The precise biochemical mediators of human trophoblast-uterine interaction are unknown. However, in order to understand the pathophysiology of early implantation failure, recurrent pregnancy loss, and premature placental separation, the critical cells and molecules which mediate adhesion to the uterus need to be studied. On a cellular level, the trophoblast shell of the early embryo establishes initial contact with the endometrial lining. Subsequently, an expanding zone of trophoblast contact with the uterine extracellular matrix (ECM) establishes the uteroplacental junction. Previous studies examining trophoblast behavior in vitro have suggested an important role for fibronectin in modulating trophoblast-ECM interactions. Recently, we have observed further evidence to suggest that a specific class of trophoblast fibronectins-those containing the oncofetal domain- may function as a tropho-uteronectin (TUN), or trophoblast-uterine connecting protein during placental development. As an interesting correlate to the in vivo situation, isolated human cytotrophoblasts in primary culture also synthesize, secrete, and deposit a TUN-like protein at cell-ECM contact sites. Furthermore, synthesis of this protein in culture is regulated by both stimulatory and inhibitory factors. We hypothesize that TUN has a significant role in modulating normal trophoblast-uterine adhesive processes. Our experimental approaches in this application utilize primary cultured human trophoblasts from first trimester and term placentae, as well as the SPA-26 trophoblast cell line.
In Specific Aim #1, we will determine whether cellular interactions with endogenous TUN are required for trophoblast adhesive activity. Our approach will be to block both RGD and non-RGD integrin binding domains with adhesion-perturbing peptides and monoclonal antibodies. Since the IIICS region contains two non-RGD cell binding sites, blocking peptides and antibodies directed against adhesion sites in this alternatively spliced fibronectin domain will be specifically assayed.
In Specific Aim #2, endogenous TUN synthesis will be blocked with antisense fibronectin oligonucleotides, permitting us to study the effects of this inhibition on trophoblast adhesive behavior. We will extend these studies by attempting to """"""""knock-out"""""""" TUN subsets containing specific IIICS domains.
In Specific Aim #3, we propose to continue our studies of TUN regulation to compare the effects of transforming growth factor beta (TGFbeta) and leukemia inhibitory factor (LIF) on primary trophoblast and SPA-26 cell cultures. Since we have hypothesized that particular domains in the IIICS region may be important for trophoblast adhesiveness, we will also determine whether TGFbeta and/or LIF specifically induce synthesis of CS1+ and CS5+ TUN isoforms. As potential paracrine regulators of trophoblast function, decidual and endometrial cells will be assayed for their TUN modulating activity. Our overall goal, by Investigating TUN function and regulation in early and late trophoblasts, is to characterize the precise molecular events required for normal trophoblast-ECM interaction throughout pregnancy.
