GABA A receptor π subunit promotes apoptosis of HTR-8/SVneo trophoblastic cells: Implications in preeclampsia

Abstract

Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter through its receptors in the mature central nervous system. The GABA type A receptor π subunit (GABRP) has been identified in the tissues of the reproductive system, particularly in the uterus. In addition, we have previously detected GABRP expression in both human and mouse placentas. To examine the role of GABRP in trophoblastic cell invasion, we constructed a pIRES2-GABRP-EGFP plasmid which was used for the transfection of a human placental cell line derived from first trimester extravillous trophoblasts (HTR-8/SVneo). The number of invaded cells was decreased by GABRP overexpression. Notably, the decrease in the invasive cell number may be due to the increased apoptosis of the HTR-8/SVneo cells following GABRP transfection, which was further confirmed by flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Based on the increased apoptosis of trophoblastic cells in pregnancies complicated by preeclampsia (PE) and the fact that GABRP promotes the apoptosis of trophoblastic cells, we hypothesized that GABRP expression is increased in the placental tissues from patients with PE compared with that in the normal groups and this hypothesis was confirmed by RT-qPCR and immunohistochemical analysis. Taken together, these findings imply that GABRP plays an important role in placentation and this pathway may be a promising molecular target for the development of novel therapeutic strategies for PE.

Figure 1

Immunolocalization of gamma-aminobutyric acid type A receptor π subunit (GABRP) in HTR-8/SVneo cells. GABRP expression was detected in the cytoplasm and the nuclear membrane of the HTR-8/SVneo cells. NEG, negative controls in which normal IgG was used in place of primary antibody. Original magnification ×200.

Figure 2

Gamma-aminobutyric acid type A receptor π subunit (GABRP) overexpression significantly inhibits the invasion of HTR-8/SVneo cells. (A) Confirmation of GABRP overexpression using RT-qPCR and western blot analysis. GAPDH was used as an internal control in RT-qPCR and a loading control in western blot analysis. (B) Representative images of filters containing invaded cells in Matrigel invasion assay. The statistical bar graphs show the summary of three independent experiments (t-test, ^*P<0.05).

Figure 3

Effects of gamma-aminobutyric acid type A receptor π subunit (GABRP) on cell viability and apoptosis of HTR-8/SVneo cells. (A) A CCK-8 assay was performed to demonstrate a decrease in cell viability of the HTR-8/SVneo cells 24, 48 and 72 h after GABRP transfection (t-test, ^*P<0.05). (B) Flow cytometric analysis showed an increase in apoptosis of the HTR-8/SVneo cells 72 h after GABRP transfection. A representative image from three independent experiments is shown. UL, upper left panel (necrotic cells); UR, upper right panel (late apoptotic cells); LL, lower left panel (live cells); LR, lower right panel (early apoptotic cells).

Figure 4

Regulators of HTR8/SVneo cell apoptosis following gamma-aminobutyric acid type A receptor π subunit (GABRP) overexpression for 72 h. (A) RT-qPCR revealed that the mRNA levels of the anti-apoptotic Bad and Bax and the pro-apoptotic Bcl-2 were increased following GABRP overexpression. (B) Western blot analysis showed that the anti-apoptotic Bad and Bax and pro-apoptotic Bcl-2 protein levels, were increased following GABRP overexpression. (C) Western blot analysis demonstrated that the effector caspase-3 and its active form, cleaved caspase-3, were increased following GABRP overexpression. GAPDH was used as an internal control for RT-qPCR and as a loading control for western blot analysis. (t-test, ^*P<0.05).

Figure 5

Differential expression of gamma-aminobutyric acid type A receptor π subunit (GABRP) in placental tissues from patients with preeclampsia (PE) or normal pregnancy. (A) The immunostaining of GABRP was mainly localized in the cytotrophoblasts (CTBs) and was increased in the PE group compared with that in the control group. Negative placental tissues of the control group and the PE group are shown. NEG, negative controls in which normal IgG was used in place of primary antibody; STB, syncytiotrophoblast. (B) RT-qPCR revealed an increase in the mRNA level of GABRP in the PE group compared with that in the control group (t-test, ^*P<0.05). Original magnification ×400.

Figure 6

Schematic depicting the effect of gamma-aminobutyric acid type A receptor π subunit (GABRP) on members of the Bcl-2 family in the apoptosis cascade and the role of Bcl-2 in balancing the apoptosis and differentiation process. (A) GABRP maintains the balance between pro-apoptotic Bad and anti-apoptotic Bcl-2 under normal conditions. However, the balance may be lost and biased towards the pro-apoptotic Bad pathway when GABRP is overexpressed. Thereafter, Bax is activated which causes cytochrome c release and activation of the caspase-mediated apoptosis cascade. (B) There is a differentiation-dependent pattern of Bcl-2 expression in the placenta whereby a signal balance between apoptosis and differentiation is achieved and hence, trophoblast mass is preserved during pregnancy.