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The G12/13-RhoA signaling pathway contributes to efficient lysophosphatidic acid-stimulated cell migration

Abstract

The membrane redistribution and phosphorylation of focal adhesion kinase (FAK) have been reported to be important for cell migration. We previously showed that Lysophosphatidic acid (LPA) induced FAK membrane redistribution and autophosphorylation in ovarian cancer SK-OV3 cells and the signaling pathway consisting of Gi-Ras-MEKK1 mediated LPA-induced FAK membrane redistribution but not FAK autophosphorylation. We also showed that the disruption of the Gi-Ras-MEKK1 pathway led to a significant reduction in LPA-stimulated cell migration. These findings raised the question of whether LPA-induced FAK autophosphorylation was required for LPA-stimulated cell migration and what signaling mechanism was involved in LPA-induced FAK autophosphorylation. In this study, we expressed the membrane anchored wild-type FAK (CD2-FAK) in SK-OV3 cells and found that the expression of CD2-FAK greatly rescued LPA-stimulated cell migration in Gi or Ras-inhibited cells. However, Gi inhibitor pertussis toxin or dominant-negative H-Ras still significantly inhibited LPA-stimulated cell migration in cells expressing the membrane anchored FAK containing a mutation in the autophosphorylation site [CD2-FAK(Y397A)]. These results suggest that FAK autophosphorylation plays a role in LPA-stimulated cell migration. With the aid of p115RhoGEF-RGS, G12 and G13 minigenes to inhibit G12/13, we found that the G12/13 pathway was required for LPA-induced FAK autophosphorylation and efficient cell migration. Moreover, LPA activated RhoA and Rho kinase (ROCK) in a G12/13-dependent manner and their activities were required for LPA-induced FAK autophosphorylation. However, Rho or ROCK inhibitors displayed no effect on LPA-induced FAK membrane redistribution although they abolished LPA-induced cytoskeleton reorganization. Our studies show that the G12/13-RhoA-ROCK signaling pathway mediates LPA-induced FAK autophosphorylation and contributes to LPA-stimulated cell migration.

Introduction

Lysophosphatidic acid (LPA), which is generated from precursors in the plasma membrane, has numerous cellular effects including cell proliferation, calcium homeostasis, cytoskeleton reorganization, cell adhesion/migration and ion transport regulation (Moolenaar 1999; van Leeuwen et al., 2003a; Sengupta et al., 2004). LPA exerts its biological activities through its interaction with four identified LPA receptors, namely LPA1, LPA2, LPA3 and LPA4, which additionally activate the Gi, G12/13 and Gq subfamilies of G proteins (Moolenaar et al., 2004). While LPA1 is expressed ubiquitously, recent studies have shown that the levels of LPA2 and LPA3 are elevated in various tumors such as ovarian, colon, breast and prostate cancers (Daaka, 2002; Fujita et al., 2003; Kitayama et al., 2004; Shida et al., 2004). High concentrations of LPA was found in both the ascites and plasma of ovarian cancer patients (Xu et al., 1995a, 1995b; Shen et al., 1998) and this has led researchers to investigate the potential role of LPA in ovarian cancer development (Fang et al., 2000; Xie et al., 2002). Several lines of evidences have convincingly linked LPA to ovarian cancer progression and metastasis: (1) LPA is produced by ovarian cancer cells but not normal ovary surface epithelial cells (Eder et al., 2000; Luquain et al., 2003); (2) LPA can promote the expression/activation of various invasion-associated proteases including urokinase plasminogen activator and metalloproteinases in ovarian cancer cells (Pustilnik et al., 1999; Fishman et al., 2001); (3) LPA induces cyclooxygenase-2 expression to promote aggressive behavior in ovarian carcinoma cells (Symowicz et al., 2005); (4) LPA enhances proangiogenic factor production (VEGF, IL-6 and 8) by ovarian cancer cells and thus promotes ascites formation and ovarian cancer-associated angiogenesis (Schwartz et al., 2001; Fujita et al., 2003; Hu et al., 2003; Fang et al., 2004); and (5) LPA confers ovarian cancer cells with chemoresistances (Frankel and Mills, 1996). The importance of LPA in cancer development is further supported by the recent studies that disrupting LPA function by preventing LPA–LPA receptor interaction or reducing LPA production by cancer cells significantly reduces tumor progression and metastasis in experimental animals (Mukai et al., 1999; Tanyi et al., 2003).

Diverse signaling mechanisms have been reported to mediate LPA-stimulated cell migration. For example, LPA activates Rac1 to promote cell spreading, lamellipodium formation and cell migration in fibroblasts (van Leeuwen et al., 2003b). In pancreatic tumor cells, Erk activation is indicated to play a critical role for LPA-stimulated cell migration (Stähle et al., 2003). LPA-induced squamous cell carcinoma motility is mediated by EGF receptor transactivation (Gschwind et al., 2002). However, these studies have yet to identify which particular G proteins act downstream of LPA receptors and how they are involved in LPA-stimulated cell migration. Using the invasive ovarian cancer SK-OV3 line as our model system, we showed that LPA induced strong FAK autophosphorylation and membrane redistribution (Bian et al., 2004). Interception of the Gi-Ras-MEKK1 signaling pathway blocked LPA-induced FAK membrane redistribution and greatly inhibited LPA-stimulated cell migration; however, it did not alter LPA-induced FAK autophosphorylation (Bian et al., 2004). These observations raised question of whether FAK autophosphorylation was important for LPA-stimulated cell migration and if so, what signaling mechanism was responsible for LPA-induced FAK autophosphorylation.

GTPases of the Rho family play an important role in the regulation and coordination of cytoskeleton remodeling during cell migration (Etienne-Manneville and Hall, 2002; Raftopoulou and Hall, 2004). GTPases cycle between an active GTP-bound state and an inactive GDP-bound state. When in an active conformation, the GTPase can interact with and activate a variety of effector proteins. Of the Rho family members, Cdc42 is involved in the establishment of cell polarity during directed cell migration, Rac1 drives cell migration by promoting the extension of lamellipodia at the leading edge of the cell and RhoA promotes the formation of actin stress fiber and focal adhesion (Etienne-Manneville and Hall, 2002). The RhoA effector proteins, Rho kinases (ROCKs), are important for the RhoA-mediated events in cytoskeleton reorganization and focal adhesion formation (Riento and Ridley, 2003). In addition, RhoA and ROCK also control actin–myosin contractility and the retraction of the rear of the cell, thus driving translocation of the cell body (Riento and Ridley, 2003). Studies conducted in both fibroblasts and neuronal cells have shown that LPA activates RhoA through the G12/13 subunits of G proteins (Kranenburg et al., 1999; Yuan et al., 2003).

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase, which plays an essential role in cell migration (Sieg et al., 2000). FAK-deficient fibroblasts display refractory responses to motility-promoting stimuli (Ilic et al., 1995). In the course of cell migration, FAK is involved in focal adhesion turnover thus controlling the process of cell attachment and detachment, which are required for cell migration (Webb et al., 2004). FAK can be activated by many stimuli including integrin clustering, growth factors and even mechanical action (Schaller, 2003). Its activation starts with autophosphorylation at tyrosine 397 and thereby creating an SH2 binding motif to recruit Src family kinases as well as adaptor proteins such as p130Cas and Grb7 into a focal contact-associated signaling complex (Cary et al., 1998; Han and Guan, 1999). Recent evidences have demonstrated the importance of both FAK autophosphorylation and plasma membrane localization for FAK to facilitate cell migration (Schaller, 2003).

The goal of this study was to determine whether LPA-induced FAK autophosphorylation was required for LPA-stimulated cell migration. We showed that membrane-anchored wild-type FAK rescued LPA-stimulated cell migration in the Gi-Ras pathway-perturbed SK-OV3 cells, further supporting our previous finding that the Gi-Ras signaling participates in cell migration by facilitating LPA-induced FAK membrane redistribution. Interestingly, membrane-anchored FAK containing a mutation in the autophosphorylation site was unable to effectively rescue LPA-stimulated cell migration, suggesting that LPA-induced FAK autophosphorylation was necessary for efficient LPA-stimulated cell migration. To identify the signaling mechanism responsible for LPA-induced FAK autophosphorylation, p115RhoGEF-RGS and GRK2-RGS were expressed in SK-OV3 cells to respectively block G12/13 and Gq-associated signaling pathways. We found that G12/13-, but not Gq-associated signaling pathway mediated LPA-induced FAK autophosphorylation and was important for efficient LPA-stimulated cell migration. Further studies also showed that LPA activated RhoA in a G12/13-dependent manner and inhibiting RhoA activity inhibited LPA-induced FAK phopshorylation and cell migration without compromising LPA-induced FAK membrane redistribution. Finally, we provided evidence that ROCK was the downstream effector of RhoA to mediate LPA-induced FAK autophosphorylation.

Results

Expression of membrane-anchored wild-type FAK rescues LPA-stimulated cell migration in Gi-Ras pathway-perturbed SK-OV3 cells

We previously showed that the Gi-Ras-MEKK1 signaling pathway mediated LPA-stimulated cell migration by facilitating FAK membrane redistribution (Bian et al., 2004). We reasoned that if FAK membrane redistribution was necessary for LPA-stimulated cell migration, forcing membrane localization of FAK should rescue cell migration in the Gi-Ras pathway-perturbed cells. To test this possibility, a mammalian expression vector encoding the membrane-anchored CD2-FAK was cotransfected into SK-OV3 cells with pCMV-Puro. The CD2-FAK-expressing cells were obtained after 2-day selection with puromycin and subsequently analysed for their ability to respond to LPA for cell migration in Gi- or Ras-inhibited conditions. CD2-FAK-expressing cells displayed greater basal motility compared to CD2-expressing cells (control) (Figure 1a), presumably due to the greater basal kinase activity (Chan et al., 1994; Frisch et al., 1996); LPA stimulation resulted in approximately five-fold increase of cell migration over the unstimulated cells in both control and CD2-FAK-expressing cells (Figure 1a). The treatment of pertussis toxin or the expression of dominant-negative H-Ras greatly inhibited LPA-stimulated cell migration in CD2-expressing control cells (90 and 81% respectively) (Figure 1a). In contrast, pertussis toxin and dominant-negative H-Ras caused only a statistically insignificant 25 and 16% inhibition in LPA-stimulated cell migration in CD2-FAK-expressing cells (Figure 1a), respectively, indicating that the expression of CD2-FAK resulted in over 70% rescue of LPA-stimulated cell migration in Gi or Ras-inhibited cells. These results are consistent with our previous findings that LPA regulates cell migration by facilitating FAK membrane redistribution (Bian et al., 2004).

Figure 1
figure1

Membrane-anchored FAK can rescue LPA-stimulated cell migration in Gi or Ras-inhibited SK-OV3 cells. (a) CD2, CD2-FAK(wt) or CD-FAK(Y397A) plasmids were cotransfected into SK-OV3 cells with pCMV-puro for 24 h and then selected with 10 μg/ml puromycin for 2 days to obtain CD2 or CD-FAK expressing cells. The transfected cells were either infected with Ad containing H-Ras(−) for 24 h or treated with 2 μg/ml pertussis toxin and then analysed for LPA-stimulated cell migration. Data are the mean±s.e. of triplicates. n=3. *P<0.001 versus no LPA stimulation. #P<0.005 versus no LPA stimulation. P<0.05 versus no LPA stimulation. (b) The transfected cells were lysed and the cell lysates subjected to immunoblotting to detecting FAK with anti-FAK mAb. The membrane was stripped and reprobed with anti-actin polyclonal antibody to ensure equal protein loading. PTX: pertussis toxin. (c) The transfected cells were plated on coverslips overnight and then subjected to immunostaining to visualize FAK as described in ‘Material and methods’. (d) The transfected cells were starved for 24 h in serum-free medium and then stimulated with 10 μ M LPA for 1 h. Cells were lysed, cell lysates immunoprecipitated with anti-FAK mAb and the immunoprecipitates subjected to immunoblotting to detect autophosphorylated FAK with anti-FAK (phosphor-Tyr397) polyclonal antibody. The membrane was stripped and reprobed with anti-FAK polyclonal antibody to detect both endogenous and membrane anchored FAK.

LPA has been shown to induce FAK autophosphorylation at tyrosine 397 in ovarian cancer cells (Sawada et al., 2002a). We thus determined the importance of FAK autophosphorylation in LPA-stimulated cell migration by examining whether membrane-anchored FAK with a mutation at tyrosine 397 could rescue cell migration in Gi or Ras-inhibited cells. CD2-FAK(Y397A) expression vector was cotransfected into SK-OV3 cells with pCMV-puro, then CD2-FAK(Y397A)-expressing cells obtained by puromycin selection and analysed for LPA-stimulated cell migration in Gi or Ras-inhibited condition. Cells expressing CD2-FAK(Y397) exhibited reduced basal cell migration compared to the control (CD2-expressing cells) (Figure 1a). Pertussis toxin treatment or dominant-negative H-Ras inhibited 72 and 66% of LPA-stimulated cell migration in cells expressing CD2-FAK(Y397A) (Figure 1a), and this represented a marginal 20 and 18% rescue of LPA-stimulated cell migration. The difference observed between cells expressing CD2-FAK and CD2-FAK(Y397A) was not due to levels of their expression or amount distributed on the plasma membrane since immunoblotting displayed similar levels of CD2-FAK and CD2-FAK(Y397A) proteins in the transfected cells (Figure 1b) and immunostaining indicated similar staining intensity of CD2-FAK and CD2-FAK(Y397A) proteins distributed in the plasma membrane in these cells (Figure 1c). To determine whether membrane-anchored FAK could be autophosphorylated, cells transfected with plasmids encoding CD2, CD2-FAK or CD2-FAK(Y397A) were treated with LPA and then assayed for FAK phosphorylation. Immunoblotting with anti-phospho-FAK(Tyr397) polyclonal antibody showed that LPA elevated the levels of autophosphorylation of CD2-FAK while no antophosphorylation was detected in CD2-FAK(Y397A) (Figure 1d). Taken together, these results suggest that autophosphorylation at tyrosine 397 of FAK is important for efficient LPA-stimulated cell migration.

The G12/13 signaling pathway mediates LPA-induced FAK autophosphorylation and contributes to efficient LPA-stimulated cell migration

Since FAK autophosphorylation was required for efficient LPA-stimulated cell migration (Figure 1), we sought to identify the signaling pathway responsible for LPA-induced FAK autophosphorylation by examining the potential involvement of G12/13 and Gq using their specific inhibitors, p115RhoGEF-RGS (specifically blocking G12/13) (Kozasa and Ye, 2004) and GRK2-RGS (specifically blocking Gq) (Kozasa and Ye, 2004). To evaluate the effectiveness of these two inhibitors, we examined how they affected the well-characterized G12/13-mediated serum response element (SRE) promoter activation and Gq-mediated PLCβ1 activation in LPA-stimulated SK-OV3 cells. We found that p115RhoGEF-RGS abolished LPA-induced SRE promoter activation while GRK2-RGS abrogated LPA-induced PLCβ1 activation (Figure 2a), thus confirming the effectiveness and specificity of p115-RhoGEF-RGS and GRK2-RGS to respectively block G12/13 and Gq-associated signaling pathways. In the subsequent study, we conducted experiments to determine the time course of LPA-induced FAK autophosphorylation. SK-OV3 cells were stimulated with 10 μ M LPA for various times, then lysed and cell lysates immunoprecipitated with anti-FAK mAb. Immunoblotting with anti-phospho-FAK(Tyr397) polyclonal antibody showed that LPA induced FAK autophosphorylation as early as 10 min and the greatest levels of FAK autophosphorylation occurred at 1 h of LPA stimulation (Figure 2b). Expression of p115RhoGEF-RGS, but not GRK2-RGS, effectively blocked LPA-induced FAK autophorylation (Figure 2c). The difference between p115RhoGEF-RGS and GRK2-RGS was not due to their expression since immunoblotting with anti-myc mAb showed similar levels of both proteins (Figure 2d). Taken together, these results indicate that the G12/13-associated signaling mediates LPA-induced FAK autophosphorylation. In the subsequent experiment, we further determined the importance of G12/13-associated signaling for LPA-stimulated cell migration. SK-OV3 cells were infected with control Ad or Ad containing p115RhoGEF-RGS or GRK2-RGS and subsequently analysed for LPA-stimulated cell migration. The expression of p115RhoGEF-RGS, but not GRK2-RGS, inhibited over 50% of LPA-stimulated cell migration (Figure 2e). These results thus indicate the requirement of FAK autophosphorylation for efficient LPA-stimulated cell migration.

Figure 2
figure2

G12-13-associated signaling is involved in LPA-induced FAK autophosphorylation and efficient LPA-stimulated cell migration. (a) SK-OV3 cells were infected with 5000 viral particles/cell control Ad or Ad containing p115RhoGEF-RGS or GRK2-RGS for 24 h and switched to serum-free medium for another 24 h. Cells were stimulated with 10 μ M LPA for either 1 h (for SRE promoter activation) or 5 min (for PLCβ1 activation) and subsequently assayed for SRE promoter activity or PLCβ1 activity as described in ‘Material and methods’. Data are the mean±s.e. of triplicates. n=2. *P<0.001 versus no LPA stimulation. #P<0.01 versus no LPA stimulation. The PLCβ1 activity at one-fold is 3600±420 d.p.m. (b) SK-OV3 cells were starved overnight and then stimulated with 10 μ M LPA for various times. Cells were lysed and cell lysates immunoprecipitated with anti-FAK-conjugated agarose. After boiling, the immunoprecipitates were subjected to immunoblotting to detect FAK autophosphoration (FAK phosphorylation at Tyr397). The membrane was stripped and reprobed anti-FAK mAb. (c) SK-OV3 cells were infected with 5000 viral particles/cell control Ad or Ad containing p115RhoGEF-RGS or GRK2-RGS for 24 h and then switched to serum-free medium for another 24 h. Cells were stimulated with 10 μ M LPA for 1 h and cell lysates subjected to the analysis of FAK autophosphorylation. (d) SK-OV3 cells were infected with 5000 viral particles/cell control Ad or Ad containing p115RhoGEF-RGS or GRK2-RGS for 48 h, then lysed and cell lysates subjected to immunoblotting with anti-myc mAb. The membrane was stripped and reprobed with anti-actin polyclonal antibody to ensure equal protein loading. (e) SK-OV3 cells were infected with 5000 viral particles/cell control Ad or Ad containing p115RhoGEF-RGS or GRK2-RGS for 24 h and then switched to serum-free medium for another 24 h. Cells were detached with PBS containing 10 mM EDTA and analysed for LPA-stimulated cell migration. Data are the mean±s.e. of triplicates. n=3. *P<0.001 versus no LPA stimulation. #P<0.01 versus no LPA stimulation.

Although p115RhoGEF is usually regulated by G12/13-associated signaling, recent studies have also suggested that p115RhoGEF may also regulated by other signals distinct from G12/13 such as PKC and CD44 (Bourguignon et al., 2003; Holinstat et al., 2003) and we thus further investigated the involvement of G12 and G13 in LPA-induced FAK autophorylation and cell migration by specifically inhibiting G12 or G13. SK-OV3 cells were transiently transfected with either G12 or G13 minigenes, which contain amino acids 326–379 of G12 subunit and amino-acid 321–377 of G13 subunit, respectively, to specifically block the respective G protein/GPCR interaction, and subsequently analysed for LPA-induced FAK autophosphorylation. Expression of the G12 minigene blocked the majority of LPA-induced FAK autophosphorylation (Figure 3a) and combined expression of both G12 and G13 minigenes almost completely blocked LPA-induced FAK autophosphorylation (Figure 3a). Moreover, combined expression of both G12 and G13 minigenes also resulted in a similar degree of the inhibition in LPA-stimulated cell migration observed in cells expressing p115RhoGEF-RGS (Figure 3b). These results indicate that endogenously expressed G12 and G13 are responsible for LPA-induced FAK autophosphorylation and contribute to LPA-stimulated cell migration.

Figure 3
figure3

The involvement of G12 and G13 in LPA-induced FAK autophosprylation and cell migration. (a) G12 and/or G13 minigene plasmids were cotransfected into SK-OV3 cells with pCMV-puro for 24 h and then selected with 10 μg/ml puromycin for 2 days to obtain G12 or G13 mingene expressing cells. The transfected cells were then starved for 24 h and then stimulated with 10 μ M LPA for 1 h. Cells were lysed, cell lysates immunoprecipitated with anti-FAK mAb and immunoprecipitates subjected to immunoblotting to detect autophosphorylated FAK. The membrane stripped and reprobed with anti-FAK polyclonal antibody to detect FAK. The ratios of used plasmids are 9:1 for both G12 minigene/pCMV-puro and G13 minigene/pCMV-puro and 4.5:4.5:1 for G12 minigene/G13 minigene/pCMV-puro. (b) SK-OV3 cells expressing both G12 and G13 minigenes were starved for 24 h and then analysed for LPA-stimulated cell migration. Data are the mean±s.e. of triplicates. n=3. *P<0.005 versus no LPA stimulation. #P<0.05 versus no LPA stimulation.

LPA induces RhoA activation in a G12/13-dependent manner and RhoA activity is required for LPA-induced FAK autophosphorylation and efficient LPA-stimulated cell migration

RhoA has been described as the primary signaling mediator for many G12/13-mediated events (Buhl et al., 1995) and known to regulate cell migration by facilitating cytoskeleton reorganization (Etienne-Manneville and Hall, 2002). Therefore, it is likely that RhoA may serve as a downstream effector of G12/13 to facilitate LPA-induced FAK autophosphorylation. To test this possibility, we first examined the effect of LPA on RhoA activation in SK-OV3 cells. Cells were starved overnight and then stimulated with 10 μ M LPA for various times. Cells were lysed and cell lysates assayed for RhoA activity. LPA activated RhoA as early as 2 min and peaked at 5 min (Figure 4a). To determine the role of Gi, G12/13 and Gq in LPA-induced RhoA activation, SK-OV3 cells were either pretreated with pertussis toxin or infected with Ad containing p115RhoGEF-RGS prior to LPA stimulation. The expression of p115RhoGEF-RGS, but not the treatment of pertussis toxin, abolished LPA-induced RhoA activation (Figure 4b). These results suggest that LPA signals through G12/13, rather than Gi or Gq, for RhoA activation.

Figure 4
figure4

LPA signals through G12/13 for RhoA activation. (a) SK-OV3 cells were starved overnight and then stimulated with 10 μ M LPA for various times. Cells were lysed and cell lysates assayed for RhoA activity as described in ‘Material and methods.’ (b) SK-OV3 cells were infected with control Ad or Ad containing p115RhoGEF-RGS for 24 h and then starved for another 24 h. Cells were stimulated with 10 μ M LPA for 5 min, then lysed and cell lysates analysed for RhoA activity. In a parallel experiment, overnight starved cells were treated with 2 μg/ml pertussis toxin or left untreated for 2 h prior to 5 min LPA stimulation. PTX: pertussis toxin.

In the next experiment, we investigated the importance of RhoA activity in LPA-induced FAK autophosphorylation. SK-OV3 cells were infected with control Ad or Ad containing dominant-negative mutant of RhoA (T19N) or treated with Rho inhibitor C3 toxin and then assayed for LPA-induced FAK autophosphorylation. The expression of dominant-negative RhoA or the treatment of C3 toxin abolished LPA-induced FAK autophosphorylation (Figure 5a). In contrast, treatment with pertussis toxin to inhibit the Gi-associated signaling pathway did not significantly alter the levels of LPA-induced FAK autophosphorylation (Figure 5a). These results suggest that RhoA works downstream of G12/13 to mediate LPA-induced FAK autophosphorylation. In the parallel experiment, we also determined how inhibiting RhoA activity would affect LPA-stimulated SK-OV3 cell migration. The expression of dominant-negative RhoA or the treatment of C3 toxin led to 52 and 58% of reduction in LPA-stimulated cell migration while pertussis toxin inhibited approximately 90% of LPA-stimulated cell migration (Figure 5b). These results suggest that the efficient LPA-stimulated cell migration requires signal from the G12/13-RhoA pathway even though the Gi-Ras pathway may play a more dominant role in LPA-stimulated cell migration.

Figure 5
figure5

RhoA activity is required for LPA-induced FAK autophosphorylation and the efficient LPA-stimulated cell migration. SK-OV3 cells were infected with control Ad and Ad containing dominant-negative RhoA for 24 h and then starved for another 24 h. In a parallel experiment, cells were treated with 5 μg/ml C3 toxin for 24 h or 2 μg/ml pertussis toxin for 2 h or left untreated. (a) To determine LPA-induced FAK autophosphorylation, 10 μ M LPA was added to cells for 1 h and cell lysates analysed for the levels of FAK (phosphor-Tyr397) on the immunoblotting with the specific antibody. (b) To analyse LPA-induced cell migration, cells were added into the upper chambers of the transwells and then allowed to migrate toward the underwells for 4 h. The underwells contained serum-free medium in the absence or the presence of 10 μ M LPA. Data are the mean±s.e. of triplicates. n=3. *P<0.005 versus no LPA stimulation. #P<0.01 versus no LPA stimulation.

ROCK is RhoA downstream effector mediating LPA-induced FAK autophosphorylation

ROCK has been previously shown to act as the RhoA downstream effector to regulate cytoskeleton reorganization (Riento and Ridley, 2003). To determine whether ROCK was involved in LPA-stimulated FAK autophosphorylation, we first examined the effect of LPA on ROCK activity by analysing its ability to phosphorylate MLC, a known ROCK substrate. SK-OV3 cells were starved overnight and then stimulated with 10 μ M LPA for various times. Cells were lysed, cell lysates immunoprecipitated with anti-ROCK mAb and the immunoprecipitates analysed for their ability to phosphorylate MLC. LPA-induced ROCK activation as early as 5 min and the greatest ROCK activity observed at 10 min of LPA stimulation (Figure 6a). The increased MLC phosphorylation was not caused by the enhanced expression of ROCK because LPA treatment did not affect the levels of ROCK protein (Figure 6a). To determine the role of G12/13 and RhoA in LPA-induced ROCK activation, p115RhoGEF-RGS or dominant-negative RhoA was expressed in SK-OV3 cells with the aid of recombinant Ad. Both p115RhoGEF-RGS and dominant-negative RhoA significantly inhibited LPA-induced ROCK activation (Figure 6b). These results suggest that the G12/13-RhoA signaling pathway mediates LPA-induced ROCK activation.

Figure 6
figure6

LPA activates ROCK in a G12/13-RhoA-dependent manner and ROCK activity is required for LPA-induced FAK autophosphorylation. (a) SK-OV3 cells were starved overnight and then stimulated with 10 μ M LPA for various times. Cells were lysed and cell lysates immunoprecipitated with anti-ROCK mAb. After several washes, the immunoprecipitates were subjected to immunocomplex kinase assay to measure ROCK activity using GST-MLC as the substrate as described in ‘Material and methods.’ (b) SK-OV3 cells were infected with control Ad or Ad containing p115RhoGEF-RGS or dominant-negative RhoA for 24 h and then starved for another 24 h. Cells were stimulated with 10 μ M LPA for 10 min, then lysed and the cell lysate used to analyse ROCK activity. (c) Overnight-starved SK-OV3 cells were treated with 10 μ M Y-27632 for 2 h or 5 μg/ml C3 toxin for 24 h and then stimulated with 10 μ M LPA for 1 h. Cells were then lysed and cell lysates used to detect the levels of FAK autophosphorylation with anti-FAK(phosphor-Tyr397) polyclonal antibody.

To define the role of ROCK in LPA-induced FAK autophosphorylation, SK-OV3 cells were treated with the selective ROCK inhibitor Y-27632 for 2 h followed by 1 h of LPA stimulation. Immunoblotting with anti-phospho-FAK (Tyr397) showed that similar to C3 toxin, the treatment of Y-27632 almost completely diminished LPA-induced FAK autophosphorylation (Figure 6c). These results thus place ROCK as the downstream effector of RhoA for mediating LPA-induced FAK phosphorylation.

RhoA and ROCK activities are not required for LPA-induced FAK membrane redistribution

RhoA and ROCK are known to regulate cytoskeleton reorganization (Riento and Ridley, 2003; Raftopoulou and Hall, 2004). We thus determined whether RhoA or ROCK played any role in LPA-induced FAK cellular localization. SK-OV3 cells were treated with C3 toxin for 24 h, pertussis toxin or Y-27632 for 2 h and then stimulated with 10 μ M LPA for 1 h followed by immunostaining to determine the cellular distribution of FAK and actin reorganization. In unstimulated control cells, FAK was mainly seen in a diffused pattern throughout the cells and F-actin detected in the inner surface of the plasma membrane (Figure 7a). The treatment of pertussis toxin, C3 toxin or Y-27632 did not cause significant change in either FAK cellular localization or F-actin staining pattern in these cells (Figure 7a). After exposure to LPA, FAK was redistributed to the inner surface of plasma membrane and dramatic actin reorganization (formation of stress fiber) readily observed in over 90% of the control cells (Figure 7b). The treatment of pertussis toxin completely blocked LPA-induced FAK membrane redistribution (Figure 7b). In contrast, LPA-induced FAK membrane redistribution was not significantly affected in either C3 toxin or Y-27632-treated cells although LPA-induced actin reorganization was greatly diminished in these cells (Figure 7b). These results suggest that (1) LPA-induced FAK membrane redistribution is not regulated by G12/13-RhoA signaling pathway and (2) FAK membrane translocation is independent of cytoskeleton reorganization.

Figure 7
figure7

RhoA and ROCK activity are not required for LPA-induced FAK membrane redistribution. (a) SK-OV3 cells were plated on collagen-coated coverslips overnight, and then treated with 5 μg/ml C3 toxin for 24 h, 2 μg/ml pertussis toxin or 10 μ M Y-27632 for 2 h or left untreated. Cells were stimulated with 10 μ M LPA for 1 h followed by immunostaining to determine FAK cellular localization and actin reorganization as described in ‘Material and methods.’ (a) unstimulated cells, (b) LPA-stimulated cells.

Gi and G12/13-associated pathways are required for LPA-stimulated cell migration in various ovarian cancer cell types

In addition to the SK-OV3 line, we previously showed that LPA was also capable of efficiently stimulating cell migration in ovarian cancer OVCAR5, SW626 and IGROV1 lines. To determine the importance of Gi and G12/13-associated pathways in LPA-stimulated cell migration in these lines, the cells were either treated with 2 μg/ml pertussis toxin or infected with Ad containing p115RhoGEF-RGS or GRK2-RGS followed by the analysis of LPA-stimulated cell migration. The treatment of pertussis toxin and expression of p115RhoGEF-RGS inhibited approximately 90 and 50% LPA-stimulated cell migration in all three lines while no significant alteration in LPA-stimulated cell migration was detected in cells expressing GRK2-RGS (Figure 8). These results suggest that coordinated Gi and G12/13-associated signaling may be a general mechanism for LPA-stimulated cell migration, at least in ovarian cancer cells.

Figure 8
figure8

Gi and G12/13-associated signaling pathways are important for various ovarian cancer cell lines. OVCAR5, SW626 and IGROV1 lines were either treated with 2 μg/ml pertussis toxin or infected with Ad containing p115RhoGEF-RGS or GRK2-RGS and subsequently analysed for LPA-stimulated cell migration as described in ‘Material and methods’. Data are the mean±s.e. of triplicates. n=3. *P<0.001 versus no LPA stimulation. #P<0.01 versus no LPA stimulation.

Discussion

One of the most crucial components of cancer cell invasion and metastasis is cancer cell migration. We previously showed that LPA significantly stimulated ovarian cancer cell migration and that the Gi-Ras-MEKK1 signaling pathway was involved in LPA-stimulated cell migration by facilitating FAK membrane redistribution (Bian et al., 2004). Since LPA-induced FAK autophosphorylation in a Gi-Ras-MEKK1-independent mechanism, we thus investigated the importance of FAK phosphorylation in LPA-stimulated cell migration by determining whether membrane-anchored wild type and autophosphorylation-defective mutant FAK could rescue LPA-stimulated cell migration in Gi or Ras-inhibited SK-OV3 cells. Expression of membrane-anchored FAK (CD2-FAK) rescued over 70% of the LPA-stimulated cell migration in pertussis toxin-treated or dominant-negative H-Ras(−)-expressing SK-OV3 cells (Figure 1); in contrast, only marginally rescued cell migration (approximately 20%) was observed in cells expressing CD2-FAK(Y397A) (Figure 1). These results suggest that FAK autophosphorylation is required for efficient LPA-stimulated cell migration.

In addition to Gi-associated signaling, G12/13 and Gq-associated signaling pathways have also been shown to mediate LPA-induced cellular events. For example, Gq is necessary for LPA-induced protein kinase D activation in fibroblasts (Yuan et al., 2003). LPA induces neurite retraction through G12/13 (Kranenburg et al., 1999). In our studies, we found that the G12/13-associated signaling pathway was involved in LPA-induced FAK autophosphorylation and efficient LPA-stimulated cell migration (Figures 2 and 3). A recent study showed that both Gi and G12/13 are involved in sphingoline-1-phosphate-stimulated endothelial cell migration (Panetti et al., 2000). We thus considered the possibility that coordinated Gi and G12/13-associated signaling may also be a common mechanism for cell migration induced by other GPCR ligands.

The G12/13 subclass of G proteins has been implicated in Rho activation (Buhl et al., 1995) although evidence exists for the participation of Gq and Gi (Dutt et al., 2002; Zeng et al., 2002; Sah et al., 2005). We showed that LPA induced RhoA activation in a G12/13-dependent manner (Figure 4a and b). Blocking RhoA activity with dominant-negative RhoA or C3 toxin prevented LPA-induced FAK autophosphorylation and efficient LPA-stimulated cell migration (Figure 5a and b), suggesting that RhoA works downstream of G12/13 to mediate FAK autophosphorylation and to facilitate efficient LPA-stimulated cell migration. Our results are consistent with an early report that G12 and G13 can stimulate tyrosine phopsphorylation of FAK in a Rho-dependent mechanism in human embryonic kidney 293 cell line (Needham and Rozengurt, 1998). Furthermore, our finding also agrees well with a recent study showing alendronate inhibits LPA-stimulated cell migration by attenuating RhoA activation (Sawada et al., 2002b).

RhoA is the primary mediator of cellular contractility, stress fiber formation, and the establishment of focal adhesion (Etienne-Manneville and Hall, 2002; Raftopoulou and Hall, 2004). A principle effector of RhoA is ROCK, which modulates the actin cytoskeleton through pathways involving myosin phosphatase and LIM kinase (Riento and Ridley, 2003). In our studies, we showed that LPA activated ROCK through the G12/13-RhoA signaling pathway (Figure 6a and b). We further showed that similar to the RhoA inhibitor C3 toxin, the treatment of ROCK inhibitor Y-27632 blocked LPA-induced FAK autophosphorylation (Figure 6c) but not FAK membrane redistribution (Figure 7a and b). These results suggest that ROCK is the downstream effector of RhoA for mediating LPA-induced FAK autophopshorylation. Previous studies showed that sphingosine-1-phosphate and muscarinic receptor agonists induced FAK tyrosine phosphorylation in a Rho-dependent mechanism (Wang et al., 1997; Linseman et al., 2000). A recent study also reported that integrin-linked kinase-regulated cell migration is mediated through FAK in a RhoA-ROCK-dependent manner (Khyrul et al., 2004). All these findings pinpoint the importance of FAK autophosphorylation for efficient cell migration.

FAK plays a critical role in focal adhesion turnover thus controlling cell migration. Both FAK phosphorylation and membrane redistribution are implicated to be essential for FAK-mediated focal adhesion turnover (Schaller, 2003; Webb et al., 2004). In studies reported by various researchers, Rac1, ERK and EGF receptor transactivation were found to be involved in LPA-stimulated cell migration in various cell types (Gschwind et al., 2002; Stähle et al., 2003; van Leeuwen et al., 2003b). How FAK is linked to these reported diverse mechanisms remains unclear. A recent study showed that FAK was required for GPCR agonist-induced Rac1 activation (Sundberg et al., 2003), thus indicating that Rac1 may participate in LPA-stimulated cell migration by serving as a FAK downstream effector. Another study reported that Erk directly phosphorylated paxillin on serine 83 and that the mutation in this site disrupted paxillin/FAK association and FAK-facilitated cell migration (Ishibe et al., 2004), thus indicating Erk may be involved in cell migration by regulating FAK-paxillin interaction. Finally, FAK has been found to integrate both growth-factor and integrin signals to promote cell migration (Sieg et al., 2000), and EGF receptor activation has been shown to induce FAK phosphorylation (Hunger-Glaser et al., 2003). It is likely that the EGF receptor transaction may facilitate LPA-induced FAK autophosphorylation and cell migration.

We previously showed that the Gi-Ras-MEKK1 signaling pathway regulated LPA-induced FAK membrane translocation (Bian et al., 2004). In this study, we further showed that the G12/13-RhoA-ROCK signaling pathway was essential for FAK autophosphorylation. Taken together our previous and recent findings, we conclude that the Gi-Ras-MEKK1 and G12/13-RhoA-ROCK signaling pathways may work in concert to promote LPA-stimulated ovarian cancer cell migration by regulating FAK membrane redistribution and autophosphorylation respectively.

Materials and methods

CD2-FAK, G12/G13 minigene plasmids, reagents and cell line

Membrane-anchored FAK expression plasmids (CD2-FAK, CD2-FAK(Y397A)) or control CD2 plasmid were previously described (Chan et al., 1994; Frisch et al., 1996). G12 and G13 minigene expression plasmids were prepared by subcloning cDNA encoding HA-tagged C-terminal of human G12 (amino acids 326-379) and G13 (amino acids 321-377) into pRK5 vector. LPA (18:1) was purchased from Avantis Lipid (Alabaster, AL, USA). Pertussis toxin was obtained from List Laboratory (Campbell, CA, USA). C3 toxin and Y-27632 were purchased from BIOMOL (Plymoth Meeting, PA, USA). The antibodies used in the study are as follows: anti-RhoA and anti-ROCK mAb from BD Transduction Laboratory (Lexington, KY, USA); anti-FAK mAb and anti-phospho-FAK (Tyr397) polyclonal antibody from Upstate Biotechnology (Charlottesville, VA, USA); antiactin polyclonal antibody from Sigma (St Louis, MO, USA); anti-PLCβ1 and anti-myc mAb from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ovarian cancer SK-OV3, OVCAR5, SW626 and IGROV1 cell lines were maintained in DMEM containing 10% fetal calf serum at 37°C in a humidified 5% CO2 incubator.

Recombinant adenoviruses (Ad)

Recombinant Ad encoding myc-tagged p115RhoGEF-RGS, myc-tagged GRK2-RGS, dominant-negative RhoA(N19), and H-Ras(N17) have been described elsewhere (Han et al., 2002; Li et al., 2005). The viruses were purified using ViraBind™ Adenovirus Purification Kit (Cell Biolabs, San Diego, CA, USA) and quantitated by QuickTiter™ Adenovirus Quantitation Kit (Cell Biolabs). The dose of Ad used in this study was 5000 viral particles/cell.

Transwell migration assay

Cell migration was performed using transwells as previously described (Bian et al., 2004). Briefly, the undersurface of the transwell was coated with 10 μg/ml collagen I overnight at 4°C. Cells were added to the upper chamber of the transwell and allowed to migrate for 4 h. LPA (10 μ M) was added into the medium in the underwell to stimulate cell migration. To determine the potential signaling pathways important for LPA-stimulated cell migration, the conditions of each treatment prior to assay for cell migration were: 2-h treatment of 2 μg/ml pertussis toxin to block Gi; 24-h infection of Ad containing p115RhoGEF-RGS or GRK2-RGS to inhibit G12/13 and Gq, respectively; 24-h infection with Ad encoding dominant-negative H-Ras(N17) or RhoA(N19) to inhibit Ras and RhoA, respectively; and 24-h incubation with 5 μg/ml C3 toxin to inhibit Rho activity. To determine how the expression of membrane-anchored FAK affected LPA-stimulated cell migration, expression vector encoding CD2-FAK, CD2-FAK(Y397A) or CD2 were cotransfected into SK-OV3 cells with pCMV-puro for 24 h using PolyFect (Qiagen). Cells were selected with 10 μg/ml puromycin (Sigma) for 2 days to kill untransfected cells and the remaining cells used to analyse LPA-stimulated cell migration. To determine how inhibiting G12 or G13 subunit affected LPA-stimulated cell migration, expression vector encoding G12 or G13 minigene was cotransfected into SK-OV3 with pCMV-puro for 24 h following by 48-h puromycin treatment to kill untranscted cells. The remaining cells were analysed for LPA-stimulated cell migration.

Analysis of RhoA activity

RhoA activity was determined by measuring the levels of GTP-bound RhoA as previously described (Han et al., 2002). Briefly, SK-OV3 cells were starved overnight in serum-free medium and 10 μ M LPA then added to cells for various times (0, 2, 5, 10 and 30 min). Cells were lysed in Mg2+ lysis buffer and cell lysates analysed for the RhoA activity by determining the amount of RhoA pulled-down by Rhotekin Rho binding domain (GTP-bound RhoA). To determine the effect of pertussis toxin on LPA-stimulated RhoA activity, SK-OV3 cells were pretreated with 2 μg/ml pertussis toxin for 2 h prior to 10 min of LPA stimulation. To determine the effect of p115RhoGEF-RGS on LPA-induced RhoA activity, SK-OV3 cells were infected with Ad containing p115RhoGEF-RGS for 24 h, then starved for 24 h, followed by 10 min of LPA stimulation.

Analysis of FAK phosphorylation

SK-OV3 cells were starved overnight and then stimulated with 10 μ M LPA for various length of time (0, 5, 10, 30 and 60 min). Cells were lysed with radioimmunoassay buffer (RIPA), and cell lysates immunoprecipitated with anti-FAK mAb-agarose beads. The beads were washed with RIPA and proteins separated by electrophoresis. The levels of FAK phosphorylation at tyrosine 397 was determined by immunoblotting with anti-phospho-FAK(Tyr397) polyclonal antibody. To determine the effect of pertussis toxin, C3 toxin and Y27632 on LPA-induced FAK phosphorylation (Tyr397), cells were treated with 2 μg/ml pertussis toxin or 10 μ M Y-27632 for 2 h or 5 μg/ml C3 toxin for 24 h followed by 1 h LPA stimulation. To determine the involvement of G12/13, Ras and RhoA involvement in LPA-induced FAK phosphorylation, cells were infected with control Ad or Ad containing p115RhoGEF-RGS, dominant-negative H-Ras(N17) or RhoA(N19) for 24 h, then starved for 24 h followed by 1 h LPA stimulation.

Analysis of SRE promoter and PLCβ1 activities

To analyse SRE promoter activity, SK-OV3 cells were transfected with SRE promoter-luciferase plasmid (Stratagene, San Diego, CA, USA) for 24 h using Effectene (Qiagen), and then infected with control Ad or Ad containing p115RhoGEF-RGS or GRK2-RGS for 24 h followed by serum starvation for 24 h. The cells were stimulated with 10 μ M LPA for 1 h, then lysed and cell lysates measured for luciferase activity using Luciferase Activity System kit (Promega). To analyse PLCβ1 activity, SK-OV3 cells were infected with control Ad or Ad containing p115RhoGEF-RGS or GRK2-RGS for 24 h and then switched to serum-free medium for 24 h. Cells were treated with 10 μ M LPA for 5 min, then lysed in RIPA buffer and cell lysates immunoprecipitated with anti-PLCβ1 mAb followed by incubation with γ-bind beads (Amersham). The PLCβ1 activity was determined by measuring IP3 production as described previously (Martelli et al., 1992). Briefly, the PLCβ1 immunoprecipitates were washed several times with PLCβ1 assay buffer (100 mM Tris-HCl, pH 7.0, 150 mM NaCl, 0.06% taurodeoxycholate, and 10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mM sodium orthovanadate and 1 mM PMSF), and then incubated in 100 μl PLCβ1 assay buffer containing 3 nmol 3H-PIP2 (specific activity 30 000 dpm/nmol) at 37°C for 30 min. Assays were terminated by adding 200 μl 10% trichloroacetic acid to the reaction on ice followed by the addition of 100 μl of BSA (10 mg/ml). The reaction was centrifuged at 2000 g for 10 min to separate unhydrolysed 3H-PIP2 (pellet) from 3H-IP3 (supernatant). The IP3 production was measured by counting the radioactivity of the supernatants with a scintillation counter.

Analysis of ROCK activity

SK-OV3 cells were starved overnight and treated with 10 μ M LPA for various lengths of time (0, 5, 10, 30, 60 and 120 min). Cells were harvated in ice-cold RIPA, and the lysates incubated with anti-ROCK mAb for 2 h. The gamma bind beads (Amersham, Piscataway, NJ, USA) were added to the lysates for 1 h followed by four washes with RIPA buffer. The beads were resuspended in 10 μl of 1 × kinase buffer (25 mM tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2 and 1 mM DTT) and then mixed with 8 μl of 5 × kinase buffer, 5 μg of recombinant GST-myosin light chain (MLC) protein, 2 μl 1 mM ATP, and 2 μl of 10 μCi/μl [γ-32P]ATP (3000 Ci/mmol) in a total reaction volume of 50 μl. The mixtures were incubated at 37°C for 30 min, and then 4 × SDS sample buffer added to the reaction. The samples were boiled and separated on 10% SDS-polyacrylamide gel. The gel was dried and exposed to X-ray film. To determine the involvement of the G12/13-RhoA pathway in LPA-induced ROCK activation, SK-OV3 cells were infected with control Ad or Ad containing p115RhoGEF-RGS or dominant-negative RhoA(N17) for 24 h, then starved for 24 h followed by 10 min of LPA stimulation.

Immunostaining

SK-OV3 cells were cultured on 10 μg/ml collagen I-coated coverslips overnight and then treated with 10 μ M LPA for 1 h. Cells were fixed in 3% paraformaldehyde, then permeabilized with 1% Triton X-100 and blocked with 5% BSA. Anti-FAK mAb (1:100 dilution) and rhodamine-conjugated phalloidin (Molecular Probe, Eugene, OR, USA) were added to the cells for 1 h followed by a 1-h incubation with FITC-conjugated rabbit-anti-mouse secondary antibody (Molecular Probe). FAK and polymerized actin were visualized by fluorescence microscopy (Axiovert 200M, Zeiss). To determine the role of RhoA and ROCK in LPA-induced FAK membrane redistribution and actin reorganization, SK-OV3 cells were treated with 5 μg/ml C3 toxin for 24 h or 10 μ M Y-27632 for 2 h prior to 1 h LPA stimulation. To determine the role of Gi in LPA-induced FAK membrane redistribution, SK-OV3 cells were treated with 2 μg/ml pertussis toxin for 2 h prior to 1 h LPA stimulation.

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Acknowledgements

We are grateful to Joan Gausepohl for preparation of this manuscript. This study was supported by the National Institute of Health Grant R01 CA93926. This is paper IMM-17465 from the Scripps Research Institute.

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Bian, D., Mahanivong, C., Yu, J. et al. The G12/13-RhoA signaling pathway contributes to efficient lysophosphatidic acid-stimulated cell migration. Oncogene 25, 2234–2244 (2006). https://doi.org/10.1038/sj.onc.1209261

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Keywords

  • LPA
  • FAK
  • cell migration

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