![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 60, Issue 2, 363-372, August 2001
o, Gi1, Gi2, and Gi3 and Sequestration of G
Institut National de la Santé et de la Recherche Médicale U482, Hôpital Saint-Antoine, Paris, France (S.F., K.R., Q.-D.N., S.E., C.G.); and The Laboratory of Experimental Cancerology, Ghent University, Ghent, Belgium (E.B., M.M.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results and Discussion
References
|
|---|
It was shown previously that platelet-activating factor receptors
(PAF-Rs) inhibit invasiveness of colonic and kidney epithelial cells
induced by the src and Met oncogenes via a pertussis toxin-sensitive mechanism. Therefore, Madin-Darby canine kidney (MDCKts.src) cells were
stably transfected with constitutively activated forms of G
o,
Gi1, Gi2, Gi3 (AGo/i), two G
sequestering proteins [C-terminal end of -adrenergic receptor kinase (ct-ARK) and the
Gt subunit of retinal G-protein transducin], and G1-G2 subunits alone or in combination. Cellular invasion induced by src,
Met, and leptin was abrogated by the AGo/i, ct-ARK, and Gt-positive clones, but was induced by coexpression of G12. In
contrast, invasion stimulated by the trefoil factors (TFFs) pS2 and
intestinal trefoil factor in MDCKts.src cells or human colonic
epithelial cells PCmsrc and HCT8/S11 was insensitive to PAF, AGo,
AGi1, and AGi2, but was abolished by AGi3 and the protease-activated receptor-1 (PAR-1) agonist thrombin
receptor-activating peptide. Depletion of free G
heterodimers by ct-ARK resulted in a remarkable decrease of cellular
adhesion and spreading on collagen matrix. Our data demonstrate the
following: 1) PAF-Rs impair cellular invasion induced by src, Met, and
leptin via the activation of Go and Gi1 to -3; 2) invasion
induced by TFFs is selectively inhibited by PAR-1 receptors and Gi3
activation; and 3) G dimers are required as positive effectors of
invasion pathways induced by oncogenes and epigenetic factors. Thus,
redistribution of Go/Gi and G/ heterotrimeric G-proteins by
PAF-R and PAR-1 exert differential functions on positive and negative
signaling pathways involved in cellular invasion and may serve as
potential targets for anticancer therapy.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
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).
ARK fusion gene, kidney
cells were grown on autoclaved glass slides (VWR, West Chester, PA) and
processed at 70 to 80% confluence. Cells were rinsed with
phosphate-buffered saline (PBS) at room temperature, fixed for 30 min
in phosphate buffer containing 4% formaldehyde, and washed 3 times in
phosphate buffer. Cells were then made permeable with Triton X-100 in
PBS for 10 min, blocked with 3% goat serum at room temperature for 30 min, and incubated overnight at 4°C with primary mAb anti-c-myc
(Santa Cruz, 1:500). Then, cells were rinsed with PBS and incubated for
1 h at room temperature with a fluorescein-conjugated goat
anti-mouse IgG (1:150) from Immunotech (Roissy, France). Stained
cells were rinsed again in PBS, fixed with a solution of glycerol in
PBS (9:2, v/v), and viewed by conventional epifluorescence on an
Olympus BH2 microscope (Olympus, Tokyo, Japan).
Total RNA was extracted from parental and Gt-transfected MDCKts.src
cells (clones 3-5) using the Trizol reagent (Invitrogen). RT-PCR was
performed using the Superscript detection kit (Invitrogen) according to
the manufacturer's protocol. The experiment comprised one cycle of 30 min at 55°C and one of 2 min at 94°C, followed by 35 cycles of
30 s at 94°C, 30 s at 65°C, and 1 min at 72°C, with a
final extension time of 10 min at 72°C. For human Gt, we used the
following sense and antisense oligonucleotides:
5'-GGCAGACACTATCGAGGAGGGCACGATGCC-3' and
5'-GAGGTGC-GCCTTCTTGACCTTCTCGAAGAAGAC-3'. PCR products were then
separated by electrophoresis in 1.5% agarose gel and detected under UV
light. The Gt primers (Invitrogen) amplified the 519-base-pair fragment specific of the human G-protein transcript.
Glyceraldehyde-3-phosphate dehydrogenase messengers (574 base pairs)
were also analyzed by RT-PCR to verify the integrity of RNA preparations.
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.
|
|
|
|
|
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.
ARK protein in MDCKts.src-ct-ARK cells (clones 3-5).
The signal was not detected by immunoblotting in parental MDCKts.src
cells and their transfected counterparts, the MDCKts.src-ct-ARK
cells (clones 1 and 2). Strong expression of the G subunit
inhibitor was confirmed by indirect immunofluorescence in
MDCKts.src-ct-ARK clone 3, whereas a much weaker signal was present
in MDCKts.src-ct-ARK clone 1 cells, at barely detectable levels. We
therefore examined the invasive properties of the low- and
high-expressing MDCKts.src-ct-ARK cells (clones 1 and 3). In the
MDCKts.src-ct-ARK clone 1 expressing very low levels of
CD8-ct-ARK, cellular invasion induced by HGF was reduced by 50%
(Fig. 7A) compared with nontransfected
parental MDCKts.src cells. This residual activity was still sensitive
to PAF inhibition, whereas src-induced cellular invasion was unaffected in MDCKts.src-ct-ARK clone 1 (Fig. 7B). In contrast, high
expression of the G scavenger leads to total inhibition of the
invasive phenotype controlled by HGF and/or src in the
MDCKts.src-ct-ARK clone 3 (Fig. 7, A and B). These findings suggest
that the G subunits are required as potential links between
G-protein-coupled receptors and positive cellular invasion pathways
induced by the src and Met oncogenes. Similarly, the G inhibitor
CD8-ct-ARK abolished leptin-, pS2- and ITF-induced cellular
invasion in MDCKts.src cells (not shown). Over the last few years, a
number of interesting connections between invasion pathways and
cellular adhesion to extracellular matrix proteins have emerged.
Increasing evidence shows multiple collaborations between cell-cell and
cell-substratum interactions, assembly of the actin cytoskeleton, and
signal transduction pathways involved in invasion and metastasis. These
interactions, together with recent reports suggesting activation by
G subunits of the Rho family GTPase including Ras, Rho, and Rac
(Harhammer et al., 1996; Ueda et al., 2000), prompted us to examine the
adhesive properties of MDCKts.src cells transfected with the G
sequestrant CD8-ct-ARK. The interdependence of these pathways is
further illustrated by the sequential role of Rac and Rho in the
initiation of new adhesion sites and their maturation into focal
adhesions.
|
|
subunits impaired cell adhesion
and spreading to collagen gels and filopodia formation in
MDCKts.src-ct-ARK clone 3 activated by the HGF/Met and src
oncogenes compared with the low-expressing ct-ARK clone 1. Cell
adhesion to extracellular matrix proteins is responsible for cell
spreading through integrin receptors, which promotes integrin
clustering and cytoskeletal reorganization and induces cells to spread.
Disruption of the gene encoding the G-protein -subunit impaired the
regulation of the actin cytoskeleton at cell-yeast particle adhesion
sites during phagocytosis and abrogated chemotaxis in the
Dictyostelium discoideum amebae (Peracino et al., 1998),
suggesting that G is intimately involved in signal transduction
networks linking cytoskeletal responses to chemoattractants. The
G-proteins may also be associated with adhesion and/or morphological changes (Hansen et al., 1994). In the present study, inhibition of G signaling effectively induced cell rounding and
alterations of spreading in MDCKts.src-ct-ARK clone 3 that was
refractory to activation of src and Met (Fig. 8B).
|
sequestrant vector Gt transducin also prevented cellular
invasion induced by HGF, leptin, and pS2, either alone (A) or combined
with activation of the src oncogene (B) in MDCKts.src-Gt cells
(clone 5). Similar results were obtained in MDCKts.src-Gt cells
(clones 3 and 4) established from transfected MDCKts.src cells
expressing the human Gt transducin protein by immunoblot (data not
shown) and RT-PCR (Fig. 9, inset). Because G subunits have been
implicated in the regulation of Rho GTPases that are dynamic regulators
of the actin cytoskeleton (Harhammer et al., 1996; Hall, 1998; Ueda et al., 2000), including the Rho/Rac interplay, our data on the sequestering peptides ct-ARK and Gt transducin are consistent with the direct implication of G subunits in several signaling pathways involved in cellular invasion and switch between cellular rounding/retraction and spreading/cellular adhesion.
|
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.
|
or G subunits exert divergent signaling functions,
depending on the functional status and relative distribution of the
resident G and G subunits and dimers interacting with a given
signal transduction system. In the G complexes, the G subunits
consist of seven subtypes with high amino acid sequence identity from
80 to 90% for G1- to -4 and 52% identical for G5 (Watson et
al., 1994). Thus, G2 subunit was expressed together with G1 in
MDCKts.src cells after cotransfection of MDCKts.src-G1 cells (clone
3) with the G2 (3 µg) and pcDNA3.1/Hygro (0.3 µg) expression
vectors and selection for 2 weeks in the presence of hygromycin (0.5 mg/ml). As shown in Fig. 11, immunoblot
analysis identified two MDCKts.src-G2-positive cells (clones 4 and
11) expressing the ectopic G2 protein (7 kDa) after subsequent
transformation of MDCKts.src-G1 cells by the G2 vector. The G2
subunit protein was not detected in MDCKts.src cells. Coexpression of
both G1 and G2 subunits in MDCKts.src cells resulted in a
remarkable induction of cellular invasion in MDCKts.src-G12 cells
(Fig. 11B; clones 4 and 11) and potentiation of invasiveness induced by
HGF (Fig. 11, B and C), and src (Fig. 11C). Furthermore, invasion induced by overexpressed G12 proteins in MDCKts.src cells
(invasion index, 5.2% and 4.8% in clones 4 and 11, respectively) is
abrogated by activation of the PAF-R and PAR-1. Our data therefore
indicate that formation of the G12 complex in MDCKts.src-G
cells is associated with induction and potentiation of cellular
invasion pathways. This is in agreement with our demonstration that
cellular invasion induced by src, Met, and epigenetic factors was
abrogated by sequestration of free G subunits by ct-ARK and
Gt. Thus, PTx neutralizes HGF-induced invasion (Fig. 1) and reverses
inhibition of cellular invasion controlled by activated PAF-R and
PAR-1, suggesting that PTx abolishes 1) G-signaling pathways
involved in the induction of cellular invasion by HGF, and 2)
Go/i-mediated inhibition of cellular invasion induced by PAF/TRAP.
Alternatively, it should be stressed that Gi subunits are also
activators of invasion pathways controlled by src and PI3'-K,
for example.
|
o/i subunits in response to multiple invasion pathways induced by
oncogenes and epigenetic factors (Fig.
12). Thus, the Gi/o subunits exert a
dominant invasion-suppressor role in our assay. In contrast, G
subunits that are liberated from PTx-sensitive G subunits are
critical mediators and links between positive cellular invasion and
adhesion pathways. In this scenario, G dimers can induce
activation of multiple signaling pathways that are critical components
of cellular invasion, namely Rho-like small G-proteins, the /
isotypes of PI3'-K, and the phospholipase C/protein kinase C cascade
(Rickert et al., 2000). The individual functions of the paired G
proteins and the PTx-insensitive G subunits associated with PAF-R
and PAR-1 may provide an alternative pathway by which these
heterotrimeric G-proteins may exert opposing effects on cellular
invasion (i.e., positive and negative signals). Precisely how the
balance between these positive and negative signals is regulated in the
integration of the cellular responses remains to be elucidated.
Disruption of this equilibrium might have important biological
consequences, because we have shown that PAF and PAR-1 receptors exert
a dominant negative function on cellular invasion. Deactivation of
Go/i subunits by PTx in the present study can be physiologically
induced and mimicked by the RGS proteins that negatively control these
PTx-sensitive G-proteins and Gz, G12/13 subunits, and PAF-R
phosphorylation as well. In contrast, we found that overexpressing
G subunits was sufficient to induce invasiveness. Thus, RGS
signals might abolish the dominant negative control exerted by Go/i
subunits on cellular invasion and trigger a permissive action on
positive invasion pathways governed by G. These results are
analogous to the negative cross-talk exerted by Go on the Gq
pathway and its downstream effectors (Hajdu-Cronin et al., 1999).
Abnormalities in the expression, protein structure, and constitutive
activation of G-protein subunits (G, Gi2, Gi3) have been
reported in human pathologies, including tumors of the ovary,
neuroendocrine tumors, and experimental models of colonic neoplasms
induced by azoxymethane in rats (Lyons et al., 1990; Bolt et al., 1998;
Farfel et al., 1999). Recently, mutants of the exchange factor cdc24
for the Rho-like GTPase Cdc42 defective in binding to the G-protein
G subunit have been described (Nern and Arkowitz, 1998) in the
context of the cytoskeletal reorientation. Therefore, activation or
inactivation of the molecular components of the PTx-sensitive and
-insensitive trimeric G-proteins in familial and sporadic tumors
(Rodrigues et al., 2001) should be considered in view of their
differential localization and function in basolateral and cytoskeletal
domains, including focal adhesion and stress fibers in polarized
epithelial cells in kidney and intestine (Hansen et al., 1994; Bolt et
al., 1998; Saha et al., 1998). Further studies will shed more light for
understanding the relative contribution and functioning of heterotrimeric G-proteins G and G in positive and negative signaling pathways involved in cellular adhesion and invasion in the
context of neoplasia and metastasis.
|
| |
Acknowledgments |
|---|
We are grateful to Professor P. Comoglio, Professor P. de Mazancourt, Dr. E. Peralta, Dr. L. Thim, Professor R. Weitzker, and Professor B. Westley for providing reagents.
| |
Footnotes |
|---|
Received December 4, 2000; Accepted April 26, 2001
This work was supported by the Institut National de la Santé et de la Recherche Médicale, Research Grants from la Fondation pour la Recherche Médicale, France (to S.F.), l'Association de la Recherche sur le Cancer, France (to C.G. and S.E.), the Fortis Bank, Verzekeringen and the FWO-Vlaaderen, Belgium.
S.F. and K.R. contributed equally to this work.
Dr. Christian Gespach, INSERM U482: Signal Transduction and Cellular Functions in Diabetes and Digestive Cancers, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France. E-mail: gespach{at}st-antoine.inserm.fr
| |
Abbreviations |
|---|
PAF, platelet-activating factor;
TFF, trefoil
factor;
PAF-R, platelet-activating factor receptor;
MDCK, Madin-Darby
canine kidney;
PTx, pertussis toxin;
ITF, intestinal trefoil factor;
AGo/i, activated forms of G0, Gi1, Gi2, and Gi3;
ct-ARK, C-terminal end of the -adrenergic receptor kinase;
Gt, transducin
G subunit;
PAR-1, protease-activated receptor-1;
TRAP, thrombin
receptor-activating peptide;
HGF, hepatocyte growth factor;
RT, reverse
transcriptase;
PCR, polymerase chain reaction;
pAb, polyclonal
antibody;
mAb, monoclonal antibody;
PBS, phosphate-buffered saline;
ECL, enhanced chemiluminescence;
PI3'-K, activated form of
phosphatidylinositol 3-kinase;
RGS, regulators of G-protein
signaling.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
-catenin complex in cells transformed with a temperature-sensitive v-SRC gene.
J Cell Biol
120:
757-766 subunits in normal rat colon and in azoxymethane-induced colonic neoplasms.
Gastroenterology
115:
1494-1503[Medline]. and phospholipid interaction with G protein-coupled receptor kinase 2.
J Biol Chem
275:
10443-10452 subunits.
Nature (Lond)
369:
418-420[Medline]. subunits.
Nature (Lond)
356:
159-161[Medline].o and Gq in Caenorhabditis elegans: the RGS protein EAT-16 is necessary for Go signaling and regulates Gq activity.
Genes Dev
13:
1780-1793 subunit to focal adhesions and associated stress fibers.
J Cell Biol
126:
811-819 dimers with small GTP-binding proteins of the Rho family.
FEBS Lett
399:
211-214[Medline].5 prevents the RGS7-Go interaction through binding to a distinct G-like domain found in RGS7 and other RGS proteins.
Proc Natl Acad Sci USA
96:
2503-2507 is a mediator of G-dependent Jun kinase activation.
J Biol Chem
273:
2505-2508 subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation.
J Biol Chem
272:
4637-4644 subunit-mediated activation of mitogen-activated protein kinases.
J Biol Chem
271:
19443-19450 subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton.
J Cell Biol
141:
1529-1537 subunits of G proteins in targeting the -adrenergic receptor kinase to membrane-bound receptors.
Science (Wash DC)
257:
1264-1267i- and Gq- coupled receptors is mediated by G exchange.
Proc Natl Acad Sci USA
96:
10626-10631 and its lipid products.
Trends Cell Biol
10:
466-473[Medline].i2-deficient mice.
Nat Genet
10:
143-150[Medline].i2 in the maintenance and biogenesis of epithelial cell tight junctions.
J Biol Chem
273:
21629-21633 subunit-like domain shared between RGS11 and other RGS proteins specifies binding to G
5 subunits.
Proc Natl Acad Sci USA
95:
13307-13312-subunits to pleckstrin homology domains.
J Biol Chem
269:
10217-10220 subunits induce stress fiber formation and focal adhesion assembly in a rho-dependent manner in HeLa cells.
J Biol Chem
275:
2098-2102-catenin in human colon cancer cells.
Cancer Res
55:
4722-4728-subunit family. Expression in brain and activation of the 2 isotype of phospholipase C.
J Biol Chem
269:
22150-22156This article has been cited by other articles:
|
|
 |
|
  G. P. Prevost, M. O. Lonchampt, S. Holbeck, S. Attoub, D. Zaharevitz, M. Alley, J. Wright, M. C. Brezak, H. Coulomb, A. Savola, et al. Anticancer Activity of BIM-46174, a New Inhibitor of the Heterotrimeric G{alpha}/G{beta}{gamma} Protein Complex. Cancer Res., September 15, 2006; 66(18): 9227 - 9234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. L. Minnear, L. Zhu, and P. He Sphingosine 1-phosphate prevents platelet-activating factor-induced increase in hydraulic conductivity in rat mesenteric venules: pertussis toxin sensitive Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H840 - H844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mukhopadhyay and A. C. Howlett Chemically Distinct Ligands Promote Differential CB1 Cannabinoid Receptor-Gi Protein Interactions Mol. Pharmacol., June 1, 2005; 67(6): 2016 - 2024. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Steinhoff, J. Buddenkotte, V. Shpacovitch, A. Rattenholl, C. Moormann, N. Vergnolle, T. A. Luger, and M. D. Hollenberg Proteinase-Activated Receptors: Transducers of Proteinase-Mediated Signaling in Inflammation and Immune Response Endocr. Rev., February 1, 2005; 26(1): 1 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Miyamoto, J. Yamauchi, N. Mizuno, and H. Itoh The Adaptor Protein Nck1 Mediates Endothelin A Receptor-regulated Cell Migration through the Cdc42-dependent c-Jun N-terminal Kinase Pathway J. Biol. Chem., August 13, 2004; 279(33): 34336 - 34342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mareel and A. Leroy Clinical, Cellular, and Molecular Aspects of Cancer Invasion Physiol Rev, April 1, 2003; 83(2): 337 - 376. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q.-D. NGUYEN, S. FAIVRE, E. BRUYNEEL, C. RIVAT, M. SETO, T. ENDO, M. MAREEL, S. EMAMI, and C. GESPACH RhoA- and RhoD-dependent regulatory switch of G{alpha} subunit signaling by PAR-1 receptors in cellular invasion FASEB J, April 1, 2002; 16(6): 565 - 576. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) on cellular invasion
pathways governed by src, HGF/Met, the epigenetic factors leptin and
TFFs, and the G