Citation

Lysophosphatidic acid (LPA) exerts a variety of biological re- sponses through specific receptors: three subtypes of the EDG-family receptors, LPA1, LPA2, and LPA3 (formerly known as EDG-2, EDG-4, and EDG-7, respectively), and LPA4/GPR23, structurally distinct from the EDG-family receptors, have so far been identified. In the present study, we characterized the action mechanisms of 3-(4-[4-([1-(2-chlorophenyl)ethoxy]car- bonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425) on the EDG-family LPA receptors. Ki16425 in- hibited several responses specific to LPA, depending on the cell types, without any appreciable effect on the responses to other related lipid receptor agonists, including sphingosine 1-phosphate. With the cells overexpressing LPA1, LPA2, or LPA3, we examined the selectivity and mode of inhibition by Ki16425 against the LPA-induced actions and compared them with those of dioctyl glycerol pyrophosphate (DGPP 8:0), a recently identified antagonist for LPA receptors. Ki16425 inhib- ited the LPA-induced response in the decreasing order of LPA1 ≥ LPA3 >> LPA2, whereas DGPP 8:0 preferentially inhib- ited the LPA3-induced actions. Ki16425 inhibited LPA-induced guanosine 5′-O-(3-thio)triphosphate binding as well as LPA receptor binding to membrane fractions with a same pharma- cological specificity as in intact cells. The difference in the inhibition profile of Ki16425 and DGPP 8:0 was exploited for the evaluation of receptor subtypes involved in responses to LPA in A431 cells. Finally, Ki16425 also inhibited LPA-induced long- term responses, including DNA synthesis and cell migration. In conclusion, Ki16425 selectively inhibits LPA receptor-mediated actions, especially through LPA1 and LPA3; therefore, it may be useful in evaluating the role of LPA and its receptor subtypes involved in biological actions.

Lysophosphatidic acid (LPA) has been shown to elicit di- verse biological actions, including Ca2+ mobilization, change in cAMP accumulation, change in cell shape and motility in association with actin rearrangement, and proliferation in a variety of cell types (Moolenaar, 1999; Contos et al., 2000; Ye et al., 2002). Extracellular LPA has also been shown to be involved in certain diseases, such as atherosclerosis and can- cer (Xu et al., 1995, 2001; Siess et al., 1999; Maschberger et al., 2000). LPA was first thought to be released from acti- vated platelets; however, a major part of extracellular LPA has been shown to be produced from lysophosphatidylcholine by lysophospholipase D, which was previously called auto- taxin (Sano et al., 2002; Tokumura et al., 2002; Umezu-Goto et al., 2002). The concentration of plasma LPA is about 100 nM, and its serum concentration can be as high as 5 µM (Sano et al., 2002). LPA increases low-density lipoprotein during its oxidation, activates endothelial cells (Siess et al., 1999; Maschberger et al., 2000), and has also been identified as a growth-promoting factor for cancer cells in malignant ascites of patients suffering from ovarian cancer (Xu et al., 1995; Xu et al., 2001). These cellular responses to LPA are mediated through G-protein-coupled receptors, and several subtypes of LPA receptors, including LPA1/EDG-2, LPA2/ EDG-4, LPA3/EDG-7, and LPA4/GPR23, a non-EDG-family LPA receptor, have been identified (Hecht et al., 1996; An et al., 1998a,b; Bandoh et al., 1999; Moolenaar, 1999; Contos et al., 2000; Im et al., 2000; Ye et al., 2002; Noguchi et al., 2003), although a novel intracellular mechanism through peroxi- some proliferator-activated receptor γ was recently demon- strated (McIntyre et al., 2003). These LPA receptor subtypes are expressed and function in a variety of cell types; however, the precise role of each LPA receptor subtype has not yet been fully characterized.

Receptor antagonists are very useful tools for evaluating the role of LPA and its receptors in biological actions and for controlling specific diseases (Tigyi, 2001). Based on their ability to inhibit Ca2+ response to LPA in A431 cells or LPA-responsive cells, we have screened 150,000 low-molecu- lar-weight compounds developed by the Kirin Brewery Co. Ltd, for LPA receptor antagonists, and found that some isox- azole derivatives showed such an inhibitory activity against the LPA action. We therefore synthesized several isoxazole derivatives and finally selected Ki16425 as the best candi- date compound (Ueno et al., 2001). In the present study, we examined the pharmacological properties of Ki16425 and compared it with that of DGPP 8:0, a recently identified LPA antagonist (Fischer et al., 2001). We found that Ki16425 inhibited LPA-induced actions in a manner highly specific to LPA and LPA receptor subtypes. Such differences in the selectivity of Ki16425 and DGPP 8:0 were successfully ap- plied to discriminate the receptor subtypes in A431 cells. Thus, the novel antagonist Ki16425 seems to be a useful tool for investigating physiological and pathophysiological roles of LPA and its receptors.