©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G Requirement for Thrombin-stimulated Gene Expression and DNA Synthesis in 1321N1 Astrocytoma Cells (*)

(Received for publication, May 30, 1995; and in revised form, June 26, 1995)

Anna M. Aragay (1) Lila R. Collins (2)(§) Ginell R. Post (2)(¶) A. John Watson (1) James R. Feramisco (2) Joan Heller Brown (2) Melvin I. Simon (1)(**)

From the  (1)Division of Biology, California Institute of Technology, Pasadena, California 91125 and (2)Department of Pharmacology, University of California, San Diego, La Jolla, California 92093

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of Galpha increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of Galpha subunit was microinjected into 1321N1 cells. Microinjection of the anti-Galpha resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of Galpha(o) did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-Galpha antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for Galpha in the mitogenic response to thrombin in human astroglial cells.


INTRODUCTION

Thrombin is a potent mitogen for fibroblasts, astrocytes, and other cell lines (Van Obberghen-Schilling et al., 1985; Cavanaugh et al., 1990; Hung et al., 1992; LaMorte et al., 1993a, 1993b). Thrombin cleaves and activates a seven-transmembrane-spanning receptor to trigger G-protein-mediated stimulation of downstream effectors (Vu et al., 1991). The actions of thrombin in 1321N1 human astrocytoma cells have been well characterized. In these cells, thrombin stimulates phospholipase C activity leading to mobilization of intracellular calcium, diglyceride generation, and redistribution of protein kinase C (Jones et al., 1989; Nieto et al., 1994). Thrombin receptor activation also leads to a biphasic increase in c-jun mRNA, an associated increase in AP-1 DNA binding activity, and a marked increase in AP-1-mediated gene expression and DNA synthesis (Trejo et al., 1992; LaMorte et al., 1993b). Ras function is required for the mitogenic effect of thrombin as well as thrombin-induced AP-1 transcriptional activity (LaMorte et al., 1993b).

There is substantial information about the signaling pathways downstream of thrombin receptor activation; however, it is not known which of the heterotrimeric G-proteins couples thrombin to the mitogenic pathway. In fibroblasts, thrombin-stimulated DNA synthesis is sensitive to pertussis toxin (PTX) (^1)(Chambard et al., 1987; van Corven et al., 1993). Experiments using microinjected antibodies against Galpha(o) or Galpha(i) suggested that the mitogenic effect of thrombin in CCL39 and in 3T3 fibroblasts is mediated through these G-proteins (LaMorte et al., 1993a; Baffy et al., 1994). However, in 1321N1 astrocytoma cells, thrombin-stimulated DNA synthesis and thrombininduced gene expression are not inhibited by pertussis toxin treatment, (^2)suggesting that G-proteins of the G(i)/G(o) family do not mediate these responses. The PTX-insensitive G-proteins G(q) and/or G have also been reported to function in mitogenic signaling by thrombin and bradykinin in fibroblasts (LaMorte et al., 1993a; Baffy et al., 1994). However, studies comparing muscarinic and thrombin receptor signaling mechanisms in astrocytes and CCL39 fibroblasts demonstrate that, although both receptors interact with G(q)/G to stimulate phospholipase C, release calcium, and activate protein kinase C, only thrombin can activate Ras and elicit cell proliferation (Trejo et al., 1992; Seuwen et al., 1990).^2 These data suggest that activation of G(q) and phospholipase C is insufficient to account for the full mitogenic effects of thrombin and that other signaling pathways are involved.

The Galpha and Galpha subunits constitute a family of G-proteins distantly related to the other G-protein alpha subunits (Strathmann and Simon, 1991). Both Galpha and Galpha lack the cysteine residue that renders these proteins susceptible to ADP-ribosylation by PTX. The signaling pathways regulated by these G-proteins have not been identified. Activation of these G-proteins has nonetheless been linked to cell growth in several cell systems. Overexpression of Galpha in NIH3T3 cells leads to neoplastic transformation (Chan et al., 1993), and expression of a GTPase-deficient mutant form of Galpha and Galpha can efficiently transform NIH3T3 cells (Jiang et al., 1993; Xu et al., 1993) and Rat-1 fibroblasts (Voyno-Yasenetskaya et al., 1994a). In addition, epidermal growth factor-stimulated mitogen-activated protein kinase activity is enhanced in fibroblasts that express the activated mutants of Galpha and Galpha (Voyno-Yasenetskaya et al., 1994b). Therefore, these G-proteins appear to be good candidates to be involved in PTX-insensitive signaling pathways leading to cell growth.

In the present study we show that the activated mutant form of Galpha induces AP-1-mediated transcriptional activation when transfected in 1321N1 cells, and this effect requires Ras function. Furthermore, we show that microinjection of an antibody against Galpha can specifically block the mitogenic effect of thrombin. Taken together, these results suggest that Galpha functions as a PTX-insensitive mediator of thrombin-induced DNA synthesis in 1321N1 cells.


EXPERIMENTAL PROCEDURES

Cell Culture and Microinjection

1321N1 human astrocytoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Cellgrow) supplemented with 5% fetal bovine serum (Gemini) at 37 °C in an atmosphere containing 10% CO(2). For microinjection, cells were plated at 50-60% confluence on 12-mm coverslips, grown for 1 day, and rendered quiescent by serum deprivation for 24 h prior to microinjection. All microinjection experiments were performed using an automatic manipulator (Eppendorf, Fremont, CA). Microinjection needles were pulled on a vertical pipette puller (Kopf, Tujunga, CA). Affinity-purified CT95 (anti-Galpha), CT112 (anti-Galpha(o)), and nonimmune IgG antibodies were dissolved in microinjection buffer containing 20 mM sodium phosphate and 50 mM NaCl and injected into the cytoplasm of quiescent cells (at the concentration indicated in the figure). Three hours postinjection the cells were stimulated by either 0.5 unit/ml thrombin (Sigma), 5% fetal calf serum, or 20 ng/ml basic fibroblast growth factor (bFGF) as indicated in the legends. The thymidine analog bromodeoxyuridine (BrdUrd) (Amersham Corp.) was added to the incubation medium. Twenty-four hours after stimulation the cells were fixed in 95% ethanol and 5% acetic acid and processed for immunofluorescence.

Immunofluorescence Staining

To identify injected cells, the coverslips were washed with phosphate-buffered saline containing 0.1% Tween 20 and incubated for half an hour at 37 °C with a fluorescein-conjugated anti-rabbit antibody (Cappel) at 20 µg/ml in phosphate-buffered saline containing 1 mg/ml bovine serum albumin, 0.5% Nonidet P-40. Cells that had incorporated BrdUrd into synthesized DNA were detected by indirect immunofluorescence as described (LaMorte et al., 1993b). Briefly coverslips were incubated with a monoclonal anti-BrdUrd antibody (Amersham Corp.) followed with a rhodamine-conjugated anti-mouse antibody (Cappel) at 1:100 dilution. The cells were analyzed and photographed with a Zeiss Axiophot fluorescent microscope.

Antibody Purification

Antibodies against Galpha subunits G and G (CT95 and CT112, respectively) were made in rabbits using synthetic peptides conjugated to keyhole limpet hemocyanin. The Galpha C-terminal peptide sequence used to raise the CT95 antibody is CQENLKDIMLQ, and the Galpha(o) C-terminal peptide sequence (CT112) is CANNLRGCGLY. The antibodies were purified by affinity chromatography using the synthetic peptides coupled to an Affi-Gel 15 (Bio-Rad) matrix. Elution was performed with 100 mM glycine, pH 2.5. The eluants were dialyzed against 20 mM phosphate buffer, concentrated, aliquoted, and stored at -70 °C until used.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot

SDS-polyacrylamide gel electrophoresis was performed as described (Laemmli, 1970). COS-7 cells were transiently transfected with the plasmids: pCISG12, pCISG13, pCISGq, or control vector pCISLacZ. Transfected COS-7 and 1321N1 membranes were prepared as described previously (Aragay et al., 1992). One volume of SDS-loading buffer was added to the cell extracts, boiled for 5 min, centrifuged, and loaded onto a 12% SDS-acrylamide gel. Gels were electrotransferred to nitrocellulose membranes, immunostained with Galpha antibodies, and visualized using the ECL method (Amersham Corp.).

Transfection of 1321N1 Cells

Human 1321N1 astrocytoma cells were plated 1 day before transfection and were transfected by the calcium phosphate coprecipitation method (Trejo et al., 1992). Each 60-mm plate received a total of 12 µg of DNA. Varying amounts of pCISG12Q229L or pCISGqR183C or pCDNA1GiR179C or the corresponding control vectors pCIS or pCDNAI were transfected with 4 µg of an AP-1-responsive reporter plasmid (2 TRE) consisting of two consensus TRE sequences (recognized by the AP-1 complex) upstream of the minimal prolactin promoter linked to the luciferase reporter gene (2 TRE-LUC). In some cases control vector was added to achieve a final DNA concentration of 12 µg per plate. For the dominant negative Ras studies, cells were cotransfected with 4 µg of 2 TRE and 4 µg of Ala-15 Ras (Powers et al., 1989) or its control vector, pZIP, and 4 µg of either pCISG12Q229L, pCISGqR183C, or pCISLacZ. 48 h later cells were lysed in 100 mM potassium phosphate, pH 7.9, containing 1% Triton X-100 and 1 mM dithiothreitol. Luciferase activity was measured in 20 µl of lysate as described previously (Trejo et al., 1992).


RESULTS AND DISCUSSION

The thrombin receptor is a member of the G-protein-coupled receptor superfamily and has been shown to couple to G(i), G(o), and G(q) proteins in different signaling pathways (Vu et al., 1991; LaMorte et al., 1993a; Baffy et al., 1994). In addition, the thrombin receptor has been shown to couple to G in platelets (Offermanns et al., 1994). In 1321N1 cells thrombin stimulates the DNA binding activity of the transcription complex AP-1 and results in an associated increase in AP-1-mediated gene expression (Trejo et al., 1992). This response cannot be explained by activation of either G(i) or G(q) alone.^2 To test the possible involvement of Galpha in AP-1-mediated gene expression we examine the ability of an activated GTPase-deficient form of Galpha to transactivate an AP-1-responsive reporter plasmid (2 TRE-LUC). 1321N1 cells were transiently co-transfected with plasmids containing the constitutively activated (GTPase-deficient) mutant forms of Galpha (Q229L), Galpha(q) (R183C), or Galpha(i) (R179C) or with a control plasmid along with the 2 TRE-LUC reporter. The activated Galpha caused a 6-7-fold increase in luciferase activity relative to control transfected cells (Fig. 1A). Only a modest (<2-fold) stimulation was seen with the activated G(q) and G(i) mutants. The marked stimulatory effect of the activated Galpha mutant suggests that G protein may couple thrombin-receptor activation to the induction of the expression of AP-1-responsive target genes.


Figure 1: Effect of Galpha on 2 AP-1-luciferase gene expression. Subconfluent 1321N1 cells were transiently transfected as described under ``Experimental Procedures.'' A, cells were transfected with 3 or 9 µg of either pCISGalpha12Q229L, pCISGalphaqR183C, or pCISGalphaiR179C or backbone vector and assayed for luciferase activity. B, cells were transfected with either pCISGalpha12Q229L or pCISGalphaqR183C in the presence of Ala-15 Ras or its backbone vector pZIP and harvested 48 h later. The cells were subsequently assayed for luciferase activity. The -fold induction is calculated by comparison with cells transfected with the control plasmid and normalized to total protein. Each bar represents the mean ± S.E. of data from three separate experiments, each containing three to four replicates.



To examine the involvement of Ras in the stimulation of AP-1 gene expression, the Ala-15 dominant inhibitory mutant of Ras (Powers et al., 1989) was coexpressed with the activated forms of either Galpha or Galpha(q) and the AP-1-sensitive luciferase reporter plasmid (Fig. 1B). The Galpha(q)-induced increase in AP-1 transcriptional activity was not affected by Ala-15 Ras. However, expression of the Ala-15 Ras mutant led to a 70% reduction in the Galpha-induced luciferase activity. These results demonstrate that Galpha stimulation of AP-1-dependent gene expression at least partially requires Ras. Together with our previous observation that Ras is required for thrombin stimulation of AP-1 activity and mitogenesis (LaMorte et al., 1993b), these results suggest that Galpha may couple the thrombin receptor to Ras activation.

To more specifically examine the involvement of Galpha in thrombin-stimulated mitogenesis, we microinjected inhibitory Galpha antibodies into intact 1321N1 astrocytoma cells. The specificity of the antiserum was first verified by Western blot using extracts of COS-7 cells transiently transfected with plasmids encoding Galpha, Galpha, Galpha(q), and LacZ (Fig. 2). The antiserum raised against a peptide corresponding to the 11 C-terminal amino acids of Galpha (referred to as CT95 or anti-Galpha antibody) recognized a protein of the expected size in COS-7 cells expressing Galpha but did not detect other G-protein alpha subunits. The CT95 antiserum also recognized a protein of the expected size in 1321N1 astrocytoma cells.^2


Figure 2: Identification of Galpha in 1321N1 astrocytoma cells by Western analysis. Extracts of COS-7 cells transiently transfected with pCISGalpha12, pCISGalpha13, pCISGalphaq, and control plasmid (pCISLacZ) and 1321N1 membrane extracts were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotted with CT95 antibody.



Serum-deprived 1321N1 cells were microinjected with either the anti-Galpha antibody, nonimmune IgG, or anti-Galpha(o) antibody (CT112). Three hours later, the cells were treated with 0.5 unit/ml thrombin. The nucleotide analog BrdUrd was added to the medium, and 24 h later the cells were fixed. Injected cells were simultaneously assessed for the presence of injected antiserum and for the nuclear incorporation of BrdUrd by immunofluorescence microscopy (Fig. 3). Thrombin stimulated DNA synthesis in 50 ± 3% of the cells while only 10 ± 2% of unstimulated cells synthesized DNA (Table 1). Injection of the CT95 antibody (10 mg/ml) reduced the proportion of cells in which thrombin stimulated DNA synthesis to 23 ± 3% (Table 1). There was a direct correlation between the amount of antibody injected and the degree of inhibition of BrdUrd incorporation (Fig. 4). Microinjection of 15 mg/ml CT95 antibody resulted in a complete abolition of thrombin-stimulated DNA synthesis but had no effect on basal BrdUrd incorporation in unstimulated cells. Preincubation of the CT95 antibody with the peptide immunogen resulted in loss of the ability of the anti-Galpha antibody to block thrombin-induced DNA synthesis (data not shown). There was no inhibition of the response to thrombin in cells injected with equivalent concentrations of nonimmune IgG. Furthermore, microinjection of an anti-Galpha(o) did not reduce the number of cells undergoing DNA synthesis in response to thrombin (Table 1). These results clearly demonstrate that inhibitory antibodies directed against Galpha can specifically block thrombin-induced DNA synthesis in 1321N1 cells.


Figure 3: Microinjection of CT95 antibody against Galpha in thrombin-stimulated 1321N1 cells. Cells were plated on glass coverslips and starved 24 h prior to injection. Random areas of quiescent cells were injected with control IgG (10 mg/ml) or CT95 antibody (10 mg/ml). Cells were stimulated with 0.5 unit/ml thrombin in DMEM. Representative examples of injected cells are shown in panelsa (IgG) and b (CT95). Panels c, e and d, f represent the same field as a and b, respectively. a, b, phase contrast micrograph; c, d, fluorescent photomicrographs depicting injected cells; e, f, fluorescent photomicrographs depicting injected cells stained for BrdUrd incorporation. Arrows indicate the injected cells.






Figure 4: Percent of DNA synthesis in thrombin-stimulated 1321N1 cells injected with CT95 antibody. Cells were plated on glass coverslips and starved 24 h prior to injection. Quiescent cells were injected in random fields with CT95 and IgG at the concentrations indicated in the lowerpanel. Cells were stimulated with 0.5 unit/ml thrombin in DMEM. DNA synthesis was statistically analyzed using the standard error of proportion comparing injected cells and uninjected cells from the same coverslips. Fields of 200-1200 cells were counted for each point. The results presented represent the means of at least two experiments. Errorbars represent the 95% confidence interval calculated by using the standard error of proportion.



To further demonstrate the specificity of the blocking effect of the CT95 antibody we examined the effect of this antibody on induction of DNA synthesis by bFGF. The actions of bFGF are presumed to be mediated through tyrosine phosphorylation and do not utilize G-protein signaling pathways. Quiescent 1321N1 cells were injected with either the anti-Galpha antibody or control nonimmune IgG and subsequently stimulated with 20 ng/ml bFGF or with 0.5 unit/ml thrombin. bFGF induced DNA synthesis in 22 ± 4% of the cells (Table 2). Microinjection of the anti-Galpha antibody (10 mg/ml) did not inhibit the bFGF-stimulated DNA synthesis. These results indicate that bFGF does not utilize Galpha for mitogenic signaling and that the anti-G antibody does not exert a generalized inhibitory effect upon DNA synthesis. These data further strengthen the argument for the specificity of action of the anti-G antibody on the response to thrombin.



We additionally examined the effect of the CT95 antibody on the serum stimulation of DNA synthesis. The addition of 5% fetal calf serum to serum-deprived 1321N1 cells stimulated DNA synthesis in 76-90% of the cells (Table 2). Microinjection of the anti-Galpha antibody reduced the response to serum by 45%. A similar effect was likewise observed by LaMorte et al.(1992) with an anti-Galpha(i) antibody in serum-stimulated fibroblasts where the thrombin response is PTX-sensitive. The inhibitory effect of the anti-Galpha antibody suggests that serum-induced DNA synthesis in 1321N1 cells is mediated in part through receptor(s) that function via Galpha. This observation argues that G is a necessary cellular transducer for certain types of mitogens in addition to thrombin.

Our previous work^2 demonstrated that the mitogenic response to thrombin in 1321N1 cells was pertussis toxin-insensitive, and we suggested that an additional pathway besides the G(q)/phospholipase C pathway was required. The results presented here suggest that G is the other transducer mediating the mitogenic effect of thrombin in the 1321N1 cells. The inhibition of thrombin-stimulated DNA synthesis by the G antibody coupled with the activation by G of AP-1-dependent transcription reported here provides the first evidence of a signaling pathway in which G links a G-protein receptor to cell growth. It also suggests a new linkage between a G-protein pathway and the oncogene ras.

The mechanism by which G affects cell growth is still not clear. Recent data demonstrate that beta released from stimulation of G(i)-coupled receptors can mediate the activation of MAP kinases acting on a Ras-dependent pathway (Winitz et al., 1993; Albas et al., 1993; Faure et al., 1994; Crespo et al., 1994; Koch et al., 1994). beta released from G by thrombin activation could mediate the mitogenic response. However, the experiments reported here demonstrate that the alpha subunit of G alone is able to induce AP-1-mediated gene expression. Other studies show that Galpha or the constitutively activated Galpha subunits are able to cause cell transformation (Chan et al., 1993; Jiang et al., 1993). It seems likely therefore that there is a pathway activated by the alpha subunit of G, which can lead to cell growth in some systems.

The direct effector of Galpha is not known. Responses shown to be indirectly mediated via G are the activation of phospholipase A(2) (Xu et al., 1993) and of a Na-H exchanger (Dhanasekaran et al., 1994), which is also regulated by G (Voyno-Yasenetskaya et al., 1994c). Since the activation of the Na-H exchanger involves protein kinase C (Dhanasekaran et al., 1994) it is possible that the activation of a particular isoform of protein kinase C by the G pathway may contribute to the mitogenic effect. In addition, Galpha-mediated gene expression is at least partially Ras-dependent, and thus Galpha may regulate proteins responsible for Ras activation. Further work will be necessary to clarify the G effector pathway and the steps leading to mitogenesis.


FOOTNOTES

*
This research was supported in part by National Institutes of Health Grants GM36927 (to J. H. B.) and GM34236 (to M. I. S.). The microinjection facilities were supported by National Institutes of Health Grants CA50528 and CA58689 (to J. R. F.). 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.

§
Supported by National Institutes of Health Predoctoral Fellowship GM17277.

Supported by National Institutes of Health Postdoctoral Training Grant HL07444.

**
To whom correspondence should be addressed: Division of Biology, 147-75, California Institute of Technology, Pasadena, CA 91125.

(^1)
The abbreviations used are: PTX, pertussis toxin; DMEM, Dulbecco's modified Eagle's medium; bFGF, basic fibroblast growth factor; BrdUrd, bromodeoxyuridine; TRE, tetradecanoyl phorbol acetate (TPA) response element.

(^2)
G. R. Post, L. R. Collins, E. D. Kennedy, A. M. Aragay, D. Goldstein, and J. H. Brown, submitted for publication.


ACKNOWLEDGEMENTS

We thank David Goldstein and Carolan Buckmaster for expert technical assistance and Nadia Al-Alawi, Vickie LaMorte, and Vlad Slepak for helpful advice and discussions.


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