PARENT SESSION
SLIDE SESSION 12: IMPLANTATION & EARLY DEVELOPMENT
Chairs: Richard Leach, Carol Brenner, Jeff Reese (Trainee)
Univ Ottawa-Monpetit 202
1:30 PM-3:30 PM

TROPHOBLAST ADHESION TO THE MATERNAL EXTRACELLULAR MATRIX DURING IMPLANTATION IS REGULATED BY INTEGRIN-MEDIATED MOBILIZATION OF INTRACELLULAR CALCIUM.

Wang, Jun¹, Armant, D. Randall¹, ¹

ABSTRACT- During mouse blastocyst implantation, the endometrial extracellular matrix modulates trophoblast adhesion in an intercellular exchange that coordinates the maternal and embryonic developmental programs. Weak integrin-mediated fibronectin (FN)-binding activity expressed on the apical surface of adhesion-competent mouse blastocysts is strengthened by sustained contact with FN through an unknown mechanism. FN-binding ₁- and ₃-class integrins activated this process when ligated by proteins containing an accessible Arg-Gly-Asp integrin recognition sequence. We have examined the role of cytoplasmic free Ca²⁺ as a second messenger in the signaling pathway regulating integrin-matrix adhesion. Treatment of blastocysts with 50 g/ml FN-120, a FN fragment containing the Arg-Gly-Asp site, elevated intracellular Ca²⁺ levels within minutes and increased FN-binding activity after 1 h. Pretreatment with 10 M of the intracellular Ca²⁺ chelator, BAPTA-AM, blocked up regulation of FN-binding activity. Elevation of cytoplasmic Ca²⁺ levels with 5 M ionomycin up regulated adhesion comparably to FN-120, suggesting that Ca²⁺ signaling is not only required, but sufficient for strong adhesion to FN. Ca²⁺-dependent proteins, protein kinase C and calmodulin, were required downstream in this signaling pathway, based on the ability of 10 M calphostin C or 10 M W7, respectively, to inhibit up regulation of adhesion induced by either FN-120 or ionomycin. Integrins elevate free Ca²⁺ through influx or release from intracellular stores, depending on cell type. FN-120-induced signaling was not inhibited with a panel of Ca²⁺ channel blockers or by chelation of extracellular Ca²⁺ using BAPTA, providing no evidence of Ca²⁺ influx. However, FN-120-induced Ca²⁺ transients and up regulation of FN-binding activity were both inhibited by antagonists of phospholipase C (10 M U73122) or the IP3 receptor (1 M xestospongin C). We conclude that integrin signaling mobilizes stored intracellular Ca²⁺ through phosphoinositide metabolism and activation of the IP3 receptor. Through its ability to activate numerous downstream pathways, Ca²⁺ may impact other trophoblast functions in addition to adhesion, which may coordinate cellular events during subsequent development. Supported by NIH grant HD36764

KEY WORDS: implantation , extracellular matrix, calcium, adhesion