Progesterone-induced blocking factor differentially regulates trophoblast and tumor invasion by altering matrix metalloproteinase activity

Abstract

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while-based on our previous data-PIBF might control trophoblast invasion by suppressing proinvasive genes.

Fig. 1

The effect of PIBF knock-down on invasiveness of trophoblast and tumor cells. The HTR-8/SVneo trophoblast cell line and primary trophoblast cells isolated from first-trimester abortion material (a) as well as HT-1080 (fibrosarcoma), A549 (lung carcinoma), HCT-116 (colorectal cancer), PC-3 (prostate cancer) tumor cell lines (b) were transfected with PIBF siRNA, or scrambled oligonucleotides (scr). Wells were seeded with control (scr) or PIBF siRNA-treated cells. Invasion of cells into the detection zones after 72 h is shown. Cells were stained with calcein AM. Images were captured using multiarea scan by a confocal microscope (bar 2 mm). c Quantification of area closure (%) calculated from measured areas of cell invasion at 72 h. Data are presented as mean ± SEM from 12 wells (HTR-8/SVneo, HT-1080) or six wells (other cell lines and primary trophoblast cells) per condition (asterisk indicates significant difference from scrambled at p < 0.05). d The efficiency of PIBF knock down (KD) was controlled by measuring PIBF levels (90-, 67-, 50-, and 34-kDa isoforms) using Western blotting at each individual experiment. Statistical analysis of Western blots are shown as mean ± SEM; asterisk indicates significant difference from scrambled at p < 0.05

Fig. 2

Xenotransplantation of PIBF-silenced trophoblast (a) and tumor (b) cells into the yolk sac of Tg(fli1:EGFP) zebrafish embryos. HTR-8/SVneo and HT-1080 cells were transfected with PIBF siRNA (PIBF KD) or scrambled (scr) oligonucleotides (control). The cells were then labeled with DiI (shown as red) and microinjected into the yolk sac of 48-h post-fertilization zebrafish embryos expressing EGFP under an endothelial promoter (green vasculature). Pictures were taken 5 days post-injection. Cells invaded the caudal part of zebrafish embryos. Arrows indicate distant micrometastases

Fig. 3

PIBF modulates MMP-2 and MMP-9 activity. a Supernatants from the invasion assay were subjected to gelatin zymography to detect MMP-2 and MMP-9 activity. b Densitometric evaluation of three separate zymograms. Data are represented as mean ± SEM (asterisk indicates significant difference from the control at p < 0.05). c Changes in relative expression of MMP-9 and TIMP-1 in PIBF-treated (24 h) HTR-8/SVneo trophoblast and HT-1080 fibrosarcoma cells (protein array, n = 2). d The expression of MMP-9 and TIMP-1 was also analyzed at mRNA level in PIBF knock down (KD) cells by real-time PCR. Cells were analyzed at 48 h post-transfection with siRNA. Changes in relative mRNA levels are shown. Data are represented as mean ± SEM

Fig. 4

PIBF signals via the IL-4Rα/PIBFR complex in trophoblast and tumor cells. a Serum-starved HTR-8/SVneo and HT-1080 cells were treated with PIBF (100 ng/ml) for the indicated time points. Total STAT6 and phosphorylated STAT6 levels were detected by Western blotting. b Invasion of control (scrambled, scr), IL-4Rα, or PIBF siRNA (KD)-treated HTR8/SVneo and HT1080 cells. Invasion of cells into the detection zones (2 mm) after 72 h is shown. Cells were stained with calcein AM. Images were captured using multiarea scan by a confocal microscope. c Quantification of area closure (%) calculated from measured areas of cell invasion at 72 h. Data are presented as mean ± SEM from 12 wells per condition (asterisk indicates significant difference from scrambled at p < 0.05). d Efficiency of PIBF or IL-4Rα knock down (KD) was determined by Western blotting. Statistical analysis of Western blots are shown as mean ± SEM; asterisk indicates significant difference from scrambled at p < 0.05

Fig. 5

PIBF-induced signaling in trophoblast and tumor cells. a Serum-starved HTR-8/SVneo and HT-1080 cells were treated with PIBF (100 ng/ml) for the indicated time points. Phospho(Ser473)-Akt, total Akt, phospho(Thr202/Tyr204)-ERK, total ERK, phospho(Ser727)-STAT3, phospho(Tyr705)-STAT3, and total STAT3 levels were detected by Western blotting. b Densitometric evaluation of the Western blots. The bars indicate mean ± SEM of three separate experiments (*p < 0.05)

Fig. 6

PIBF-regulated proteins. a IL-6 secretion of PIBF knock-down HTR-8/SVneo and HT-1080 cells (cytometric bead array). Supernatants were analyzed at 48 h post-transfection with siRNA. Data are represented as mean ± SEM of three separate experiments (*p < 0.05). b EGF and HB-EGF expression in control and 24-h PIBF-treated HTR-8/SVneo and HT-1080 cells in a protein array (n = 2). c EGF and HB-EGF expression in control (scrambled) and PIBF knock-down HT-1080 cells in a protein array (n = 2). d The effect of 24 h PIBF treatment on STAT3 activation in control (Scr) and HB-EGF knock down HT-1080 cells. Upper panel Western blot for phospho-STAT3 (Tyr). Lower panel mean ± SEM of densitograms from three separate experiments (asterisk p < 0.05). e Efficiency of HB-EGF knock down was analyzed by Western blotting. Statistical analysis of Western blots is shown as mean ± SEM; asterisk indicates significant difference from scrambled at p < 0.05

Fig. 7

PIBF binds to the promoter of IL-6, HB-EGF, and EGF. a Subcellular localization of PIBF in HTR-8/SVneo and HT-1080 cells. (1) Cells were labeled with anti-PIBF antibody, and reacted with anti-rabbit Ig-Cy3 (shown as red). Nuclei were counterstained with Hoechst (shown as blue). Merged images taken by confocal microscope. (2) Control, without anti-PIBF antibody. b PCR of non-immunoprecipitated (input) and PIBF or control IgG immunoprecipitated DNA from ChIP assay of HTR-8/SVneo and HT-1080 cells, using primers to the promoter of IL-6, HB-EGF, EGF, and PlGF. PlGF ChIP was used as a negative control. c Detection of PIBF isoforms by Western blotting on the eluted protein/DNA complex immunoprecipitated by anti-PIBF

Fig. 8

Transcriptional regulation of tumor invasion by PIBF. PIBF activates not only the Jak1/STAT6 pathway but also the PI-3 K/Akt and ERK cascades through the IL-4Rα/PIBFR complex. Moreover, PIBF is able to enter the nucleus where it binds the promoter regions of EGF, IL-6, and HB-EGF and induces their transcription. The expressed and secreted EGF then binds its own receptor and further triggers the PI-3 K/Akt, MAPK, and Jak/STAT3 pathways, resulting in proliferation, survival, and increased MMP-9 expression, thus increased invasive behavior. (ECM extracellular matrix)