Basic Fibroblast Growth Factor and N-Cadherin Maintain Rat Granulosa Cell and Ovarian Surface Epithelial Cell Viability by Stimulating the Tyrosine Phosphorylation of the Fibroblast Growth Factor Receptors

Abstract

Both granulosa cells (GCs) and ovarian surface epithelial cells undergo apoptosis in vivo. Although basic fibroblast growth factor (bFGF) and N-cadherin-mediated cell contact inhibit GC apoptosis, little is known about the factors that influence rat ovarian surface epithelial (ROSE) cell apoptosis. The present studies were designed to determine whether bFGF and N-cadherin maintain the viability of both GC and ROSE cells by stimulating separate signaling pathways. For the GC studies, large GCs were collected from immature rat ovaries after Percoll gradient centrifugation and placed in serum-free culture for 24 h. These studies confirmed that about 10% of the aggregated GCs and more than 50% of single GCs were apoptotic after culture. bFGF reduced the percentage of apoptotic single GCs, but did not influence aggregated GCs. A neutralizing antibody to bFGF blocked bFGF’s antiapoptotic action, but did not alter the percentage of apoptotic aggregated GCs. The antibody to N-cadherin not only increased the percentage of aggregated apoptotic GCs, but also blocked bFGF’s ability to maintain the viability of single GCs. The effect of the FGF receptor antibody was similar to that of the N-cadherin antibody. Like GCs, ROSE cells also undergo apoptosis in serum-free medium. Exposure to either the N-cadherin or FGF receptor antibody, even in the presence of serum, increased the percentage of apoptotic aggregated ROSE cells. As tyrosine kinase activity is involved in maintaining cell viability, the pattern of tyrosine-phosphorylated proteins was examined after culture in control (ascites) or N-cadherin antibody-supplemented medium. Exposure to the N-cadherin antibody altered the pattern of tyrosine-phosphorylated proteins, decreasing the tyrosine phosphorylation of proteins in the 130- to 180-kDa range and increasing the tyrosine phosphorylation of one or more proteins of about 50 kDa. The identity of the 50-kDa protein is unknown. However, immunoprecipitation studies demonstrated that the N-cadherin antibody reduced the amount of tyrosine-phosphorylated FGF receptor in both GCs and ROSE cells by 50%. This decrease corresponds to an increase in apoptosis among aggregated cells. Taken together, these data suggest that homophilic N-cadherin binding and bFGF-FGF receptor binding activate signal transduction pathways that converge at the level of the FGF receptor and subsequently promote the viability of both GC and ROSE cells.