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Identification and Regulation of the IGFBP-4 Protease and Its Physiological Inhibitor in Human Trophoblasts and Endometrial Stroma: Evidence for Paracrine Regulation of IGF-II Bioavailability in the Placental Bed during Human Implantation

Department of Gynecology and Obstetrics (L.C.G., G.H.F., K.I., I.S., B.I., L.-F.S., J.C.I.), Stanford University Medical Center, Stanford, California 94305; Endocrine Research Laboratory (C.A.C., L.B.), The Mayo Foundation, Rochester, Minnesota 55905; The Statens Serum Institut (M.C.), DK-2300 Copenhagen, Denmark; and Department of Molecular and Structural Biology (M.T.O., C.O.), University of Aarhus, DK-8000 Aarhus, Denmark

Address all correspondence and requests for reprints to: Linda C. Giudice, Ph.D., M.D., Division of Reproductive Endocrinology and Infertility, Center for Research on Women’s Health and Reproductive Medicine, Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317. E-mail: . giudice{at}stanford.edu

Abstract

The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using ¹²⁵I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean ± SEM levels of 172.4 ± 32.8 mIU/liter·10⁵ cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1–100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 ± 3.6 mIU/liter·10⁵ cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 ± 308 mIU/liter·10⁵ cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu²⁷ IGF-II, and Des (1–6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70–90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.

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