G12 Requirement for Thrombin-stimulated Gene Expression and DNA Synthesis in 1321N1 Astrocytoma Cells

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 G12 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 Gα12 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 G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of Gα12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-Gα12 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 Gαo did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-Gα12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for Gα12 in the mitogenic response to thrombin in human astroglial cells. 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 G12 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 Gα12 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 G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of Gα12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-Gα12 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 Gαo did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-Gα12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for Gα12 in the mitogenic response to thrombin in human astroglial cells. INTRODUCTIONThrombin 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) 1The abbreviations used are: PTXpertussis toxinDMEMDulbecco's modified Eagle's mediumbFGFbasic fibroblast growth factorBrdUrdbromodeoxyuridineTREtetradecanoyl phorbol acetate (TPA) response element. (Chambard et al., 1987; van Corven et al., 1993). Experiments using microinjected antibodies against Gαo or Gαi 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, 2G. R. Post, L. R. Collins, E. D. Kennedy, A. M. Aragay, D. Goldstein, and J. H. Brown, submitted for publication. suggesting that G-proteins of the Gi/Go family do not mediate these responses. The PTX-insensitive G-proteins Gq and/or G11 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 Gq/G11 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 Gq and phospholipase C is insufficient to account for the full mitogenic effects of thrombin and that other signaling pathways are involved.The Gα12 and Gα13 subunits constitute a family of G-proteins distantly related to the other G-protein α subunits (Strathmann and Simon, 1991). Both Gα12 and Gα13 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 Gα12 in NIH3T3 cells leads to neoplastic transformation (Chan et al., 1993), and expression of a GTPase-deficient mutant form of Gα12 and Gα13 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 Gα12 and Gα13 (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 Gα12 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 Gα12 can specifically block the mitogenic effect of thrombin. Taken together, these results suggest that Gα12 functions as a PTX-insensitive mediator of thrombin-induced DNA synthesis in 1321N1 cells.EXPERIMENTAL PROCEDURESCell Culture and Microinjection1321N1 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% CO2. 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-Gα12), CT112 (anti-Gαo), 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 StainingTo 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 PurificationAntibodies against Gα subunits G12 and Goa (CT95 and CT112, respectively) were made in rabbits using synthetic peptides conjugated to keyhole limpet hemocyanin. The Gα12 C-terminal peptide sequence used to raise the CT95 antibody is CQENLKDIMLQ, and the Gαo 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 BlotSDS-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 Gα antibodies, and visualized using the ECL method (Amersham Corp.).Transfection of 1321N1 CellsHuman 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 DISCUSSIONThe thrombin receptor is a member of the G-protein-coupled receptor superfamily and has been shown to couple to Gi, Go, and Gq 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 G12 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 Gi or Gq alone.2 To test the possible involvement of Gα12 in AP-1-mediated gene expression we examine the ability of an activated GTPase-deficient form of Gα12 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 Gα12 (Q229L), Gαq (R183C), or Gαi (R179C) or with a control plasmid along with the 2 × TRE-LUC reporter. The activated Gα12 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 Gq and Gi mutants. The marked stimulatory effect of the activated Gα12 mutant suggests that G12 protein may couple thrombin-receptor activation to the induction of the expression of AP-1-responsive target genes.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 Gα12 or Gαq and the AP-1-sensitive luciferase reporter plasmid (Fig. 1B). The Gαq-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 Gα12-induced luciferase activity. These results demonstrate that Gα12 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 Gα12 may couple the thrombin receptor to Ras activation.To more specifically examine the involvement of Gα12 in thrombin-stimulated mitogenesis, we microinjected inhibitory Gα12 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 Gα12, Gα13, Gαq, and LacZ (Fig. 2). The antiserum raised against a peptide corresponding to the 11 C-terminal amino acids of Gα12 (referred to as CT95 or anti-Gα12 antibody) recognized a protein of the expected size in COS-7 cells expressing Gα12 but did not detect other G-protein α subunits. The CT95 antiserum also recognized a protein of the expected size in 1321N1 astrocytoma cells.2Figure 2:Identification of Gα12 in 1321N1 astrocytoma cells by Western analysis. Extracts of COS-7 cells transiently transfected with pCISGα12, pCISGα13, pCISGαq, and control plasmid (pCISLacZ) and 1321N1 membrane extracts were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotted with CT95 antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Serum-deprived 1321N1 cells were microinjected with either the anti-Gα12 antibody, nonimmune IgG, or anti-Gαo 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-Gα12 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-Gαo 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 Gα12 can specifically block thrombin-induced DNA synthesis in 1321N1 cells.Figure 3:Microinjection of CT95 antibody against Gα12 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 as a and contrast c, d, fluorescent injected fluorescent injected cells for BrdUrd the injected Large Image Figure ViewerDownload Hi-res image Download in a Figure of DNA synthesis in thrombin-stimulated 1321N1 cells injected with CT95 Cells were plated on glass coverslips and starved 24 h prior to injection. cells were injected in with CT95 and IgG at the concentrations indicated in the Cells were stimulated with 0.5 unit/ml thrombin in DMEM. DNA synthesis was analyzed using the of proportion comparing injected cells and cells the of cells were for The results represent the of at least two represent the 95% by using the of Large Image Figure ViewerDownload Hi-res image Download demonstrate the specificity of the effect of the CT95 antibody we examined the effect of this antibody on induction of DNA synthesis by The actions of are to be mediated through and do not G-protein signaling 1321N1 cells were injected with either the anti-Gα12 antibody or control nonimmune IgG and stimulated with 20 ng/ml or with 0.5 unit/ml thrombin. DNA synthesis in ± of the cells (Table 2). Microinjection of the anti-Gα12 antibody (10 mg/ml) did not the DNA synthesis. These results that not Gα12 for mitogenic signaling and that the antibody not a inhibitory effect DNA synthesis. These data the for the specificity of of the antibody on the response to in a We examined the effect of the CT95 antibody on the serum stimulation of DNA synthesis. The of 5% fetal calf serum to 1321N1 cells stimulated DNA synthesis in of the cells (Table 2). Microinjection of the anti-Gα12 antibody reduced the response to serum by effect was observed by LaMorte et with an antibody in fibroblasts the thrombin response is The inhibitory effect of the anti-Gα12 antibody suggests that DNA synthesis in 1321N1 cells is mediated in through that function This observation that G12 is a for of in to previous that the mitogenic response to thrombin in 1321N1 cells was pertussis and we suggested that an the C was The results suggest that G12 is the other the mitogenic effect of thrombin in the 1321N1 cells. The inhibition of thrombin-stimulated DNA synthesis by the G12 antibody coupled with the activation by G12 of AP-1-dependent transcription reported the first of a signaling in which G12 a G-protein receptor to cell also suggests a between a G-protein and the by which G12 cell growth is not data demonstrate that stimulation of receptors can mediate the activation of on a Ras-dependent et al., 1993; et al., 1993; et al., et al., et al., 1994). G12 by thrombin activation mediate the mitogenic However, the experiments reported demonstrate that the α subunit of G12 is to AP-1-mediated gene expression. studies show that Gα12 or the constitutively activated Gα12 subunits are to cell transformation (Chan et al., 1993; et al., 1993). that is a activated by the α subunit of which can to cell growth in some direct of Gα12 is not shown to be mediated G12 are the activation of phospholipase et al., 1993) and of a et al., which is also regulated by (Voyno-Yasenetskaya et al., the activation of the protein kinase C et al., it is possible that the activation of a of protein kinase C by the G12 may to the mitogenic In addition, gene expression is at least partially and Gα12 may proteins for Ras be to the G12 and the leading to INTRODUCTIONThrombin 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) 1The abbreviations used are: PTXpertussis toxinDMEMDulbecco's modified Eagle's mediumbFGFbasic fibroblast growth factorBrdUrdbromodeoxyuridineTREtetradecanoyl phorbol acetate (TPA) response element. (Chambard et al., 1987; van Corven et al., 1993). Experiments using microinjected antibodies against Gαo or Gαi 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, 2G. R. Post, L. R. Collins, E. D. Kennedy, A. M. Aragay, D. Goldstein, and J. H. Brown, submitted for publication. suggesting that G-proteins of the Gi/Go family do not mediate these responses. The PTX-insensitive G-proteins Gq and/or G11 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 Gq/G11 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 Gq and phospholipase C is insufficient to account for the full mitogenic effects of thrombin and that other signaling pathways are involved.The Gα12 and Gα13 subunits constitute a family of G-proteins distantly related to the other G-protein α subunits (Strathmann and Simon, 1991). Both Gα12 and Gα13 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 Gα12 in NIH3T3 cells leads to neoplastic transformation (Chan et al., 1993), and expression of a GTPase-deficient mutant form of Gα12 and Gα13 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 Gα12 and Gα13 (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 Gα12 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 Gα12 can specifically block the mitogenic effect of thrombin. Taken together, these results suggest that Gα12 functions as a PTX-insensitive mediator of thrombin-induced DNA synthesis in 1321N1 cells.