guanosine-triphosphate and lysophosphatidic-acid

CD97, an adhesion-linked G-protein-coupled receptor (GPCR), is induced in multiple epithelial cancer lineages. We address here the signaling properties and the functional significance of CD97 expression in prostate cancer. Our findings show that CD97 signals through Gα12/13 to increase RHO-GTP levels. CD97 functioned to mediate invasion in prostate cancer cells, at least in part, by associating with lysophosphatidic acid receptor 1 (LPAR1), leading to enhanced LPA-dependent RHO and extracellular signal-regulated kinase activation. Consistent with its role in invasion, depletion of CD97 in PC3 cells resulted in decreased bone metastasis without affecting subcutaneous tumor growth. Furthermore, CD97 heterodimerized and functionally synergized with LPAR1, a GPCR implicated in cancer progression. We also found that CD97 and LPAR expression were significantly correlated in clinical prostate cancer specimens. Taken together, these findings support the investigation of CD97 as a potential therapeutic cancer target.

Topics: Antigens, CD; Cell Line, Tumor; Disease Progression; Extracellular Signal-Regulated MAP Kinases; GTP-Binding Protein alpha Subunits, G12-G13; Guanosine Triphosphate; Humans; Lysophospholipids; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; rhoA GTP-Binding Protein; Signal Transduction

We investigated the regulation of T-type channels by Rho-associated kinase (ROCK). Activation of ROCK via the endogenous ligand lysophosphatidic acid (LPA) reversibly inhibited the peak current amplitudes of rat Ca(v)3.1 and Ca(v)3.3 channels without affecting the voltage dependence of activation or inactivation, whereas Ca(v)3.2 currents showed depolarizing shifts in these parameters. LPA-induced inhibition of Ca(v)3.1 was dependent on intracellular GTP, and was antagonized by treatment with ROCK and RhoA inhibitors, LPA receptor antagonists or GDPssS. Site-directed mutagenesis of the Ca(v)3.1 alpha1 subunit revealed that the ROCK-mediated effects involve two distinct phosphorylation consensus sites in the domain II-III linker. ROCK activation by LPA reduced native T-type currents in Y79 retinoblastoma and in lateral habenular neurons, and upregulated native Ca(v)3.2 current in dorsal root ganglion neurons. Our data suggest that ROCK is an important regulator of T-type calcium channels, with potentially far-reaching implications for multiple cell functions modulated by LPA.

Topics: Animals; Blotting, Western; Calcium Channels, T-Type; Electrophysiology; Ganglia, Spinal; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Mutagenesis, Site-Directed; Neurons; Patch-Clamp Techniques; Phosphorylation; Protein Serine-Threonine Kinases; Rats; Retinoblastoma; rho-Associated Kinases; Thionucleotides

Lysophosphatidic acid (LPA), a major G protein coupled receptor (GPCR)-activating ligand present in serum, elicits growth factor like responses by stimulating specific GPCRs coupled to heterotrimeric G proteins such as G(i), G(q), and G12/13. Previous studies have shown that the overexpression of wild-type Galpha12 (Galpha12WT) results in the oncogenic transformation of NIH3T3 cells (Galpha12WT-NIH3T3) in a serum-dependent manner. Based on the potent growth-stimulating activity of LPA and the presence of LPA and LPA-like molecules in the serum, we hypothesized that the serum-dependent neoplastic transformation of Galpha12WT-NIH3T3 cells was mediated by the stimulation of LPA-receptors (LPARs) by LPA in the serum. In the present study, using guanine nucleotide exchange assay and GST-TPR binding assay, we show that the treatment of Galpha12WT-NIH3T3 with 2 muM LPA leads to the activation of Galpha12. Stimulation of these cells with LPA promotes JNK-activation, a critical component of Galpha12-response and cell proliferation. We also show that LPA can substitute for serum in stimulating JNK-activity, DNA synthesis, and proliferation of Galpha12WT-NIH3T3 cells. LPA-mediated proliferative response in NIH3T3 cells involves Galpha12, but not the closely related Galpha13. Pretreatment of Galpha12WT-NIH3T3 cells with suramin (100 microM), a receptor-uncoupling agent, inhibited LPA-stimulated proliferation of these cells by 55% demonstrating the signal coupling between cell surface LPAR and Galpha12 in the neoplastic proliferation of NIH3T3 cells. As LPA and LPAR mediated mitogenic pathways have been shown to play a major role in tumor genesis and progression, a mechanistic understanding of the signal coupling between LPAR, Galpha12, and the downstream effectors is likely to unravel additional targets for novel cancer chemotherapies.

Topics: 3T3 Cells; Animals; Cell Proliferation; Cell Transformation, Neoplastic; Enzyme Activation; GTP-Binding Protein alpha Subunits, G12-G13; Guanosine Diphosphate; Guanosine Triphosphate; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Mice; Pertussis Toxin; Receptors, Lysophosphatidic Acid; Signal Transduction; Suramin

The mevalonate metabolic pathway is necessary for the isoprenylation of a number of small GTPases. We have previously presented that Rho plays a pivotal role in 1-oleoyl-lysophosphatidic acid (LPA)-induced invasion of rat ascites hepatoma MM1 cells. Herein we report the effect of HMG-CoA reductase inhibitors, fluvastatin and lovastatin, on the in vitro invasion of MM1 cells. Fluvastatin and lovastatin inhibited LPA-induced MM1 cell invasion in a dose-dependent manner. Fluvastatin inhibited LPA-induced translocation of RhoA protein from the cytosol to the membrane and RhoA activation which was measured by pull-down assay for GTP-bound RhoA. Fluvastatin also inhibited the translocation of both endogenous and dominant-active RhoA from the cytosol to the membrane, actin stress fiber assembly and in vitro invasion of the cells expressing dominant-active RhoA (Val14-RhoA). These results indicate that HMG-CoA reductase inhibitors have the potential to reduce RhoA activation and cancer cell invasion by targeting the Rho protein isoprenylation.

Topics: Actins; Animals; Cell Division; Cell Line, Tumor; Cell Membrane; Cytosol; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fluvastatin; Guanosine Triphosphate; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Lovastatin; Lysophospholipids; Microscopy, Fluorescence; Mutation; Protein Transport; Rats; rhoA GTP-Binding Protein; Time Factors