9. Herb, A., Burnashev, N., Werner, P., Sakmann, B., Wisden, W. & Seeburg, P. H.(1992) Neuron 8, 775-785 [Medline] [Order article via Infotrieve]
  10. Hille, B.(1992) Neuron 9, 187-195 [Medline] [Order article via Infotrieve]
  11. Keinänen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoorn, T. A., Sakmann, B. & Seeburg, P. H.(1990) Science 249, 556-560 [Medline] [Order article via Infotrieve]
  12. Lee, S. W., Tsou, A.-P., Chan, H., Thomas, J., Petrie, K., Eugui, E. M. & Allison, A. C.(1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1204-1208 [Abstract]
  13. Lee, W. S., Kao, C., Bryant, G. O., Liu, X. & Berg, A. J.(1991) Cell 67, 365-376 [Medline] [Order article via Infotrieve]
  14. Liu, F. & Green, M. R.(1990) Cell 61, 1217-1224 [Medline] [Order article via Infotrieve]
  15. Liu, F. & Green, M. R.(1994) Nature 368, 520-523 [CrossRef][Medline] [Order article via Infotrieve]
  16. Liu, K., Bergson, C., Levenson, R. & Schmauss, C.(1994) J. Biol. Chem. 269, 29220-29226 [Abstract/Free Full Text]
  17. Marshall, J. & Howe, J. R.(1994) Biophys. J. 66, 436 (abstr.)
  18. Mayo, E. K., Warren, R. & Palmiter, R. D.(1982) Cell 29, 99-108 [Medline] [Order article via Infotrieve]
  19. Nouer, L.(1991) in Heat Shock Response (Nouer, L., ed) pp. 167-220, CRC Press, Boca Raton, FL
  20. Partin, K. M., Patneau, D. K., Winters, C. A., Mayer, M. C. & Buonanno, A.(1993) Neuron 11, 1069-1082 [Medline] [Order article via Infotrieve]
  21. Raymond, L. A., Blackstone, C. D. & Huganir, R. L.(1993) Nature 361, 637-641 [CrossRef][Medline] [Order article via Infotrieve]
  22. Ruether, J. E., Maderious, A., Lavery, D., Logan, J., Fu, S. M. & Chen-Kiang, S.(1986) Mol. Cel. Biol. 6, 123-133 [Medline] [Order article via Infotrieve]
  23. Schmauss, C., Haroutunian, V., Davis, K. L. & Davidson, M.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8942-8946 [Abstract]
  24. Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L. & Schwartz, J.-C.(1990) Nature 347, 146-151 [CrossRef][Medline] [Order article via Infotrieve]
  25. Sokoloff, P., Andrieux, M., Besancon, R., Pilon, C., Martres, M.-P., Giros, B. & Schwartz, J.-C.(1992) Eur. J. Pharmacol. 225, 331-337 [CrossRef][Medline] [Order article via Infotrieve]
  26. Stern-Bach, Y., Bettler, B., Hartley, M., Sheppard, P. O., O'Hara, P. J. & Heinemann, S. F.(1994) Neuron 13, 1345-1357 [Medline] [Order article via Infotrieve]
  27. Van der Elsen, P., de Pater, S., Houweling, A., van der Veer, J. & van der Erb, A.(1982) Gene(Amst.) 18, 175-185 [CrossRef][Medline] [Order article via Infotrieve]
  28. Van der Elsen, P., Houweling, A. & van der Erb, A.(1983) Virology 128, 377-390 [Medline] [Order article via Infotrieve]
  29. Wang, L.-Y., Taverna, F. A., Huang, X.-P., MacDonald, J. F. & Hampson, D. R.(1993) Science 259, 1173-1175 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.