Expression of Pro- and Anti-Apoptotic Proteins during Placental (Trophoblast) Differentiation

Abstract 278 Poster Session I, Saturday, 5/1 (poster 139)

Development and maintenance of a differentiated phenotype depend on a balance among cell proliferation, maturation and apoptosis. Apoptosis is the morphologic description of programmed cell death. In human placenta, apoptosis ↑s to term in the syncytiotrophoblast, which forms the anatomical maternal-fetal interface, and in the trophoblast of the fetal membranes (chorion laeve). Placental apoptosis appears to play a critical role in embryonic development, parturition and fetal wastage. However, the molecular mechanisms controlling cell death during placental maturation are unknown. Therefore, we investigated the role of pro- and antiapoptotic proteins during trophoblast differentiation. Highly purified (>95%) cytotrophoblasts (CTBs) were isolated from third trimester placentae and maintained in monolayer culture (0-4 days). Under these conditions, CTBs exit the cell cycle, terminally differentiate, ↑ expression of placental glycoprotein hormones (assayed by Northern blot) and, by d 3, fuse into syncytia. Using FACS analysis, we determined that cultured CTBs become quiescent on d 1. After d 3, large numbers of cells enter apoptosis (hypodiploid population). Induction and apoptosis was confirmed by morphologic criteria (membrane blebbing, cellular shrinkage, condensation and margination of nuclear chromatin) using confocal microscopy and by the presence of DNA internucleosomal cleavage. Since heteromers of the Bcl-2-related gene products are critical regulatory components of the apoptotic pathway, we also examined trophoblast expression of pro- and antiapoptotic Bcl-2 family members. Expression of Bcl-2-related proteins was determined by multiprobe ribonuclease protection assays and by Western blots of CTB RNA and cell lysates, respectively. CTBs expressed Bcl-2 and Bak mRNAs at low levels and Bax, Bcl-x_Land Mcl-1 more robustly. Transcript levels of Bax, an apoptosis promotor, ↓d slightly and Bax protein ↓d several-fold by d 4. Although steady-state mRNA levels for Bcl-2, Bcl-x_L, and Mcl-2 remained relatively constant from d 1→4, immunoblots showed that these antiapoptotic protiens ↑d significantly on d 3-4 (Bcl-2 > Bcl-x_L and Mcl-2). These data indicate that the balance between Bax and Bcl-2 expression shifts to apoptosis activation during trophoblast terminal differentiation in vitro. ↑s in proapoptotic Bax mRNA/protein precede both morphologic evidence of apoptosis and genomic DNA fragmentation. In contrast, the d 4 rise in antiapoptotic proteins coincident with ↑d apoptosis was unexpected, and may indicate emergence of an apoptosis-resistant population of trophoblasts. Immunohistochemistry of placental villi and placental bed tissue indicates that extravillous (uterine) trophoblast is the principal site for such growth-arrested, nonapoptotic cells. We speculate that protection from apoptosis in extravillous trophoblast may a critical mechanism for establishing and maintaining the uteroplacental microenvironment during pregnancy. (Supported by HD11343).