An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling

Jen-Feng Lai,1 Shin-Hun Juang,2 Yi-Mei Hung,2 Hsin-Yuan Cheng,1 Tzu-Ling Cheng,1 Keith E. Mostov,3 and Tzuu-Shuh Jou1

1Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, and 2Division of Cancer Research, National Health Research Institutes, Taipei 100, Taiwan, Republic of China; and 3Department of Anatomy and Cardiovascular Research Institute, University of California, San Francisco, California 94143

Submitted 19 February 2003 ; accepted in final form 3 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulated expression systems are invaluable for studying gene function, offer advantages of dosage-dependent and temporally defined gene expression, and limit possible clonal variation when toxic or pleiotropic genes are overexpressed. Previously, establishment of inducible expression systems, such as tetracycline- and ecdysone-inducible systems, required assessment of the inducible characteristics of individual clones by tedious luciferase assays. Taking advantage of a green fluorescent protein (GFP) reporter controlled by tetracycline- or ecdysone-responsive element and fluorescence-activated cell sorting, we propose a simple and efficient strategy to select highly inducible cell lines according to their fluorescence profiles after transiently transfecting the candidate cell pools with a surrogate GFP reporter. We have demonstrated that tetracycline- and ecdysone-inducible systems could be set up in Madin-Darby canine kidney and HEK-293 cells by employing this selection scheme. Importantly, this dual regulatory expression system is applied in studying the complex interplay between two Ras-related small GTPases, Cdc42 and Rac1, on detachment-induced apoptosis. Furthermore, establishment of two tightly regulated expression systems in one target cell line could be of great advantage for dissecting small GTPase Rac1-transduced signaling pathways by using global gene expression approaches such as proteomic assays.

fluorescence-activated cell sorting; green fluorescent protein; Ras small GTPases; anoikis


FUNCTIONAL STUDIES OF GENES usually depend on phenotypic observation after expression of the genes of interest in cell lines. However, uncontrolled expression of genes with pleiotropic effects conceivably can result in selecting cell lines with biased characteristics, which confound interpretation of the experimental results. Tightly regulated gene expression is thus an appealing way to solve these problems. Recently, several versions of regulatory expression systems have become available, including the tetracycline regulatory (Tet) (8) and ecdysone-inducible systems (18). The original Tet system has enjoyed its popularity as the "Tet-off" system, because tetracycline-regulated transactivator (tTA) falls off the tetracycline-responsive promoter and gene expression is shut down in the presence of tetracycline in a controllable way. The Tet system has since evolved into a new format by substitution of four amino acids in tTA to generate reverse tTA (rtTA), which requires tetracycline for binding to the tetracycline-responsive promoter and subsequent activation of the downstream genes (9). This transformed descendant is known as the Tet-on system and is expected to be more useful than Tet-off in developmental studies and gene therapy because of its favorable pharmacokinetics (6). Ecdysone triggers insect metamorphosis by activating the interaction between ecdysone receptor proteins and the ecdysone-responsive elements (EcREs) in the Drosophila genome. By transferring an engineered form of ecdysone receptor gene into mammalian cells, a gene of interest placed downstream of EcRE could be expressed upon the addition of an ecdysone analog into the culture medium. The tetracycline- and ecdysone-inducible systems offer several benefits over traditional expression systems based on constitutive promoters: 1) controlled expression during the selection process greatly reduces the possibility of selecting stable clones harboring a suppressing mutation of exogenously expressed genes; 2) the physiological range of protein expression avoids cellular toxicity elicited by overexpression of genes with pleiotropic effects; and 3) when the transferred gene is not induced, the cell population serves as a genetically matched control for the experimental condition. Although regulatory expression systems appear to be ideal tools for examining gene functions, it is not straightforward to create inducible expression systems on demand.

As a first step to employ the power of these gene expression systems, a plasmid encoding the regulatory protein must be transfected into the target cell line. To select a stable clone possessing the most desirable characteristic, this process frequently involves intensive cloning, cell culturing, and testing of each candidate clone and is very time consuming. Because of the effort demanded for screening for useful clones by the conventional strategy, large-scale screening cannot be performed easily, and this makes a successful outcome uncertain. Therefore, extensive application of inducible gene expression systems, although desirable for studying gene functions, has been limited.

Independent control of two target genes by two inducible gene expression systems would be invaluable in many applications. Although such an idea has been tested and successfully executed (7, 16, 17, 27), more general application has been hindered by the technical limitations of setting up inducible cells as described above. Here we present a simple and efficient fluorescence-activated cell sorter (FACS)-based scheme to generate inducible expression cells without going through the tedious single clone selection procedure. With this selection strategy, a population of highly inducible cells is enriched in which ecdysone- and tetracycline-controlled expression systems function together to facilitate dissection of Rho family GTPase signaling pathway's involvement in regulating anoikis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and antibodies. Plasmids expressing tTA, rtTA, and its mutant rtTA-M2 (pUHrT16-1) were gifts from Dr. Hermann Bujard (University of Heidelberg) and Wolfgang Hillen (University of Erlangen). pM2-IRES-Puro, a plasmid expressing rtTA-M2 followed by an internal ribosome entry site (IRES)-linked puromycin selection marker, was constructed by subcloning rtTA-M2 encoding sequence from pUHrT16-1 into pIRESpuro2 (Clontech). pVgRXR, a plasmid expressing an engineered ecdysone heterodimeric receptor, was from Invitrogen. pTet-TS, a plasmid expressing tetracycline-controlled transcriptional silencer (tTS), was from Clontech. Inducible green fluorescent protein (GFP) reporter plasmids pIND-GFP and pTRE-GFP were constructed by inserting GFP cDNA into the parental vectors pIND (Invitrogen) and pTRE (Clontech), respectively, using standard molecular cloning procedure. Ecdysone-responsive luciferase reporter pIND-Luc was constructed by inserting luciferase-expressing cDNA into pIND. Tetracycline-responsive luciferase reporter pTRE-Luc was from Clontech. Ecdysone-responsive {beta}-galactosidase reporter pIND-LacZ was from Invitrogen. Plasmid expressing constitutively active Rac1 gene under the control of a tetracycline-regulated promoter (pU-MRac1-V12) was a gift of Rong-Guo Qiu (21). Coding sequence expressing dominant negative (DN) mutant of inhibitory kB{alpha} (I{kappa}B{alpha}) was subcloned from pRCMV-I{kappa}B{alpha}(DN) (gift of Yen-Shen Lu) to the ecdysone-responsive vector to make pIND-I{kappa}B{alpha}(DN). All engineered constructs were confirmed by DNA nucleotide sequencing. Rabbit anti-I{kappa}B{alpha} antibody was from Santa Cruz Biotechnology. Mouse anti-Rac1 and Cdc42 antibodies were from Upstate Biotechnology.

Cell culture and transfection. Madin-Darby canine kidney (MDCK) cells and human embryonic kidney 293 (HEK-293) cells were grown in DMEM containing 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For luciferase reporter assay, one million MDCK or HEK-293 cells were transfected with 3.6 µg of pIND-Luc or pTRE-Luc and 0.4 µg of pRL-TK (Promega), a Renilla luciferase-expressing construct driven by thymidine kinase promoter as an internal control to normalize the transfection efficiency. One million MDCK or HEK-293 cells were transfected with 4 µg of the fluorescent reporter plasmids pIND-GFP and pTRE-GFP. To obtain enough cellular lysates for protein two-dimensional gel electrophoresis, we used 16 µg of pU-MRac1-V12 and 16 µg of pIND-I{kappa}B{alpha}(DN) to transfect 10 million cells for each experiment. To enrich ecdysone-regulated clones, 20 million MDCK and HEK-293 cells were transfected with 80 µg of pVgRXR and selected in the presence of 300 or 150 µg/ml zeocin, respectively. Tetracycline-inducible cells were established by selecting pM2-IRES-Puro and pTet-TS (at a ratio of 1:15)-transfected cells in 2.5 µg/ml puromycin.

Flow cytometry. One to ten million cells were sorted by Vantage flow cytometer (Becton-Dickinson). The fluorescence profiles were analyzed and processed using CellQuest v3.3 software.

Fluorescence microscopic examination, photographic presentation, and immunoblotting. Detailed experimental procedures were performed as described previously (10, 11).

Luciferase and LacZ assays. One million MDCK or HEK-293 cells were transfected with the reporter constructs as described in Cell culture and transfection. The cells were trypsinized 6 h after transfection and plated onto 24-well plates with 1 µg/ml doxycycline (Sigma) for the tetracycline-inducible system or 5 µM ponasterone (Stratagene) for the ecdysone-inducible system added to half of the wells to activate the reporter. Inducers were absent from the other half of the wells as noninduced controls. The cells were incubated for 40 h before being harvested for dual luciferase assays (dual firefly and Renilla luciferase reporter assay; Packard BioScience). Promoter activities were measured in a Wallac's luminometer (1420 multilabel counter) and expressed by dividing the firefly luciferase activity with Renilla luciferase activity; each value was the mean of a triplicate measurement. Every assay was repeated at least three times. When pIND-LacZ was included in the reporter assay, its activity was evaluated using the Galactostar kit (Tropix).

Cell death ELISA assay. MDCK cells were induced to express transgenes for 24 h as an attached culture. They were subsequently trypsinized and cultured in suspension on ultralow-attachment plates (Costar) at a density of 5 x 104 cells/ml for 16 h before being processed for measurement of DNA-histone complex by using the Cell Death ELISA kit (Roche Molecular Biomedicals).

Two-dimensional gel electrophoresis. Cells were extracted in 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% 1,4-dithioerythritol (DTE), and 40 mM Tris for 1 h at room temperature and centrifuged at 200,000 g for 1 h at 22°C, and protein was normalized by Bradford assay before being loaded into Immobiline DryStrip (pH 3–10NL, 18 cm; Pharmacia). The strips were rehydrated with 100 µg of cell lysate in 8 M urea, 2% CHAPS, 2% immobilized pH gradient (IPG) buffer (pH 3–10; Pharmacia), 2 mM tributylphosphine, and trace bromphenol blue for 4–6 h at 0 V and 12 h at 50 V. Isoelectric focusing was performed at 20°C by using the IPGphor IEF system (Pharmacia) at 300 V for 3 h, a gradient of 300–3,500 V for 3 h, 3,500 V for 3 h, and constant 5,000 V. After the focusing was completed after 125,000 Vh, strips were incubated in equilibration buffer (50 mM Tris, pH 6.8, 6 M urea, 2% SDS, and 30% glycerol), first with 2% DTE for 15 min and then with 2.5% iodoacetamide and bromphenol blue for another 15 min. Equilibrated strips were inserted onto 9–16% gradient SDS-PAGE gels and run at 40 mA for 1 h, 50 mA for 3 h per gel at 12°C in Protean IIxi Multi-Cell (Bio-Rad). Analytical gels were silver stained and preserved by air drying between sheets of moistened cellophane.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A standard protocol for selecting an inducible cell line starts with transfecting a cell line with a plasmid expressing the regulatory component (e.g., tTA or rtTA for the Tet system or heterodimeric ecdysone receptor for the ecdysone-inducible system). After candidate clones are established by drug selection, each clone is then individually expanded and tested by reporter assay under induced and noninduced conditions. This critical step, although essential to the success of generating a regulatory expression system, usually involves intensive cloning, cell culturing, and testing procedures that are very time and labor consuming. In contrast to the conventional way of selecting an optimal responder using individual cloning and laborious reporter assays, we propose a selection strategy based on mass cell culture and an inducible fluorescent reporter. A GFP reporter is constructed under the control of the regulatory promoter unit and used to transfect candidate clones that have been pooled and expanded after a drug selection procedure. Because the pattern and the degree of responsiveness of the inducible GFP reporter should reflect the profiles of luciferase reporter assay among different clones, the inducible GFP reporter could substitute for traditional luciferase reporter for assessing the candidate clones as a population. Furthermore, unlike the luciferase assay, FACS of GFP-expressing cells does not necessitate destruction of the cells being examined. The fact that the cells after sorting are still viable makes possible repetitive selection and expansion to efficiently enrich responders.

Establishing ecdysone-inducible lines in MDCK and HEK-293 cells by mass cell culture and cell sorting based on a fluorescent reporter. To illustrate the effectiveness of the proposed strategy, we report the application of this strategy to generate an ecdysone-inducible expression system in an MDCK cell line. Twenty million MDCK cells were transfected with pVgRXR. About 500 transfectants survived selection of zeocin and were pooled together. This candidate pool was then transiently transfected with pIND-GFP, a GFP reporter plasmid constructed under the control of an ecdysone-responsive element, and divided into two pools immediately after transfection. One pool of the cells was kept in medium to which 5 µM ponasterone, a synthetic ecdysone analog, was added, whereas the other pool was maintained as noninduced control. Two days later, cells were subjected to FACS. The difference in fluorescence profiles under induced and noninduced conditions revealed a window for optimal responders to be positively sorted (Fig. 1A). These selected cells were then expanded in the absence of ponasterone until enough cells had been grown for another round of positive selection. This strategy was very effective in enriching clones that apparently had the characteristics of ecdysone-inducible expression of GFP reporter. However, as the percentage of inducible cells increased after each round of positive selection, progressively more cells were found in the selected pool that possessed unregulated expression characteristics. To eliminate these leaky responders, cells after the fourth round of positive selection were expanded and transfected again with pIND-GFP and left in medium without ponasterone for negative selection. We noted that there were two populations of cells: the first with fluorescence even under noninduced conditions and the second with no fluorescence (S+4 cells in Fig. 1A). To better appreciate the dynamic change of ecdysone inducibility in this cohort of cells, we retained aliquots of cells before each round of positive FACS selection and after final negative selection, and we transfected these cells with pIND-GFP at the same time for direct observation under a fluorescence microscope (Fig. 1B). FACS was so effective in purging the leaky clones that only a few cells still possessed leaky expression characteristics after a single round of negative selection (S+4-1 cells in Fig. 1B).



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Fig. 1. A: fluorescence profiles of candidate Madin-Darby canine kidney (MDCK) cells before and after each round of fluorescence-activated cell sorting (FACS) selection. Zeocin selection established a pool of candidate clones, S+0, that was transiently transfected with pIND-GFP and subjected to FACS. The difference in the fluorescence profiles of cells grown in the absence (solid blue line) and presence of 5 µM ponasterone (solid green line) served as a window to indicate the portion of cells to be sorted (S+1, red horizontal line). S+1 cells were expanded and sequentially enriched by the same selection scheme 3 more times (designated S+2, S+3, and S+4, respectively). After the 4th round of positive selection, S+4 cells were transiently transfected with pIND-GFP again and subjected to FACS under noninduced condition. The fluorescence profile was compared with that of nontransfected parental cells (black dotted line) so that a window could be chosen to negatively select the tightly regulated cells (S+4-1) from the leaky responders. B: sequential enrichment of ecdysone-inducible MDCK cells by positive and negative FACS. Aliquots of cells before and after each sorting procedure were retained and transiently transfected with pIND-GFP and grown in the absence or presence of 5 µM ponasterone for direct visual comparison under a fluorescence microscope. Cells were counterstained with Hoechst 33342 (blue) for better appreciation of the percentage of cells expressing green fluorescence.

 

To quantitatively demonstrate the efficiency of this inducible GFP reporter and FACS-based selection strategy, we transfected pIND-Luc into aliquots of MDCK cells collected before and after each round of selection. The candidate clones capable of ecdysone inducibility were enriched after each round of positive selection as demonstrated by gradually increased luciferase activity and activation factor after each round of positive selection (Fig. 2A). Although the absolute induced luciferase activity seemed to decrease after one round of negative selection, the relative induction factor actually did not change significantly.



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Fig. 2. Improving responsiveness of ecdysone-inducible MDCK and HEK-293 cell lines by FACS. Aliquots of MDCK (A) and HEK-293 cell lines (B) were collected before and after each performance of FACS, as described in Fig. 1, and analyzed by luciferase reporter assay. After transfection with pIND-Luc, cells were grown in the absence (open bars) or presence of 5 µM ponasterone (solid bars) for 40 h before being harvested for dual luciferase assays (luciferase activities are in log scales). Values are arithmetic means ± SE of 3 independent assays. The degree of activation (shaded bars) is expressed as the ratio of paired luciferase activities under induced vs. noninduced conditions.

 

We also applied a similar selection strategy to sort HEK-293 cells that had been stably transfected with pVgRXR. The selection was so efficient, presumably resulting from the transfection-friendly nature of HEK-293 cells, that just two rounds of positive selection permitted successful collection of a pool of cells (293.Ec) possessing an impressive degree of ecdysone-inducible expression (Fig. 2B).

A tetracycline-regulated activator and silencer system potentiates selection of inducible cell lines based on a fluorescent reporter. Because the FACS-based selection was very efficient in establishing the ecdysone-inducible expression system, we tried to apply a similar strategy to set up a tetracycline-inducible expression system in 293.Ec, which was selected for ecdysone-regulated expression. When pTRE-GFP, a GFP reporter plasmid constructed under tetracycline-responsive promoter, was transfected into HEK-293 cells stably expressing tTA (293.Ec.1), the fluorescence profiles under induced and noninduced conditions were so similar that it was hard to identify a window to positively select the inducible cells by FACS (Fig. 3A). That window became even more inconspicuous when pTRE-GFP was transfected into another population of HEK-293 cells that stably expressed rtTA (293.Ec.2) (Fig. 3A). Such a dilemma is not related to the fact that ecdysone-inducible system has been established in these particular HEK-293 strains, because similar fluorescence profiles were also observed when pTRE-GFP was transfected into HEK-293 cell strains that do not express ecdysone receptor proteins (data not shown). The main reason for this failure is the transient status of transfected GFP plasmids. Under such conditions, the reporter plasmids are usually multicopied per cell and exist in an episomal form, which precludes the chromatin repression effect and therefore elicits gene expression by the interaction between endogenous transcriptional machinery and the minimal CMV promoter element in the tetracycline-regulated promoter (1, 4).



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Fig. 3. A: ecdysone-regulated HEK-293 cells (293.Ec) were established by drug selection to stably express tetracycline-regulated transactivator (tTA; 293.Ec.1), reverse tTA (rtTA; 293.Ec.2), or rtTA-M2 plus tetracycline-controlled transcriptional silencer (rtTA-M2 + tTS; 293.Ec.3). They were transiently transfected with pTRE-GFP and incubated in the absence or presence of 1 µg/ml doxycycline (Dox) for 40 h before GFP fluorescence was assessed by flow cytometry. B and C: an enriched pool of ecdysone and tetracycline double-inducible HEK-293 cells (293.Ec.3.+2-2) was cotransfected with pIND-LacZ and pTRE-Luc and then analyzed by reporter assay. B: for the tetracycline system, the luciferase activities were measured in the presence (solid bars) or absence of 1 µg/ml Dox (open bars) as controls. The activation factors (shaded bars) were calculated by dividing the luciferase activities under 1 µg/ml Dox with those under no Dox. The assays were performed when the ecdysone promoter-driven reporter was either turned off (–Pon) or on (+Pon) for comparison. C: for the ecdysone system, the {beta}-galactosidase reporter activities were measured in the presence of 5 µM ponasterone (solid bars) or vehicles (open bars) as controls. The activation factors (shaded bars) were calculated by dividing the {beta}-galactosidase activities under 5 µM ponasterone with those under no ponasterone. The assays were performed when the tetracycline promoter was either turned off (–Dox) or on (+Dox) for comparison. Each result (mean ± SE) was from a triplicate experiment. Every assay was repeated at least 3 times.

 

To solve this problem, we adopted the tetracycline-controlled activation/repression system that had been demonstrated to reduce unwanted background expression of tetracycline promoter-regulated transgene (3, 4, 22). By fusing KRAB silencing domains to TetR, a tetracycline-controlled silencer (tTS) is generated that binds to the operator sequence within the tetracycline-regulated promoter and actively shields the promoter from transcriptional activation in the absence of tetracycline. Addition of tetracycline releases tTS from the promoter and at the same time activates the promoter via rtTA (4). Furthermore, we adopted an engineered version of rtTA, rtTA-M2, in this transactivator/transrepressor system because rtTA-M2, which possesses five amino acid substitutions (S12G, E19G, A56P, D148E, and H179R) of rtTA, had been proved to display a considerably lower background activity and higher range of induction than rtTA (13, 25). The HEK-293 line, which had been installed with the ecdysone-inducible expression system (293.Ec; Fig. 2), was transfected with pM2-IRES-Puro and pTet-TS, followed by puromycin selection. Initial assay of the selected cells (293.Ec.3) confirmed the efficiency of the tetracycline-controlled activation/repression system for reducing background activity of transiently transfected pTRE-GFP (Fig. 3A). We then processed 293.Ec.3 for two rounds of positive selections and two rounds of negative selection using the FACS strategy described above and designated the resulting cells as 293.Ec.3.+2-2. Luciferase activity assay showed that the reporter gene could be efficiently regulated by doxycycline in this cell line; the inducibility of the reporter was up to two orders of magnitudes (Fig. 3B). Furthermore, its expression was independent of the expression status of the {beta}-galactosidase reporter gene controlled by the ecdysone-inducible system (Fig. 3B). Similarly, the expression of ecdysone promoter-driven {beta}-galactosidase reporter was also independent of the status of tetracycline system-controlled luciferase reporter (Fig. 3C).

A tetracycline and ecdysone double-inducible system assists delineation of signaling hierarchy of Ras-related small GTPases involved in regulating anoikis. To demonstrate the usefulness of a double-regulated expression system in dissecting complex signaling pathways within a cellular context, we set up a stable MDCK cell line capable of inducible expression of two related small GTPases, Cdc42 and Rac1, under ecdysone- and tetracycline-regulated expression systems, respectively. We first introduced the ecdysone-inducible system, using the FACS-based selection strategy described, into a Rac1 dominant negative mutant (Rac1N17)-expressing MDCK cell line that had been demonstrated to express the mutant GTPase tightly and efficiently under a tetracycline-repressible system (10, 11). We named this population of ecdysone-inducible cells Rac1N17.1. A constitutively active Cdc42 mutant (Cdc42V12)-expressing plasmid was then constructed under the control of the ecdysone-inducible promoter. This plasmid was stably transfected into the Rac1N17.1 to generate a MDCK clone in which myc-tagged Rac1N17 and FLAG-tagged Cdc42V12 mutants could be tightly, efficiently, and independently expressed under tetracycline- and ecdysone-regulated expression systems, respectively (Fig. 4A). Rac1 and Cdc42 both belong to the RhoA family of the Ras superfamily of GTPases. Cdc42 has been demonstrated to act upstream of Rac1, and Rac1 upstream of RhoA, in controlling actin cytoskeleton organization in fibroblasts (19). To examine whether the same signaling cascade also plays a role in anoikis (5), a detachment-induced apoptotic process, we took advantage of the Rac1N17 and Cdc42V12 double-expressing cell line and observed how differential expression of these two transgenes affected apoptosis of MDCK cells kept in suspension condition. Figure 4B shows expression of Rac1N17-enhanced anoikis of MDCK cells, as demonstrated previously (2), whereas Cdc42V12 inhibited anoikis of MDCK cells, and expression of both mutants resulted in an enhancing effect on anoikis. This finding implies that Rac1 acts downstream to Cdc42 along the signaling pathway governing detachment-induced apoptosis in MDCK cells.



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Fig. 4. Effect of expressing Ras-related small GTPases on detachment-induced apoptosis in MDCK cells. A: myc-tagged Rac1N17 and FLAG-tagged Cdc42V12 expression is controlled by tetracycline-repressible and ecdysone-inducible systems, respectively, in a MDCK stable clone. Expression levels of transgenes and the endogenous proteins were assessed by Western blot analysis, using antibodies recognizing both the endogenous and the epitope-tagged mutant GTPases (denoted by an asterisk). B: apoptosis was initiated by maintaining MDCK cells in suspension condition for 16 h, and fragmented DNA-histone complex was quantified as an index of apoptosis. Each result (mean ± SE) was from a quadruplicate experiment. Every assay was repeated at least 3 times.

 

A double-inducible system facilitates identification of signaling molecules relaying complex signaling pathways by proteomic assays. Besides its effect on actin cytoskeleton organization, the Rac1 small GTPase also regulates gene expression through its effect on transcriptional factors (26). Rac1 has been shown to regulate NF-{kappa}B activity in response to physiological and pathological stimuli such as mechanical stretch and hypoxic stress (12, 20, 24). Although Rac1 has been demonstrated to activate MEKK1, which activates JNK, and JNK in turn activates NF-{kappa}B by stimulating degradation of I{kappa}B (15), there might be other unknown pathways linking Rac1 and I{kappa}B. Taking advantage of the ecdysone and tetracycline double-regulated 293.Ec.3.+2-2 cell line, we transiently expressed constitutively active Rac1 and dominant negative I{kappa}B genes and explored the possibility of using this double-inducible system to dissect the signaling pathways linking Rac1 and I{kappa}B. We transfected constitutively active Rac1 gene to 293.Ec.3.+2-2 under the control of tetracycline-regulated promoter (pU-MRac1-V12). By Western blot, we demonstrated that this myc epitope-tagged Rac1 mutant could be tightly regulated by doxycycline (Fig. 5A). In the mean time, a truncated mutant missing the first 70 amino acids of I{kappa}B{alpha} was cloned downstream to the ecdysone-responsive promoter to make pIND-I{kappa}B{alpha}(DN). Deletion of the NH2-terminal 70 amino acids of I{kappa}B removes the potential phosphorylation sites at serine 32 and 36 positions by I{kappa}B kinase and has been demonstrated to inhibit subsequent activation of endogenous NF-{kappa}B in a dominant negative fashion (personal communication, Yen-Shen Lu). The expression of this transgene was robustly induced with the addition of 5 µM of ponasterone, whereas the transgene was not detected under noninduced conditions (Fig. 5B). We obtained cellular lysates for two-dimensional gel electrophoresis under three different conditions: 1) neither Rac1V12 nor I{kappa}B(DN) was expressed; 2) only Rac1V12 was expressed; and 3) both Rac1V12 and I{kappa}B(DN) were expressed. After comparing protein expression profiles for each condition, we could detect several types of changes in expression pattern [Fig. 5, C–F; please also refer to the Supplemental Material1 for this article (published online at the American Journal of Physiology-Cell Physiology web site) to view whole images of the silver-stained gels]. There were spots on silver-stained two-dimensional gels that were not visible under condition 1 but that became visible under conditions 2 and 3 (Fig. 5C). In contrast, there were spots that appeared only under condition 2 (Fig. 5D). The former likely represent Rac1 downstream genes that are not activated through NF-{kappa}B, whereas the latter represent Rac1 downstream genes that are dependent on NF-{kappa}B activation. In addition, we also detected spots with changes in relative intensity or distribution, and these possibly represent proteins whose posttranslational modification status are affected by the activities of Rac1 and NF-{kappa}B (Fig. 5, E and F). These findings disclose latent candidates along Rac1 signaling pathways and exemplify the potential of applying this double-regulated expression system in proteomic studies to dissect complex signaling pathways.



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Fig. 5. Western blot analysis for transgene expression in tetracycline and ecdysone double regulated HEK-293 cells. pU-MRac1-V12 and pIND-I{kappa}B{alpha}(DN) were cotransfected into 293.Ec.3.+2-2. Cell lysates were harvested under conditions in which tetracycline- and ecdysone-inducible expression systems were differentially operated. A: endogenous Rac1 as well as Rac1V12 expression was detected by a mouse anti-Rac1 monoclonal antibody. Comparable endogenous Rac1 signal indicates equal loading of cell lysates. B: both endogenous I{kappa}B (asterisk) and the truncated I{kappa}B dominant negative mutant (arrow) were detected with I{kappa}B-specific antibody. C–F: HEK-293.Ec.3.+2-2 double-regulated cells were cotransfected with pU-MRac1-V12 and pIND-I{kappa}B{alpha}(DN) and split into 3 aliquots after transfection. The first aliquot was added with 1 µg/ml Dox to induce the expression of constitutive active Rac1, the second aliquot was added with 1 µg/ml Dox and 5 µM Pon to induce both constitutive active Rac1 and dominant negative I{kappa}B, and the third aliquot was left without any inducer as a negative control. Representative regions of parallel processed and silver-stained gels are shown, and spots of interest are denoted by filled arrowheads (C), double filled arrowheads (D), arrows (E), and open arrowheads (F). Whole images of the silver-stained gels may be viewed online as supplemental material to this article.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major challenge for biomedical researchers in the post-genomic era is to identify which gene(s), among thousands of candidates found by methods such as library subtraction, differential display, and DNA microarrays, are truly important in disease pathogenesis and are genuine molecular targets for drug development. Although the above-described molecular genetic tools are sensitive enough to pick up potential candidates in terms of their differential expression levels under different physiological or pathological conditions, whether they are directly linked to pathogenesis still awaits further functional assays in a cellular or organismal context. One powerful way of studying gene functions is to analyze the phenotypic changes as the expression of the gene of interest is varied in a precisely controlled manner. Fine-tuning of gene activity is of particular importance for metabolic pathway or developmental switching when quantitative parameters usually define ultimately different outcomes. Furthermore, if the genes of interest are potentially toxic while overexpressed, such as those involved in the cell death signaling, there can be tremendous difficulties in generating useful stable cell clones. Regulated gene expression system is thus an appealing solution to these problems.

Although regulated gene expression system is such a goal for cell biologists, it is by no means easily attainable using an individual cell cloning strategy. Besides the conventional screening strategy using luciferase assay, there are alternative strategies such as screening clones with integrated tetracycline-regulated transactivator gene by PCR amplification of the DNA extracted from the candidates or screening them by using tTA- or rtTA-specific antibodies. Although these methods offer alternative options for luciferase assay, these strategies still rely on individual cell cloning and are therefore very time and effort demanding. Furthermore, positive signals from either a PCR or Western blot result could poorly correlate with the inducibility of the selected clones. In fact, strong expression of regulatory proteins such as tTA or rtTA seems to be negatively selected because they may squelch endogenous transcriptional factors (8). The traditional strategy of selecting optimal responder is very demanding, in that it could cost an experienced researcher ~6 mo to assessing 230 clones before finally identifying an ideal responder (personal communication, Y. Altschuler, Hebrew University of Jerusalem). In contrast, our new strategy routinely screens 500–1,000 candidate clones collectively at the same time, thanks to the conjugated use of inducible GFP reporters and FACS. On average, each round of positive or negative selection takes 7–10 days, depending on the recovery percentage of sorted cells, and the full time course for enriching a useful population of inducible cells takes just a few months. This advantage certainly enhances the possibility of selecting tightly regulable and highly inducible clones. In addition, we have not observed any drifting of the induction/suppression phenotypes of the double-regulated MDCK and HEK-293 cell lines after more than 20 passages, which demonstrates the long-term applicability of this system.

During preparation of this article, we noted that a similar FACS selection strategy had been proposed for generating an inducible expression system (22, 23). Whereas tetracycline- and ecdysone-inducible systems could be established efficiently in retrovirus-transduced cells, the retrovirus-based GFP reporters are stably integrated in the genome of the established cell lines and may interfere with some application, e.g., subcellular localization by immunofluorescence. Although the inducible GFP reporters used in our FACS-based strategy might also integrate into the genome of some host cells after repeated transient transfections, there were still cells possessing inducible expression characteristics without having the GFP reporter integrated in their chromosomes (data not shown). Furthermore, the major difference between our work and the retroviral approach resides in our attempt to extend this FACS-based strategy to set up a tetracycline-inducible system and final success in combining ecdysone- and tetracycline-inducible systems together for broader application.

Although we generated the ecdysone and tetracycline expression systems sequentially by FACS-based sorting of GFP fluorescent cells, it is theoretically possible to set up a double inducible system simultaneously. Substitution of one of the two inducible GFP reporters, pIND-GFP or pTRE-GFP, with construct expressing red fluorescence-emitting protein such as DsRed would permit establishment of ecdysone and tetracycline systems at the same time by two-color fluorescence sorting (14).

The combination of tetracycline-regulated and ecdysone-inducible expression systems offers an experimental modality of inducing two genes of interest independently in a temporal and dose-controlled manner. This system could be combined with research tools with a molecular precision at a genomic scale, such as DNA microarray or proteomic assay, to dissect complex signaling pathways. Although thousands of genes could be analyzed simultaneously by these global approaches, the candidates' number might be just too elusive for meaningful target genes to be identified precisely. The advantage of two independent inducible expression systems allows massively parallel studies of the interaction of two candidate genes in a homogenous cellular environment. Consider the example of Rac1 and NF-{kappa}B for illustration of the potential of this dual regulatory expression system. Rac1 is known to activate NF-{kappa}B, but Rac1 does not directly link to NF-{kappa}B. To search for the possible molecules connecting Rac1 to NF-{kappa}B along the pathway, constitutively active Rac1 (Rac1V12) and dominant negative I{kappa}B [I{kappa}B-(DN)] could be inducibly expressed independently using the system described above. Conceivably, there are three groups of proteins (groups X, Y, and Z) whose expression patterns could be affected by the expression of Rac1V12. Assuming that group X directly relays the signaling between Rac1 and NF-{kappa}B, group Y is indirectly activated by Rac1 through the influence of NF-{kappa}B, whereas group Z proteins are specifically affected by Rac1 but not related to the activation of NF-{kappa}B. If we induce the expression of I{kappa}B-(DN) on top of Rac1V12 expression, group Y proteins would be back to their baseline expression pattern (as in Fig. 5D) and group Z proteins would be not affected at all (as in Fig. 5C), whereas the expression of group X proteins might be affected in a complicated pattern (as in Fig. 5, E and F). Therefore, interesting spots could be pursued by sensitive assay such as microcapillary liquid chromatography followed by mass spectrometric identification.

In summary, a dual regulatory expression system, when combined with global expression approaches such as DNA microarrays or proteomic assays, could be an invaluable research tool for signaling transduction studies.


    DISCLOSURES
 
This work was supported by National Science Council, Taiwan, R.O.C. Grants NSC 89-2323-B-002-010 and NSC 90-2323-B-002-009 (to T.-S. Jou), National Taiwan University Hospital Grant NTUH 92-S014 (to T.-S. Jou), and National Institutes of Health grants (to K. E. Mostov).


    ACKNOWLEDGMENTS
 
We express gratitude to Dr. Hermann Bujard for sending the plasmids encoding tTA and rtTA and to Dr. Wolfgang Hillen for sending plasmid encoding the rtTA mutant M2. We also appreciate the technical aid from Hsiao-Hui Lee and Chi-Feng Cho.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. S. Jou, Dept. of Internal Medicine, National Taiwan Univ. Hospital and National Taiwan Univ. College of Medicine, No. 7 Chung-Shan S. Road, Taipei 100, Taiwan, ROC (E-mail: jouts{at}med.mc.ntu.edu.tw).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supplemental material for this article may be found online at http://ajpcell.physiology.org/cgi/content/full/00064.2003/DC1. Back


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