GROWTH-FACTOR ACTIVITY OF ENDOTHELIN-1 IN PRIMARY ASTROCYTES MEDIATEDBY ADHESION-DEPENDENT AND ADHESION-INDEPENDENT PATHWAYS

Endothelin-1 (ET-1) has been shown to induce DNA synthesis in primary astrocytes by stimulating the extracellular signal-regulated kinase (E RK) pathway. To clarify the mechanisms responsible for the anchorage-d ependent growth of astrocytes, the relationships between cell adhesion and ERK activation were investigated. Here it is reported that ET-1 p romotes the formation of stress fibers and focal adhesions and the tyr osine phosphorylation of focal adhesion kinase (FAK) and paxillin, as well as Src activation and association of phosphorylated FAK with Grb2 . Pretreatment of astrocytes with cytochalasin D or C3-transferase, wh ich inhibits actin polymerization or Rho activity, respectively, preve nted the activation/phosphorylation of Src, FAK, and paxillin after ET -1 stimulation; by contrast, the ERK pathway was not significantly aff ected. This differential activation of FAK/Src and ERK pathways was al so observed with astrocytes 10 and 60 min after replating on poly-L-or nithine-precoated dishes. Collectively these findings indicate that ac tivation of FAK and Src is dependent on actin cytoskeleton integrity, Rho activation, and adhesion to extracellular matrix, whereas ERK acti vation is independent of these intracellular events and seems to corre late with activation of the newly identified protein tyrosine kinase P YK2. Induction of DNA synthesis by ET-1, however, was reduced dramatic ally in astrocytes pretreated with either cytochalasin D or C3-transfe rase. This study provides a demonstration of Rho-and adhesion-dependen t activation of FAK/Src, which collaborates with adhesion-independent activation of PYK2/ERK for DNA synthesis in ET-1-stimulated astrocytes .