EFFECTS OF INHIBITORS OF INTEGRIN BINDING ON CELLULAR OUTGROWTH FROM BOVINE INNER CELL MASSES IN VITRO

Abstract

Bovine inner cell masses (ICM) cultured on fibronectin give rise to extensive cellular outgrowths containing endoderm. Peptides with the Glu-Ile-Leu-Asp-Val (EILDV) and Arg-Gly-Asp (RGD) sequences inhibit cell migration on fibronectin by binding to the fibronectin-recognition site in several integrins. To identify integrins involved in endodermal cell outgrowth on fibronectin and vitronectin, the effects of the EILDV and RGD peptides were evaluated in vitro. In experiment 1, ICM were cultured on fibronectin in medium containing 0.5 or 1.0 mg/ml EILDV or RGD (or both). Compared with 0 mg/ml, 0.5 mg/ml EILDV suppressed (P<0.10) outgrowth area overall, and 1.0 mg/ml EILDV reduced (P<0.05) outgrowth area after 72 h of culture. Compared with 0 mg/ml, 0.5 and 1.0 mg/ml RGD reduced (P<0.05) outgrowth area after 72 h of culture. Plasminogen activator activity in conditioned medium increased (P<0.05) in 0.5 mg/ml RGD but decreased (P<0.10) in 1.0 mg/ml RGD compared with 0 mg/ml RGD. In experiment 2, bovine ICM were cultured on vitronectin in medium containing 0.5 or 1.0 mg/ml RGD. Neither concentration of RGD (P>0.10) affected the extent of cellular outgrowth on vitronectin. Bovine endodermal cell migration on fibronectin can be modulated by the RGD and EILDV peptides. Despite inhibition, neither peptide completely prevented outgrowth on fibronectin. In contrast, cellular outgrowth on vitronectin was unaffected by RGD. The persistence of cellular outgrowth on fibronectin and the absence of inhibition by RGD for ICM cultured on vitronectin suggests that bovine endodermal cells can use alternative cellular adhesion systems, such as nonintegrin receptors, during outgrowth.