Mitogenic Signaling by Lysophosphatidic Acid (LPA) Involves Ga12

Mitogenic signaling by lysophosphatidic acid (LPA) involves Ga12

V Radhika1, Ji Hee Ha1, Muralidharan Jayaraman1, Siu-Tai Tsim1 and N Dhanasekaran*,1

1Fels Institute for Cancer Research and Molecular Biology, Temple University School Medicine, Philadelphia, PA 19140, USA

Lysophosphatidic acid (LPA), a major G protein coupled heterotrimeric G proteins such as Gi,Gq,andG12/13 receptor (GPCR)-activating ligand present in serum, (Van Corven et al., 1989; Moolenaar, 1999; Anliker and elicits growth factor like responses by stimulating specific Chun, 2004). To date, four distinct LPA receptors GPCRs coupled to heterotrimeric G proteins such as Gi, (LPARs), namely LPA1 (Edg-2), LPA2 (Edg-4), LPA3 Gq, and G12/13. Previous studies have shown that the (Edg-7), and LPA4, have been identified (Fischer et al., overexpression of wild-type Ga12 (Ga12WT) results in the 1998; Kranenburg and Moolenaar, 2001; Lynch, 2002). oncogenic transformation of NIH3T3 cells (Ga12WT- While LPA1 is expressed ubiquitously, other LPARs are NIH3T3) in a serum-dependent manner. Based on the expressed only at very low levels in a tissue-specific potent growth-stimulating activity of LPA and the manner. However, recent studies have identified that the presence of LPA and LPA-like molecules in the serum, expression of LPA receptors LPA2 and LPA3, are we hypothesized that the serum-dependent neoplastic greatly increased in various tumors such as ovarian and transformation of Ga12WT-NIH3T3 cells was mediated colon cancers (Pustilnik et al., 1999; Fang et al., 2002; by the stimulation of LPA-receptors (LPARs) by LPA in Mills and Moolenaar, 2003). It has also been observed the serum. In the present study, using guanine nucleotide that ovarian tumors synthesize and secrete LPA, thereby exchange assay and GST-TPRbinding assay, we show increasing LPA levels in the ascitic fluid and serum of that the treatment of Ga12WT-NIH3T3 with 2 lM LPA ovarian cancer patients (Xu et al., 1995; Andre et al., leads to the activation of Ga12. Stimulation of these cells 2002). Furthermore, the growth factor like activity of with LPA promotes JNK-activation, a critical component LPA in stimulating cancer cell proliferation, migration, of Ga12-response and cell proliferation. We also show that and invasiveness has been well documented (Moolenaar LPA can substitute for serum in stimulating JNK-activity, et al., 2004). Considering the importance of such LPA DNA synthesis, and proliferation of Ga12WT-NIH3T3 and LPAR-mediated autocrine and paracrine signaling cells. LPA-mediated proliferative response in NIH3T3 pathways in tumor etiology and progression, there has cells involves Ga12, but not the closely related Ga13. been a major focus in identifying the G protein that Pretreatment of Ga12WT-NIH3T3 cells with suramin couples LPARs to cell proliferation (Anliker and Chun, (100 lM), a receptor-uncoupling agent, inhibited LPA- 2004). stimulated proliferation of these cells by 55% demonstrat- The ability of LPARs to stimulate different physio- ing the signal coupling between cell surface LPARand logical responses has primarily been ascribed to their Ga12 in the neoplastic proliferation of NIH3T3 cells. As coupling to the a-subunits of different G-proteins (Van LPA and LPARmediated mitogenic pathways have been Corven et al., 1989; Moolenaar et al., 2004). While Gaq/ shown to play a major role in tumor genesis and 11 have been reported to mediate PLC activation in progression, a mechanistic understanding of the signal response to LPA, Ga13 as well as Gaq has been shown to coupling between LPAR, Ga12, and the downstream activate the cytoskeletal changes in response to LPA effectors is likely to unravel additional targets for novel (Kranenburg et al., 1999; Van Leeuwen et al., 2003; cancer chemotherapies. Yuan et al., 2003). However, the identity of the a- Oncogene (2005) 24, 4597–4603. doi:10.1038/sj.onc.1208665 subunit that transduces mitogenic signals from LPARs Published online 18 April 2005 has remained unresolved. While LPA-mediated proliferation has been shown to be associated with Gi-family Keywords: G protein; LPA; Ga13; cell-proliferation; of G proteins in neuronal cells (Tigyi et al., 1996), the oncogene; transformation weak mitogenic-activity of Gai (Radhika and Dhanase- karan, 2001) casts uncertainties on the role of Gai as the mediator of LPA-mediated mitogenic responses. In this context, the strong mitogenic activity and the ability to Lysophosphatidic acid (LPA) is a single chain glycer- couple to LPARs points to Ga12 as a potential candidate ophospholipid that acts as an extracellular growth for transducing cell proliferation signals from LPAR to factor by activating specific seven transmembrane, G intracellular effectors. While previous studies have protein coupled receptors (GPCRs) that are coupled to demonstrated that LPA can stimulate Ga12 (Gohla et al., 1998), evidence linking LPA and LPARs to Ga12- *Correspondence: N Dhanasekaran; E-mail: [email protected] mediated mitogenic responses has not been established. Received 1 October 2004; revised 22 February 2005; accepted 22 Using NIH3T3 cells stably expressing Ga12, here we February 2005; published online 18 April 2005 demonstrate that LPA activates Ga12 by stimulating LPA stimulation of Ga12 V Radhika et al 4598 GDP/GTP exchange and that LPA promotes JNK- activation, a critical component of Ga12-response and cell proliferation. In addition, we show that LPA- mediated proliferative response in NIH3T3 cells involves Ga12, but not Ga13, and that LPA can substitute for serum in stimulating JNK-activity, DNA synthesis, and proliferation of Ga12WT-NIH3T3 cells. Further- more, the observation that suramin, an agent that uncouples ligand-cell surface receptor interaction, specifically inhibits LPA-stimulated proliferation of Ga12WT-NIH3T3 cells demonstrate the signal coupling between LPA receptor and Ga12 in stimulating the proliferative responses of NIH3T3 cells. These results, identifying Ga12 as the signal transducer for LPA- mediated mitogenic pathway, is likely to have far reaching implications in defining the signaling mechanisms underlying the LPA-mediated autocrine/paracrine control of tumor cell growth and progression. NIH3T3 cells stably expressing Ga12WT show increased cell proliferation when grown in medium containing 5% calf serum (Figure 1a, b). Previous studies have shown that the proliferation and subse- quent transformation of Ga12WT-NIH3T3 cells are fully contingent upon the presence of serum, whereas the

mutational activation of Ga12 abrogates such serum- Figure 1 Expression of Ga12WT stimulates the proliferation of requirement (Chan et al., 1993; Dermott and Dhanase- NIH3T3 cells: expression of Ga12 in NIH3T3 cells and their karan, 2002). This has been attributed to a growth proliferative response were monitored in comparison with the factor that stimulates the expressed Ga WT in these pcDNA3-vector control cells. (a) Expression of wild-type Ga12 was 12 monitored by immunoblot analysis of lysates (50 mg) from NIH3T3 cells (Dermott and Dhanasekaran, 2002; Dermott et al., cells stably expressing pcDNA3-vector and Ga12QL-NIH3T3 cells 2004). Since LPA is a major GPCR-activating growth using antibodies to Ga12 (a) following previously published factor present in the serum (Moolenaar et al., 2004), we procedures (Dermott et al., 2004). The blot was stripped and re- investigated whether LPA, through its respective probed with GAPDH antibody to monitor equal loading or proteins. Autoradiogram shows the expression of Ga12WT cells in GPCRs, stimulates Ga12 in mediating the proliferation NIH3T3 cells. (b) Proliferation of NIH3T3 cells expressing of these cells. Ga12WT was determined by quantifying the increase in cell 5 To demonstrate that LPA stimulates Ga12 in these numbers. NIH3T3-Ga12WT and NIH3T3-pcDNA3 (3 Â 10 ) were cells, we sought to determine the ability of LPA to plated on 60-mm dishes. After synchronizing the cells by serum- catalyse the exchange of guanine nucleotides in Ga . starvation for 24h, their growth in medium containing 5% calf 12 serum was monitored by counting cells at 24h using a hemacyt- Activated GPCRs including LPARs catalyse the ex- ometer. The histogram shows the increase in the number of cells in change of guanine nucleotides in the a-subunits of the Ga12WT expressing NIH3T3 cells as compared to vector control heterotrimeric G protein through which it transmits the cells. Mean7s.e. values were determined from three independent signal. Therefore, analysis of GTP-loading in the experiments candidate a-subunit, in response to the ligand of interest, has been used to identify the specific coupling between the GPCR and the a-subunit of interest (Offermanns et al., 1994; Barr et al., 1997; Windh and primary role of LPA in Ga12-mediated neoplastic Manning, 2002). To carry out such an analysis, the growth of NIH3T3 cells. plasma membranes prepared from Ga12WT- or vector Recently, it has been shown that the activated forms 35 control NIH3T3 cells were preincubated with S- of Ga12 and Ga13 bind to the tetratricopeptide repeat GTPgS for 5 min and stimulated with 2 mM of LPA. (TPR) domain of Ser/Thr phosphatase type 5 (Yama- Ga12 was immunoprecipitated from the membranes and guchi et al., 2002, 2003). Based on this observation, it the 35S-radiolabeled-GTP-gS bound to the immunopre- has been demonstrated that the interaction between the cipitated Ga12 was quantified in a scintillation counter. glutathione S-transferase (GST)-fused TPR domain of Results from these experiments also indicated that the PP5 (GST-TPR) and activated Ga12 or Ga13 can be used incubation of membranes with 2 mM LPA stimulated to determine the activation status of either of these a- GTP-loading in Ga12 (Figure 2b), suggesting the subunits (Yamaguchi et al., 2003). Therefore, to further coupling between a putative LPAR and Ga12 in these validate the stimulation of Ga12 by LPA in these cells, cells. These results, in conjunction with the previous we examined the ability of GST-TPR to pull down Ga12 findings that LPA and LPA-like molecules form the from the lysates from LPA-stimulated (2 mM for 1 min) major group of GPCR-ligands present in the serum and Ga12WT-NIH3T3 cells following the methods of Ya- the LPARs, LPA1 and LPA2, are highly expressed in maguchi et al. (2003). Lysates from Ga12WT-NIH3T3 NIH3T3 cells (Fischer et al., 2001), highlight the cells exposed to 5% calf serum and Ga12QL-NIH3T3

Oncogene LPA stimulation of Ga12 V Radhika et al 4599 cells that stably expresses mutationally activated form of downstream activation of JNKs in NIH3T3 cells. Ga12 (Ga12Q229L or Ga12QL) were used as positive Lysates from cells stimulated with 5% serum or 2 mM controls. The presence of Ga12, pulled down by GST- LPA were subjected to an in vitro solid phase kinase TPR bound to glutathione-Sepharose beads, was assay using glutathione-Sepharose bound GST-c-Jun identiﬁed by immunoblot analysis using antibodies to (1–79) as a substrate. When Ga12WT-NIH3T3 cells were Ga12. Results from these experiments indicated that the stimulated with 5% serum for 10 min, they showed the treatment of cells with LPA readily activated Ga12 as potent activation of JNK similar to our previously indicated by the increased levels of Ga12 pulled-down by published results. More interestingly, stimulating GST-TPR-Sepharose beads in lysates from LPA-stimu- Ga12WT-NIH3T3 cells with 2 mM LPA resulted in the lated cells compared to those of unstimulated control activation of JNK similar to that of serum stimulation groups (Figure 2b, lane 5 versus lane 2 and lane 4). (Figure 3c, d). Increasing concentration of LPA showed Together with the observation that the levels of LPA- a concentration-dependent increase in JNK activity activated Ga12 (Figure 2b, lane 5) are comparable to (Figure 3b). This increase is quite comparable to the those of serum-stimulated (Figure 2b, lane 6) or concentration-dependent increase in JNK activity in mutationally activated Ga12 (Figure 2b, lane 7), our response to 5% calf serum (Figure 3a), thereby results further validate our conclusion (Figure 2a) that demonstrating the functional activation of Ga12 by LPA readily stimulates Ga12 in these cells. LPA/LPAR. In addition to establishing the functional To establish the functional coupling between LPA- coupling between LPA-LPAR and Ga12 in JNK stimulated LPAR (LPA-LPAR) and Ga12, we decided response, these results also indicate that LPA can to examine whether the stimulation of LPA can be effectively substitute for serum in activating JNK in correlated with a previously established Ga12-speciﬁc Ga12-NIH3T3 cells. functional response. Previous studies from our labora- A role for pertussis toxin (PTX)-sensitive Gai has tory as well as others have shown Ga12-mediated cell been recently proposed in LPA-stimulated cell prolif- proliferation and transformation involves the activation eration (Tigyi et al., 1996). However, the pretreatment of Jun-kinases (JNKs) (Prasad et al., 1995; Collins et al., of Ga12WT-NIH3T3 or pcDNA3-NIH3T3 cells 1996; Mitsui et al., 1997). Therefore, we investigated with 15 U of PTX failed to inhibit LPA-stimulated whether LPA-stimulation of Ga12WT leads to its proliferation of these cells (number of cells in

Figure 2 LPA stimulates Ga12 in NIH3T3 cells. Stimulation of Ga12 by LPA was monitored by LPA-mediated quanine nucleotide exchange in Ga12 as well as by the assessing the levels of activated Ga12 binding to GST-TPR. (a) LPA stimulated guanine nucleotide exchange in Ga12 was monitored using the membranes prepared from NIH3T3 cells expressing Ga12WT and vector pcDNA3 following the previously published methods (Wong et al., 1995). The membrane pellet was resuspended in 100 ml of lysis buffer. LPA-stimulated guanine nucleotide exchange in Ga12 was monitored using [35S]GTPgS loading assay following previously published procedures (Windh and Manning, 2002). Membrane proteins (20 mg), prepared from NIH3T3 cells stably expressing Ga12WT and pcDNA3-vector, were resuspended in 55 mlof20mM Tris-HCl, pH 7.4, 3 mM MgCl2,1mM EDTA, 100 mM NaCl and 1mM of GDP and incubated with 1 mCi of [35S]GTPgS(1mCi, 5nM) for 5 min at 301C in the presence or absence of 2 mM LPA or 5% CS. The reaction was stopped by the addition of 600 mlof immunoprecipitation buffer containing 50 mM Tris HCl, pH 7.5, 20 mM MgCl2, 150 mM NaCl, 0.5% NP40, 1% aprotinin, 100 mM GDP, and 100 mM GTP. Ga12 in the reaction mixture was immunoprecipitated using Ga12-antibodies and protein-A Sephar- ose beads. Preimmune serum was used as a control for immunorecipitation. [35S]GTP binding stimulated by 5% calf serum and 2 mM LPA was quantiﬁed in a scintillation counter. Results shown are the mean7s.e.m. values of three experiments and * denotes Po0.01 compared to unstimulated control value. (b) GST-TPR binding assay was carried out following the methods of Yamaguchi et al. (2003). Ga12WT-NIH3T3, Ga12QL-NIH3T3, or pcDNA3-NIH3T3 were stimulated with either LPA (2 mM)or bovine serum (5%) for 1 min, rinsed twice with phosphate-buffered saline, and lysed with 500 ml of the ice- cold cell lysis buffer (25 mM HEPES, 0.1% Triton X-100, 300 mM NaCl, 20 mM b-glyceropho- sphate, 1.5 mM MgCl2, 0.2 mM EDTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM PMSF, 1 mM Na3VO4). Cell lysates were then centrifuged for 10 min at 15 000 Â g at 41C, and the supernatants were incubated with GST-TPR bound to glutathione-Sepharose beads for 3 h at 41C. After washing the beads with the ice-cold cell lysis buffer, the bound proteins were fractionated by SDS–PAGE and subjected to immunoblot analysis with antibodies to Ga12

Oncogene LPA stimulation of Ga12 V Radhika et al 4600 5 Ga12WT-NIH3T3 þ LPA ¼ 16.070.5 Â 10 compared LPA was monitored with appropriate controls. A 5 to 13.671.1 Â 10 cells in Ga12WT þ LPA þ PTX duplicate group of cells treated with 5% calf serum group), suggesting little or no role for Gai in LPA- was used as positive control. The ability of LPA to mediated mitogenic pathway in these cells. Our results, stimulate cell growth was determined by monitoring the presented here thus far, have established that LPA, DNA synthesis in these cells in response to LPA using presumably through its responsive LPARs, stimulates 3H-thymidine incorporation into DNA as an index for Ga12 and the downstream signaling events regulated by DNA synthesis (Vara Prasad et al., 1994). When cells Ga12 such as the activation of JNK (Prasad et al., 1995; expressing Ga12 along with vector control group was Collins et al., 1996; Voyno-Yasenetskaya et al., 1996). stimulated with 2 or 20 mM of LPA, there was four- to Next, we examined whether LPA can stimulate Ga12- six-fold increases in DNA synthesis compared to vector mediated proliferative response of NIH3T3 cells. If control cells (Figure 4), indicating that LPA stimulates LPA-LPAR preferentially couples to Ga12, it can be cell proliferation via Ga12. To rule out the possibility reasoned that LPA stimulates cell proliferation more that the observed coupling between LPA-LPAR and potently in cells that overexpresses Ga12. To establish Ga12 in stimulating mitogenic pathway was due to the such a correlation, the proliferation of NIH3T3 cells overexpression of Ga12, we also examined the effect of stably expressing Ga12WT in response to 2 or 20 mM of LPA on the mitogenic responses of NIH3T3 cells overexpressing wild-type Ga13. Results from such analysis indicated that LPA-LPAR stimulated the mitogenic responses only in Ga12-expressing Ga12WT- NIH3T3 cells and not in Ga13-expressing Ga13WT- NIH3T3 cells (Figure 4). These results, while clearly establishing that the observed LPA/LPAR-coupling to Ga12 in eliciting mitogenic response is not due to the forced promiscuous coupling by the overexpressed a- subunit, further demonstrates that LPA-LPARs couples speciﬁcally to Ga12 to stimulate mitogenic responses. Recently, it has been shown that LPA can exert its effect through an intracellular receptor belonging to peroxisome proliferator-activated receptor family (McIntyre et al., 2003). To establish that LPA stimulates Ga12-mediated mitogenic responses through the cell surface LPARs coupling to Ga12, we investigated the effects of suramin on LPA-mediated proliferation of Ga12WT-NIH3T3 cells. It has been shown that suramin

Figure 3 LPA stimulates JNK activity in NIH3T3 cells expressing Ga12WT: lysates from Ga12WT-NIH3T3 and pCDNA3-NIH3T3 stimulated with 5% calf serum and 2 mM LPA for 15 min were subjected to JNK assay using GST-c-Jun (1–79) beads (Prasad et al., 1995). The cells were stimulated with 5% CS and 2 mM LPA for 24h. In total, 100 mg of the lysate protein was incubated with 10 ml of 50% GST-c-Jun (1–79)-bound glutathione-Sepharose beads as substrate for 2 h at 41C in a rotator. At the end of the incubation, the beads were washed with JNK lysis buffer followed by two washes with JNK reaction buffer. The beads were resuspended in 40 ml of JNK buffer containing 20 mM [g-32P]ATP (5000 cpm/pmol) and the kinase reaction was carried out for 20 min at 301C. The reaction was stopped by addition of Laemmli’s buffer. Phosphorylated proteins were resolved in SDS–polyacrylamide gel and visualized by autoradiogram. The bands were quantiﬁed for JNK activity. JNK activity is presented as fold stimulation over the unstimulated control (US). (a) Graph shows the fold stimulation of JNK activity in response to different concentrations of calf serum in pCDNA3 and Ga12WT expressing NIH3T3 cells over the unstimulated control. (b) Graph shows the fold stimulation of JNK activity in response to different concentrations of LPA in pCDNA3-NIH3T3 and Ga12WT-NIH3T3 cells over the unstimulated control. (c) Autoradiogram showing phospho-JNK from Ga12WT-NIH3T3 and pCDNA3-NIH3T3 cells stimulated by 5% CS and 2 mM LPA. (d) Histogram showing the fold increase of JNK activity in Ga12WT and pCDNA3 expressing NIH3T3 cells over the unstimulated control. JNK activity in Ga12WT is high as compared to the vector control-pCDNA3 in response to both CS and LPA. Results presented as bar diagram were from three independent experiments (mean7s.e.) whereas the results presented as the autoradiogram is from a typical experiments

Oncogene LPA stimulation of Ga12 V Radhika et al 4601

Figure 5 Cell proliferation of Ga12WT is stimulated by LPA receptors. Cell proliferation was assayed by [3H]Thymidine incorporation. Ga12WT-NIH3T3 and pCDNA3-NIH3T3 was Figure 4 LPA stimulates proliferation of NIH3T3 cells expressing stimulated by 20 mM LPA in the presence or absence of 100 mM Ga12WT: proliferation of NIH3T3 cells expressing wild-type Ga12, Suramin. Histograms show the fold increase in [3H]Thymidine Ga13, or empty vector in response to LPA was monitored by M 3 incorporation over the unstimulated control in response to 20 m quantifying the incorporation of H-labeled thymidine in DNA. To LPA and Suramin. [3H]Thymidine incorporation was reduced in monitor, DNA synthesis, NIH 3T3 cells expressing vector, Ga12, 4 Ga12WT-NIH3T3 cells in the presence of Suramin indicating the and Ga13 were seeded in 24-well plates (5 Â 10 cells/well) and cell proliferation of Ga WT is stimulated by LPA receptors. 1 12 grown for 24h at 37 C in DMEM with 5% CS. The cells were Mean7s.e. values were determined from three independent serum-starved for 24h by replacing serum media with 0.2% BSA experiments and 20 mM HEPES in DMEM to obtain quiescence. Cells were then stimulated with 2 mM LPA, 20 mM LPA, 5% calf serum or none for 9 h, following which 1 mCi of 3H-labeled thymidine (20 Ci/ mmol) was added to each well and incubated for an additional 6 h as a diagnostic or prognostic marker for ovarian cancers at 371C. Cells were then washed with PBS and solubilized in 0.1% underscore the need to understand the signaling SDS. DNA was precipitated with 10% chilled TCA and the mechanisms regulated by LPA receptors in tumor cell precipitate was collected by filtration on GF/C Whatman filters. growth (Xu et al., 1998; Mills et al., 2002). In this The TCA precipitable counts were determined by scintillation counting. Results presented as bar diagram are derived from a set context, our results demonstrating the ability of LPA to of three independent experiments (mean7s.e.) stimulate Ga12-medaited mitogenic response is of great- er significance, considering the potent mitogenic and oncogenic activity of Ga12. Previous studies have shown that PTX-sensitive Gi inhibits the interaction of mitogenic ligands such as LPA family of G proteins as well as PTX-insensitive Gq and by disrupting their interactions with their specific cell G12 family of G proteins can couple LPARs to surface receptors (Chan et al., 1993). Therefore, we mitogenic responses (Mills and Moolenaar, 2003). investigated the effect of suramin on LPA-stimulated However, the activated mutants of the a-subunits of DNA synthesis in Ga12WT-NIH3T3 cells by determin- Gi or Gq family of G proteins are extremely weak in ing the rate of3H-Thymidine incorporation into the stimulating mitogenic signaling pathways in any of the DNA. As shown in Figure 5, treatment of Ga12WT- cell types that have been tested so far (Radhika and transfectants with 100 mM suramin for 1 h inhibited Dhanasekaran, 2001). Even Ga13, which shares 67% LPA-stimulated DNA synthesis by 55%. These results amino-acid identity with Ga12, is relatively weak in clearly demonstrated that the stimulation of DNA eliciting mitogenic response (Vara Prasad et al., 1994). synthesis by LPA in NIH3T3 cells expressing wild-type In contrast, the ability to stimulate mitogenic response Ga12 requires the activation of Ga12 by LPA through its by serum-stimulated Ga12-WT as well as the activated cognate receptor(s). mutant of Ga12 (Ga12QL) is quite potent (Vara Prasad Aberrant activation of oncogenes or inactivation of et al., 1994; Dermott and Dhanasekaran, 2002). It is tumor suppressor genes has been implicated in the significant to note that the transforming activity of Ga12 genesis and progression of different cancers (Hanahan is comparable only to that of the activated mutants of and Weinberg, 2000). Recent studies have indicated that H-ras (Vara Prasad et al., 1994). Therefore, it is more growth promoting GPCRs such as LPARs and their likely that LPAR couples to Ga12 in stimulating coupled G proteins play a potential role in cancer mitogenic responses. The results presented here firmly pathophysiology (Mills and Moolenaar, 2003; Radeff- support such view. However, it should be noted here Huang et al., 2004). Studies evaluating the role of LPA that our results do not negate the ability of LPA to

Oncogene LPA stimulation of Ga12 V Radhika et al 4602 stimulate Ga13 or any other a-subunits in a different LPA2 receptors. It has been shown that NIH3T3 cells cellular or physiological context. Results from different express LPA1 and LPA2 (Fischer et al., 2001). The laboratories, including ours, indicate that the stimula- concentration-dependent activation of JNK by LPA in tion of a specific Ga-subunit by LPA is dependent on the Ga12WT-NIH3T3 cells suggest that LPA1 and/or LPA2 cell-type or as well as the physiological context, receptors couple to Ga12 in these cells. presumably determined by the type of LPAR expressed Previous studies have shown that LPA1-receptor (Tigyi et al., 1996). While LPA readily activates Ga12 in specifically couples to PTX-dependent signaling path- NIH3T3 cells, it has little or no effect on Ga13.In way (Tigyi et al., 1996). Since PTX-treatment failed contrast, it appears that LPA stimulation is specific to to have any effect on LPA-stimulated proliferation Ga13 in HEK293 cells (Yamaguchi et al., 2003; Ha and of Ga12WT-NIH3T3 cells (number of cells in Ga12WT þ Dhanasekaran, unpublished results). Nonetheless, taken LPA ¼ 16.070.5 Â 105 compared to 13.671.1 Â 105 in together with the previous studies that have shown that Ga12WT þ LPA þ PTX group), it is more likely that the the transforming activity of Ga13 is extremely weak LPAR coupled to Ga12 in stimulating mitogenic compared to Ga12 and the loss of Ga13 does not affect response is LPA2-receptor. The observation that the the mitogenic response of Ga13À/À MEFs, our studies increased expression of LPA1 induces apoptosis (Furui presented here point to a role for Ga12 – and not to Ga13 et al., 1999), whereas the expression of LPA2 promotes – in LPA-mediated mitogenic response. the proliferation of ovarian cancer cell lines (Goetzl Our demonstration that LPA activates Ga12 and et al., 1999) further substantiates such pairing of LPA2 stimulates the proliferation of Ga12-expressing NIH3T3 receptor and Ga12. This conclusion gains more sig- cells in a suramin-sensitive manner provides a direct nificance in light of the observation that LPA2 is one of evidence for signal coupling between cell surface LPAR the receptors whose expression is amplified in certain and Ga12 – in contrast to other intracellular LPA- types of cancers (Mills and Moolenaar, 2003). Thus, our binding receptors – in mitogenic responses. The ability studies presented here discuss one branch of the LPA of LPA to substitute serum in stimulating Ga12- signal transduction pathway involving Ga12 in activat- mediated JNK-response and DNA synthesis further ing cell proliferation. Therefore, a mechanistic under- supports the view that LPA is the primary factor standing of the molecules involved in LPA-mediated underlying the previously identified serum-dependent mitogenic signaling pathway regulated by Ga12,as neoplastic transformation by Ga12 (Chan et al., 1993; elucidated here, may offer the possibility to selectively Dermott and Dhanasekaran, 2002). Previous studies disrupt or restore specific arms of the LPA signaling from different laboratories including ours have shown cascade. that the stimulation of the wild-type Ga12 or overexpression of constitutively activated Ga12 results in the robust activation of JNK in different cell types (Prasad Abbreviations et al., 1995; Collins et al., 1996; Voyno-Yasenetskaya LPA, lysophosphatidic acid; G protein, guanine nucleotide binding protein; GPCR, G protein coupled receptor; JNK, Jun et al., 1996). A requirement for JNK in Ga12-mediated mitogenic responses has also been shown (Mitsui et al., N-terminal kinase; LPAR, lysophosphatidic acid receptor; PTX, pertussis toxin.. 1997; Tolkacheva et al., 1997). Similarly, the ability of LPA to stimulate the activation of JNK has been shown in many different cell types. More interestingly, studies Acknowledgements from LPA receptor knockout mice have revealed that We thank Professor M Negishi, Kyoto University, Japan, for the generous gift of GST-TPR encoding pGEX-4T-2-GST- JNK was activated by LPA in wild type, lpa1À/À,and TPR construct. Helpful discussions and critical reading of the lpa2À/À mouse embryonic fibroblasts but not in the manuscript by Mr Zachariah G Goldsmith, Ms Rashmi cells derived from double knockout lpa1À/À lpa2À/À Kumar, and Ms Kimia Kashef are gratefully acknowledged. (Contos et al., 2002). These findings establish that the This work was supported by grants from the National stimulation of JNK by LPA involves either LPA1 or Institutes of Health (GM49897).

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