Lysophosphatidic acid induces ME180 cell migration via its putative receptor GPR87

We have previously proposed that the G protein-coupled receptor, GPR87, may be an LPA receptor. This study evaluated whether GPR87 was functionally involved in the LPA response using ME180 cervical carcinoma cells. Our in vitro wound healing assay revealed that LPA-induced cell migration was suppressed by the LPA antagonist Ki16425. RT-PCR analyses indicated that ME180 cells expressed LPA1 receptor and GPR87, both of which are antagonized by Ki16425. The ME180 cells pretreated with gpr87-specific siRNAs significantly reduced LPA-induced cell migration. These results suggest that GPR87 is activated by LPA and the activation of GPR87 leads cell migration. Abbreviations: LPA, lysophosphatidic acid; GPCR, G proteincoupled receptor; MAPK, mitogen-activated protein kinase; PI3-K, phosphoinositide3-kinase; EGFR, epidermal growth factor receptor; EMT, epithelial mesenchymal transition; MMP, matrix metalloprotease; NSCLC, non-small-cell lung cancer Introduction Lysophosphatidic acid (LPA) is one of the lysophospholipids produced by autotoxin enzymatic activity invascular endothelialcells and acts as an extracellular signaling molecule [1,2]. LPA increases the phosphorylation of mitogen-activated protein kinase (MAPK), phosphoinositide3-kinase (PI3-K), and low molecular weight G-proteinsby binding to at least six specific G-protein coupled receptors (GPCRs) known as LPA receptors [1–6]. LPA1-3 receptors sharing >50% homology are known to be “endothelial differentiation gene” (EDG) members, while three additional LPA receptors LPA [46] are members of the P2Y purinergic family. These receptors have significantly different encoding sequences than LPA receptors [1-3], yet still bind and mediate LPA signaling effects [3]. These receptors are expressed in various mammalian cells and tissues [3]. We previously reported that the intracellular Ca2+ concentration in CHO cells expressing a GPR87–Gα16 fusion protein increased with exposure to a low concentration of LPA and proposed that GPR87, a member of P2Y family, was an LPA receptor [4]. Glatt et al. reported that GPR87 may contribute to the viability and proliferation of several human tumor cells [5]. Recently, Yan et al. predicted that GPR87 was involved with the regulation of the proliferation and migration of epithelial cancer cells expressing the cancer stem cell-marker CD133 [6]. Moreover, we previously reported that colony dispersal of human A431 epithelial cells was induced by LPA through the activation of GPR87 and the LPA1 receptor [7]. Cell colony dispersal has been known as the epithelial mesenchymal transition (EMT) that occurs in cancer cell migration and transition [8,9]. In general, cell migration and EMT are both regarded as important factors in cancer progression and malignancy development. Importantly, patients with bladder cancer cells that expressed GPR87 had shorter survival times than patients whose cancer did not express GPR87 [10]. Similar results were reported for patients with lung cancer [11]. Because induction of the migration ability of cancer cells is thought to be important for cancer cell progression to malignancy, GPR87 may be a key molecule in this process. However, definitive evidence showing that GPR87 is an LPA receptor has been lacking. It is well documented that various cell lines migrate in response to LPA through the activation of diverse signaling pathways [12-15]. Ullich showed that LPA receptor activation transactivates epidermal growth factor receptor (EGFR) through the activation of a matrix metalloprotease (MMP) and the release of an EGF-like peptide [1618]. Recently, Muthusami et al. reported that the treatment of human cervical carcinoma ME180 cells with EGF induced the nuclear accumulation of transcription activating factor2 downstream of Erk, increasing cell migration and EMT [19]. Because ME180 cells have been shown to express GPR87 [5], the cells can be expected toinduce cell motility following GPR87 activation depending on GPCR-mediated EGFR transactivation. GPCRs have attracted a great deal of research interest because of their numerous physiological roles, although more than 120 GPCRs exist as orphan receptors with unknown endogenous stimulating ligands [20]. Deorphanization of orphan GPCRs has been expected to lead to a better understanding of their physiological functions and suggest new therapeutic targets in drug discovery. However, the rate at which orphan GPCRs are deorphanized has recently decreased. Initially some 10 GPCRs were deorphanized each year, whereas very few GPCRs have been deorphanized since 2004 [21]. Thus, matching GPR87 to LPA should impact the current understanding of the physiological Correspondence to: Norihisa Fujita, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Tel: +81-77-561-2783, Fax: +81-77-561-5203; E-mail: [email protected]