Effects of Y-27632, a rho-associated kinase ROCK inhibitor, on adhesion, survival, syncytium formation and hCG secretion of term human cytotrophoblasts

Abstract

s / Placenta 35 (2014) A1eA112 A101 P2.128-N. EFFECTS OF Y-27632, A RHO-ASSOCIATED KINASE ROCK INHIBITOR, ON ADHESION, SURVIVAL, SYNCYTIUM FORMATION AND HCG SECRETION OF TERM HUMAN CYTOTROPHOBLASTS Kenichiro Motomura , Naoko Okada , Akio Matsuda , Haruhiko Sago , Hirohisa Saito , Kenji Matsumoto a National research institute for child health and development, Tokyo, Japan; National center for child health and development, Tokyo, Japan Objectives: Several different signal transduction pathways are known to participate in the multiple steps of placenta formation. Rho kinase reportedly promotes first-trimester extravillous trophoblast migration, but the effects of Rho-associated kinase on the cytotrophoblasts themselves remain obscure. In the present study, we investigated the effects of Y27632, a Rho-associated kinase ROCK inhibitor, on several functions of term human cytotrophoblasts. Methods: Primary human trophoblasts (PHT) were isolated from term uncomplicated placentas by trypsin, dispase and DNase I treatment, percoll density gradient centrifugation and negative selection using anti-HLA class I mAb-coated immunomagnetic beads. Purified PHT were suspended in 10% FBS-containing medium supplemented with EGF in the presence and absence of Y-27632 and cultured in non-coated plates for up to 96 hours. Cell viability was measured by WST-8 assay. Adhesion was assessed by counting the number of cell nuclei on the culture plate after staining with an immunofluorescent dye. Syncytium formation and trophoblast maturation were assessed by qPCR assay of syncytin1/syncytin2 and betahCG mRNA expression, respectively. Results: Y-27632 significantly increased PHT viability. Y-27632 increased PHT adhesion to the non-coated culture plate in a concentration-dependent manner. Y-27632 enhanced expression of mRNA for such syncytialization markers as syncytin1 and syncytin2. Y-27632 also enhanced expression of beta-hCG mRNA. Conclusion: Rho-associated kinase is likely to play critical roles in several functions of PHT. The mechanisms regulating Rho-associated kinase in cytotrophoblasts should be studied further. In addition, intracellular signals downstream of Rho-associated kinase should be clarified. P2.129-N. TO INVADE OR NOT TO INVADE: DEFINING THE ROLE OF MTA3 IN TROPHOBLAST DIFFERENTIATION AND MIGRATION Mariko Horii, Matteo Moretto-Zita, Yingchun Li, Mana M. Parast Department of Pathology, University of California San Diego, La Jolla,