Nuclear factor-kappa B localization and function within intrauterine tissues from term and preterm labor and cultured fetal membranes

Abstract Background The objective of this study was to quantify the nuclear localization and DNA binding activity of p65, the major transactivating nuclear factor-kappa B (NF-kappaB) subunit, in full-thickness fetal membranes (FM) and myometrium in the absence or presence of term or preterm labor. Methods Paired full-thickness FM and myometrial samples were collected from women in the following cohorts: preterm no labor (PNL, N = 22), spontaneous preterm labor (PTL, N = 21), term no labor (TNL, N = 23), and spontaneous term labor (STL, N = 21). NF-kappaB p65 localization was assessed by immunohistochemistry, and DNA binding activity was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based method. Results Nuclear p65 labeling was rare in amnion and chorion, irrespective of clinical context. In decidua, nuclear p65 labeling was greater in the STL group relative to the TNL cohort, but there were no differences among the TNL, PTL, and PNL cohorts. In myometrium, diffuse p65 nuclear labeling was significantly associated with both term and preterm labor. There were no significant differences in ELISA-based p65 binding activity in amnion, choriodecidual, and myometrial specimens in the absence or presence of term labor. However, parallel experiments using cultured term fetal membranes demonstrated high levels of p65-like binding even the absence of cytokine stimulation, suggesting that this assay may be of limited value when applied to tissue specimens. Conclusions These results suggest that the decidua is an important site of NF-kappaB regulation in fetal membranes, and that mechanisms other than cytoplasmic sequestration may limit NF-kappaB activation prior to term

Trophoblast Cell Fusion and Differentiation Are Mediated by Both the Protein Kinase C and A Pathways

<div><p>The syncytiotrophoblast of the human placenta is an epithelial barrier that interacts with maternal blood and is a key for the transfer of nutrients and other solutes to the developing fetus. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. BeWo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Dysferlin, a syncytiotrophoblast membrane repair protein, is up-regulated in BeWo cells induced to fuse by treatment with forskolin; this fusion is thought to occur through cAMP/protein kinase A-dependent mechanisms. We hypothesized that dysferlin may also be up-regulated in response to fusion through other pathways. Here, we show that BeWo cells can also be induced to fuse by treatment with an activator of protein kinase C, and that this fusion is accompanied by increased expression of dysferlin. Moreover, a dramatic synergistic increase in dysferlin expression is observed when both the protein kinase A and protein kinase C pathways are activated in BeWo cells. This synergy in fusion is also accompanied by dramatic increases in mRNA for the placental fusion proteins syncytin 1, syncytin 2, as well as dysferlin. Dysferlin, however, was shown to be dispensable for stimulus-induced BeWo cell syncytialization, since dysferlin knockdown lines fused to the same extent as control cells. The classical trophoblast differentiation marker human chorionic gonadotropin was also monitored and changes in the expression closely parallel that of dysferlin in all of the experimental conditions employed. Thus different biochemical markers of trophoblast fusion behave in concert supporting the hypothesis that activation of both protein kinase C and A pathways lead to trophoblastic differentiation.</p> </div

The time course for quantitative PCR analysis of mRNA expression for syncytin 1, syncytin 2, and DYSF in response to treatment of BeWo cells with 10 nM PMA, 20 µM FK, or a combination of FK+ PMA.

<p>All mRNA expression was normalized to RPLPO. Treatment of BeWo cells with PMA resulted in increased expression of mRNAs for syncytin 1, syncytin 2, and DYSF. However, these increases were not as high as those found with FK treatment. Simultaneous treatment of cells with PMA + FK led to dramatic increases in mRNAs for syncytin 2 and DYSF that peaked at 48 h of treatment. Increases in syncytin 1 mRNA was not as dramatic as for syncytin 2. Results are the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 (vs. control), ^## P < 0.01; ^### P < 0.001 (vs. 48 h FK+PMA) by one-way ANOVA/Bonferroni.</p

Bis I inhibited PMA-induced DYSF expression in a dose-dependent manner.

<p>Cells were treated with 0.25% DMSO (CTRL), 10 nM PMA, or 10 nM PMA plus 0.1 or 1.0 µM Bis I for 72 h. Cell lysates were generated and immunoblots were probed with anti-DYSF. Each lane received an equal concentration of protein and detection of GAPDH served as an additional loading control. Results are representative of three independent experiments.</p

Foskolin-induced differentiation was augmented by PMA.

<p>(A) An immunoblot showing the dose-response for FK in the presence or absence of 10 nM PMA. Note that PMA augments the expression of DYSF at each concentration of FK tested. Each lane received an equal concentration of proteins and detection of GAPDH served as an additional loading control. Results are representative of three independent experiments. (B) The time course for DYSF expression in the presence of 10 nM PMA, 20 µM FK, or the combination of FK and PMA. Note that there was increased DYSF expression with the combination of FK and PMA at each time point tested when compared to PMA or FK alone. Also with the combination of FK and PMA, DYSF expression was evident by 24 h while it lagged behind with PMA or FK alone under these conditions. Results are representative of three independent experiments. (C) Immunofluorescence localization of DYSF (red) and E-cadherin (green) following 72 h treatment with 10 nM PMA, 20 µM FK, or a combination of FK and PMA; the nuclei were stained with DAPI (blue). Note the enhanced fluorescence signal for DYSF in the FK + PMA sample when compared to FK or PMA alone. Bar = 50 µm. (D) The immunofluorescence assays were quantified and reported as percentage of nuclei in syncytia (% fusion). Results are the mean ± SD (n = 3). **P < 0.01; ***P < 0.001 (vs. control), ^### P < 0.001 (48 h PMA <i>vs</i>.48 h FK+PMA; 48 h FK vs. 48 h FK+PMA), ^†††P < 0.001 (72 h PMA <i>vs</i>. 72 h FK+PMA), ns: not significant (72 h FK <i>vs</i>.72 h FK + PMA) by one-way ANOVA/Bonferroni. (E) The time course for the expression of cell-associated βhCG protein in response to 10 nM PMA, 20 µM FK, or the combination of PMA and FK is shown. Control cells do not have detectable βhCG. While PMA does induce the expression of βhCG, it is at a modest level when compared to treatment with FK. The stimulation of BeWo cells with PMA and FK simultaneously induces higher levels of βhCG than FK alone; this is most evident at 24 h treatment. The immunoblot and immunofluorescence data in this figure are representative of at least three independent experiments. (F) The time course for βhCG secretion in response to 10 nM PMA, 20 µM FK, and a combination of PMA and FK is shown. Each treatment induced βhCG secretion with PMA + FK > FK > PMA. The results are the mean ± SD (n = 3). *P < 0.05; ***P < 0.001 (vs. control), ^#P < 0.05; ^## P < 0.01 (48 h FK <i>vs</i>.48 h FK+PMA; 48 h PMA vs. 48 h FK+PMA), ^††P < 0.01; ^†††P < 0.001 (72 h FK <i>vs</i>. 72 h FK+PMA; 72 h PMA <i>vs</i>. 72 h FK+PMA) by one-way ANOVA/Bonferroni.</p

Morphology change following myoferlin depletion in MDA-MB-231 cells.

<p>Atomic force and scanning electron microscopy images showing the spindle, elongated shape of lentiviral control (LTV-ctrl) cells and the more flat and circular morphology of myoferlin depleted (MYOF-KD) cells. AFM imaging shows pronounced actin stress fibers (black arrows) oriented along the long axis being evident in the control but not in the MYOF depleted cells. Cytoplasmic poles, lamellipodia and filopodia are observable in the SEM images. White arrowheads indicate the leading edge of cells.</p

Myoferlin depletion attenuates MMP1 expression and collagen I invasion capacity of MDA-MB-231 cells.

<p>(A) Representative (<i>n</i> = 3) immunoblotting results of secreted MMP1 in 24 h serum starved supernatant of MDA-MB-231 lentiviral control (LTV-ctrl) and myoferlin knockdown (MYOF-KD) cells. Recombinant human matrix metalloproteinase-1 was used as a standard. Verification of myoferlin knockdown was done in the corresponding cell lysates with GAPDH as a loading control. (B) Secreted pro-MMP1 was evaluated in 231^LTV-ctrl and 231^MYOF-KD cells (<i>n</i> = 2) using ELISA (mean ± s.d., Kruskal-Wallis test/Dunn's multiple comparison analysis). (C) Results from Boyden chamber invasion assays using a coating of 3 mg/ml of rat tail collagen I to evaluate the invasive capacity of 231^LTV-ctrl and 231^MYOF-KD cells (<i>n</i> = 3, mean ± s.d., Kruskal-Wallis test/Dunn's multiple comparison analysis).</p

Myoferlin depletion reduces invasive but not migratory capacity of MDA-MB-231s control (LTV-ctrl) and MYOF depleted cells (MYOF-KD).

<p>(A) Boyden chamber migration assay of MDA-MB-231 cells moving across 8 µm porous membranes towards a 10% serum gradient for 24 h (mean ± s.d., <i>n</i> = 3, unpaired 2-tailed t-test). (B) 24 h Boyden chamber invasion results of MDA-MB-231 cells across a 100% Matrigel coated 8 µm porous membrane towards a 10% serum chemoattractant (mean ± s.d., <i>n</i> = 3, unpaired 2-tailed t-test).</p

Morphology change following myoferlin depletion in MDA-MB-231 cells.

<p>Immunofluorescence micrographs showing the morphology of MDA-MB-231 wild type (WT), lentiviral transduction control (LTV-ctrl), and myoferlin knockdown (MYOF-KD) stable cell lines in culture. Note the more epithelial morphology of MYOF-KD cells compared to the more mesenchymal appearance of the WT and LTV-ctrl cells. Bar = 50 µm.</p