calpain and lysophosphatidic-acid

Calcium-mediated proteolysis plays an important role in cell migration. Lysophosphatidic acid (LPA), a lipid mediator present in serum, enhances migration of carcinoma cells. The effects of LPA on calpain-mediated proteolysis were, therefore, examined in PC-3, a human prostate cancer cell line.. Cultured PC-3 cells were used in studies utilizing pharmacologic interventions, immunoblotting, and confocal immunolocalization.. Focal adhesion kinase (FAK), a tyrosine kinase involved in cell adhesion, is rapidly proteolyzed in serum-starved PC-3 cells exposed to the calcium ionophore, ionomycin; Nck, p130CAS, PKCα, and Ras-GAP are also degraded. Thapsigargin, which causes more moderate increases in intracellular calcium, induces partial proteolysis of these proteins. Calpain inhibitors block the proteolytic responses to ionomycin and thapsigargin. Ionomycin does not induce proteolysis in cells maintained in serum, suggesting a protective role for growth factors contained in serum. LPA causes minor FAK proteolysis when added alone, but protects against ionomycin-induced proteolysis in a time-dependent manner. LPA also protects against the cell detachment that eventually follows ionomycin treatment. The response to LPA is blocked by an LPA receptor antagonist. A similar effect of LPA is observed in ionomycin-treated Rat-1 fibroblasts. In PC-3 cells, the protective effects of LPA and serum are correlated with phosphorylation and redistribution of paxillin, suggesting roles for phosphorylation-mediated protein-protein interactions.. The complex effects of LPA on calpain-mediated proteolysis of FAK and other adhesion proteins are likely to play a role in the ability of LPA to promote attachment, migration, and survival of prostate cancer cells.

Topics: Adenocarcinoma; Animals; Calpain; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Drug Screening Assays, Antitumor; Fibroblasts; Focal Adhesion Protein-Tyrosine Kinases; Humans; Ionomycin; Isoxazoles; Lysophospholipids; Male; Paxillin; Phosphorylation; Propionates; Prostatic Neoplasms; Proteolysis; Rats; Thapsigargin

Lysophosphatidic acid receptor (LPA(1)) signaling initiates neuropathic pain through demyelination of the dorsal root (DR). Although LPA is found to cause down-regulation of myelin proteins underlying demyelination, the detailed mechanism remains to be determined. In the present study, we found that a single intrathecal injection of LPA evoked a dose- and time-dependent down-regulation of myelin-associated glycoprotein (MAG) in the DR through LPA(1) receptor. A similar event was also observed in ex vivo DR cultures. Interestingly, LPA-induced down-regulation of MAG was significantly inhibited by calpain inhibitors (calpain inhibitor X, E-64 and E-64d) and LPA markedly induced calpain activation in the DR. The pre-treatment with calpain inhibitors attenuated LPA-induced neuropathic pain behaviors such as hyperalgesia and allodynia. Moreover, we found that sciatic nerve injury activates calpain activity in the DR in a LPA(1) receptor-dependent manner. The E-64d treatments significantly blocked nerve injury-induced MAG down-regulation and neuropathic pain. However, there was no significant calpain activation in the DR by complete Freund's adjuvant treatment, and E-64d failed to show anti-hyperalgesic effects in this inflammation model. The present study provides strong evidence that LPA-induced calpain activation plays a crucial role in the manifestation of neuropathic pain through MAG down-regulation in the DR.

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Demyelinating Diseases; Disease Models, Animal; Enzyme Activation; Leucine; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Associated Glycoprotein; Neurotoxins; Peripheral Nervous System Diseases; Receptors, Lysophosphatidic Acid; Sciatic Neuropathy; Sensory Receptor Cells; Spinal Nerve Roots