| This study examined the role of osteopontin (OPN), a phosphorylated secreted glycoprotein, in the promotion of trophoblastic cell migration and adhesion, the two major components of the embryo implantation process. The first aim was to purify and characterize the OPN forms expressed in human biological fluids such as milk and urine. Using the biochemical properties of OPN, I was able to design a successful purification strategy based on DEAE-Sephacel chromatography followed by affinity chromatography. It also was established that the major form of OPN present in urine is highly phosphorylated, which is consistent with its expression in milk and with the phosphates' role in inhibiting calcification in biological fluids (Chapter 2). Human choriocarcinoma cells, namely JAR, BeWo and JEG-3, were treated with variants of OPN differing in the extent of phosphorylation following sequential dephosphorylation with tartrate-resistant acid phosphatase (TRAP), and their migratory and adhesive responses were measured. The highly phosphorylated human milk OPN (OPN-1) strongly triggered migration in these cell lines, whereas the less phosphorylated variants, OPN-2a and OPN-2b, did not trigger this response. Interestingly, the opposite effect was observed in relation to adhesion of JAR cells to these forms. The dephosphorylated OPN-2a and OPN-2b favored adhesion and spreading of these cells while OPN-1 did not (Chapter 3). Using broad spectrum protein kinase profiling, p70 S6 kinase was identified as a major signal transduction pathway activated by OPN-1 during the migratory response in JAR cells. This activation was blocked completely by rapamycin and LY294002, thus, demonstrating that OPN-1-stimulated migration is mediated through mTOR and PI3K, respectively. PD98059 had no effect on the activation of p70 S6 kinase by OPN-1, thus, demonstrating that this response does not involve the Ras/MAPK signaling cascade (Chapter 4). Together, these data show that the highly phosphorylated OPN-1 plays a key role in trophoblastic cell migration, through recruitment of actin filaments to the cellular membrane, and provide evidence for the involvement of a new signaling PI3K/mTOR/p70 S6 kinase pathway in the JAR cells response to OPN-1. These observations also imply that extracellular phosphatases such as TRAP may attenuate OPN-1-driven cell migration. |
