Opposing actions of hepatocyte growth factor and basic fibroblast growth factor on cell contact, intracellular free calcium levels, and rat ovarian surface epithelial cell viability

Previous studies demonstrated that cell-to-cell contact stimulates a tyrosine phosphorylation signal transduction pathway that prevents rat ovarian surface epithelial (ROSE) cells from undergoing apoptosis. Hepatocyte growth factor (HGF), also know as scatter factor (SF), is expressed by ovarian stromal and thecal cells and has been shown to reduce cell contact in nonovarian tissues. The present studies were designed to determine whether HGF/SF promotes ROSE cells to dissociate and subsequently become apoptotic. Because an increase in intracellular free calcium ([Ca2+](i)) is often an early event in the apoptotic cascade, the effects of HGF/SF on [Ca2+](i) levels were also assessed. ROSE cells were cultured in serum-free medium with HGF/SF, basic fibroblast growth factor (bFGF), thapsigargin, Bay K, actinomycin D, cycloheximide, and/or BAPTA depending on the experimental design. Cell contact was assayed by time-lapse photography; [Ca2+](i) levels were measured with Fluo-3, and apoptosis was assessed by in situ DNA staining. HGF/SF decreased cell contact within 1 h, increased [Ca2+](i) levels by 3 h, and induced apoptosis by 6 h of culture. bFGF inhibited these HGF/SF-induced responses. The increase in [Ca2+](i) appears to represent a point in the apoptotic cascade that commits ROSE cells to die. This concept is based on the observations that: 1) in the presence of the calcium chelator BAPTA, HGF/SF decreased cell contact but did not increase [Ca2+](i) or apoptosis; 2) bFGF blocked HGF/SF-induced increase in [Ca2+](i); 3) bFGF did not attenuate HGF/SF's apoptetic action if exposed to cells after the increase in [Ca2+](i); and 4) RNA and protein synthesis were required for HGF/SF to increase [Ca2+](i), whereas the thapsigargin- and Bay K-induced increase in [Ca2+](i) and apoptosis were independent of RNA/protein synthesis. These observations indicate that the components of the apoptotic cascade distal to the increase in [Ca2+](i) are present within ROSE cells and are activated by a sustained elevation of [Ca2+](i). The present studies also show that when ROSE cells establish contact with 3T3 cells that express N-cadherin, [Ca2+](i) levels are maintained at low basal levels. In contrast, cell contact with 3T3 cells that do not express N-cadherin results in elevated [Ca2+](i) levels. Similarly, a synthetic N-cadherin peptide, which inhibits hemophilic N-cadherin binding, increases [Ca2+](i) levels. Taken together, these data indicate that hemophilic N-cadherin binding between adhering cells plays an important role in maintaining calcium homeostasis. Further, these data support the concept that HGF/SF's ability to promote the dissociation of ROSE cells accounts in part for its ability to increase [Ca2+](i) levels.