Introduction

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Persistent dysregulation of signal transduction pathways promote the acquisition of anarchic functions in digestive epithelial cells, adjacent tissues, and immune cells, leading to local inflammation and further susceptibility to the neoplastic progression. Strong experimental and clinical data suggest a close relationship between inflammatory disease and the occurrence of solid tumors in intestine, breast, and prostate (Rudolph et al., 1995a; Emami et al., 2001). Local mediators, cytokines, and inflammatory agents, such as platelet-activating factor (PAF), thrombin, and trefoil factors (TFFs) are involved in the cellular responses to injury and wound repair during human inflammatory processes in the gastrointestinal tract, including peptic ulceration and colitis, Crohn's disease, pancreatitis, and biliary disease (Cirino et al., 1996; Kotelevets et al., 1998; Emami et al., 2001). We have shown previously that functional and specific PAF receptors (PAF-R) are expressed in normal human colonic epithelial crypts, colonic adenoma, and adenocarcinoma cell lines (Kotelevets et al., 1998). Moreover, we found that src- and Met-induced invasion of collagen gels was abrogated by PAF-R activation in Madin-Darbin canine kidney (MDCKts.src) and colonic PCmsrc cells via a pertussis toxin (PTx)-sensitive pathway, suggesting that Go/Gi and G/ heterotrimeric G-proteins are involved in this process. PTx, which is specific for Go and Gi subunits, prevents the catalysis of GDP-GTP exchange by the receptor, blocking both G and G signaling. The preferred substrates of PTx are Go/i subunits associated with G complexes, whereas monomeric G subunits are very poor substrates (Rudolph et al., 1995b). Redistribution and functional activity of G subunits released from PTx-sensitive proteins and serpentine receptors are important components of exocytosis and scaffold of molecular complexes at the plasma membrane level and subcellular compartments, including the actin network (Hamm, 1998). We also presented evidence that leptin and the TFFs pS2, spasmolytic polypeptide, and intestinal trefoil factor (ITF) are positive and potent effectors of cellular invasion in premalignant and src-transformed kidney and colonic epithelial cells (Attoub et al., 2000; Emami et al., 2001). Leptin and TFFs are expressed in epithelial cells along the gastrointestinal tract and are considered local and paracrine regulators of mucosal integrity, renewal, inflammation, and neoplastic progression.

This study was therefore conducted to identify the subtypes and cellular functions of the heterotrimeric G-protein subunits linked to the negative control exerted by the serpentine PAF-R on positive invasion pathways controlled by src, Met, and the intestinal mediators leptin and TFFs. For this purpose, we stably transfected MDCKts.src cells by constitutively activated GTPase-deficient forms of Go, Gi1, Gi2, Gi3 (AGo/i), the chimeric C-terminal end of the -adrenergic receptor kinase (ct-ARK) scavenging G subunits (Pitcher et al., 1992; Crespo et al., 1994), and transducin G subunit (Gt), another agent known to sequester free G-protein dimers (Federman et al., 1992). To examine further the role of G subunits on cellular invasion, G1 was also overexpressed, either alone or with G2. The relative contribution of PAF-R, AGo/i, and G subunits on cellular invasion and adhesion was then examined with the use of these experimental models, as well as with the human colorectal cell lines PCmsrc and HCT8/S11, which are derived from familial and sporadic tumors, respectively (Behrens et al., 1993; Vermeulen et al., 1995; Empereur et al., 1997). Because trefoil peptides are overexpressed in digestive epithelial cells during inflammatory situations and cancer progression (Emami et al., 2001), we next investigated whether PAF-R can control cellular invasion in MDCKts.src and HCT8/S11 cells stably transfected by the human pS2 cDNA. Results were compared with the functional activity of the thrombin PAR-1 receptor that is specifically activated by the thrombin receptor-activating peptide (TRAP) (Seiler et al., 1996). The PAR-1 receptor has multiple-signaling capacity and is also coupled to the PTx-sensitive G-proteins Go, Gi1, Gi2, and Gi3. We report that activated Go/i subunits exert an invasion-suppressor role, whereas the G subunits are required as critical and positive mediators of cellular invasion pathways induced by oncogenes and epigenetic factors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

DNA Constructs. The rat cDNAs encoding the constitutively activated forms of the PTx-sensitive G protein subunits AGo/i were generously provided by Dr. E. Peralta (Harvard University, Cambridge, MA). They were cloned in frame into the eukaryotic expression vector pcDNA3.1 recombined with the neo resistance gene (Invitrogen BV, Breda, The Netherlands). The AGo/i cDNAs were inserted at the cloning sites EcoRI/XhoI (Q205L-Go) or EcoRI/XbaI (Q204L-Gi1, -2, and -3). These mutationally activated forms of GTPase-deficient G-proteins were further designated as AGo/i1, -2, and -3. The structure and function of the expression vectors encoding AGo/i was checked by direct DNA sequencing. The capture and sequestration of G subunits was induced by the myc-tagged expression vector pcDNA3 encoding a chimeric protein comprising the membrane-bound CD8 receptor and ct-ARK, as described previously (Pitcher et al., 1992). The human transducin Gt subunit cDNA cloned into the pcDNA3.1 expression vector was purchased from the American Type Culture Collection (Manassas, VA). The bovine G1 and G2 cDNAs were cloned from the pcDM8-1 vector into the pcDNA3.1 expression vector (Crespo et al., 1994). The G sequestrant vector ct-ARK and expression vectors encoding G1 and G2 were generously provided by Dr. R. Weitzker (Klinikum der Friedrich-Schiller-Universität, Jena, Germany).

Cell Lines and Culture Conditions. MDCK epithelial cells (MDCKts.src) transformed by a temperature-sensitive mutant of v-src (MDCKts.src, clone 2) were cultured in Dulbecco's modified Eagle's medium (Invitrogen SARL, Cergy Pontoise, France) supplemented with 10% fetal calf serum (Roche Molecular Biochemicals, Meylan, France) plus L-glutamine and antibiotics (Invitrogen), as described previously (Behrens et al., 1993). MDCKts.src cells display an invasive phenotype at the permissive temperature of 35°C for src activity and are not invasive at the restrictive temperature 40°C. The human colorectal cell line PCmsrc was grown in 6-cm diameter Petri dishes. After transfer of the activated c-src oncogene in the premalignant PC/AA/C1 cell line, PCmsrc cells became tumorigenic in the athymic nude mice and are invasive upon addition of hepatocyte growth factor (HGF) (Empereur et al., 1997). The MDCKp110* cell line stably transfected with a constitutively activated form of bovine p110* by addition of the C-terminal farnesylation signal from Ha-Ras (Khwaja et al., 1997) was a generous gift from Dr. J. Downward (Imperial Cancer Research Fund, London, UK). Kidney MDCKts.src-pS2 cells (clone 2) and colonic epithelial cells HCT-8/S11-pS2 cells (clone 2) stably transfected by the human full-length hpS2 were cultured under standard conditions (Emami et al., 2001).

Stable Transfection of Kidney Epithelial Cells. Approximately 3 × 10⁶ MDCKts.src cells were stably transfected by the activated forms of the PTx-sensitive G-protein subunits AGo/i1, -2, and -3 using the corresponding pcDNA3.1 expression plasmids (3 µg) and 18 µl of the LipofectAMINE Plus reagent (Invitrogen). Control transfections were performed using the empty vector pcDNA3.1. MDCKts.src cells were also transfected under the same conditions using either the G sequestrant vectors CD8-ct-ARK or Gt, and the vectors encoding bovine G1 and G2 (see above). After 48 h, cultures were selected for 2 weeks in 1 mg/ml neomycin or 0.5 mg/ml hygromycin (Invitrogen) and MDCKts.src-resistant colonies were ring-cloned as individual colonies or pooled for analysis of ectopic expression of the AGo/i, CD8-ct-ARK, Gt, G1, and G2 proteins by immunoblot analysis, indirect immunocytochemistry, and additional functional characterization.

Western Blot Analyses, Immunocytochemistry, and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis. For immunoblotting, cultured cells were homogenized at 4°C in radioimmunoprecipitation buffer containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 100 µM benzamidine, and 100 µM Na₃VO₄ as protease inhibitors. Insoluble material was removed by centrifugation for 15 min at 4°C and 12,000g. Proteins were resolved using nonreducing conditions in 12.5% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride or Hybond-C Extra membranes (Amersham Pharmacia Biotech, Orsay, France). Membranes were blocked overnight in Tris-buffered saline (20 mM Tris-HCl, pH 8, 150 mM NaCl) containing 5% dried skimmed milk. The blots were then probed for 4 h at room temperature with one of the following primary polyclonal or monoclonal antibodies at the indicated dilutions: the rabbit pAb specific for Go and Gi3 (1:2000) was from Euromedex (Souffelweyersheim, France); the rabbit pAb AS for Gi1 and -2 (1:200) was a generous gift from Professor P. Mazancourt (Hôpital Raymond Poincaré, Garches, France) the rabbit pAb raised against the C-terminal part of Gi3 (1:2500) was from Calbiochem (Meudon, France); the rabbit pAbs against Gt transducin K-20 (1:500), G1 C-16 (1:100), and G2 A-16 (1:500), and the mAb raised against the c-myc epitope (1:1000) were purchased from Santa Cruz Biochemicals (Santa Cruz, CA). Membranes were then washed in Tris-buffered saline containing 0.1% Tween 20 and probed for 90 min with either a donkey anti-rabbit IgGs pAb (1/2000, Amersham), or a goat anti-mouse IgGs pAb (1/2000, Santa Cruz Biochemicals), and then revealed by enhanced chemiluminescence Western detection (ECL; Amersham).

Collagen Invasion and Cell Adhesion Assays. For invasion of collagen gels by renal and colorectal epithelial cells, Petri dishes were filled with 1.35 ml of neutralized type I collagen and incubated overnight at 37°C and allowed to gel. Cells were harvested using Moscona buffer and trypsin/EDTA and were seeded on top of collagen gels. Cultures were incubated for 24 h at the indicated temperature in the presence or absence of the indicated effectors. The depth of cell migration inside the gels was measured with the use of an inverted microscope (Vleminckx et al., 1991). Invasive and superficial cells were counted in 12 fields of 0.157 mm². The invasion index is the number of cells invading the gel expressed as a percentage of the total number of cells.

Peptides and Reagents. HGF scatter factor was a generous gift from Professor Paolo Comoglio (University of Turin, Italy). Purified recombinant human ITF and hpS2 were a generous gift from Dr. L. Thim (Novo Nordisk, Bagsvaerd, Denmark) and Professor B. Westley (University of Newcastle upon Tyre, UK). Leptin was from R & D Systems Europe (Oxford, United Kingdom). PTx, PAF, thrombin, and phenylmethylsulfonyl fluoride were from Sigma (Saint Quentin Fallavier, France). The PAR-1 agonist TRAP (SFLLRN) was from Bachem Biochimie (Voisins-le-Bretonneux, France). Collagen type I was from Upstate Biotechnology (Lake Placid, NY).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

Negative Control of Cellular Invasion Pathways by PAF-R, PAR-1, and the Activated Forms AGo/i of the PTx-Sensitive G-Proteins. We have recently shown that PAF receptors exert a negative control on src- and HGF-induced cellular invasion in kidney and colonic epithelial cells (Kotelevets et al., 1998). We first re-examined the effect of PTx on positive and negative invasion pathways induced by G-protein -coupled receptors and other transduction elements, including Met, src, and the activated form of phosphatidylinositol 3-kinase (PI3'-K) (p110*). As shown in Fig. 1A, 200 ng/ml PTx fully reversed the inhibitory effect of PAF on invasion induced by src in MDCKts.src cells incubated at the permissive temperature 35°C (invasion index, 7.5 ± 0.6%). The A protomer of PTx is a single peptide that ADP-ribosylates cysteine residues of membrane Go/i subunits, blocking the heterotrimeric complex in the inactive GDP-bound state and preventing dissociation of G and G. The toxin uncouples the activation of the heterotrimeric complex by PAF-R. In contrast, the same concentrations of PTx (20-200 ng/ml) produced the inverse effect and abolished HGF-induced cellular invasion in MDCKts.src cells incubated at the nonpermissive temperature 40°C (Fig. 1B), directly implicating substrate G-protein -subunits and released G subunits in signaling through the Met tyrosine kinase. Similarly, other signals mediated by tyrosine kinase-associated receptors, including epidermal growth factor, insulin-like growth factor-1, basic fibroblast growth factor, erythropoietin Epo receptors, and insulin receptors, can be inhibited or induced by PTx treatment and G sequestrant because of direct and indirect associations with Gi, G, and G subunits (Luttrell et al., 1997; Hallak et al., 2000). Indirect cross-talk and trans-activation of the epidermal growth factor receptors was also induced by the G-protein-coupled receptors for thrombin and lysophosphatidic acid (Prenzel et al., 1999). Note that PTx did not abolish invasion induced by the tyrosine kinase src and constitutively activated PI3'-K in MDCKts.src and MDCKp110* cells (Fig. 1, C and D), leptin, and TFFs in kidney and colonic epithelial cells. These findings raise the possibility that the PTx-sensitive trimeric subunits on one hand and the G subunits on the other are selectively and, respectively involved in negative and positive invasion pathways. To explore this possibility, we stably transfected MDCKts.src cells with GTPase-deficient mutants of Go/i encoding-activated forms (AGo/i) of these four G PTx-sensitive subunits. To confirm that AGo/i forms are expressed in MDCKts.src cells, immunoblot analysis in Fig. 2 identified several positive clones overexpressing Go (clones 1 and 4), Gi1 (clone 6), Gi2 (clones 1, 7, 8, and 10) and Gi3 (clones 1-3). For example, densitometry analysis revealed that the Gi3 protein was overexpressed 2- and 3-fold in clones 1 and 2, respectively. As shown in Fig. 3, A and B, stable expression of AGo/i completely abolished cellular invasion induced by HGF and src, suggesting that all four PTx-sensitive G subunits are candidate-signaling elements in the negative control of cellular invasion by activated PAF-R. To gain insight into this possibility, we next determined whether PAF and PAR-1 thrombin receptors have similar activities on cellular invasion induced by the trefoil factors pS2 and ITF. The PAR-1 receptor is a seven-transmembrane domain G-protein-coupled receptor that is also connected with the PTx-sensitive proteins Go/i (Seiler et al., 1996). As shown in Fig. 4, cellular invasion induced by ITF in kidney MDCKts.src and colonic PCmsrc epithelial cells is resistant to PAF (A and B), and the same situation is observed in kidney MDCKts.src-pS2 and colonic HCT8/S11-pS2 epithelial cells stably transfected by the trefoil factor pS2 (C and D). In contrast, the PAR-1 agonist TRAP abolished ITF- and pS2-induced invasion in the four models, suggesting that the Go/i subunits exert a differential control on invasion. In agreement with this interpretation, we observed that thrombin or the exogenous agonist TRAP completely inhibited cellular invasion induced by HGF in MDCKts.src cells and activated PI3-K in MDCKp110* cells in a PTx-sensitive manner (data not shown). Pertussis toxin (200 ng/ml) also completely reversed the inhibitory effect of thrombin or TRAP on HGF-, ITF-, and src-mediated invasion in MDCKts.src cells (not shown). We therefore examined the relative contribution of the activated forms of G-proteins AGo/i on the negative control exerted by PAF-R and PAR-1 receptors on ITF- and pS2-induced invasion. As shown in Fig. 5, all forms of AGo/i blocked invasion induced by leptin and HGF in MDCKts.src cells. In contrast, only MDCKts.src cells stably transfected by AGi3 (clones 1 and 2) seem incapable of responding to ITF and pS2, suggesting that only this Gi3 subunit can mediate the negative control exerted by activated PAR-1 receptors on cellular invasion induced by the TFFs.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Differential control by PTx of cellular invasion pathways induced by src, HGF/Met, and the activated form p110* of PI3'-K in MDCK cells. MDCKts.src cells were incubated for 24 h in the presence or absence of the indicated effectors [PAF (0.1 µM) or HGF (10 units/ml)] alone or with increasing concentration of PTx (20-200 ng/ml). The percentage of invasive cells in collagen type I gels was measured either at the permissive temperature 35°C for src activation (A and C) or at the restrictive temperature 40°C (B). D, PTx was tested at 37°C in kidney MDCK cells stably transfected by the constitutively activated form of PI3'-K (MDCKp110*). Data are means ± S.E. of three or four separate experiments.

View larger version (68K): [in this window] [in a new window]   Fig. 2.   Expression by Western blot of the constitutively activated forms of the AGo/i1 to -3 subunits in stably transfected MDCKts.src cells. Parental MDCKts.src cells (P) were stably transfected by the pcDNA3.1 expression vector recombined with the GTPase-deficient forms of Go-Q205L (AGo), Gi1-Q204L, Gi2-Q204L, and Gi3-Q204L (AGi1-3). Positive MDCKts.src clones expressing given AG subunits were identified by immunoblot analysis using the antibodies specified under Materials and Methods. Immunoblots were revealed by the ECL Western detection system and quantified by the use of a densitometer. The G subunits migrate at 41 kDa (arrow). Results are representative of one other independent experiment.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Negative control of src- and HGF-induced invasion of collagen gels by constitutively activated forms of the AGo/i1-3 subunits in MDCKts.src cells. Cellular invasion was compared in MDCKts.src cells before (parental cell line) and after stable expression of the constitutively activated forms of Go (clone 1), Gi1 (clone 6), Gi2 (clone 1), and Gi3 (clone 2). The effect of the AGo/i1-3 subunits was tested on invasion induced by HGF at 40°C in MDCKts.src cells (A) and by src in MDCKts.src cells incubated at the permissive temperature 35°C (B). The percentage of invasive cells was determined as described under Materials and Methods. Data are means ± S.E. of three separate experiments.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Differential signaling of PAF-R and PAR-1 thrombin receptors toward cellular invasion pathways induced by trefoil peptides in kidney and colonic epithelial cells. The effect of PAF (0.1 µM) and the PAR-1 agonist TRAP (10 µM) was tested on invasion of collagen gels induced by ITF (100 nM) in kidney MDCKts.src and colonic PCmsrc epithelial cells (A and B). Results were compared with the effects of PAF and TRAP on constitutive cellular invasion induced by overexpression of the pS2 cDNA in kidney MDCKts.src-pS2 and human colonic HCT/8S11-pS2 epithelial cells (C and D). The percentage of invasive cells was determined as indicated under Materials and Methods. Data are means ± S.E. of three separate experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Differential signaling of activated AGo/i1-3 subunits toward cellular invasion pathways induced by leptin, HGF, and trefoil factors in kidney and colonic epithelial cells. Cellular invasion induced by leptin (100 ng/ml), HGF (10 units/ml), and the trefoil factors ITF or pS2 (100 nM) was measured at 40°C in MDCKts.src cells before (parental cell line) and after stable expression of the constitutively activated forms of Go (clones 1 and 4), Gi1 (clone 6), Gi2 (clone 1), Gi3 (clones 1 and 2). The percentage of invasive cells was determined as indicated under Materials and Methods. Data are means ± S.E. of three or four separate experiments.

Depletion of Free G Subunits by the -Sequestering Peptides ct-ARK and Gt: Consequences for Cellular Invasion and Adhesion. It is becoming increasingly evident that signaling through the G heterodimers and the individual  and  subunits released upon activation of PTx-sensitive G-proteins Go/i plays a key role in multiple-signaling pathways. For example, inhibition or activation of specific isoforms of adenylate cyclase (Hamm, 1998), G-protein-coupled receptor kinases GRK1 to -3 involved in receptor desensitization (Pitcher et al., 1992; Carman et al., 2000), membrane-targeting of G subunits and binding to pleckstrin homology domains (Touhara et al., 1994), G exchange between Gi-coupled and Gq-coupled receptors (Quitterer and Lohse, 1999), src- and ras-dependent activation of mitogen-activated protein kinases, G-sensitive PI3'-K and Jun kinases, and phospholipase C- are concerned with G signaling (Luttrell et al., 1996; Lopez-Ilasaca et al., 1998; Rickert et al., 2000). Recently, release of G from activated insulin-like growth factor I receptor (Hallak et al., 2000) and direct interactions between G and the Rho family of GTPases, Rho and Rac, have been documented (Harhammer et al., 1996; Ueda et al., 2000). The role of G signaling in either cellular invasion or adhesion is unknown. Accordingly, we have established MDCKts.src cell lines stably expressing the G sequestrant vector comprising the CD8 receptor and ct-ARK.

View larger version (76K): [in this window] [in a new window]   Fig. 6.   Western blot and immunofluorescence analysis of the G sequestrant peptide ct-ARK in MDCKts.src cells stably transfected the CD8-ct-ARK expression vector. Top, expression of the myc-tagged -adrenergic receptor kinase (ct-ARK) peptide in MDCKts.src cells transfected by the CD8-ct-ARK vector (clones 1-5). Immunoblotting of total cell lysates was performed using the c-myc mAb and revealed by the ECL Western detection system. Bottom, stable expression of the G sequestrant was analyzed by indirect immunofluorescence in MDCKts.src-ct-ARK cells (clones 1 and 3). Cells were processed with the mAb against the c-myc epitope, followed by fluorescein-conjugated goat anti-mouse IgG as secondary antibody, as described under Materials and Methods.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Negative control of src- and HGF-induced invasion of collagen gels by the G sequestrant peptide ct-ARK in MDCKts.src cells stably transfected with the CD8-ct-ARK expression vector. Cellular invasion index was determined in parental and ct-ARK-transfected MDCKts.src cells (clones 1 and 3) incubated at the nonpermissive temperature 40°C for src activation (A) or at the permissive temperature 35°C (B). MDCKts.src-ct-ARK cells were treated with 10 units/ml HGF alone or with 0.1 µM PAF and compared with their respective control cells (no effector). The percentage of invasive cells was determined as described under Materials and Methods. Data are means ± S.E. of three separate experiments.

View larger version (94K): [in this window] [in a new window]   Fig. 8.   Effect of overexpressed G sequestrant peptide CD8-ct-ARK on adhesive properties and spreading of MDCKts.src cells on collagen gels. Adhesion to collagen type I gels was compared in MDCKts.src-ct-ARK cells (clones 1 and 3 in A and B, respectively) incubated for 24 h at 35°C or 40°C in the presence or absence of HGF (10 units/ml). Cell were seeded at the density of 105 cells/ml. The white arrows in A underline the src-dependent filopodia formation in MDCKts.src-ct-ARK clone 1 incubated in the presence or absence of HGF. In contrast, the MDCKts.src-ct-ARK clone 3 showed a remarkable reduction of filopodia protrusions and cellular spreading induced by src (B). Most interestingly, ct-ARK caused shape change and cellular rounding in MDCKts.src-ct-ARK clone 3 incubated at the nonpermissive temperature for src activation (B), leading to a loss of cell adhesion, even in the presence of the Met activator HGF (black arrows).

View larger version (28K): [in this window] [in a new window]   Fig. 9.   Negative control of src- and HGF-induced invasion of collagen gels by the G sequestrant peptide Gt in MDCKts.src cells stably transfected by the Gt transducin expression vector. Cellular invasion index was determined in parental and Gt-transfected MDCKts.src cells cultured at 40°C (A) or at the permissive temperature 35°C for src activation (B). Stably transfected MDCKts.src-Gt cells (clone 5) were treated with either 10 units/ml HGF, 100 ng/ml leptin, or 100 nM pS2 and compared with their respective control cells (no effector) and parental MDCKts.src cells incubated in the presence or absence (Control) of 100 ng/ml leptin. The percentage of invasive cells was determined as described under Materials and Methods. Data are means ± S.E. of three separate experiments performed in clone 5 and are representative of another experiment performed in clones 3 and 4.

Activation of Cellular Invasion by Coexpression of G Subunits. Our data on the G sequestering proteins ct-ARK and Gt support the hypothesis that free G endogenous dimers released from activated PTx-sensitive G-proteins are required for initiation and activation of positive cellular invasion pathways controlled by oncogenes and epigenetic factors. To explore further such a possibility, we next examined the effect of ectopic overexpression of G1 alone or combined with G2 in MDCKts.src cells. First, we stably transfected MDCKts.src cells with G1 and selected four G418-resistant colonies (Fig. 10A). Immunoblot analysis identified two MDCKts.src-G1 cells (clones 1 and 3) overexpressing the G1 protein (36 kDa). As shown in Fig. 10B, HGF-induced invasion of collagen gels was completely blocked by overexpression of G1 in clone 3, whereas src-induced activation was unaffected (Fig. 10C). Thus, we examined the G1-dependence of the negative control exerted by PAF-R and PAR-1 on src-induced invasion. As a consequence of G1 overexpression, there was a substantial attenuation of PAF-R-mediated inhibition of cellular invasion (Fig. 10C), but no significant effect on the negative control exerted by PAR-1 was found. Our data are therefore consistent with the possibility that overexpression of G1 alone exerts a selective inhibition of the PAF-R and HGF/Met signaling pathways. This possibility is consistent with the recent finding that molecular complexes between G subunits and several regulators of G-protein signaling (RGS) have been identified, because structural domains in RGS proteins exhibit striking homologies to G subunits (Snow et al., 1998; Levay et al., 1999; Sowa et al., 2000). Most importantly, some G/RGS heterodimers behave as GTPase-activating proteins for certain G subunits, such as Go (Snow et al., 1998). The RGS activity and selectivity for PTx-sensitive or -insensitive G subunits can be directly or indirectly determined through their interactions with different G subunits, G-protein-coupled receptors, or other binding partners. For example, RGS1, RGS3, RGS4, and GAIP stimulate the GTPase activity of Gi family members but are ineffective against Gs. Thus, overexpression of G1 can selectively mimic, at least in part, PTx-induced deactivation of the PAF-R and Met signaling pathways involved in cellular invasion, as shown in Fig. 1.

View larger version (28K): [in this window] [in a new window]   Fig. 10.   Effect of overexpressed G1 subunit on positive and negative invasion pathways in stably transfected MDCKts.src-G1 cells. A, expression of the G1 subunit was analyzed by Western blot in parental MDCKts.src cells (P) and transfected MDCKts.src-G1 cells (G418-resistant clones 1-4), using the G1 pAb. The G1 subunit migrates at 36 kDa (arrow). Cellular invasion was then quantified in the MDCKts.src-G1-positive clone 3 incubated at 40°C (B) or 35°C (C). Parental MDCKts.src and MDCKts.src-G1 cells were treated with either HGF (10 units/ml), PAF (0.1 µm), or TRAP (10 µm) and compared with their respective control cells (no effector). The percentage of invasive cells was determined, as described under Materials and Methods. Data are means ± S.E. of three separate experiments.

View larger version (38K): [in this window] [in a new window]   Fig. 11.   Positive control of cellular invasion after ectopic coexpression of G1 and G2 subunits in stably transfected MDCKts.src-G12 cells. Reversion by PAF-R and PAR-1 activation. A, expression of the G2 subunit in MDCKts.src-G1 cells (P) subsequently transfected by the G2 vector. Immunoblotting of total cell lysates was performed using the affinity-purified rabbit pAb G2 (A-16) and revealed two positive MDCKts.src-G12 cells (clones 4 and 11) by the ECL Western detection system. B and C, cellular invasion index was determined in parental MDCKts.src cells incubated in the presence or absence of 100 ng/ml leptin and in MDCKts.src-G12 cells (clones 4 and 11) incubated at 40°C (B) or at the permissive temperature 35°C for src activation (C). Stably transfected MDCKts.src-G12 cells and parental MDCKts.src cells were treated with either 10 units/ml HGF, PAF (0.1 µM), or the PAR-1 agonist TRAP (10 µM) and compared with their respective control cells (no effector). The percentage of invasive cells was determined as described under Materials and Methods. Data are means ± S.E. of three separate experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 12.   Model depicting the negative and positive invasion pathways induced by activated PAF-R and PAR-1 in kidney and colonic epithelial cells. Activation of Go/i subunits by serpentine PAF-R and PAR-1 exerts a dominant negative control () on cellular invasion pathways governed by src, HGF/Met, the epigenetic factors leptin and TFFs, and the G subunits (+) that are released from these PTx-sensitive G-proteins. The G subunits, on the other hand, are invasion promoters, according to their connections with src, phospholipase C-, PI3'-kinases, and the Rho-like GTPases that induce the formation of filopodia and lamellipodia (Cdc42 and Rac), focal adhesions, and stress fibers (Rho). The Go/Gi downstream targets remains to be identified (?) in the context of the integration of the positive and negative signaling pathways implicated in cellular spreading, migration, local invasion, and distant metastasis.