MULTIFUNCTIONAL PHOSPHATIDIC-ACID SIGNALING IN MAMMARY EPITHELIAL-CELLS - STIMULATION OF PHOSPHOINOSITIDE HYDROLYSIS AND CONVERSION TO DIGLYCERIDE

We have shown previously that phosphatidic acid esterified to polyunsa turated fatty acids is mitogenic for primary cultures of mouse mammary epithelial cells embedded within collagen gels. We hypothesized that this mitogenic competence resulted from the ability of this phospholip id to activate multiple signal transduction pathways in mammary epithe lium. A closer examination of this hypothesis was undertaken by examin ing the effect of exogenous phosphatidic acid on phosphoinositide (PI) hydrolysis and its intracellular metabolism to diglyceride, an activa tor of protein kinase C. For assays of phosphoinositide-specific phosp holipase C activation, mammary epithelial cells from virgin Balb/c mic e were isolated by collagenase dissociation of mammary glands and cult ured on the surface of Type I collagen-coated culture dishes. Phosphat idic acid (PA) stimulated a sustained increase in inositol phosphates and caused inositol phospholipid depletion when added to cells in whic h inositol phospholipids were prelabeled with H-3-myoinositol. This ef fect was specific for PA among phospholipids tested. Neither lineoleic acid, that can be released from PA, nor prostaglandin E(2) affected P I hydrolysis. When mammary epithelial cells were cultured inside colla gen gels in the presence of exogenous PA or phosphatidylcholine (PC) r adiolabeled with H-3-glycerol, PA was found to persist intracellularly and be dephosphorylated to diglyceride (an activator of protein kinas e C) to a greater extent than PC, a nonmitogenic phospholipid. In cont rast to PA, epidermal growth factor (EGF) only slightly stimulated PI hydrolysis, showing that these two different growth-promoting factors do not actively couple to the same signal transduction pathways in mam mary epithelial cells. These results show that PA may activate multipl e pathways in mammary epithelial cells either directly or via its meta bolism to diglyceride. (C) 1995 Wiley-Liss, Inc.