Wnt-5a is a representative ligand that activates a B-cateninindependent pathway in the Wnt signaling. Although abnormal activation of B-catenin-dependent pathway is often observed in human cancer, the relationship between Bcatenin-independent pathway and tumorigenesis is not clear. We sought to clarify how Wnt-5a is involved in aggressiveness of gastric cancer. Abnormal expression of Wnt-5a was observed in 71 of 237 gastric cancer cases by means of immunohistochemistry. The positivity of Wnt-5a expression was correlated with advanced stages and poor prognosis of gastric cancer. Wnt-5a had the abilities to stimulate cell migration and invasion in gastric cancer cells. Wnt-5a activated focal adhesion kinase and small GTP-binding protein Rac, both of which are known to play a role in cell migration. Cell migration, membrane ruffling, and turnover of paxillin were suppressed in Wnt-5a knockdown cells. Furthermore, anti-Wnt-5a antibody suppressed gastric cancer cell migration. These results suggest that Wnt-5a stimulates cell migration by regulating focal adhesion complexes and that Wnt-5a is not only a prognostic factor but also a good therapeutic target for gastric cancer.

Brain microglia are a major source of inflammatory cytokines, such as tumor necrosis factor-␣ (TNF-␣), which have been implicated in the progression of neurodegenerative diseases. Recently, microglia were revealed to be highly responsive to ATP, which is released from nerve terminals, activated immune cells, or damaged cells. It is not clear, however, whether released ATP can regulate TNF-␣ secretion from microglia. Here we demonstrate that ATP potently stimulates TNF-␣ release, resulting from TNF-␣ mRNA expression in rat cultured brain microglia. The TNF-␣ release was maximally elicited by 1 mM ATP and also induced by a P2X 7 receptorselective agonist, 2Ј-and 3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate, suggesting the involvement of P2X 7 receptor. ATP-induced TNF-␣ release was Ca 2ϩ -dependent, and a sustained Ca 2ϩ influx correlated with the TNF-␣ release in ATP-stimulated microglia. ATP-induced TNF-␣ release was inhibited by PD 098059, an inhibitor of extracellular signal-regulated protein kinase (ERK) kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of p38 mitogen-activated protein kinase. ATP rapidly activated both ERK and p38 even in the absence of extracellular Ca 2ϩ . These results indicate that extracellular ATP triggers TNF-␣ release in rat microglia via a P2 receptor, likely to be the P2X 7 subtype, by a mechanism that is dependent on both the sustained Ca 2ϩ influx and ERK/p38 cascade, regulated independently of Ca 2ϩ influx. Key Words: Microglia-Tumor necrosis factor-␣-ATP-P2X 7 receptor-Ca 2ϩ -Mitogenactivated protein kinase. J. Neurochem. 75, 965-972 (2000).

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