The functional role of Rho and Rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosi nophils. Very little is known about the eotaxin signaling in eosinophils ex cept the activation of the mitogen-activated protein (MAP) kinase family. T he p21 G protein Rho and its substrate Rho-associated coiled-coil forming p rotein kinase (ROCK) regulate the formation of stress fibers and focal adhe sions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity , indicating that ROCK is downstream of Rho. We then examined the role of R ho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemot axis was significantly inhibited by exoenzyme C3 or Y-27632. Because extrac ellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whet her Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-276 32 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactiv e oxygen species production. There is a dichotomy of downstream signaling p athways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosi nophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.