Lysophosphatidic acid (LPA) exerts a variety of biological responses through specific receptors: three subtypes of the EDG-family receptors, LPA 1 , LPA 2 , and LPA 3 (formerly known as EDG-2, EDG-4, and EDG-7, respectively), and LPA 4 /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]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425) on the EDG-family LPA receptors. Ki16425 inhibited 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 LPA 1 , LPA 2 , or LPA 3 , 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 inhibited the LPA-induced response in the decreasing order of LPA 1 Ն LPA 3 Ͼ Ͼ LPA 2 , whereas DGPP 8:0 preferentially inhibited the LPA 3 -induced actions. Ki16425 inhibited LPA-induced guanosine 5Ј-O-(3-thio)triphosphate binding as well as LPA receptor binding to membrane fractions with a same pharmacological 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 longterm responses, including DNA synthesis and cell migration. In conclusion, Ki16425 selectively inhibits LPA receptor-mediated actions, especially through LPA 1 and LPA 3 ; 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 diverse biological actions, including Ca 2ϩ 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 cancer (Xu et al., 1995(Xu et al., , 2001Siess et al., 1999;Maschberger et al., 2000). LPA was first thought to be released from activated platelets; however, a major part of extracellular LPA has been shown to be produced from lysophosphatidylcholine by lysophospholipase D, which was previously called autotaxin (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 M (Sano et al., 2002). LPA increases low-density lipoprotein during its oxidation, activates endothelial cells (Siess et al., This work was supported in part by a research grant grants-in-aid for scientific research from the Japan Society for the Promotion of Science and by research gr...

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In bone metastatic lesions, osteoclasts play a key role in the development of osteolysis. Previous studies have shown that macrophage colony-stimulating factor (M-CSF) is important for the differentiation of osteoclasts. In this study, we investigated whether an inhibitor of M-CSF receptor (c-Fms) suppresses osteoclast-dependent osteolysis in bone metastatic lesions. We developed small molecule inhibitors against ligand-dependent phosphorylation of c-Fms and examined the effects of these compounds on osteolytic bone destruction in a bone metastasis model.

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Fas (APO-1/CD95) and its ligand have been identified as important signal-mediators of apoptosis (1). The structural organization of Fas (APO-1/CD95) indicates that it is a member of the tumor necrosis factor receptor superfamily, which also includes the p75 nerve growth factor receptor (2), the T-cellactivation marker CD27 (3), the Hodgkin-lymphoma-associated antigen CD30 (4), the human B cell antigen CD40 (5), and T cell antigen OX40 (6). Genetic mutations of both Fas and its ligand have been associated with lymphoproliferative and autoimmune disorders in mice (7,8). Furthermore, alterations of Fas expression level have been implicated in the induction of apoptosis in T-cells infected with human immunodeficiency virus (9). Several Fas-interacting signal transducing molecules, have been identified using yeast two-hybrid and biochemical approaches, including Fas-associated phosphatase-1 (FAP-1) 1 (10), FADD/MORT1/CAP-1/CAP-2 (11-13), and RIP (14). All but FAP-1 associate with the functional cell death domain of Fas and overexpression of FADD/MORT1, or RIP induces apoptosis in cells transfected with these proteins. In contrast, FAP-1 is the only protein that associates with a negative regulatory domain (C-terminal 15 amino acids) (15) of Fas and that inhibits Fas-induced apoptosis.FAP-1 (PTPN13) has several alternatively-spliced forms that are identical to PTP-BAS/hPTP1E/PTPL1 (16 -18), and contains a membrane-binding region similar to those found in the cytoskeleton-associated proteins, ezrin (19), radixin (20), moesin (21), neurofibromatosis type II gene product (NFII) (22), and protein 4.1 (23), as well as in the PTPases PTPH1 (24), PTP-MEG (25), and PTPD1 (26). FAP-1 intriguingly contains six PDZ (GLGF/DHR) domains that are thought to mediate intra-and inter-molecular interactions among protein. The third PDZ repeat of FAP-1 was first identified as a domain showing the specific interaction with the C terminus of Fas receptor (10). In the present study, we first demonstrated that the C-terminal three amino acids (SLV) of human Fas were necessary and sufficient for its interaction with the third PDZ domain of FAP-1. More important, we were able to induce Fas-mediated apoptosis in a colon cancer cell line by the direct cytoplasmic microinjection of this tripeptide (Ac-SLV). MATERIALS AND METHODSConstructions of Libraries and Screenings-To create numerous mutations in a restricted DNA sequence, PCR mutagenesis with degenerate oligonucleotides was employed according to a protocol described elsewhere (27). Based on the homology between human and rat, two palindromic sequences were designed for construction of a semi-random library. The two primers used were 5Ј-CGGAATTCNNNNNNNNNAA-CAGCNNNNNNNNNAATGAANNNCAAAGTCTGNNNTGAGGATC-CTCA-3Ј and 5Ј-CGGAATTCGACTCAGAANNNNNNAACTTCAGA-NNNNNNATCNNNNNNNNNGTCTGAGGATCCTCA-3Ј. Briefly, the two primers (200 pmol each), purified by high pressure liquid chromatography, were annealed at 70°C for 5 min and cooled at 23°C for 60 min. A Klenow fragment (5 units) was used for filling in with a ...