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Molecular Pharmacology Laboratory, Guthrie Research Institute, Sayre, Pennsylvania
Received September 25, 2003; accepted January 28, 2004
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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q/11 signaling is terminated by transient expression of the COOH-terminal fragment of phospholipase C (PLC-
1ct), the NH2-terminal fragment of G protein-coupled receptor kinase, or the regulator of G protein signaling 2. Pretreatment of all cells expressing wild-type or mutant Rac1 proteins with edelfosine, a phosphatidylinositol-specific PLC inhibitor, or Go 6976, which inhibits conventional protein kinase C (PKC) isoforms, diminishes the M3 mAChR's ability to inhibit proliferation. Our results identify Gq/11, PLC, and PKC as participants in the M3 mAChR-mediated inhibition of cell proliferation. These findings indicate that in the context of high Rac1 activity, but not RhoA activity, M3 mAChR-mediated activation of these participants triggers cell death.
). In contrast, activation of endogenous M3 mAChRs inhibits DNA synthesis in several small-cell lung carcinoma cell lines (Williams, 2003a). These contradictory responses also occur in cells stably transfected with mAChRs. Transfected M3 mAChRs can function as both agonist-dependent oncogenes (Gutkind et al., 1991) and receptor-mediated tumor suppressors (Felder et al., 1993) in fibroblasts. Activation of transfected M3 mAChRs inhibits or stimulates cell proliferation, depending on the cellular environment (Nicke et al., 1999; Burdon et al., 2002). Thus, activation of mAChRs initiates diverse proliferative responses, which may be unique to individual cellular environments. The delineation of the signaling pathways participating in the mAChR-mediated inhibition of proliferation will clarify the role of mAChRs in pathophysiological processes, including aberrant cell proliferation.
Events mediated by G protein-coupled receptors (GPCRs), including changes in cell proliferation, often involve the activation of small GTPases of the Rho family (Marinissen and Gutkind, 2001). Signals generated by Rac1 and RhoA can provide both stimulatory and inhibitory signals for cell proliferation (Aznar and Lacal, 2001). Microinjection of Rac1 or RhoA into quiescent fibroblasts stimulates cell-cycle progression and subsequent DNA synthesis (Olson et al., 1995). The expression of dominant-negative Rac1 is associated with arrest in the G2/M phase (Moore et al., 1997) and diminished proliferative responses to GPCR activation (Burstein et al., 1998). However, activation of Rac and Rho proteins may also trigger cell death (Esteve et al., 1998). Therefore, Rac1 or RhoA may act to stimulate cell proliferation or apoptosis depending on the cell context and environment (Aznar and Lacal, 2001).
Our findings that Rac1 and RhoA participate in M3 mAChR-initiated events (Strassheim et al., 1999; Ruiz-Velasco et al., 2002) prompted us to investigate the role of Rac1 and RhoA in the M3 mAChR-mediated inhibition of cell proliferation. Using Chinese hamster ovary (CHO) cells stably expressing M3 mAChRs (CHO-m3) and coexpressing mutant or wild-type Rac1 (Ruiz-Velasco et al., 2002) or RhoA (Strassheim et al., 1999) proteins, we show that Rac1, but not RhoA, participates in the M3 mAChR-mediated inhibition of cell proliferation. Interestingly, activation of the M3 mAChR in cells overexpressing wild-type Rac1 or expressing constitutively active Rac1Val12 not only suppresses cell proliferation but also induces cell death.
We also tested whether Gq/11, which couples to M3 mAChRs (Wess, 1996), is involved in the M3 mAChR-mediated effects on proliferation. We found that transient transfection with cDNA constructs encoding proteins that inhibit Gq/11 activity completely abrogated the M3 mAChR-induced inhibition of proliferation in all cell lines tested. These results indicate that the antiproliferative effects of mAChR activation require the presence of active Gq/11. Consistent with this observation, we show that inhibition of the Gq/11 signaling cascade using pharmacological inhibitors of phospholipase C (PLC), Ca2+ mobilization, or protein kinase C (PKC) diminishes the antiproliferative effects of M3 mAChR activation.
This study identifies Gq/11, PLC, and PKC as participants in the M3 mAChR-induced inhibition of proliferation. Our results indicate that in the context of high Rac1 activity, but not RhoA activity, the M3 mAChR-mediated activation of these participants triggers cell death. These findings suggest that specific intracellular cues, such as high Rac activity, can escalate the M3 mAChR-mediated inhibition of cell proliferation to cell death.
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Materials and Methods |
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Cell Lines. The CHO-K1 sublines stably transfected with the M1, M2, or M3 mAChR are referred to as CHO-m1, CHO-m2, and CHO-m3, respectively. Although CHO cells express endogenous Rac1 (Ruiz-Velasco et al., 2002) and RhoA (Miao et al., 2002), we have used clonal sublines of CHO-m3 cells stably coexpressing HA-tagged wild-type or mutant Rac1 (Ruiz-Velasco et al., 2002) or RhoA proteins (Strassheim et al., 1999) for our studies. The m3WTRho-1, m3CARho-4, and m3DNRho-2 cells are CHO-m3 sublines stably expressing HA-RhoA, constitutively active HA-RhoAVal14, and dominant-negative HA-RhoAAsn19, respectively (Strassheim et al., 1999). The m3WTRac, m3CARac, and m3DNRac cells are CHO-m3 sublines stably expressing HA-Rac1, constitutively active HA-Rac1Val12, and dominant-negative HA-Rac1Asn17, respectively (Ruiz-Velasco et al., 2002). The m3Zeo-2 cells are CHO-m3 cells stably transfected with the pZeoSV2 plasmid. All cells were maintained in complete medium consisting of Ham's F-12 medium supplemented with heat-inactivated fetal calf serum (5%), glutamine (0.3 µg/ml), penicillin (20 U/ml), and streptomycin sulfate (20 µg/ml).
Assay of RhoA Activity. The bacterial expression vector coding for the Rho binding domain of rhotekin tagged to glutathione S-transferase (GST) was generously provided by Dr. Martin Schwartz (Scripps Research Institute, La Jolla, CA). The assays were performed as described previously (Varker et al., 2003). Cells were permeabilized by a freeze-thaw cycle and incubated in reaction buffer (50 mM HEPES, 100 mM NaCl, 2 mM EDTA, and 6 mM MgCl2, pH 7.4) in the presence or absence of 100 µM carbachol (CCh) or 100 µM GTP
S for 30 s at 37°C. The cells were then solubilized in ice-cold lysis buffer (50 mM Tris, 10 mM MgCl2, 500 mM NaCl, 0.2% SDS, 2% Triton X-100, 1% sodium deoxycholate, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.2) and centrifuged (14,000g for 10 min at 4°C). The resulting supernatants were incubated with the bacterially expressed, GST-tagged Rho binding domain of rhotekin followed by precipitation with glutathione-Sepharose 4B beads, as described previously (Varker et al., 2003). The precipitated HA-tagged RhoA GTP was detected by ECL Western blotting using antibody to HA.
Assay of Rac1 Activity. The bacterial expression vector coding for the GST-tagged p21 binding domain of the p21-activated kinase (GST-PBD) was generously provided by Dr. Gary Bokoch (Scripps Research Institute). The assays were performed as described previously (Ruiz-Velasco et al., 2002). Cells were subjected to a freeze-thaw cycle, incubated with 100 µM CCh or 100 µM GTPS for 3 min at 37°C, and lysed in lysis buffer. The lysates were incubated with bacterially expressed GST-PBD followed by precipitation with glutathione-Sepharose 4B beads, as described previously (Ruiz-Velasco et al., 2002). The precipitated HA-tagged Rac1 GTP was detected by ECL Western blotting using antibody to HA.
Transfection of Cells with cDNA Constructs. The cDNA constructs coding for green fluorescent protein (GFP), GFP-PLC-1ct and GFP-RGS2 in the pEGFP-C1 vector and GFP-GRK-nt in the pEGFP-N1 vector (all from BD Biosciences Clontech, Palo Alto, CA), were a generous gift of Dr. Stephen Ikeda (National Institutes of Health, Bethesda, MD). Cells were transfected by suspension in Ham's F-12 medium (6 x 106 cells/200 µl medium) containing 15 µg of the indicated cDNA and electroporated by a single electric pulse (200 V for 50 ms) using a BTX Electro Square Porator (Genetronics Inc., San Diego, CA). The electroporated cells were transferred to tissue culture plates and incubated for 24 h (at 37°C in 5% CO2) in complete Ham's F-12 medium.
BrdU Uptake. BrdU incorporation was measured using a modification of a previously described assay (Olson et al., 1995). Cells electroporated with different cDNA constructs were plated onto glass coverslips in complete CHO medium (5 x 104 cells/ml medium). After 1 h, media with or without CCh (final concentration, 10 µM) were added to the wells, and the cells were incubated for an additional 16 to 24 h before being incubated with BrdU, according to the manufacturer's specifications (Amersham Biosciences) for 3 h (at 37°C). The cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (for 15 min at 4°C) and permeabilized with acid/alcohol (90% ethanol, 5% distilled water, and 5% acetic acid) (90 s at 25°C). After incubating with PBS containing 1% bovine serum albumin (30 min at 25°C), the cells were incubated with mouse antibody to BrdU (90 min at 25°C), followed by incubation with TRITC-labeled anti-mouse IgG (1 h at 25°C). The cells were mounted in PBS containing 90% glycerol and 0.1% p-phenylenediamine and examined using an Optiphot fluorescence microscope (Nikon, Melville, NY).
To assess the percentage of BrdU-positive cells, the cells were examined by an investigator without knowledge of the treatment of the cells or the identity of the GFP-tagged protein expressed by the cells. In each assay, investigators examined at least 90 different cells transfected with the same cDNA and subjected to the same treatment.
DNA Fragmentation Assay. A DNA fragmentation assay to indicate apoptosis was performed using a modification of a previously reported protocol (Mandlekar et al., 2000). Cells were plated (2 x 106 cells/plate) in complete CHO medium. After 1 h, media with or without CCh (final concentration, 10 µM) were added to the plates, and the cells were incubated for an additional 12 or 24 h. Detached cells suspended in culture supernatants were collected and pooled with adherent cells that were harvested by trypsinization. The cells were washed with PBS and resuspended in a buffer of 10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.5% Triton X-100 to lyse the cells. The cell lysates were incubated on ice for 60 min. DNA was extracted by adding an equal volume of neutral phenol/chloroform/isoamyl alcohol mixture, pH 8.0 (Fisher Scientific Co., Fair Lawn, NJ) and precipitated with 2 volumes of 100% ethanol and 0.1 volume of 5 M NaCl at -20°C overnight. The precipitates were dissolved in 10 mM Tris, pH 8.0, and 1 mM EDTA and treated with 1 µg/ml Rnase A at 37°C for 2 h. DNA fragments were resolved by electrophoresis in a 2% agarose gel and visualized by ethidium bromide staining.
[3H]Thymidine Uptake. Uptake of [3H]thymidine by cells was used as an indicator of DNA synthesis as described previously (Varker et al., 2003). Cells were plated in 96-well plates with or without the indicated drugs in complete growth medium to promote exponential proliferation. One hour after plating, additional media with or without the stated concentrations of CCh and [3H]thymidine were added. The cells were incubated for the indicated times in a humidified atmosphere of 5% CO2/95% air. Before harvesting, the cells were incubated (20 min at 37°C) with PBS containing 5 mM EDTA and 5 mM EGTA to induce detachment of the cells from the microtiter plates. The cells were washed and lysed with distilled water and collected on filters using an automatic cell harvester (Skatron, Sterling, VA). The filters were placed in ScintiSafe Econo 1 scintillation fluid (Fisher Scientific) and counted with the use of an LS-6000 -counter (Beckman Coulter, Inc., Fullerton, CA).
Immunofluorescence Assays. Immunofluorescence assays were conducted by a modification of a previously described protocol (Ruiz-Velasco et al., 2002). Cells electroporated with different cDNA constructs were plated onto glass coverslips in complete CHO medium (5 x 104 cells/ml medium). After 2 days in culture, the media were replaced with fresh media with or without CCh (final concentration, 10 µM), and the cells were incubated for an additional 30 min. The cells were fixed in 4% paraformaldehyde in PBS (15 min, 4°C) and permeabilized in Triton X-100 (0.2%, 10 min at 25°C). After incubating with PBS containing 1% bovine serum albumin (30 min at 25°C), the cells were incubated with mouse antibody to HA (1 h at 25°C) followed by incubation with TRITC-labeled anti-mouse IgG (1 h at 25°C). The cells were mounted and examined as described above. Digital images of the cells were collected using a Kodak DC 290 zoom digital camera (Eastman Kodak, Rochester, NY) and Adobe Photoshop software (Adobe Systems, Mountain View, CA).
Cell-Cycle Analysis. Cells were plated at 106 cells/10 cm dish with or without 30 µM edelfosine or 2 µM Go 6976. One hour after plating, additional media with or without CCh (final concentration, 10 µM) were added, and the cells were incubated for 24 h. The cells were harvested, washed, and resuspended in 5 mM EDTA in PBS. The cells were fixed in 50% ethanol, incubated with Rnase cocktail (500 U/ml Rnase A, 20,000 U/ml Rnase T1) for 30 min, and stained with propidium iodide. The samples were analyzed using a fluorescence-activated cell sorter (FACSCalibur, BD Biosciences, San Jose, CA).
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Results |
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q/11 subunits, but not by activation of mAChRs, which couple to Gi/o.
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Mobilization of Intracellular Ca2+ Participates in the M3 mAChR-Induced Inhibition of Proliferation. The mobilization of intracellular Ca2+ is one of the earliest responses to the M3 mAChR-mediated activation of Gq/11 (Wess, 1996). The effects of Ca2+ mobilization on M3 mAChR-induced inhibition of CHO cell proliferation was investigated using BAPTA/AM, a chelator of intracellular Ca2+ (Billman, 1993), and 2-APB, which inhibits inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ fluxes (Maruyama et al., 1997). Interestingly, preincubation of CHO-m3 cells with 25 µM BAPTA/AM (Fig. 2A) or 50 µM 2-APB (Fig. 2B) reduces the ability of the M3 mAChR to inhibit cell proliferation. Thus, the reduction of intracellular Ca2+ levels by chelating intracellular Ca2+ or preventing IP3-induced Ca2+ mobilization diminishes but does not completely abrogate M3 mAChR-induced inhibition of proliferation.
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Activation of the M3 mAChR Activates Rac1 and RhoA. Activation of some GPCRs that couple to Gq/11 has been shown to result in the activation of small GTPases of the Rho subfamily, including RhoA and Rac1 (Strassheim et al., 1999; Marinissen and Gutkind, 2001; Ruiz-Velasco et al., 2002). The effects of M3 mAChR activation on the activities of HA-tagged Rac1 and HA-tagged RhoA were investigated by precipitating the GTP-bound forms of the proteins using GST-PBD or the Rho-binding domain of rhotekin, respectively. The precipitates were examined by ECL Western blotting using antibody to HA (Fig. 3A). We found that the association of HA-Rac1 with GST-PBD and the association of HA-RhoA with the Rho binding domain of rhotekin are increased by M3 mAChR activation (Fig. 3A). These associations are also increased by the nonspecific activation of GTPases with GTPS (Fig. 3A). Thus, the stimulation of M3 mAChR increases GTP binding to both Rac1 and RhoA, indicating activation of these GTPases.
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Overexpression of Rac1, or the Expression of Constitutively Active Rac1Val12 Enhances the Ability of the M3 mAChR to Inhibit Cell Proliferation. We investigated the participation of Rac1 and RhoA in the M3 mAChR-induced inhibition of proliferation using CHO-m3 cell lines stably expressing HA-tagged mutant or wild-type Rac1 or RhoA (Strassheim et al., 1999; Ruiz-Velasco et al., 2002). The m3Zeo-2 cells are CHO-m3 cells stably transfected with the pZeoSV2 plasmid. These cells do not exhibit any detectable proliferative differences from the parental CHO-m3 cell line under basal conditions or upon activation of the M3 mAChR. In the absence of drugs, cells expressing wild-type or mutant HA-RhoA or HA-Rac1 proteins exhibit similar rates of proliferation (Fig. 3, B, C, and D), indicating that the transfected GTPases do not alter normal proliferation. Activation of M3 mAChR significantly diminishes the proliferation of all cell lines investigated (Fig. 3, B and C). Incubation with CCh reduces [3H]thymidine uptake to the same extent in control m3Zeo-2 cells and in cells expressing wild-type RhoA (m3WTRho-1 cells) or mutant RhoA proteins (m3CARho-4 or m3DNRho-2 cells) (Fig. 3B). These findings indicate that RhoA does not participate in the M3 mAChR-induced inhibition of proliferation. In contrast, the expression of HA-tagged wild-type Rac1 in m3WTRac cells or the expression of constitutively active HA-Rac1Val12 in m3CARac cells enhances the M3 mAChR-induced inhibition of proliferation compared with that seen in control m3Zeo-2 cells (Fig. 3, C and D). The expression of dominant-negative HA-Rac1Asn17 in m3DNRac cells diminishes the ability of the M3 mAChR to inhibit proliferation compared with control m3Zeo-2 cells (Fig. 3, C and D). These findings suggest that Rac1 participates in the antiproliferative effect of M3 mAChR activation.
Gq/11 Activity Is Required for the M3 mAChR-Mediated Inhibition of Proliferation. The M3 mAChR is known to signal to Gq/11 subunits (Wess, 1996). Signaling by Gq/11 can be terminated by proteins such as the PLC-1ct, which binds to Gq/11 (Paulssen et al., 1996), RGS2, which acts as a GTPase-activating protein (Heximer et al., 1997), and GRK-nt, which has an RGS2 domain, allowing it to bind to Gq/11 and inactivate it (Carmen et al., 1999). We investigated the involvement of Gq/11 in M3 mAChR-induced inhibition of proliferation using m3Zeo-2 (Fig. 3A), m3WTRac (Fig. 3B), m3CARac (Fig. 3C), and m3DNRac (Fig. 3D) cells transiently transfected with cDNA constructs coding for GFP or GFP-tagged PLC-1ct, GFP-tagged GRK-nt, or GFP-tagged RGS2. Cells transiently transfected with cDNA coding for GFP exhibit mAChR-induced inhibition of DNA synthesis, as indicated by the decrease in the percentage of BrdU-positive cells (Fig. 4). Transient expression of cDNA constructs coding for GFP-tagged PLC-ct, GRK-nt or RGS2 abrogates the M3 mAChR-mediated inhibition of BrdU incorporation in all cell lines examined (Fig. 4). These findings indicate the M3 mAChR-induced inhibition of DNA synthesis, and the resulting decrease in cell proliferation, is dependent on Gq/11 signaling.
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Inhibition of Gq/11 Signaling Diminishes the M3 mAChR-Mediated Translocation of Rac1 to Cell Junctions. Activation of M3 mAChR initiates numerous Rac1-dependent cellular responses, including the translocation of Rac1 from membrane ruffles to cell junctions, which is an indication of Rac1 activation (Ruiz-Velasco et al., 2002). We investigated the role of Gq/11 signaling in the activation of Rac1 by examining the M3 mAChR-induced changes in the intracellular distribution of HA-Rac1 proteins. Incubation of m3CARac cells with CCh for 30 min induces the accumulation of constitutively active HA-Rac1Val12 at cell junctions during mAChR-induced cell-cell compaction (Ruiz-Velasco et al., 2002) (Fig. 5D). Transfection of m3CARac cells with the cDNA construct coding for GFP-tagged GRK-nt diminishes M3 mAChR-induced cell-cell compaction and the translocation of HA-Rac1Val12 to cell junctions (Fig. 5H). In contrast, there is strong junctional localization of HA-Rac1Val12 in CCh-treated cells not expressing GRK-nt (*, Fig. 5H). The expression of GFP-tagged PLC-1ct or GFP-tagged RGS2 by m3CARac cells also diminishes the M3 mAChR-induced translocation of HA-Rac1Val12 to cell junctions (data not shown). These findings indicate that Gq/11 signaling is involved in the M3 mAChR-mediated translocation of HA-Rac1 proteins, suggesting that Gq/11 participates in Rac1 activation by the M3 mAChR.
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M3 mAChR Activation Induces Apoptosis. The M3 mAChR-induced reduction of [3H]thymidine uptake could be caused by a diminished entry or progression through S phase, an increase in cell death, or a combination of both responses. We investigated the possibility that the M3 mAChR-mediated inhibition of proliferation might be caused by the induction of apoptosis by examining DNA fragmentation in cells exposed to CCh for 12 h (Fig. 6, lanes 1-8) or 24 h (Fig. 6, lanes 9-16). Exposure of m3WTRac cells (Fig. 6, lane 4) or m3CARac cells (Fig. 6, lane 6) to CCh for 12 h results in large increases in cell death, primarily caused by apoptosis. The response of the m3Zeo-2 cells to the apoptotic effects of CCh is less marked (Fig. 6, lane 2); no evidence of apoptosis is seen in m3DNRac cells exposed to CCh for 12 h (Fig. 6, lane 8). DNA fragmentation is evident in all cell lines after 24 h of CCh treatment (Fig. 6, lanes 10, 12, 14, and 16). These findings indicate that M3 mAChR activation induces apoptosis in all cell lines studied. This response is most marked in the m3WTRac and m3CARac cells, which are the cell lines most responsive to mAChR-induced inhibition of proliferation (Fig. 3, C and D).
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PI-PLC Participates in the M3 mAChR-Induced Inhibition of Proliferation. M3 mAChR stimulation alters the activity of numerous enzymes that may participate in the inhibition of proliferation. We examined the participation of different proteins in the M3 mAChR-induced inhibition of proliferation by treating m3Zeo-2, m3WTRac, m3CARac, and m3DNRac cells with a number of selective antagonists (Table 1). Treatment of all the sublines with a range of concentrations of inhibitors of phospholipase A2 (PLA2), phosphatidylcholine-specific PLC (PC-PLC), Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), myosin light chain kinase, mitogen-activated protein kinase kinase 1 and 2 (MEK1/2), and phosphatidylinositol 3 kinase do not affect the M3 mAChR-induced inhibition of proliferation.
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Agonist binding to the M3 mAChR causes Gq/11 to associate with PLC-, resulting in activation of the enzyme (Marinissen and Gutkind, 2001). The PI-PLC inhibitor edelfosine (Powis et al., 1992) was used to investigate the role of PLC- in the inhibition of proliferation induced by M3 mAChR activation. Preincubation of all cell lines with edelfosine diminishes the mAChR-induced inhibition of proliferation (Fig. 7). Although this response occurs at all tested concentrations of CCh, edelfosine is most effective at inhibiting the antiproliferative effects of 10 µM CCh (Fig. 7, C, F, I, and L). The "rescue" effect is greatest in the cell lines most susceptible to CCh-induced inhibition of proliferation, the m3WTRac cells, which overexpress wild-type Rac1 (Fig. 7, D-F), and the m3CARac cells, which express constitutively active Rac1Val12 (Fig. 7, G-I). These results indicate that PI-PLC enzymes participate in the M3 mAChR-mediated inhibition of proliferation.
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To examine the effects of mAChR activation on cell-cycle progression, we defined the proportion of the cell population in each phase of the cell cycle and in the sub-Go/G1 cell population after incubation of the cells with 10 µM CCh for 24 h (Fig. 8). Treatment of m3Zeo-2 and m3DNRac cells with CCh significantly depresses the proportion of the cell population in the S phase (Fig. 8, B and N) compared with that seen in untreated cells (Fig. 8, A and M). Very little CCh-induced increase in the proportion of these cells in the sub-Go/G1 population is observed (Fig. 8, B and N). These results indicate that in the m3Zeo-2 and m3DNRac cell lines, activation of the M3 mAChR results in inhibition of DNA synthesis and not in a concomitant massive increase in cell death, although some apoptosis is evident (Figs. 6 and 8, B and N). In contrast, incubation of m3WTRac and m3CARac cells with CCh for 24 h results in a massive increase in cells in the sub-Go/G1 population, indicating a large increase in cell death (Fig. 8, F and J) compared with that of untreated cells (Fig. 8, E and I). The proportion of CCh-treated m3WTRac cells (Fig. 8F) and m3CARac cells (Fig. 8J) in the S phase is greatly reduced compared with cells incubated without CCh (Fig. 8, E and I) and compared with CCh-treated m3Zeo-2 (Fig. 8B) and m3DNRac (Fig. 8N) cells. Thus, activation of the M3 mAChR diminishes cell-cycle progression in the m3Zeo-2 cells (Fig. 8B) and m3DNRac cells (Fig. 8N); in contrast, activation of the M3 mAChR induces cell death in the m3WTRac (Fig. 8F) and m3CARac (Fig. 8J) cells.
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Treatment of cells with edelfosine increases the proportion of cells in the G2/M phase of the cell cycle (Fig. 8). This effect is greater in the m3Zeo-2 (Fig. 8C), m3WTRac (Fig. 8G), and m3CARac (Fig. 8K) cells than in m3DNRac (Fig. 8O) cells. Pretreatment of m3WTRac and m3CARac cells with edelfosine before the addition of CCh decreases the sub-Go/G1 population of cells and increases the proportion of cells in the S and G2/M phases of the cell cycle (Fig. 8, H and L). Thus, inhibition of PI-PLC activity leads to a decrease in M3 mAChR-induced cell death (Fig. 8Q).
Conventional Isoforms of PKC Participate in the M3 mAChR-Induced Inhibition of Proliferation. Activation of PI-PLC by the M3 mAChR results in the generation of diacylglycerol, which in turn activates PKC. We investigated the role of PKC in the M3 mAChR-induced inhibition of proliferation using Go 6976, an inhibitor of conventional PKC isoforms (Martiny-Baron et al., 1993). Preincubation of all the cell lines with Go 6976 diminishes the ability of M3 mAChR activation to inhibit cell proliferation (Fig. 9). Treatment of m3Zeo-2, m3WTRac, m3CARac, and m3DNRac cells with 10 µM CCh results in a reduction of [3H]thymidine uptake to 27 ± 0.9, 9 ± 0.7, 18 ± 1, and 42 ± 1.5% of control, respectively (Fig. 9, C, F, I, and L). Incubation of m3Zeo-2, m3WTRac, m3CARac, and m3DNRac cells with 2 µM Go 6976 followed by 10 µM CCh results in the reduction of [3H]thymidine uptake to 63 ± 2.5, 21 ± 1.7, 46 ± 3.7, and 60 ± 1.5% of control, respectively (Fig. 9, C, F, I, and L). These results indicate that conventional PKC isoforms may participate in the M3 mAChR-induced inhibition of proliferation.
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Cell-cycle studies indicate that treatment of cells with Go 6976 moderately diminishes the proportion of the cell population in the S phase of the cell cycle (Fig. 10, C, G, K, and O). In contrast to edelfosine, which profoundly increases the proportion of G2/M cells (Fig. 8), Go 6976 does not significantly affect G2/M progression. However, Go 6976 does protect cells from CCh-induced cell death. Preincubation of m3WTRac and m3CARac cells with 2 µM Go 6976 reduces the ability of M3 mAChR activation to induce cell death in these two cell lines (Fig. 10, H, L, and Q). Thus, inhibition of PKC activity decreases M3 mAChR-induced cell death (Fig. 7Q) in the m3WTRac and m3CARac cells.
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Discussion |
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q/11 but not by activation of the M2 mAChR that couples to Gi/o, thus confirming previous findings (Detjen et al., 1995; Burdon et al., 2002; Williams, 2003a). The mAChR-induced inhibition of proliferation was completely abrogated in cells transiently expressing PLC-1ct (Paulssen et al., 1996), RGS2 (Heximer et al., 1997), or GRK-nt (Carmen et al., 1999), which are proteins that terminate Gq/11 signaling. Many studies implicate G heterodimers in GPCR-mediated changes in cell proliferation (Marinissen and Gutkind, 2001). However, our findings indicate that the M3 mAChR signal which inhibits proliferation is transduced by the Gq/11 subunit.
Activation of PI-PLC by Gq/11 generates two signaling cascades: the IP3 signaling pathway leading to mobilization of intracellular Ca2+, and the diacylglycerol pathway that activates PKC (Wess, 1996). We have shown that buffering of Gq/11 signaling, inhibition of PI-PLC, inhibition of Ca2+ mobilization, or inhibition of PKC all serve to diminish the ability of the M3 mAChR to inhibit cell proliferation. Thus, all of these signaling components downstream of Gq/11 participate in the M3 mAChR-mediated inhibition of proliferation.
Our findings indicate that activation of M3 mAChR inhibits cell proliferation by Rac1-dependent and Rac1-independent means. Expression of dominant-negative Rac1Asn17 in m3DNRac cells lessens the M3 mAChR-induced inhibition of proliferation, indicating that Rac1 participates in this event. However, the mAChR-mediated inhibition of proliferation may also occur by a Rac1-independent mechanism, because the expression of Rac1Asn17 does not completely abrogate the antiproliferative response. Interestingly, activation of mAChRs in cells expressing increased levels of Rac1 (m3WTRac cells) or in cells expressing constitutively active Rac1Val12 (m3CARac cells) results not only in inhibition of proliferation but also in apoptosis. Thus, a single signal, activation of mAChR, induces the inhibition of proliferation in these cells. In the presence of a second signal, overexpression of active Rac1, cell death is triggered (Fig. 11). Our hypothesis that two signals are required to induce Rac1-mediated cell death is consistent with previously reported findings. Overexpression of Rac1 induces apoptosis only upon serum deprivation in NIH 3T3 fibroblasts and human erythroleukemia K562 cells (Esteve et al., 1998; Embade et al., 2000). Introduction of constitutively active OsRac1, a rice homolog of human Rac1, induces apoptosis only in cells that also carry a lesion mimic mutant of rice (Kawasaki et al., 1999). Thus, the presence of at least two cooperative signals is required for the induction of cell death.
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Interestingly, our results indicate that only Rac1 participates in the mAChR-induced inhibition of proliferation even though stimulation of M3 mAChRs activates both RhoA and Rac1. These findings are consistent with previous observations that both RhoA and Rac1 activity levels are increased after treatment of U937 cells with tumor necrosis factor-, but only Rac1 participates in the inhibition of cell growth and induction of apoptosis (Esteve et al., 1998).
We present findings that support the role of Gq/11 in regulating Rac1-mediated activity. Activation of M3 mAChR induces the accumulation of HA-Rac1Val12 at cell junctions, a process that involves the activated form of Rac1 (Ruiz-Velasco et al., 2002). In m3CARac cells transiently expressing GRK-nt, the translocation of HA-Rac1Val12 to cell junctions is greatly diminished. Our results cannot determine whether signaling through Gq/11 directly activates Rac1. However, dampening of Gq/11 activity does interfere with the ability of M3 mAChR to regulate Rac1 activity. This finding suggests that Gq/11 participates in the activation of Rac1 analogous to the Gq/11-mediated activation of RhoA (Vogt et al., 2003).
We observed that the antiproliferative effects of M3 mAChR activation are greater in cell populations expressing wild-type Rac1 than in those expressing constitutively active Rac1val12 proteins. It is intriguing to speculate that this response occurs because wild-type Rac1 is more effective than constitutively active Rac1Val12 in competitively interacting with a guanine nucleotide exchange factor (GEF) that activates other small GTPases required for cell proliferation. One such GEF may be SmgGDS (Williams, 2003b). Because GEFs only associate with the GDP-bound forms of small GTPases, GEFs should associate more often with wild-type Rac1 than with constitutively active Rac1, which remains in the GTP-bound form for prolonged periods. In the absence of mAChR stimulation, wild-type Rac1 would not compete with other small GTPases for GEFs. However, mAChR activation may stimulate the interaction of wild-type Rac1 with certain GEFs, diminishing the interaction of these GEFs with other small GTPases. If the interaction of these GEFs with other small GTPases is required for cell proliferation or survival, then mAChR activation would have a greater antiproliferative effect in m3WTRac cells than in m3CARac cells. Consistent with these findings, other studies have reported that wild-type Rac1 is more deleterious than constitutively active Rac1 under certain conditions (Esteve et al., 1998). Additional studies are needed to clarify these somewhat surprising findings.
We investigated the role of several enzymes, including PC-PLC, PI3 kinase, PLA2, CaM kinase II, and MEK1/2, which have been identified as possible participants in mAChR- or Rac1-initiated pathways, to determine whether they are involved in mAChR-induced inhibition of proliferation. PC-PLC, which is involved in some CHO cell signaling pathways (Wen et al., 1995), transduces apoptotic signals in a variety of cell types (Civone et al., 1995). Activation of PI3 kinase induces Rac-mediated events (Rodriguez-Viciana et al., 1997). PLA2 activity is necessary for the synthesis of arachidonic acid, which can initiate several Rac-mediated events such as membrane ruffling (Shin et al., 1999) and c-fos serum response element activation (Kim and Kim, 1997). CaM kinase II is necessary for activation of the Rac GEF Tiam1, (Fleming et al., 1999) and for activation of Rac itself (Lian et al., 2001). Activated Rac1 can stimulate MEK1/2 in a wide variety of cells (Eblen et al., 2002). Our studies, using a wide range of selective inhibitor concentrations, indicate that these enzymes are not required for the M3 mAChR-initiated inhibition of proliferation. This finding is intriguing because all of these enzymes have been implicated in GPCR-signaling events and have been reported to be activators or downstream targets of Rac-mediated processes.
Treatment of all the cell lines with the PI-specific PLC inhibitor edelfosine (Powis et al., 1992) diminishes the ability of the M3 mAChR to inhibit proliferation and partially rescues the m3WTRac and m3CARac cells from mAChR-induced cell death. Treatment with edelfosine slows the progression through G2/M in numerous cell types (Shafer and Williams, 2003) including the m3Zeo-2, m3WTRac, and m3CARac cells studied here. The accumulation of edelfosine-treated cells in G2/M phases may prevent them from entering another phase of the cell cycle in which they are more susceptible to the M3 mAChR-antiproliferative effects.
Interestingly, we observed a greater proportion of m3DNRac cells in the G2/M phase compared with the other cell lines (Fig. 8M). This agrees with previous reports that Rac1 regulates progression through G2/M phases (Olson et al., 1995). The m3DNRac cells are the least responsive to the M3 mAChR-mediated inhibition of proliferation. It is intriguing to speculate that the slowed progression of these cells through G2/M phases makes them less vulnerable to mAChR-induced inhibition of proliferation.
Inhibition of conventional isoforms of PKC with Go 6976 diminishes the M3 mAChR-induced inhibition of proliferation. This finding indicates that PKC activation contributes to the M3 mAChR-mediated inhibition of proliferation. PKC activity may be important for mAChR-induced inhibition of proliferation because PKC may have a role in activating Rac1. Interestingly, PKC is known to regulate the activity of small GTPases by regulating their association with RhoGDI (Mehta et al., 2001). Other investigators have shown that the PKC-mediated phosphorylation of RhoGDI causes the release of Rho from RhoGDI, thereby activating the GTPase (Mehta et al., 2001). By analogy, the PKC-mediated phosphorylation of RhoGDI may release Rac1 from RhoGDI; this is a plausible mechanism because Rac1 associates with RhoGDI in the CHO cells we used (Ruiz-Velasco et al., 2002). Inhibition of conventional isoforms of PKC by Go 6976 may prevent the release of Rac1 from RhoGDI. This event may prevent activation of Rac1 and thus diminish the mAChR-induced inhibition of proliferation.
Our findings indicate that the presence of elevated Rac1 activity alters the response of these cells to M3 mAChR stimulation. The stably transfected cells used in this study tolerate the expression of excess wild-type or mutant Rac1 proteins without affecting normal cell-cycle progression or increasing cell death. M3 mAChR activation inhibits the proliferation of cells expressing normal Rac1 levels but promotes the death of cells expressing high Rac1 levels or activity. These findings suggest that the apparent contradictory responses of different cell systems to M3 mAChR activation involve differential expression of signaling molecules such as Rac1.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor; CHO, Chinese hamster ovary; HA, hemagglutinin; CCh, carbachol; PLC, phospholipase C; GRK, G protein-coupled receptor kinase; RGS, regulator of G protein signaling; PI, phosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; PKC, protein kinase C; GPCR, G protein-coupled receptor; BrdU, bromodeoxyuridine; TRITC, tetamethylrhodamine isothiocyanate; 2-APB, 2-aminoethoxydiphenylborate; BAPTA/AM, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; GST, glutathione S-transferase; PBD, p21-activated kinase binding domain; GFP, green fluorescent protein; PBS, phosphate-buffered saline; IP3, inositol 1,4,5-triphosphate; PLA2, phospholipase A2; PC-PLC phosphatidylcholine-specific phospholipase C; CaM kinase II, Ca2+/calmodulin-dependent protein kinase II; MEK1/2, mitogen-activated protein kinase kinase 1/2; GEF, guanine nucleotide exchange factor; FACS, fluorescence-activated cell sorting; ECL, enhanced chemiluminescence; GTPS, guanosine 5'-3-O-(thio)triphosphate; Go 6976, 12-(2-cyano-ethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; AACOCF3, arachidonyl trifluoromethyl ketone; D609, tricyclodecan-9-yl-xanthogenate; KN-93, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine); W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; LY 294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; Edelfosine, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine.
Address correspondence to: Carol L. Williams, Ph.D., Molecular Pharmacology Laboratory, One Guthrie Square, Sayre, PA 18840. E-mail: Williams_carol{at}guthrie.org
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, p < 0.001; 
