A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear Among the protein kinases that have been purified from or localized to the nuclei of eucaryotic cells, the two beststudied enzymes, referred to as NI (6,39,41) and NII (6,11,31,40) activated by double-stranded RNA (33) specifically phosphorylates the translation factor EIF-2a (9). The enzyme isolated from mouse spleen nuclei by Ohtsuki et al0was stimulated up to fivefold by DNA specifically for phosphorylation of two small nonhistone proteins (26-28), and histone phosphorylation by the chromatin-associated cGMP-dependent kinase is also stimulated by DNA (12); in both of these cases, however, the stimulation results from interaction of DNA with the substrate rather than with the enzyme (12,26

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.

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 (125)I-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.10(5) 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.10(5) 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.10(5) 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, Leu(27) 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 ...