Vascular smooth muscle migration and proliferation in response to lysophosphatidic acid (LPA) is mediated by LPA receptors coupling to Gq

Cellular Signalling 18 (2006) 1695 – 1701 www.elsevier.com/locate/cellsig

Vascular smooth muscle migration and proliferation in response to lysophosphatidic acid (LPA) is mediated by LPA receptors coupling to Gq Jihee Kim 1 , Janelle R. Keys 2 , Andrea D. Eckhart ⁎ Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA Received 7 December 2005; received in revised form 12 January 2006; accepted 16 January 2006 Available online 28 February 2006

Abstract Many G protein-coupled receptors can couple to multiple G proteins to convey their intracellular signaling cascades. The receptors for lysophosphatidic acid (LPA) possess this ability. LPA receptors are important mediators of a wide variety of biological actions including cell migration, proliferation and survival which are processes that can all have a considerable impact on vascular smooth muscle (VSM) and blood vessels. To date, confirmation of G proteins involved has mostly relied on the inhibition of Gi-mediated signaling via pertussis toxin (PTx). We were interested in the specific involvement of LPA-Gq-mediated signaling therefore we isolated aorta VSM cells (VSMCs) from transgenic mice that express a peptide inhibitor of Gq, GqI, exclusively in VSM. We detected both LPA1 and LPA2 receptor expression in mouse VSM whereas LPA1 and LPA3 were expressed in rat VSM. SM22-GqI did not alter LPA-induced migration but it was sufficient to attenuate LPA-induced proliferation. GqI expression also attenuated LPA-induced ERK1/2 and Akt activation by 40–50%. To test the feasibility of this peptide as a potential therapeutic agent, we also generated adenovirus encoding the GqI. Transient expression of GqI was capable of inhibiting both LPAinduced migration and proliferation of VSMCs isolated from rat and mouse. Furthermore, ERK activation in response to LPA was also attenuated in VSMCs with Adv-GqI. Therefore, LPA receptors couple to Gq in VSMC and mediate migration and proliferation which may be mediated through activation of ERK1/2 and Akt. Our data also suggest that both chronic and transient expression of the GqI peptide is an effective strategy to lower Gq-mediated LPA signaling and may be a successful therapeutic strategy to combat diseases with enhanced VSM growth such as occurs following angioplasty or stent implantation. © 2006 Elsevier Inc. All rights reserved. Keywords: Vascular smooth muscle; G protein coupled receptor; Restenosis; Lysophosphatidic acid; Migration; Proliferation; Mitogen

1. Introduction In the treatment of atherosclerosis, all forms of percutaneous coronary revascularization, including angioplasty and stents, impose injury on blood vessels [1]. Vascular cellular growth and/ Abbreviations: ERK, extracellular signal-regulated kinase; MAPKs, mitogen-activated protein kinases; LPA, lysophosphatidic acid; ISO, isoproterenol; EGFR, epidermal growth factor receptor; GPCR, G protein-coupled receptors; VSMCs, vascular smooth muscle cells; RTK, receptor tyrosine kinase; AngII, angiotensin II; PTx, B. pertussis toxin; BSA, bovine serum albumin. ⁎ Corresponding author. Eugene Feiner Laboratory for Vascular Biology and Thrombosis, Thomas Jefferson University, Rm 309 College Building, 1025 Walnut St., Philadelphia, PA 19107, USA. Tel.: +1 215 955 9982; fax: +1 215 503 5731. E-mail address: [email protected] (A.D. Eckhart). 1 Current address: Duke University Medical Center, Durham, NC 27710, USA. 2 Current address: University of Queensland, Brisbane, Australia. 0898-6568/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2006.01.009

or migration occur in order to repair the vessel and G proteincoupled receptors (GPCRs) are intimately involved in this process. Lysophosphatidic acid (LPA) is a naturally occurring vasoreactive agent that has been implicated in the development of this abnormal and life-threatening growth in VSM. LPA has prolific effects on numerous cells types and stimulates associated mitogenic and migratory signaling pathways. Within the vasculature, LPA is a potent bioactive lipid that can exert effects on both platelets and blood vessels [2]. LPA has been shown to accumulate in the neointima of human atherosclerotic plaques and can stimulate the proliferation and migration of VSMCs [2]. There are 3 well-characterized members of the LPA receptor family, LPA1-3 and a more recently cloned LPA4 [3]. LPA1 is the most widely expressed LPA receptor and rat VSM has been found to express both LPA1 and LPA3 [4]. LPA receptors have been linked to three major G proteins, Gi, Gq and G12/13 [2]. In an attempt to determine which receptors mediate certain

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intracellular signaling cascades, experiments were performed in mouse embryonic fibroblasts (MEFs) [5]. Results from these studies suggest that, at least in MEFs, LPA1 is mostly linked to adenylate cyclase inhibition suggesting that it couples primarily to Gi whereas LPA2 is linked to phospholipase C activation and therefore coupled primarily to Gq and less is known regarding LPA3 signaling [5]. Importantly, it remains unclear which G protein is essential to LPA-mediated intracellular signaling cascades leading to VSM migration and growth. There is considerable interest in the possibility that modulation of GPCR signaling pathways has the potential to treat pathophysiologic mechanisms fundamental to the progression of heart failure and restenosis. Instead of targeting the signaling of each individual GPCR, it would be more effective to target an entire class of G proteins. Class-specific inhibition of G protein mediated signaling has been proposed for a few families of α subunits [6–8]. A 54-amino-acid carboxyl-terminal peptide of the α subunit of Gq family (GqI) is the region that interacts specifically with the activated receptor. Expression of GqI can inhibit the activation of Gq proteins and subsequent downstream signaling events by competing with Gαq subunit for binding with the receptor [8]. This approach specifically targets the receptor– Gαq interface selectively while signaling through Gs and Gicoupled receptor is not blocked [8]. When pressure overload was surgically induced in the hearts of transgenic mice with cardiac-specific expression of GqI, the transgenic mice developed significantly less ventricular hypertrophy than control mice [8]. It is important to note that since the GqI targets the receptor–Gq interface, the dissociation of Gαq and Gβγ is inhibited thus, effectively blocking both signals. To understand the mechanisms of Gq-mediated proliferation and migration of primary cultured VSMC, we generated transgenic mice with VSM-targeted expression of GqI using SM22α promoter, SM22-GqI [9]. We also generated adenovirus expressed GqI, Adv-GqI, to induce the effective expression in primary VSMC extracted from adult rat and mouse. In this study, we were interested in determining the effect of GqI on LPA-induced VSMC migration and proliferation in VSMC from mouse and rat VSMCs with transient infection of adenovirus encoding GqI. ERK and Akt activation were also tested in the presence of GqI in VSMCs because these kinases are well known downstream effectors of Gq signaling and regulators of cell proliferation and cell survival. 2. Experimental procedures 2.1. Materials B. pertussis toxin (PTx) was from List Biologicals. PMT and rPMT were from Calbiochem. LPA, phenylephrine (PE) and angiotensin II (AngII) were purchased from Sigma. EGF was from polyclonal anti-phosphorylated ERK and AKT from Cell Signaling Technology. Polyclonal anti-ERK2 was from Santa Cruz Biotechnology. [3H] thymidine was purchased from Amersham.

2.2. Generation of VSM-specific GqI overexpressing mice The transgenic mice were created as previously described [9]. A 54-amino-acid carboxyl-terminal peptide (305–359) of

the α subunit of Gq family (GqI) was inserted in-frame behind the VSM-specific − 441 to + 40 portion (relative to transcript start) of the SM22α promoter. Transgenic mice were compared to their non-transgenic littermate controls (NLCs). 2.3. Cell culture Arterial VSMCs were obtained from mouse and rat aorta by enzymatic digestion with 0.1% collagenase II, 0.1% soybean trypsin inhibitor, and 15 U/ml elastase, following removal of the adventitial layer and endothelial cells [10]. Cells were cultured in M199 with 20% fetal bovine serum and 1% penicillin– streptomycin (Life technologies). For migration assays, VSMCs were used during passages 3–5. For other experiments, VSMCs were used during passages 3–10. The purity of these cultures was N 95% VSMC as measured by smooth muscle α-actin as previously described [10]. 2.4. PCR to determine LPA receptor expression LPA1-3 were determined using: LPA1 Fwd 3′–ATCTTT ggCTATgTTCgCCA–3′, Rev 5′–TTgCTgTgAACTCCAg CCA–3′, LPA2 Fwd 5′–TggCCTACCTCTTCCTCATg TTCCA–3′, Rev 5′–gggTCCAgCACACCACAAATgCC–3′, and LPA3 Fwd 5′–AgTgTCACTATgACAAgC–3′, Rev 5′–g CCgAgATgTTgCAg AggC–3′. GAPDH primers used were Fwd 5′–AgCCA CATCgCTCAgAACAC–3′ and Rev 5′–gA ggCATTgCTgAT gATCTTg–3′. PCR products were resolved on agarose gel and sequenced to verify accuracy of amplified product. 2.5. Adenovirus construction The adenovirus expressing GqI was generated using ADeasy system (Clontech). Control adenovirus containing the green fluorescent protein (GFP) alone or a bicistronic vector containing GFP and the GqI under control of dual CMV promoters were used at 100 : 1 multiplicity of infection (MOI) in cell culture. 2.6. Western blotting Western blotting was performed for ERK1/2 and Akt phosphorylation using specific anti-phosphorylated ERK and Akt antibodies. Cells were serum starved in M199 with 0.1% bovine serum albumin (BSA) for 72 h. Serum starved cells in 6 well dishes were stimulated with agonists for indicated times at 37 °C. After stimulation, monolayers were washed once with ice-cold phosphate buffered saline (PBS), and lysed in buffer containing with 50 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride and 10 μg/ml aprotinin. After lysis, similar amounts of proteins were electrophoresed on a 4–20% polyacrylamide/Tris–glycine gel. The antibodies were visualized using ECL and quantified by scanning laser densitometry.

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LPA2 LPA3 Fig. 1. LPA1 and LPA2 receptors are expressed in mouse VSMCs whereas LPA1 and LPA3 are expressed in rat VSMCs. Primary VSMCs were isolated and cultured from control mice and rats. Total RNA was isolated and RT–PCR was performed to determine LPA receptor expression. The positive control we used was mouse heart since this tissue expresses all three LPA receptor subtypes. CTL refers to negative control (RT–PCR with no RNA). GAPDH was used to verify equal loading of all samples (data not shown).

2.7. DNA synthesis DNA synthesis was determined as incorporation of [3H]thymidine into trichloroacetic acid (TCA)-insoluble material as described [11]. Cells were seeded at an initial density of 20,000 cells/well in 12-well dishes, and grown to 80% subconfluence (1 day). Cells were rendered quiescent by 72 h in serum-free M199 supplemented with 0.1% BSA, and stimulated by the addition of fresh medium containing the agonists and/or inhibitors. [3H]-Thymidine (0.5 μCi/ml) was added to the medium at the time of stimulation. Cells were washed twice with PBS and three times with 0.5% TCA and then cells were lysed with 1 N NaOH. Extent of cell growth was determined by counting incorporation of [3H]-thymidine into nascent DNA strands. 2.8. VSMC migration Migration was measured in Transwell chambers (Costar Corp., pore size 8 μm). Filters were coated with 10 μg/ml fibronectin (in PBS overnight at 4 °C), rinsed once with PBS, and placed in the lower chamber containing serum-free M199 supplemented with agonists. Cells suspended in serum-free M199 containing 0.1% bovine serum albumin were added to the upper chamber (1 × 105 cells/well). Cells were allowed to migrate for 4 h at 37 °C. Nonmigratory cells were removed from the top filter surface with a cotton swab. Migrated cells, attached to the bottom surface, were

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Data were presented as mean ± S.E.M. Unpaired Student's t test were used to compare treatment groups using GraphPad Prizm software. Typically, all assays were repeated 5–6 times unless otherwise noted. 3. Results 3.1. LPA receptor expression We used non-quantitative RT–PCR to determine mRNA expression of LPA1–3 receptors in mouse and rat aorta VSMCs. Adult mouse heart RNA was used as a positive control since mRNA for the 3 LPA receptors is detected in this tissue (Fig. 1) [12]. We detected the expressions of LPA1 and LPA2 in mouse VSMCs whereas LPA3 mRNA could not be detected. In contrast, we detected LPA1 and LPA3, but not LPA2, in rat VSMCs which corresponds to previously published data [4]. 3.2. LPA-induced migration is attenuated in SM22-GqI VSMCs Migration of VSMCs is thought to be important to the formation of the neointima [13,14]. LPA stimulation induced migration 2.1 ± 0.2 fold (n = 5) as compared to basal in NLC cells as represented by both the filter density (Fig. 2A) and averaged data (Fig. 2B). SM22-GqI was capable of attenuating LPAinduced migration approximately 40%. However, because basal values were also diminished the fold increase in migration with 4 h 10− 7 M LPA was similar in NLC and SM22-GqI VSMCs (Fig. 2). Both basal and LPA-induced migration in NLC and GqI aorta cells was attenuated by overnight treatment with 100 ng/mL PTx (data not shown) suggesting that in these cells, LPA-induced migration is coupled to both Gi and Gq-mediated mechanisms. To verify that there were no deficiencies in Gq-mediated signaling downstream of the receptors that could confer the GqImediated attenuation in migration, we also tested VSMCs migration in the presence of Pasteurella multocida toxin (PMT).

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Fig. 2. Gq plays a role in LPA-induced migration in mouse VSMCs. Migration was determined using Boyden chambers prior to and following LPA stimulation in the absence and presence of the inhibitor of Gq signaling. (A) Representative filters from the Boyden chambers under basal conditions and following 10− 7 M LPA for 4 h. (B) averaged data for migration assays. n = 5 for each group. †P b 0.05 vs. NLC matched group (basal or LPA) via two-tailed t-test.

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Fig. 3. LPA-induced VSMC proliferation is mediated by Gq. Thymidine incorporation was determined following LPA stimulation in NLC (n = 5 for each group) vs. SM22-GqI (n = 5 for each group). The response to PE, a primarily Gqcoupled receptor agonist was also attenuated by GqI expression whereas the response to EGF was unaltered. ⁎P b 0.05 vs. Basal, two-tailed t-test. †P b 0.05 vs. NLC-LPA group, two-tailed t-test.

PMT activates signaling pathways including phosphoinositide hydrolysis and mobilization of intracellular calcium by targeting free monomeric Gαq subunits [15,16]. In this study, SM22-GqI VSMCs were pretreated with 100 ng/ml PMT and rPMT for 24 h and loaded into upper part of transwell chamber followed by 4h incubation. Both PMT and rPMT induced SM22-GqI VSMC migration by 1.7 ± 0.1 and 1.8 ± 0.03 fold increase over basal, respectively (n = 4, P b 0.001). Therefore, Gq-mediated signaling is not compromised in these cells and it appears that GqI acts by preventing the coupling of LPA receptors to Gq.

signals from extracellular stimuli to the nucleus. One of the ubiquitous downstream targets of mitogenic signaling is MAPKs, in particular ERK1/2. Importantly, both Ras and MAPKK dependent activation of ERKs has been demonstrated to be critical for pathological intimal hyperplasia because inhibition of either limits VSM cell proliferation [18,19]. Therefore, we investigated the effect of GqI on LPA-mediated ERK activation in SM22-GqI VSMCs. Equal amounts of cell lysates from VSMCs of NLC and SM22-GqI mice were used to detect activated ERK using anti-phosphorylated ERK antibodies. Phosphorylation of ERK1/2 in NLC VSMCs were increased after 10− 5 M LPA addition by 3.2 ± 0.5 fold over basal, respectively (n = 5, P b 0.01) (Fig. 4). In SM22-GqI VSMCs, LPA mediated ERK activation was significantly inhibited by 40% compared to NLC (n = 5, P b 0.05) (Fig. 4). As a positive control for Gq signaling, α1-adrenergic stimulation with PE elicited a 3.0 ± 0.4 fold increase in NLC ERK1/2 activation which was also attenuated by 40% with SM22-GqI expression (Fig. 4A, B). In contrast, EGF signaling was unaltered by SM22GqI expression (Fig. 4A, B). 3.5. LPA-induced rat and mouse aorta VSMC migration was attenuated by Adv-GqI infection To test the therapeutic potential of GqI, we developed a GqI expressing adenovirus, Adv-GqI. We verified that we could get robust VSMC expression of GqI (Fig. 5A). We then tested whether transient expression of Adv-GqI could regulate VSMC

3.3. LPA-mediated proliferation was blocked by SM22-GqI expression

3.4. Gq-mediated ERK and Akt activation was blocked by GqI presence from SM22-GqI VSMCs In many cell types, proliferative pathways proceed via a cascade of phosphorylation events that transduce mitogenic

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Proliferation of VSMCs is implicated in the pathogenesis of hypertension, primary atherosclerosis, and restenosis [17]. LPA stimulation can induce DNA replication and mitosis and we were interested in determining the role of Gq signaling in the LPAinduced VSMC proliferation. We determined DNA synthesis by measuring the amount of thymidine incorporation. Basal levels of proliferation were similar in the VSMCs isolated from NLC and SM22-GqI mice (Fig. 3). LPA induced a 3.9 ± 0.6-fold increase (n = 5) in proliferation in NLC VSMCs. In contrast, LPA induced proliferation only 1.8 ± 0.3-fold (n = 5) in VSMCs isolated from transgenic SM22-GqI mice (Fig. 3). As a positive control for GqI, we also stimulated proliferation in VSMCs with PE, an α1-adrenergic receptor agonist. PE induced a 1.4 ± 0.1-fold increase in proliferation which was completely attenuated by GqI expression (Fig. 3). In contrast, stimulation of the tyrosine kinase growth factor receptor endothelial-derived growth factor, EGF, was not altered with GqI expression (Fig. 3). Therefore, GqI expression appears to be specific to Gq-mediated signaling. In addition, the majority of LPA-induced proliferation is due to Gq-mediated signaling.

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Fig. 4. ERK1/2 activation in response to LPA is attenuated by GqI expression as is PE but not EGF. ERK1/2 activation was determined as an increase in the ratio of phospho-ERK1/2 to total ERK1/2 after 5 min of stimulation. (A) Representative autoradiograph. (B) Histogram of averaged data. n = 5 for NLC groups and n = 3 for SM22-GqI groups. ⁎P b 0.05 vs. Basal, two-tailed t-test. † P b 0.05 vs. NLC-LPA group, two-tailed t-test.

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Fig. 5. LPA-induced migration is attenuated by adenoviral expression of GqI in both rat and mouse VSMCs. (A) Representative autoradiograph of GqI expression following Adv-GqI infection of rat VSMCs. (B) Migration as assessed on the filters in the Boyden chambers following 4 h of LPA stimulation. A bicistronic adenovirus was used that directed GFP alone or GFP and GqI. Increased GFP is indicative of increased migration into the filter. (C) Averaged data for rat VSMCs following Adv-GqI infection and LPA stimulation. n = 4 for all groups. (D) Averaged data for rat VSMCs following Adv-GqI infection and LPA stimulation. n = 4 for all groups. ⁎P b 0.05 vs. Basal, two-tailed t-test.

3.6. Adv-GqI infection attenuated DNA synthesis in rat aorta VSMCs We also examined DNA synthesis, an indicator of VSMC proliferation with acute Adv-GqI expression. LPA induced thymidine incorporation by 20% in control VSMCs with GFP

expression. There was a significant attenuation in LPA-induced migration with Adv-GqI expression (Fig. 6). As a control for Gq signaling, we also induced proliferation with AngII. 10− 6 M AngII induced a 40% increase in proliferation. This was completely attenuated by Adv-GqI (Fig. 6). Therefore, acute GqI expression in rat VSMCs was able to inhibit LPA-induced proliferation.

Thymidine Incorporation (cpm)

migration in a similar manner to chronic transgenic SM22-GqI expression. Both mouse and rat VSMCs were infected with either GFP or Adv-GqI virus and seeded into transwell chambers in the presence of 10− 7 M LPA. VSMCs were visualized by confocal microscopy after 4 h incubation (Fig. 6A). LPA induced migration 1.3 ± 0.1 fold over control (n = 4, P b 0.05) in rat VSMCs with GFP (control) expression (Fig. 5B, C). GFP-GqI expression abolished LPA-induced rat VSMC migration (Fig. 5B, C). Similarly, Adv-GqI expression in mouse VSMCs also attenuated LPA-mediated migration (Fig. 5D). Acute expression of GqI was more capable of inhibiting LPAinduced migration suggesting that perhaps level of expression was higher and therefore GqI was more effective or that with chronic transgenic expression, there is some sort of compensatory mechanism. Regardless, data from both rat and mouse VSMCs with either acute or chronic expression of GqI suggests that Gq is an essential component to LPA-induced migration.

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Fig. 6. LPA and AngII-induced VSMC proliferation is attenuated by Adv-GqI expression in rat VSMCs. Thymidine incorporation was determined in rat VSMCs infected with either GFP only or Adv-GqI. n = 5 for all groups. ⁎P b 0.05 vs. Basal, two-tailed t-test. †P b 0.05 vs. NLC-LPA group, two-tailed t-test.

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Fig. 7. ERK1/2 activation is attenuated by Adv-GqI expression in response to LPA and AngII stimulation but not EGF. (A) Representative autoradiographs. (B) Histogram of Western blots performed for phospho-ERK1/2 and total ERK1/2 and represented as a ratio. n = 5 for all groups. ⁎P b 0.05 vs. Basal, twotailed t-test. †P b 0.05 vs. NLC-LPA group, two-tailed t-test.

3.7. Adv-GqI expression inhibits LPA-induced ERK1/2 activation There was a 3.6 ± 0.4 fold increase in ERK1/2 activation in control GFP-infected VSMCs (Fig. 7). This was attenuated to a 1.6-fold increase in VSMCs infected with Adv-GqI (Fig. 7). As a positive control for Gq signaling, ERK1/2 activation by AngII was also attenuated by Adv-GqI expression whereas EGFinduced ERK1/2 activation was not (Fig. 7). Therefore, acute GqI expression in rat VSMCs was able to inhibit LPA-induced ERK1/2 activation. 4. Discussion This is the first study to address LPA-mediated signaling from the point of Gq signaling rather than Gi inhibition to determine LPA coupling specificity. It clearly illustrates and important role for Gq in LPA-induced migration, proliferation and ERK activation. The data also suggest that class-specific inhibition of Gq-mediated signaling may be a powerful tool to inhibit abnormal VSMC growth and help to prevent the development and progression of hypertrophic cardiovascular disease pathologies. Hormones, neurotransmitters and many other biologically active molecules transduce signals through G proteins. G proteins are composed of α, β, and γ subunits and transduce external signals from heptahelical receptors to intracellular effectors [20]. The α subunits are unique to each G proteins which are divided into four major families on the basis of their amino acid sequence homology. In addition, five β subunits and

at least 12 γ subunits have so far been described in the mouse and human genomes [20]. The GPCR and G protein interaction is the first step in the action of an agonist and determines the intracellular pathway that leads to the final cellular effect. Molecular diversity of GPCRs and the capability of these receptors to activate multiple types of G proteins theoretically allow the transmission of signals through multiple effectors pathways. Our aim in this study was to determine the specific role of Gq in VSM migration and proliferation in response to LPA. We found that LPA-induced proliferation is indeed mediated by Gq signaling in VSMC. Migration was not completely attenuated in the SM22GqI VSMCs but adenoviral expression of GqI was sufficient to completely attenuate LPA-induced migration. This might be due to expression levels of the GqI being greater using adenoviral expression. However, it does point out that both Gi and Gq are important in LPA-induced VSMC migration. LPA-induced activation of ERK1/2 is dependent on Gq signaling. Classspecific G protein inhibition offers potentially significant advantages over single receptor antagonists, especially in multifactorial diseases where multiple hormones or neurotransmitters may be involved such as in hypertension, myocardial hypertrophy, and vascular injury. Therefore, the GqI peptide can inhibit the activation of intracellular effectors mediated by several different Gq activators in VSMCs and can be used as therapeutic reagent for vascular diseases. Among four families of Gα subunits, we focused on Gq using a peptide that targets the receptor–Gq interface using the last 54 amino acid residues of the murine α-subunit of Gq within the region of the Gαq protein that interacts with activated receptors. Gq signaling has been implicated to play a critical role in the pathogenesis of prevalent human diseases including diabetes, hypertension, myocardial infarction, congestive heart failure, and stroke [9,21,22]. The GqI peptide specifically inhibits Gq signaling without affecting Gs or Gi signaling [8]. Subsequently, transgenic mice with cardiac-targeted GqI expression have attenuated myocardial Gq signaling as assessed by MAPK activation in response to AngII, endothelin 1, and PE [8]. Ventricular pressure overload was surgically induced in these mice by transverse aortic constriction (TAC), which in NLC mice results in a reproducible and significant left ventricular hypertrophy after 7 days while GqI TG mice developed significantly less LV hypertrophy and ERK1/2 activation compared with that of NLC mice [8,23]. This suggests that the GqI is a powerful antihypertrophic agent in both VSMCs and heart during conditions that elevate endogenous agonists that couple to Gq signaling. LPA has been known to induce cell growth and migration in various cell lines [5] and can cause dedifferentiation of VSMCs which results in proliferation and migration [4]. To elicit these effects, LPA receptors couple to at least three G proteins including Gq, Gi, and G12/13 [5,24]. In airway smooth muscle cells, LPA-induced actin reorganization was only blocked when Gαi2 and Gαq were both depleted by antisense treatment. These data indicate that LPA-induced actin reorganization, which is a fundamental process in cell motility and division, is mediated by both Gi and Gq pathways [25]. Using PTx, LPA1/2/3-Gi coupling has been implicated in cardiomyocyte hypertrophy

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[24]. Importantly, we were also able to see some component of LPA Gi-mediated signaling in the mouse VSMCs but our data suggests that at least in mouse and rat VSMCs, LPA coupling to Gq is the primary pathway that determines proliferation, migration and ERK1/2 and Akt activation. Results derived from LPA receptor knockout mice illustrate that at least in MEFs, LPA1 receptors are coupled to Gi and perhaps G12/13 whereas LPA2 is coupled to Gq [5]. Importantly, we and others have shown that rat VSMCs express only LPA1 and LPA3 [4] whereas we document LPA1 and LPA2 expression in mouse VSMCs. It is somewhat surprising that these 2 species express different LPA receptors yet our data suggests that both rat and mouse VSMCs respond similarly to GqI. According to the MEF results, our data would suggest that at least in mouse VSMCs, proliferation, migration and ERK1/2, Akt activation is mediated by LPA2 and that perhaps, this function is mediated via the less well characterized LPA3 receptor in rat VSMCs although this remains to be determined. The present study demonstrates that GqI inhibits LPAmediated migration, proliferation, ERK1/2 activation in rat and mouse VSMCs. GqI was successful in inhibiting LPA-induced VSMC growth responses with either chronic or transient expression. These data suggest that gene therapy using the peptide inhibitor of Gq or development of an inhibitor molecule of Gq signaling could be a powerful therapeutic approach to limit unwanted VSMC proliferation and migration. Acknowledgments This work was supported by a grant from the American Heart Association (0230060N ADE). References [1] R.A. Schwartz, T.D. Henry, Rev. Cardiovasc. Med. 3 (Suppl. 5) (2002) S4. [2] W. Siess, Biochim. Biophys. Acta 1582 (2002) 204. [3] K. Noguchi, S. Ishii, T. Shimizu, J. Biol. Chem. 278 (2003) 25600.

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