Insights into cAMP‐dependent molecular mechanisms regulating expression and function of LGALS16 gene in choriocarcinoma JEG‐3 cells

The human choriocarcinoma cell line JEG‐3 offers a valuable model to study galectin‐16 gene (LGALS16) expression and functions in the context of placental cell differentiation and cancer cell biology. Recent evidence indicates that cAMP‐mediated signaling pathways might be responsible for the upregulation of LGALS16; however, the underlying mechanisms are unknown. Here, we employed biochemical inhibitors of the cAMP cascade and CRISPR/Cas9 engineered cells to assess regulatory patterns and associations between cAMP‐induced trophoblast differentiation and LGALS16 expression in JEG‐3 cells. The expression of LGALS16 was significantly upregulated in parallel with human chorionic gonadotropin beta (CGB), a biomarker of syncytiotrophoblast differentiation, in response to 8‐Br‐cAMP. Inhibition of p38 MAPK and EPAC significantly altered LGALS16 expression during differentiation, while PKA inhibition failed to change LGALS16 and CGB3/5 expression in our cell model. The CRISPR/Cas9 LGALS16 knockout cell pool expressed a significantly lower amount of CGB3/5, a reduced level of CGB protein, and an unaltered cell growth rate in response to 8‐Br‐cAMP in comparison with wild‐type JEG‐3 cells. Collectively, these findings suggest that LGALS16 is required for the trophoblast‐like differentiation of JEG‐3 cells, and its expression is mediated through p38 MAPK and EPAC signaling pathway branches.

are deemed to be critical for regulating cAMP-dependent upregulation of chorionic gonadotropin beta (CGB), a biomarker of placental trophoblast cell differentiation (Burnside et al., 1985;Chen et al., 2013;Kaminker & Timoshenko, 2021), in association with at least LGALS13 (Orendi et al., 2010).Considering that all placenta-specific genes are localized within the same gene cluster on the chromosome band 19q13.2,which is proposed to be a result of ancestor gene duplication and the insertion of transposable elements (Than, Romero, et al., 2014), we sought to explore further the cAMP-dependent mechanisms of LGALS16 regulation in a model of trophoblast differentiation of choriocarcinoma JEG-3 cells which was introduced in our previous work (Kaminker & Timoshenko, 2021).
In this study, we used pharmacological inhibitors of cAMPmediated signaling pathways and CRISPR/Cas9 LGALS16 knockout (KO) JEG-3 cells to examine differences in the cellular responses (cell differentiation, proliferation, and gene expression) to 8-Br-cAMP, an inducer of trophoblastic cell differentiation.Our findings demonstrate that p38 MAPK and EPAC branches of cAMP signaling cascade may drive the expression of LGALS16, which is required for trophoblastic differentiation of JEG-3 cells.
The CRISPR/Cas9 KO cell pool was generated using the single guide RNA sequence, UUUCUACACUGAGAUGAAUG, targeting exon 3 of the LGALS16 gene near an AGG protospacer adjacent motif sequence.To isolate knockout clones, limiting dilutions of 0.5 cells/well and 1 cell/well were performed in 96-well plates (#83.3924;Sarstedt) with 100 µL cell medium per well.Cell growth was monitored over 8 weeks.At 80% confluency, cells were subcultured in 96-well Sarstedt plates to grow cells for sequencing and in 24-well Falcon plates (#3047; Becton Dickinson) to maintain the clone culture (Kaminker, 2022).

| Cell growth assay
Cells were seeded at 1 × 10 4 cells/mL (3 mL) in 35 mm dishes from Sarstedt (#83.3900) and were grown for 7 days.To prepare cells for counting, the cell culture medium was aspirated, the cell monolayer was rinsed with DPBS and treated with 1 mL of warm TrypLE for 5-6 min, and 2 mL of complete medium was added after confirming cell dissociation.Trypan blue test was used to count the number of live cells using a hemocytometer.

cell viability assay
Cells were seeded at 2 × 10 5 cells/mL in 96-well plates (100 µL per well) and given 24 h to adhere.The cell culture medium was replaced, and cells were treated with inhibitors for 36 h with the medium and inhibitor replaced every 12 h.To run the MTT assay, 20 µL of MTT solution (2.5 mg/mL) was added to each well, and cells were incubated at 37°C for 4 h.Next, the medium was aspirated, 150 µL of DMSO was added to each well to dissolve formazan crystals, and the plate was placed on a shaker for 30 min.A BioTek 800 TS Absorbance Reader from Agilent Technologies was used to measure absorbance at 570 nm.The readings were corrected considering blanks and normalized to the control untreated cells, which were set up to 100% cell viability.

| Statistical analysis
A minimum of three biological replicates were tested for each treatment.Statistical analysis was performed using GraphPad Prism 10 for Windows, version 10.0.1 (218) (GraphPad Software).One-way analysis of variance (ANOVA) and Tukey's honestly significant difference test were used to determine significant differences between means, considering p < .05 as a statistical threshold.
Pearson's correlation test was used to assess the correlation between the expression of different genes.

| Effects of biochemical modulators of cAMPmediated signaling on trophoblastic differentiation and placental galectin gene expression in JEG-3 cells
We previously demonstrated that 8-Br-cAMP-induced trophoblastic differentiation of JEG-3 cells was associated with upregulation of the LGALS16 gene (Kaminker & Timoshenko, 2021), however, molecular mechanisms behind this regulation were not explored.To start analyzing the involvement of different branches of the cAMP signaling cascade in placental cells (Kusama et al., 2018;Orendi et al., 2010), we selected 3 biochemical inhibitors against PKA (H-89), p38 MAPK (SB203580), and EPAC (ESI-09) as well as a stimulator of EPAC (8-CPT-2-Me cAMP) for our study.First, the cytotoxicity of these drugs was assessed using an MTT test for cell viability.Within the 0.6-100 µM concentration range, the cell viability did not change in the presence of SB203580 and 8-CPT-2-Me cAMP, whereas H-89 and ESI-09 were cytotoxic with IC50 values of 6.6 µM and 28.4 µM, respectively (Figure 1a).Based on these results, the following nontoxic concentrations of drugs were used in the subsequent experiments to treat JEG-3 cells: 5 µM H-89, 5 µM SB203580, 10 µM ESI-09, and 10 µM 8-CPT-2-Me cAMP.
Co-treatment of JEG-3 cells with 8-Br-cAMP (250 µM, 36 h) and any of the four tested biochemical drugs led to an upregulation of CGB3/5 expression by 15-60 fold, as an indicator of trophoblastic differentiation, with no differences from control cells except for treatment with SB203580 (Figure 1b,c).This suggests that p38 MAPK is most likely the primary signaling branch essential for the regulation of JEG-3 cell differentiation in our model.Given the 8-Br-cAMP-induced differentiation of JEG-3 cells, next, we compared the expression profiles of LGALS16 with a better characterized placental gene, LGALS13.These are closely related genes localize within the chromosome 19 placental galectin gene cluster together with The list and properties of oligonucleotide primers for quantitative polymerase chain reaction.LGALS14 (Than, Romero, et al., 2014), although the latter is not expressed in trophoblast cell lines, including JEG-3 cells (Wang et al., 2021).A significant upregulation of both LGALS13 and LGALS16 by up to sixfold was induced by 8-Br-cAMP similar to CGB3/5 (Figure 1b,c).However, the effects of biochemical drugs on these genes demonstrated some variations.To gain more insights into a possible association between these genes (CGB3/5, LGALS13, and LGALS16), we performed correlation analysis between all individual measurements using a Pearson's correlation test.This analysis revealed significant positive correlations between all these gene pairs (Table 2), which suggests common mechanisms in their regulation and a possible need of galectin members for trophoblastic differentiation and healthy placental development as known for LGALS13 (Blois et al., 2019;Sammar et al., 2019;Than, Balogh, et al., 2014).Therefore, the next step of our study was to investigate more specifically, the role of LGALS16 in regulating trophoblastic differentiation of JEG-3 cells using a genetic engineering approach.LGALS16 in the presence of 8-Br-cAMP (Kaminker, 2022).Indeed, while the upregulation of LGALS16 and CGB3/5 gene expression was significant and readily evident for WT JEG-3 cells, which served as a positive control, the LGALS16 KO clonal pool showed no responses to treatment with 8-Br-cAMP (250 µM, 36 h) for both genes (Figure 2a).
Moreover, we were not able to detect CGB at a protein level in KO cells treated with 8-Br-cAMP, whereas WT cells demonstrated an obvious accumulation of this essential marker of trophoblastic differentiation as per western blot analysis (Figure 2b).We also tested the only antibody available for galectin-16 protein from Abcam but were not able to detect galectin-16 at a protein level in our cell models (data not shown).We next assessed the growth patterns of WT and LGALS16 KO JEG-3 cells in the absence and presence of 8-Br-cAMP as a functional assessment for cell differentiation, which is usually associated with a decrease in cell growth rate.A growth curve analysis indeed confirmed a slower growth rate during 8-Br-cAMP-induced differentiation of WT JEG-3 cells in comparison with untreated cells, whereas no differences in growth rates were noticed for LGALS16 KO JEG-3 cells either untreated or treated with 8-Br-cAMP (Figure 2c).Overall, these results suggest that LGALS16 might be required at least at a gene level for maintaining trophoblastic differentiation of JEG-3 cells.

| DISCUSSION
The current knowledge about cellular functions and molecular mechanisms regulating the expression of LGALS16 is less studied than other placental galectins (Si et al., 2021;Than et al., 2009Than et al., , 2014)), and our results provide inspiring novel insights into the cell biology of this gene.First, our findings with biochemical drugs suggest that 8-Br-cAMP-induced upregulation of LGALS16 in JEG-3 cells undergoing trophoblastic differentiation is driven through cAMP signaling branches associated with p38 MAPK and EPAC.Second, the LGALS16 gene is required for trophoblastic differentiation of JEG-3 cells as CRISPR/Cas9 LGALS16 KO cells failed to produce CGB and did not change growth rate in response to 8-Br-cAMP.
Trophoblast differentiation is associated with multiple signaling pathways, including the cAMP cascade, which is highly coordinated and includes PKA-dependent and PKA-independent branches that lead to cell migration, fusion, and syncytialization (Gupta et al., 2016).
Ultimately, syncytiotrophoblasts serve as the primary producers of CGB, a biomarker of trophoblast differentiation, to maintain the corpus luteum and promote progesterone secretion for embryo implantation and growth as well as proper capillary development (Cole, 2010;Fournier et al., 2015).Many galectins are recognized as important players of these processes for placenta development and, accordingly, as potential biomarkers for gestational disorders (Blois et al., 2019).Seminal work by Than, Romero, et al. (2014) demonstrated that the mRNA expression of placental galectins (−13, −14, and −16) was upregulated an a time-dependent and cellspecific manner during differentiation of primary trophoblasts and BeWo cells, which was detected along with a delayed CGB3 upregulation.Our results extend these findings in a model of JEG-3 choriocarcinoma cells by showing that LGALS16 is not only a cAMPinduced gene in the context of trophoblast differentiation, but it might be required for CGB production.This was consistent with positive significant correlations between the expression of LGALS16 and CGB3/5 as well as between these genes and LGALS13 suggesting some common mechanisms in their regulation.This regulation may include cell signaling crosstalk between different branches of the cAMP cascade (PKA, EPAC, and p38 MAPK) because of the variations in the effects of pharmacological inhibitors.Indeed, in comparison with p38 MAPK and EPAC modulators, we did not detect an inhibitory effect of H-89 (PKA inhibitor) at a nontoxic dose of 5 µM on 8-Br-cAMP-induced upregulation of LGALS13, LGALS16, and CGB3/5 expression in JEG-3 cells.However, H-89 at a higher concentration of 10 µM was reported to inhibit both CGB3/5 and LGALS13 expression in another choriocarcinoma cell line BeWo undergoing trophoblast differentiation in response to forskolin, an activator of adenylyl cyclase (Orendi et al., 2010).Further studies are required to elaborate on these discrepancies considering the influence of different variables (cell type, H-89 concentration, type of agonists) in these systems.Remarkably, an inhibitory effect of SB203580 (p38 MAPK inhibitor) on the expression of LGALS13/ LGALS16 was consistent with an inhibition of LGALS13 in primary cultures of human cytotrophoblasts (Costa et al., 2016).Similarly, p38 MAPK was shown to regulate the expression of other galectins, for example, galectin-1 in different cell lines (Fuertes et al., 2004;Jin Lim et al., 2014).Considering that in addition to PKA (Gupta et al., 2016), other kinases can phosphorylate p38 MAPK (Cuadrado & Nebreda, 2010) (Than, Romero, et al., 2014).
However, the details of this regulation remain to be explored.
The upregulation of LGALS16 in the context of choriocarcinoma cell responses may suggest its role as a type of tumor suppressor gene regulating cell differentiation (Gutmann, 1995).In fact, there are other galectins (−4, −7, −8, −9, and −12), which are claimed as tumor suppressors (Kim et al., 2013;Nagy, 2002;Satelli et al., 2011;Wiersma et al., 2013;Yang et al., 2001).This conceptual point is important because different alterations in galectin expression, including upregulation of common, low tissue-specific galectins, were reported when comparing malignant tissues with normal tissues (Thijssen et al., 2015).However, targeting galectins with inhibitors has not achieved clinical success against cancer yet and most studies did not go beyond the initial phases of clinical trials (Laderach & Compagno, 2023).This indicates the need for developing novel conceptual paradigms, especially those that focus on the cell biology of galectins, to understand how cellular networks of widespread and tissue-specific galectins can be involved in the regulation of essential cellular processes for a better understanding of cancer development and strategic aspects of cancer therapy.
This study also leaves an unanswered question about posttranslational modification of regulatory proteins through phosphorylation and O-GlcNAcylation as these may play an essential role in processes of cellular differentiation and galectin regulation (Hart et al., 2011;Tazhitdinova & Timoshenko, 2020).Moreover, it is not clear why galectin-16 protein cannot be detected, while a strong upregulation of the LGALS16 gene is evident in JEG-3 cells treated with 8-Br-cAMP.This may represent a mechanism similar to the translational regulation and deposition of maternal mRNAs in oocytes due to cytoplasmic polyadenylation, resulting in high gene and low protein expression in cells (Winata & Korzh, 2018).

| CONCLUSION
Collectively, our findings demonstrate that LGALS16 is required for the trophoblast-like differentiation of JEG-3 cells induced by 8-Br-cAMP, and the expression of this gene is mediated through p38 MAPK and EPAC signaling pathways.
RNA isolation, cDNA synthesis, conventional PCR, and real-time quantitative polymerase chain reaction (RT-qPCR) were carried out to KAMINKER ET AL.
The 8-Br-cAMP-induced expression of LGALS13 was inhibited by SB203580 and 8-CPT-2-Me cAMP, whereas H-89 and ESI-09 did not change the expression of this gene compared to control cells (Figure1b,c).This pattern was slightly different for LGALS16, namely H-89 and 8-CPT-2-Me cAMP, which did not change the 8-Br-cAMP-induced expression of this gene, whereas SB203580 inhibited and ESI-09 augmented this change.Nevertheless, these results suggest that both p38 MAPK and EPAC might be functional branches of cAMP-mediated signaling cascades, leading to the regulation of both LGALS13 and LGALS16 genes.
Further investigations are ongoing to determine whether this happens with the regulation of LGALS16 expression, what the specific conditions activating galectin-16 translation, and how the presence of LGALS16 augments the expression of the syncytiotrophoblast marker CGB and supports cell differentiation.
(Koyanagi et al., 2021dination and interplay should not be overlooked.As to cAMP/EPAC signaling, to our knowledge, this is the first report addressing the regulation of human placental galectins and successfully demonstrating the effects of EPAC agonist and antagonist on the expression of LGALS13 and LGALS16.A similar association was noted for galectin-3 and cAMP/EPAC signaling in mouse Schwann cells in vivo(Koyanagi et al., 2021), further T A B L E 2 Pairwise Pearson's correlation coefficients for the expression of chorionic gonadotropin subunit beta and placental galectin genes in JEG-3 cells.
regulation in JEG-3 cells for future studies, which implies a crosstalk between the stimulatory p38 MAPK branch and the inhibitory EPAC branch in this context.It would be important to consider which transcription factors could be engaged by these signaling pathways to