Caroline H. Damsky

Most transformed cells have lost anchorage and serum dependence for growth and survival. Previously, we established that when serum is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts and endothelial cells (Ilić et al. 1998. J. Cell Biol. 143:547–560). The present goals are to identify survival sequences in FAK and signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. We report that binding of the SH3 domain of p130Cas to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is withdrawn. The FAK–p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4) pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but not in fibroblasts cultured on collagen, which does not. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, we confirm work of others that survival signals are transduced by FAK, phosphatidylinositol 3′-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented herein. Thus, survival signals from extracellular matrix and serum are transduced by FAK via two distinct pathways.

Establishment of the human placenta is essential for subsequent development of the embryo. Previous studies from our laboratories have demonstrated that chorionic villus cytotrophoblast stem cells undergo a stepwise differentiation process in vivo that results in their ability to invade the uterine wall. This process can be mimicked by isolated primary first-trimester cytotrophoblasts in vitro. Efforts to study the regulation of this differentiation pathway have been hampered by the inability of the isolated cytotrophoblast to replicate in culture. We therefore performed experiments to determine the suitability of the temperature-sensitive simian virus 40-transformed cell line SPA 255-26 (SPA-26), derived from early-gestation cytotrophoblasts, for studying the cytotrophoblast differentiation pathway that results in uterine invasion. Our results show that this cell line exhibits many properties of differentiated early-gestation cytotrophoblasts at both permissive and nonpermissive temperature. These cells were invasive in vitro and expressed the repertoire of hormones, adhesion molecules, and proteinases characteristic of an advanced stage of cytotrophoblast differentiation in vivo. Thus, these cells should be useful in studying the regulation of the adhesive and invasive behavior of human cytotrophoblasts.