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

Electrostatic surface analysis of the N-domain of cTnC.

<p>Depicts the N-domain of cTnC in the Ca²⁺-free state (figures a through c) and Ca²⁺-saturated states (figures d through f). The cTnC N-domain helices A through D are colored as red, green, cyan and blue respectively. (a) Transparent rendering of the electrostatic surface is shown with the protein in cartoon. (b) In the Ca²⁺-free state the loss of regulatory Ca²⁺ caused the rearrangement of helices B and C. These helices are no longer orthogonal to each other but nearly parallel. This resulted in a breach in the hydrophobic pocket that surrounded the cTnI-Rr (pointed out by the arrow). (c) The cTnC N-domain hydrophobic pocket when viewed from below the cTnC N-domain. This view shows the hydrophobic environment in which the cTnI-Rr is located. The arrows points to the gap in the hydrophobic pocket. (d) Transparent rendering of the electrostatic surface with the protein rendered as cartoon. (e) In the Ca²⁺-saturated state there is no breach in the hydrophobic pocket within which the cTnI-Rr is held. (f) The cTnC N-domain hydrophobic pocket in the Ca²⁺-saturated state when view from below.</p

FRET distance distribution between cTnT and cTnI.

<p>Distribution of the distances P(r) between cTnT residue 276 and cTnI residues 151, 160, and 167, determined in the reconstituted cTn complex at low Ca²⁺ (broken curve) and saturating Ca²⁺ (solid curve).</p

FRET distance measurements within the cardiac troponin complex.

<p>The FRET distances measured between donor and acceptor probes in the Ca²⁺-saturated and the Ca²⁺-free states are tabulated. The half-widths are parenthesized. The FRET distances and restraints were applied as NOE restraints <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087135#pone.0087135-Sheldahl1" target="_blank">[49]</a> between the Cα's of the participating amino acids. In columns 3,4 and 5,6 the experimentally measured FRET distance is tabulated along with the distance between the Cα's of the participating amino acids in the modeled structures, in the Ca²⁺-saturated states and Ca²⁺-free states respectively. In the FRET analysis, due to the ambiguity in the value of the dipole–dipole orientation factor between energy donor molecules and energy acceptor molecules and due to the dimensions of the probes attached by linkers to the side chains of the amino acid residues, the measured distance will have an uncertainty of ±10% <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087135#pone.0087135-dosRemedios1" target="_blank">[53]</a>. Although the length of probe linkers is ∼10 Å, the linker is not unidirectional but folds randomly (during folding and rotation the probe and can acquire length of ∼7 Å). Based on these above factors there is good correlation between the measured FRET distance and the model. The italicized numbers in the third column pertain to the distance between the C-alphas in the crystal structure (1J1E). Compared to X-ray crystallography technique, FRET is a low resolution structural tool, and it does not have the lattice constraints that would be present in X-ray determined structure. However, FRET can acquire structural information in a more physiological environment, particularly with time-resolved approach (as we used here) it can provide dynamic information (represented by HW of the distance distribution) associated with each measured distance. Broad distributions of our FRET distances listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087135#pone-0087135-t001" target="_blank">Table 1</a> suggest that troponin exhibits a much dynamic structure in solution than in crystal. Therefore some discrepancies in the mean FRET distances with respect to the distances measured in X-ray structure would be expected. If we consider the structural dynamics (large HWs) observed in solution samples, these differences are in reasonable range.</p

Ca²⁺-free state structure of the cardiac troponin complex.

<p>The cTn structure after 9.5²⁺-free state is depicted using CCP4MG version 2.7.3. (a) The cTn complex is positioned such as to see the dynamics of the cTnI-Md. The cTnI-Md is seen to have positioned itself close to the cTnC helix A. The cTnI-Rr held within the cTnC N-domain hydrophobic pocket has its secondary structure perturbed due to the closing of the hydrophobic pocket. (b) Depicts the cTnC N-domain wherein the loss of regulatory Ca²⁺ led to structural rearrangement of the helices B, C and D. The helices B and C are almost parallel to each other. (c) View of the N-terminal extension of cTnI above the cTn core domain complex. In this view the collapsed conformation of the cTnC N-domain hydrophobic pocket is well seen. It may be compared against the conformation of the open cTnC N-domain hydrophobic pocket in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087135#pone-0087135-g005" target="_blank">Fig. 5c</a>. (d) The averaged structure of the cardiac troponin complex in the Ca²⁺ free state from 2 ns to 9.5 ns. The first two nanoseconds were given for the system to equilibrate. (e) The secondary structure of the troponin C in the presence of FRET distance restraints are calculated from 2 ns till 11.1 ns. The cTnC N-domain helices N (residues 4–11), A (residues 14–28), B (residues 38–47), C (residues 54–61) and D (residues 74–85) experienced considerable secondary structure evolution, but in contrast, the cTnC C-domain helices E (residues 94–104), F (residues 117–123), G (residues 130–140), and H (residues 150–157) are comparatively stable. Perturbation in the structure of helix D pulls and releases the D/E linker which in turn unfolds and refolds helix E. This fluctuations cause the D/E linker to alternate between flexible and rigid conformations that effectively helps release and retract the cTnI-Ir towards and away from actin in the absence and presence of Ca²⁺. The residue numbers associated with the helices of cTnC which are given within brackets were derived from the crystal structure 1J1E.pdb.</p

Electrostatic surface analysis of the cTnC and cTnI.

<p>Depicts the electrostatics in the vicinity of cTnC helix E in the Ca²⁺-free state after 9.5 ns of simulation. The hydrophobic, negative and positive surfaces are colored white, red and blue respectively. (a) The thick arrow points to the unfolded segment in helix E. The positively charged blue residues of cTnI-Ir are seen arching (pointed to by the dotted line with arrow head) towards the unfolded cTnC helix E (pointed to by thick black arrow). The amino acids sequence of the cTnI-Ir residues is 138-KFKRLPT and the sequence of the opposing cTnC residues are 92-KSEEEL. The predominantly negative (red) cTnC helix E is attracted to the positive region of cTnI-Ir. The unfolded helix E has adopted a “U” shape (pointed to by the thick black arrow). (b) The unfolded helix E is seen in concert with cTnI and cTnT. The cTnC Glu94 is attracted to cTnI Lys141 (not seen in picture), cTnC Glu95 is attracted to the nitrogen on Leu129 of cTnI and Arg142 of cTnI, and cTnC Glu96 is attracted to Arg 267 of cTnT.</p

Secondary structure timeline of the cTnI-Rr/switch.

<p>The cTnI-Rr is held within the cTnC N-domain hydrophobic pocket. In the presence of regulatory Ca²⁺ the cTnI-Rr maintains a helical conformation. In the absence of regulatory Ca²⁺ the secondary structure is perturbed. The absence of the helical conformation would release the cTnI-Md to interact with actin in the Ca²⁺-free state whereas, the presence of the regulatory Ca²⁺ would refold the cTnI-Rr into a helix effectively retracting the cTnI-Md from actin.</p