**3. Discussion**

The early stages of pregnancy comprise the attachment of embryo to the uterine epithelium, invasion of embryo into the uterine stroma and the decidualization of the stroma. Multiple molecules forming a complex network regulate implantation. The interplay between endometrial tissue and conceptus is critical to induce uterine receptivity to implantation [52]. The pre- and early implantation periods require alterations in the gene expression both in the epithelium and in the trophoblast cells. These changes lead to the activation of signalling pathways regulating proliferation, growth, migration and invasion [53].

FKN expression and release were detected in the HEC-1A endometrial cell line [54] while the trophoblast cell line JEG-3 expressed CX3CR1 [14]. Recent publications revealed that FKN might regulate adhesion and migration of trophoblast cells at different stages of pregnancy [14,55] and the FKN-CX3CR1 axis was suggested to be implicated in the maternal-fetal communication [13]. In our study, we established JEG-3 monocultures modelling the pre-implantation period, when the trophoblast cells of conceptus interact with soluble fractalkine secreted by endometrial cells. Bilaminar co-cultures, in which JEG-3 and HEC-1A cells can get in physical contact with each other [10,42,56,57], were used to model the attachment of conceptus with uterine epithelium and the early implantation period. The aim of our study was to unravel the role of fractalkine in the regulation of implantation by the examination of a set of implantation-related genes in trophoblast cells (Figure S1).

Progesterone receptor (PR) is a crucial nuclear receptor regulating implantation and decidualization by the activation of its target genes [58]. The mediators (kinases and coactivators) of PR signalling are also important for successful embryo implantation [53]. During the pre-implantation period, PR initiates a complex signalling network to prepare the endometrium for embryo attachment and implantation that is called the window of receptivity. PR expression in the uterus is dynamically regulated by many factors [58].

We hypothesized that soluble FKN may influence PR expression in trophoblast cells; therefore, the mRNA and protein levels of PR were examined both in mono- and co-cultures. The mRNA expression of PR increased both in the mono- and co-cultures after fractalkine treatments. At protein level, PR showed a significant increase only using the highest FKN concentration at each time point of the JEG-3 monoculture. In the co-culture, PR protein expression revealed fluctuation. These results suggest a time dependent negative feedback in PR expression, and it is supposed that FKN acted in a concentration-dependent manner on PR.

We also examined SRC-1, the member of p160/steroid receptor coactivator family, the coregulatory molecule of PR at FKN treatment [59]. SRC-1 binds to the activated PR in the nucleus and triggers transcriptional response of PR [59]. SRC-1 significantly increased only in monocultured JEG-3 cells at 24 h both at mRNA and protein levels. In case of co-cultured JEG-3 cells, the increment of SRC-1 protein level at 24 h and 48 h showed parallel changes with the PR protein level suggesting the SRC-1 contributed to the PR action.

Activin receptor 1B is expressed by JEG-3 cells showed different expression levels after FKN treatments. Activin receptors are co-localized with activins and follistatins that are the regulators of activin receptor's activity [60]. Activin receptor regulates SMAD2/3 transcription factors that can be modulated by MAPKs as well [61]. In JEG-3 cells, mRNA expression of activin receptor was significantly elevated after 6 h and 24 h. The protein level of activin receptor followed the changes of the mRNA levels showing that FKN increased the activin receptor expression on the plasma membrane of JEG-3 cells. In the co-culture, the activin receptor B1 protein level showed fluctuation that may be caused by the paracrine effect of activin and follistatin produced by the endometrial cells [60].

MMPs play a critical role in the invasion of trophoblast cells. Among MMPs, MMP2 and 9 seem to be essential in the regulation of invasion and the behaviours of trophoblasts [62–64]. During the implantation, trophoblast cells release large amounts of MMP2 and 9 [65]. MMP expression is regulated by many factors e.g., activin signalling, BMP2 signalling and MAPK pathways [33–36]. FKN treatment increased MMP2 mRNA level at 6 h, MMP2 and MMP9 levels at 24 h only in the monocultured JEG-3 cells. MMP9 was elevated in the co-culture, proposing that MMP9 is more affective in the regulation of invasion than MMP2. At protein level, both MMP2 and MMP9 followed the alterations of mRNA expression in the JEG-3 cells. In the case of the co-cultured JEG-3 cells, we revealed that MMP2 protein level significantly elevated only at 48 h. The increased level of MMP9 protein supports the hypothesis that FKN-CX3CR1 axis acted as a regulator of MMP2 and MMP9 expressions.

These results raise the possibility that during the pre-implantation period (JEG-3 monoculture, at 24 h), fractalkine increases both MMP2 and MMP9 levels to preparing the embryo for implantation. Then at attachment and at early implantation period (co-culture model), MMP9 level increases continuously as the dominant enzyme regulating invasion, while MMP2 participates in the later steps of invasion.

CX3CR1 can be activated both by soluble and membrane-bound FKN. It seems that soluble FKN is important in JEG-3 monocultures acting through CX3CR1 regulating signalling pathways, PLC-PKC, PI3K/AKT/NFκB and MAPK pathways (p38, ERK1/2 and JNK) [15]. These pathways control the proliferation, growth, differentiation and apoptosis [18]. In the case of the co-cultures, both soluble and membrane bound FKN are present, which may contribute to the alterations of fractalkine receptor expression between mono- and co-cultured JEG-3 cells: elevated level in monocultured cells using F10 and 20 at 24 h, but reduced level using the same FKN concentrations at the same time point in co-cultured cells. Fluctuation of the CX3CR1 level in JEG-3 cells may be caused by MAPKs activated by the receptor. MAPKs may activate transcription factors (e.g., SP-1, AP-1, STATs) altering the transcriptional rate of CX3CR1 [66].

To determine the activity of downstream signalling pathways mediated by FKN-CX3CR1, we examined the phosphorylated forms of MAPKs (p38, ERK1/2, JNK) and AKT. The phosphorylation pattern was different in the two examined cell types. In monocultured JEG-3 cells p-ERK1/2 and p-p38 protein levels significantly increased at each time point using F20 although the rate of elevation showed decreasing phenomenon with time. On the contrary, p-JNK showed an increasing level with time, suggesting that p-JNK may replace the aforementioned enzymes when their amounts decrease. F10 exerted lower effect on the JEG-3 cells proposing a concentration dependence. In case of p-AKT, only F20 was able to increase its level. Co-cultured JEG-3 showed increased p-ERK1/2 levels at 6 h and 48 h while p-p38 and p-JNK elevated at 24 h proposing a cooperation between the kinases. In the case of p-AKT, we found similar changes to the p-p38 and p-JNK. The co-work of these three kinases may be responsible for the increased viability of co-cultured JEG-3 cells. Our results prove that FKN provides diverse effects on mono- and co-cultured trophoblasts and these alterations may be caused by the distinct impressions of soluble and membrane-bound FKN and may be influenced by other interactions between trophoblasts and endometrial cells.

It is proven that the presence of HEC-1A in the co-cultures contributes to the action of FKN on JEG-3 cells. This effect is probably mediated by membrane-bound fractalkine expressed by endometrial cells. We examined the expression of those genes and proteins that have been describe as the participants of implantation. We examined activin, follistatin and BMP2 expressions, which are the regulators of activin receptor and BMPR signalling. The activin and follistatin expression changed inversely in HEC-1A cells. The activin mRNA expression increased when the follistatin expression decreased and activin expression decreased when the follistatin mRNA levels were elevated. These results suggest that the interactions between the two cell types may influence the expressions of the activin receptor ligand activin and the binding protein of the ligand follistatin affecting receptor activity. The increasing activin level may contribute to the invasion capacity of trophoblast by acting on matrix metalloproteinase (MMP) expression [34].

Follistatins act as binding proteins for bone morphogenetic protein 2 (BMP2) as well, regulating BMP2 signalling through BMP receptors (BMPR) [67]. The BMP2 signalling inhibits the proliferation of trophoblasts but promotes trophoblast invasion by increasing metalloproteinase secretion [68]. BMP2 is

(Figure 6).

a regulator of SMAD2/3 signalling, influencing the effect of activin receptors, and contributing to increased invasiveness [35,62]. BMP2 induces activin production as well, thus increases the activity of activin receptors [69]. In our experiments, BMP2 mRNA expression was significantly higher during the whole experiment compared to the control. At protein level, BMP2 showed fluctuation suggesting that the changing level of BMP2 in HEC-1A cells contribute to the signalling processes occurring in JEG-3 cells and may increase the action of fractalkine on trophoblast cells. Maybe the increased secretion of BMP2 affects the activity of BMPR elevating MMP expression and invasiveness of trophoblast cells.

Our experiments reveal that increasing fractalkine concentrations act differently on mono- and co-cultured JEG-3 cells. In the case of the monoculture the highest FKN concentration (20 ng/mL) was the most effective in increasing the expression of implantation-related genes. In the case of the co-culture, the intercellular interactions between HEC-1A and JEG-3 cells influenced the action of fractalkine treatment and it seems that both F10 and F20 affected the expression of the examined genes. The activated signalling pathways contributed to cell proliferation and then to the increased invasiveness. The results obtained from our in vitro co-culture experiments should be further investigated in vivo. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 12 of 19 the most effective in increasing the expression of implantation-related genes. In the case of the coculture, the intercellular interactions between HEC-1A and JEG-3 cells influenced the action of fractalkine treatment and it seems that both F10 and F20 affected the expression of the examined

In this study, we examined the effect of FKN on the implantation-related genes expressed by trophoblast cells. FKN and CX3CR1 interaction is proven to be involved in the regulation of implantation influencing the expression of several genes (Figure 6). It is revealed that membrane-bound FKN and CX3CR1 receptor binding changes the expression and/or activity of PR by the activation of MAPK pathways [70]. In the co-cultures, it is revealed that HEC-1A cells alter the expressions of activin, follistatin and BMP2 regulators of activin receptor and BMP receptors. Secreted activin may bind to its receptor by paracrine way on trophoblast cells activating SMAD2/3 transcription factors that are also controlled by the BMP2/BMPR signalling pathway. SMAD2/3 pathway can upregulate MMP2 and MMP9 regulating invasion process. MAPKs activated by the FKN signal transduction pathway affect positively MMP2 and MMP9 expression, increasing the invasiveness of trophoblast cells. PR influenced by fractalkine can also modify MMP2 transcription. (Figure 6). genes. The activated signalling pathways contributed to cell proliferation and then to the increased invasiveness. The results obtained from our in vitro co-culture experiments should be further investigated in vivo. In this study, we examined the effect of FKN on the implantation-related genes expressed by trophoblast cells. FKN and CX3CR1 interaction is proven to be involved in the regulation of implantation influencing the expression of several genes (Figure 6). It is revealed that membranebound FKN and CX3CR1 receptor binding changes the expression and/or activity of PR by the activation of MAPK pathways [70]. In the co-cultures, it is revealed that HEC-1A cells alter the expressions of activin, follistatin and BMP2 regulators of activin receptor and BMP receptors. Secreted activin may bind to its receptor by paracrine way on trophoblast cells activating SMAD2/3 transcription factors that are also controlled by the BMP2/BMPR signalling pathway. SMAD2/3 pathway can upregulate MMP2 and MMP9 regulating invasion process. MAPKs activated by the FKN signal transduction pathway affect positively MMP2 and MMP9 expression, increasing the invasiveness of trophoblast cells. PR influenced by fractalkine can also modify MMP2 transcription.

**Figure 6.** Mechanism of action of fractalkine on JEG-3 trophoblast cells Fractalkine binds to its cognate receptor on the surface of trophoblast cells activating MAPK and AKT pathways. MAPKs can phosphorylate PR on serine amino acid residue increasing its activity. Secreted activin may bind to activin receptor on the surface of trophoblast cells activating SMAD2/3 transcription factors that are also the targets of the BMP2/BMPR signalling pathway. SMAD2/3 transcription factors can upregulate MMP2 and MMP9 expressions. MAPKs are activated by fractalkine signal transduction pathway, act positively on MMP2 and MMP9 transcription. PR influenced by fractalkine can also affect MMP2 synthesis. **Figure 6.** Mechanism of action of fractalkine on JEG-3 trophoblast cells Fractalkine binds to its cognate receptor on the surface of trophoblast cells activating MAPK and AKT pathways. MAPKs can phosphorylate PR on serine amino acid residue increasing its activity. Secreted activin may bind to activin receptor on the surface of trophoblast cells activating SMAD2/3 transcription factors that are also the targets of the BMP2/BMPR signalling pathway. SMAD2/3 transcription factors can upregulate MMP2 and MMP9 expressions. MAPKs are activated by fractalkine signal transduction pathway, act positively on MMP2 and MMP9 transcription. PR influenced by fractalkine can also affect MMP2 synthesis.

In summary, FKN acts in a concentration and time dependent manner on trophoblast cells. FKN

In summary, FKN acts in a concentration and time dependent manner on trophoblast cells. FKN is a positive regulator of implantation, via changing the activity of PR, activin receptor and BMPR and the expression of MMPs. The presence of endometrial cells contributes to the proper implantation as the interactions between trophoblast and endometrial cells in the co-culture changes the gene expression pattern of JEG-3 cells at fractalkine treatment. Based on our results, FKN treatment may contribute to successful attachment and implantation of the embryo.
