Pregnancy-Associated Plasma Protein A Proteolytic Activity Is Associated with the Human Placental Trophoblast Cell Membrane

Abstract

Pregnancy-associated plasma protein-A (PAPP-A) is a product of the placenta and decidua and is secreted into the maternal circulation during human pregnancy. It recently has been identified as an IGF binding protein (IGFBP)-4 protease. Presumed functions at the maternal-fetal interface are to proteolyze IGFBP-4 and thus increase IGF bioavailability locally in the placenta, to promote IGF-II-mediated trophoblast invasion into the maternal decidua, and to modulate IGF regulation of steroidogenesis and glucose and amino acid transport in the villous. Herein, we have investigated the possibility that IGFBP-4 proteolysis may occur on the trophoblast cell membrane, presumably to increase local bioavailable IGF for interactions with cognate IGF membrane receptors. Human trophoblasts were cultured, trophoblast plasma membranes were isolated and solubilized, and IGFBP-4 protease activity and PAPP-A immunoreactivity in the solubilized plasma membrane fraction were investigated. IGFBP-4 protease activity was detected in solubilized human trophoblast membranes, resulting in cleavage of recombinant human IGFBP-4 into 18- and 14-kDa fragments, detected by Western immunoblot analysis. This protease activity was dependent on the presence of IGF-II, and its metal ion dependence was demonstrated by inhibition of the protease by the metal chelators, EDTA and EGTA. The presence of PAPP-A in solubilized human trophoblast membranes was demonstrated by Western immunoblotting. Trophoblast membrane PAPP-A had a relative molecular weight of approximately 200 kDa and comigrated on (reducing) SDS-PAGE with recombinant human PAPP-A and PAPP-A secreted into media conditioned by cultured human trophoblasts. IGFBP-4 protease activity was not detected after immunodepletion of PAPP-A from the trophoblast membrane fraction with PAPP-A polyclonal antibodies, suggesting the identity of the membrane-derived IGFBP-4 protease as PAPP-A. Immunocytochemistry revealed PAPP-A on the cell membrane and in the cytoplasm of human trophoblasts in culture. Together, these data demonstrate the presence of an IGF-II- and metal-dependent IGFBP-4 protease activity in human trophoblast plasma membranes, identified as PAPP-A, which is well situated to proteolyze IGFBP-4 at the maternal-placental interface to facilitate IGF action at the villous surface and/or the invading extravillous cytotrophoblast.

Gene expression patterns in human placenta

“…2). Genes abundantly expressed in villus regions of placenta relative to other normal human tissues included insulin-like growth factor (IGF) 2 and pregnancy-associated plasma protein A (PAPP-A), which are known to be expressed in placenta (16,17). In some cases, the extraplacental expression would suggest a potential biological role.…”

IGF-binding protein-4: biochemical characteristics and functional consequences

“…IGFBPs are expressed by the maternal decidua and may regulate IGF actions during pregnancy (Han et al 1996). In this context IGFBP-4 is particularly interesting for several reasons: (i) IGFBP-4 is the second most abundant IGFBP in the placental bed (Han et al 1996); (ii) PAPP-A, an IGF-dependent IGFBP-4 protease, is secreted by human trophoblast cells and decidualized endometrial stroma , Sun et al 2002 and is markedly increased in the maternal circulation as pregnancy progresses (Byun et al 2000); and (iii) in human pregnancy serum the majority of PAPP-A (.99%) is found as a PAPP-A/proMBP complex (Oxvig et al 1993). During pregnancy, rapid placental development and fetal growth obviously increases the need for growth-promoting factors such as IGFs.…”

Cell Surface Targeting of Pregnancy-associated Plasma Protein A Proteolytic Activity

“…Although these data were carried out with polyspecific PAPP-A antibodies known to recognize different antigens (22,38), they are in agreement with our data. Furthermore, we have recently demonstrated that PAPP-A antigen present in a preparation of solubilized membranes from human trophoblasts is proteolytically active (51).…”