Mouse trophoblastic cell lines: II--Relationship between invasive potential and proteases.
The specialized interaction between embryonic and maternal tissue is unique to mammalian development. This interaction begins with the invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. Because of their highly specialized behavior invasive cells must attach to the extracellular matrix proteins, secrete proteinases, capable of degrading matrix, and migrate through the degraded matrix; invasion is partially dependent on the proteinase activity of the cells. The objective, therefore, was to study a vitro system to examine the mechanism(s) of trophoblast cell invasion and its relationship to proteinases. Since little is known about the actual mechanism(s) involved. The mouse trophoblast cell lines established from placentas of different gestational ages were chosen to study their invasive properties in vitro. To begin to understand the biochemical basis of this behavior, the chromogenic assay and the substrate gel technique was used to analyze the cell associated and secreted plasminogen activators. All lines secrete and synthesize both urokinase-type (uPA) and tissue-type (tPA) plasminogen activators. The most invasive line SM9-2, derived from mid-gestation (day 9) placenta showed the highest enzymatic activity in the conditioned medium (CM), whereas in cell extract (CE) SM-10 line derived from late gestation placenta had the highest PAs activity. Four forms of secreted PAs in CM were of 79, 72, 43 and 35 kDa molecular weights, whereas in CE only 79 kDa molecular weight form of PA was detected using substrate SDS-PAGE gels. Additional observations from cells cultured on Marrigel Invasion Chambers also showed secretion of PAs by noninvading and invading cells in a biphasic pattern suggest the involvement of these enzymes in the extracellular proteolysis. The expression of matrix metalloproteinase gelatinase B (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) were examined by RT-PCR in all the lines, however MMP-9 and TIMP-1 signals were strongly expressed in SM9-2 and SM-10 lines respectively. CM and CE were characterized by gelatin zymography, and the proteinases secreted by these cells in CM were confirmed to be metalloproteinases with approximate molecular masses between 52 to 92 kDa. Proteinases secreted by noninvading and invading cells cultured on Matrigel Invasion Chambers were not identical suggesting that specialized, temporally regulated metallopro-teinases are involved in trophoblast invasion. Trophoblast cell invasion in Matrigel Invasion Chambers was significantly inhibited in all the lines by using 1, 10-phenanthroline, an inhibitor of metalloproteinases. The results indicated that mouse trophoblast cells have matrix--degrading capabilities through metalloproteinase activity. Similar metalloproteinase activity has been reported to be necessary for human trophoblast invasion, suggesting a similar mechanism of implantation. Trophoblast culture system described here should be useful in studying some of the early events in human placentation.
Sharma
R K
RK
Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City 66103, USA.
D000818:Animals; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D017364:Collagenases / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010064:Embryo Implantation / Q000502:physiology*; D010450:Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D005260:Female; D020780:Matrix Metalloproteinase 9; D051379:Mice; D010618:Phenanthrolines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000378:metabolism; D014327:Trophoblasts / Q000166:cytology / Q000201:enzymology* / Q000502:physiology*; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000378:metabolism
D010618:Phenanthrolines; D011480:Protease Inhibitors; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D010450:Endopeptidases; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D017364:Collagenases; D020780:Matrix Metalloproteinase 9; C025205:1,10-phenanthroline