Soluble receptor (DcR3) and cellular inhibitor of apoptosis-2 (cIAP-2) protect human cytotrophoblast cells against LIGHT-mediated apoptosis

Abstract

LIGHT (tumor necrosis factor superfamily 14) is among the powerful apoptosis-inducing cytokines synthesized in human placentas. Here, we investigated mechanisms protecting cytotrophoblast (CTB) cells from LIGHT-mediated apoptosis. Viability assays and caspase-3 immunoblots using recombinant LIGHT were done to establish that CTB cells purified from term placentas resist LIGHT-induced apoptosis. Although the cells were also resistant to killing by another placental cytokine, interferon-gamma (IFN-gamma), a combination of the two induced apoptosis. Killing was prevented by DcR3-Fc fragment but not control human-Fc fragment, showing that apoptosis occurs via the LIGHT pathway and that soluble receptors provide protection. Next, two cellular inhibitors of apoptosis expressed in CTB cells, cellular inhibitor of apoptosis (cIAP)-1 and cIAP-2, were investigated for protection. Cellular IAP-1 was unchanged after stimulation with LIGHT whereas cIAP-2 mRNA and protein were elevated. The increase was abrogated by treating CTB cells with LIGHT + IFN-gamma, implying a central role for cIAP-2 in preventing LIGHT-mediated apoptosis and an ability of IFN-gamma to overcome cIAP-2 protection. Definitive evidence was provided in experiments that showed that cIAP-2 anti-sense morpholinos permit LIGHT to induce apoptosis in HT-29 cells. In summary, the data are consistent with the postulate that placental CTB cells are protected from LIGHT-mediated apoptosis by both soluble receptor, DcR3, and cIAP-2.

Figure 1

Analysis of cell viability after cytokine treatments using the MTT assay. A: CTB cells treated with rhLIGHT for 48 and 96 hours. B: CTB cells treated with rhIFN-γ (100 U/ml) for 48 and 96 hours. C: CTB cells treated with rhLIGHT + rhIFN-γ (100 U/ml) for 48 and 96 hours. D: HT-29 cells treated with rhLIGHT + rhIFN-γ (10 U/ml) for 48 and 96 hours (positive control). Data are shown as a percentage of the control values (no cytokine added). Error bars represent the means ± SEM of six preparations of CTB cells purified from six different placentas (A–C) and six different preparations of HT-29 cells (D). Each assay was performed in triplicate. *, P < 0.05; **, P < 0.01.

Figure 2

Analysis of cell viability after exposure of rhLIGHT/rhIFN-γ-treated cells to control-Fc or DcR3-Fc using the MTT assay. A: CTB cells. B: HT-29 cells (positive control). Cells treated with rhLIGHT/rhIFN-γ for 96 hours as before, were also exposed to either 4 μg/ml of control-Fc (human Fc) or the same concentration of the LIGHT-soluble receptor, DcR3-Fc. Data are shown as a percentage of the control value (no Fc fragment). Error bars represent the means ± SEM of three preparations of CTB purified from three different placentas or three different preparations of HT-29 cells. All data points represent triplicate wells. *, P < 0.05; **, P < 0.01.

Figure 3

Induction of caspase-3 fragments in CTB cells by rhLIGHT + rhIFN-γ. Cells were treated for 96 hours with (left to right): control PBS (C), rhLIGHT (100 ng/ml) (L), rhIFN-γ (100 U/ml) (I), or rhLIGHT (100 ng/ml) + rhIFN-γ (100 U/ml) (L/I), then immunoblotted to reveal caspase-3. Arrows indicate the active heterodimer (p20 and p17) as well as the inactive p24 fragment. The immunoblot is representative of three independent experiments.

Figure 4

Effects of rhLIGHT, rhIFN-γ, and rhLIGHT + rhIFN-γ on the abundance of cIAP-2 protein in CTB cells at 48 hours (A) and 96 hours (B). CTB cells were incubated with (left to right) control PBS (C), rhLIGHT (100 ng/ml) (L), rhIFN-γ (100 U/ml) (I), or rhLIGHT (100 ng/ml) + rhIFN-γ (100 U/ml) (L/I), then immunoblotted to reveal cIAP-2 protein. The blots were then incubated to reveal actin. Arrows indicate cIAP-2 and actin protein, respectively. Data gathered by scanning densitometer representing the mean fold change over control (normalized to actin) ± SEM of three preparations of CTB cells, purified from three different placentas, are shown below representative blots.

Figure 5

Immunoblot of protein from HT-29 cells treated for 96 hours with control PBS (C), rhLIGHT (100 ng/ml) (L), rhIFN-γ (10 U/ml) (I), or rhLIGHT (100 ng/ml) + rhIFN-γ (10 U/ml) (L/I), for cIAP-2 (and actin). Arrows indicate cIAP-2 and actin protein, respectively. Data gathered by scanning densitometer representing the fold change over control (normalized to actin), are shown below the blot. Representative of two independent experiments.

Figure 6

Model system for testing cIAP-2 function. A: Imaging of fluorescein-tagged morpholinos inside HT-29 cells 72 hours after ethoxylated polyethylenimine delivery. B: Immunoblot for cIAP-2 in HT-29 cells treated for 96 hours with control morpholino [control (C)] or cIAP-2 anti-sense morpholino [test (T)]. Arrows indicate cIAP-2 or actin protein, respectively. Data gathered by scanning densitometer representing the fold change over control (normalized to actin), are shown below the blot. Representative of two independent experiments. Original magnification, ×200 (A).

Figure 7

MTT analysis of HT-29 cells treated first with cIAP-2 or control morpholinos and then with PBS (control) or rhLIGHT (1 to 1000 ng/ml) for 96 hours. Data are shown as a percentage of the control values (no cytokine added). Error bars represent the means ± SEM of three preparations of CTB cells purified from three different placentas. Each assay was performed in triplicate. *, P < 0.05.

Figure 8

A schematic illustration summarizing regulation of LIGHT/IFN-γ-mediated apoptosis in human villous CTB cells.