Alpha 1 Antitrypsin Regulates Trophoblast Syncytialization and Inflammatory Factor Expression

The serine protease inhibitor alpha1-antitrypsin (A1AT) may possess protective functions of impaired organs in a manner independent of its protease inhibitor activity. A1AT expression has been shown to fluctuate in patients with pregnancy-induced hypertension, which suggests that A1AT may play a role in the syncytialization of villous trophoblasts. A1AT expression was knocked down in primary trophoblasts. RNA was extracted from these cells and subjected to RNA-sequencing analysis to determine the levels of expression of markers of syncytialization and inflammation. In addition, A1AT protein was localized in trophoblastic cells in placental tissues. Knockdown of A1AT upregulated the expression of FOSL1 and markers of syncytialization, as well as cell fusion, whereas overexpression of A1AT had the opposite effects. FOSL1 overexpression stimulated syncytialization, similar to the effects of A1AT knock down. Inhibitors of p38MAPK and JNK reduce the expression of inflammatory factors, whereas a p38MAPK inhibitor suppressed FOSL1 expression. Collectively, these findings indicated A1AT may negatively regulate inflammatory responses by controlling the activation of p38MAPK and JNK, and that p38MAPK mediates trophoblast syncytialization by altering FOSL1 expression. Therefore, a dysfunction in A1AT could be responsible for abnormal placental formation and pregnancy-associated disorders.


Introduction
Alpha-1 antitrypsin (A1AT), a member of the serpin superfamily, is a serine protease inhibitor encoded by the SERPINA1 gene. In patients with inflammatory diseases, this protein is synthesized primarily in hepatocytes as an acute-phase reaction and secreted into the sera [1]. Interestingly, a reduction in intracellular A1AT protein has been shown to exacerbate inflammatory responses at endometriosis-like lesions in mice [2] and to increase endoplasmic reticulum (ER) stress-induced adipokine production in cultured human adipocytes [3]. Furthermore, treatment with purified A1AT was found to inhibit inflammatory responses induced by proinflammatory cytokines in human endometrial cells in vitro [2], and to improve the survival rate of mice with peritonitis and sepsis in vivo [4]. Although A1AT has been reported to reduce organ damage independent of its serine protease activity [5][6][7][8], the roles of A1AT in inflammatory physiology and disorders remain incompletely understood.
The placenta is the essential organ for the maintenance of pregnancy, with functions that include the exchange of gases and nutrition, the production of various bioactive factors and the maintenance of immune tolerance of the conceptus to the maternal immune system [9,10]. The placenta is composed primarily of fetal chorion and endometrial decidua. Human trophoblasts differentiate into villous and extravillous trophoblasts (EVT) [11]. 2 of 13 During the process of syncytialization, cytotrophoblasts fuse to form multinucleate syncytiotrophoblasts, which are characterized by morphological and functional differentiation. In both primary human trophoblasts and the trophoblast-derived choriocarcinoma cell line BeWo, intracellular cyclic AMP (cAMP) mediates the production of human chorionic gonadotropin (hCG) and progesterone [12,13], and upregulates fusogenic syncytins through the transcription factors, glial cell missing 1 (GCM1), and ovo like transcriptional repressor 1 (OVOL1) [14][15][16].
Pathological conditions such as preeclampsia may be caused by insufficient invasion and syncytialization of trophoblasts [17,18]. A1AT has been shown to regulate ER stress-induced invasion of EVT [7], and urinary A1AT may be a marker of the severity of preeclampsia [19]. A1AT expression has been shown to fluctuate in patients with pregnancyinduced hypertension, which suggests that A1AT may play a role in the syncytialization of villous trophoblasts [20]. However, the roles of A1AT in placental physiology and disorders remain unknown. We therefore hypothesized that A1AT may play a role in the placental inflammatory response and trophoblast syncytialization. To test this hypothesis, we explored the role of A1AT on the expression of inflammatory cytokines and syncytialization markers in human trophoblast cells using RNA-sequencing (RNA-seq) analysis.

A1AT Knockdown Upregulates Inflammatory Response-Related Gene Expression in Trophoblasts
Immunostaining of placental tissues with antibody to A1AT showed that A1AT was localized to cytotrophoblasts and syncytiotrophoblasts (Figure 1a), and western blotting showed that isolated trophoblasts express several molecular weights of A1AT, presumably glycosylated and cleaved forms (Figure 1b). The function of A1AT in trophoblasts was investigated by A1AT knockdown, followed by RNA-seq analysis of these trophoblast cells. A total of 1334 differentially expressed genes (DEGs) were detected (Figure 1c), with gene ontology (GO) biological process analysis showing that many of the upregulated DEGs were associated with the inflammatory response ( Figure 1d).

A1AT Regulates Activation of p38MAPK and JNK and Expression of Inflammatory Cytokines in Trophoblasts
To investigate the mechanism by which A1AT regulates inflammatory responses in trophoblasts, A1AT was knocked down or overexpressed in the trophoblast BeWo cell line, and the levels of several intracellular signaling factors determined. The levels of phosphorylated JNK and p38MAPK were increased by A1AT knockdown and reduced by A1AT overexpression (Figure 2a). A1AT overexpression enhanced ERK1/2 phosphorylation, but neither A1AT knockdown nor overexpression altered the levels of phosphorylated NFKB, IKB, and CREB. To determine whether JNK and p38MAPK mediated the A1AT-induced expression of inflammatory cytokines, BeWo cells with knocked down A1AT were treated with inhibitors of JNK (SP600125) and p38MAPK (SB203580). Each inhibitor suppressed the expression of CXCL1, CXCL2, CXCL3, CCL5, IL1A, IL1B, and IL6 mRNAs, all of which were inflammatory response-related DEGs (Figure 2b). The expression of CCL3 and CCL20 was inhibited by the p38MAPK inhibitor SB203580, but not by the JNK inhibitor SP600125.

A1AT Expression Alters the Syncytialization of Trophoblasts
We next investigated whether A1AT has a significant effect on the syncytialization of trophoblasts. In the presence of a stable cAMP analog dibutyryl cAMP (Db), an inducer of syncytialization, A1AT knockdown further increased the expression of the syncytialization markers CGB, ERVFRD-1, GCM1, and OVOL1 ( Figure 3a) and enhanced cell fusion in BeWo cells (Figure 3b). Conversely, A1AT overexpression reduced the expression of Db-induced syncytialization markers and cell fusion (Figure 3c,d). The protein concentration of hCGβ, which is encoded by CGB, was also increased by A1AT knockdown and reduced by A1AT overexpression (Figure 3e).

FOSL1 Mediates A1AT Knockdown-Induced Syncytialization of Trophoblasts
To identify the transcription factors that regulate the expression of syncytialization markers, we compared the genes previously reported to regulate ERVFRD-1 or CGB expression with the DEGs upregulated in our RNA-seq data. Only one gene was identified, FOSL1 ( Figure 4a). The level of FOSL1 protein was increased by A1AT knockdown and reduced by A1AT overexpression (Figure 4b). To evaluate its effect on syncytialization, FOSL1 was overexpressed in BeWo cells (Figure 4c). FOSL1 overexpression increased the Db-induced expression of CGB and ERVFRD-1 (Figure 4d), and enhanced cell fusion (Figure 4e). In addition, FOSL1 overexpression increased the levels of expression of several inflammatory cytokines, such as CXCL2, CXCL3, CXCL8, IL6, and IL36G (Figure 4f). The effect of A1AT knockdown on the expression of FOSL1 was blocked by the inhibition of p38MAPK (Figure 4g).

A1AT Regulates Activation of p38MAPK and JNK and Expression of Inflamm Cytokines in Trophoblasts
To investigate the mechanism by which A1AT regulates inflammatory trophoblasts, A1AT was knocked down or overexpressed in the trophobl

A1AT Expression Alters the Syncytialization of Trophoblasts
We next investigated whether A1AT has a significant effect

FOSL1 Mediates A1AT Knockdown-Induced Syncytialization of Trophoblasts
To identify the transcription factors that regulate the expression of syncytialization markers, we compared the genes previously reported to regulate ERVFRD-1 or CGB ex pression with the DEGs upregulated in our RNA-seq data. Only one gene was identified FOSL1 (Figure 4a). The level of FOSL1 protein was increased by A1AT knockdown and

Relative expression
Ctrl A1AT -OE Db: FOSL1 was overexpressed in BeWo cells (Figure 4c). FOSL1 overexpression increased the Db-induced expression of CGB and ERVFRD-1 (Figure 4d), and enhanced cell fusion (Figure 4e). In addition, FOSL1 overexpression increased the levels of expression of several inflammatory cytokines, such as CXCL2, CXCL3, CXCL8, IL6, and IL36G (Figure 4f). The effect of A1AT knockdown on the expression of FOSL1 was blocked by the inhibition of p38MAPK (Figure 4g).

Discussion
The present study showed that endogenous A1AT is associated with syncytialization via FOSL1, and that A1AT suppresses the expression of inflammatory cytokines in trophoblasts. A1AT knockdown enhanced, whereas A1AT overexpression inhibited, syncytialization. This study also found that the silencing of A1AT induced the expression of a large number of DEGs in primary trophoblasts, with most of these DEGs being immunerelated genes. Knockdown of A1AT was found to stimulate the intracellular signaling proteins, p38MAPK and JNK, with their inhibitors abrogating the expression of inflammatory cytokines induced by A1AT silencing. Thus, a reduction in endogenous A1AT may lead to upregulation of the expression of FOSL1, which can promote the syncytialization of trophoblasts or the expression of several inflammatory cytokines via activation of p38MAPK ( Figure 5). ** p < 0.01. (g) Expression of mRNAs encoding inflammatory cytokines in A1AT-KD BeW treated with SP600125 (SP, 20 μM) or SB203580 (SB, 20 μM) for 24 h. GAPDH was used as the l control. Results are reported as the means ± SEMs of three independent experiments. ** p < 0

Discussion
The present study showed that endogenous A1AT is associated with syncytiali via FOSL1, and that A1AT suppresses the expression of inflammatory cytokines in oblasts. A1AT knockdown enhanced, whereas A1AT overexpression inhibited, sy alization. This study also found that the silencing of A1AT induced the expressio large number of DEGs in primary trophoblasts, with most of these DEGs being im related genes. Knockdown of A1AT was found to stimulate the intracellular sig proteins, p38MAPK and JNK, with their inhibitors abrogating the expression of in matory cytokines induced by A1AT silencing. Thus, a reduction in endogenous may lead to upregulation of the expression of FOSL1, which can promote the sync zation of trophoblasts or the expression of several inflammatory cytokines via acti of p38MAPK ( Figure 5). A1AT is a serine protease inhibitor that might interact with the enzyme high-te ature requirement A serine peptidase 1 (HTRA1) [21]. However, preliminary result our laboratory found that HTRA1 was not expressed in BeWo cells. A1AT express EVT has been reported to alter cell motility by inducing ER stress and HTRA1 in a protease activity-independent manner [7]. Neither A1AT knockdown nor overexpr altered the expression of ER stress markers in BeWo cells (data not shown). Impor the present study showed that A1AT knockdown activated p38MAPK and JNK. administration has been reported to inactivate the p21-activated kinase (PAK)/ transducer and activator of transcription 1 (STAT1)/p38MAPK signaling pathw trophoblasts from patients with preeclampsia [22,23], and S-nitrosylated A1AT was to enhance the expression of inflammatory factors by activating p38MAPK and JNK In this study, several forms of A1AT protein were detected in villous lysates. Be A1AT activity may be influenced by post-translational modification, including S-ni ation, oxidization, glycosylation, and enzyme cleavage, the profile of A1AT forms A1AT is a serine protease inhibitor that might interact with the enzyme high-temperature requirement A serine peptidase 1 (HTRA1) [21]. However, preliminary results from our laboratory found that HTRA1 was not expressed in BeWo cells. A1AT expression in EVT has been reported to alter cell motility by inducing ER stress and HTRA1 in a serine protease activity-independent manner [7]. Neither A1AT knockdown nor overexpression altered the expression of ER stress markers in BeWo cells (data not shown). Importantly, the present study showed that A1AT knockdown activated p38MAPK and JNK. A1AT administration has been reported to inactivate the p21-activated kinase (PAK)/signal transducer and activator of transcription 1 (STAT1)/p38MAPK signaling pathway in trophoblasts from patients with preeclampsia [22,23], and S-nitrosylated A1AT was found to enhance the expression of inflammatory factors by activating p38MAPK and JNK [24]. In this study, several forms of A1AT protein were detected in villous lysates. Because A1AT activity may be influenced by post-translational modification, including S-nitrosylation, oxidization, glycosylation, and enzyme cleavage, the profile of A1AT forms might change under pathological placental conditions and consequently result in the functional alteration of this protein. In addition, it is reported that several protein kinases including MAPK play a crucial role in trophoblast differentiation and onset of preeclampsia [25][26][27]. These findings are consistent with our results, showing that A1AT regulates the syncytialization and expression of inflammatory cytokines via activation of p38MAPK and JNK. FOSL1, a leucine zipper transcription factor and member of the activator protein 1 (AP-1) family, has been found to heterodimerize with JUN proteins. FOSL1 is thought to regulate fundamental cellular processes, including cell proliferation, differentiation, motility, and invasion [28]. Fosl1 knockout in rats resulted in early embryonic death [29]. Evaluation of rat trophoblast stem cells showed that FOSL1 expression was markedly increased during trophoblast differentiation, with these cells acquiring both endocrine and invasive properties [20,[29][30][31]. The role of FOSL1 in trophoblast invasion is conserved in rats and human trophoblast cells [29,31,32].
This study found that knockdown of A1AT upregulated the expression of FOSL1. Similar to A1AT knockdown, FOSL1 overexpression also increased the expression of syncytialization markers and cell fusion. Furthermore, p38MAPK inhibitor downregulated FOSL1 expression induced by A1AT knockdown. Taken together, these findings indicate that a decrease in A1AT upregulated FOSL1 expression via the activation of p38MAPK, resulting in the syncytialization of trophoblasts. FOSL1 overexpression also induced the expression of several proinflammatory cytokines, suggesting that the expression of inflammatory cytokines induced by knocking down A1AT was partially mediated by FOSL1.
In conclusion, this study demonstrated that A1AT regulated the expression in trophoblasts of inflammatory cytokines via the activation of p38MAPK and JNK, and that A1AT enhanced the syncytialization of trophoblasts via the expression of FOSL1 induced by p38MAPK. These findings suggest that optimal expression of A1AT in trophoblasts may be required for placental syncytialization and inflammatory responses, and that villous A1AT expression may be associated with hypertensive disorders of pregnancy and early abortion.

Immunohistochemistry
Placental tissues were obtained from three pregnant women during their third trimester (32 weeks of gestation) in Nippon Medical School. Informed consent was obtained from each patient who underwent cesarean section, and the study protocol was approved by the institutional review board of Nippon Medical School and the Clinical Research Ethics Committee of Tokyo University of Pharmacy and Life Sciences (#1512: approval 1 September 2015). The sections were immunostained for A1AT. Briefly, portions of the placental samples were immediately fixed in 4% paraformaldehyde in PBS, dehydrated, and embedded in paraffin. The sections were rehydrated, boiled for 20 min in 10 mM citrate buffer (pH 6.0), and incubated overnight at 4 • C with rabbit polyclonal anti-A1AT antibody (1:200; Dako, Tokyo, Japan) or, as a negative control, an equal molar concentration of normal rabbit IgG (1:100, sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), which were incubated with Histofine Simple Stain MAX-PO (Nichirei Biosciences, Tokyo, Japan). The sections were stained with DAB (Fujifilm Wako Pure Chemical Corp., Osaka, Japan), and counterstained with hematoxylin [7].

RNA-Sequencing (RNA-Seq), GO, and Pathway Analyses
For RNA-seq analysis, RNA was extracted from cultured primary trophoblast cells using Isogen II (Nippon Gene), according to the manufacturer's instructions. The cDNA libraries for RNA-seq were prepared using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA), according to the manufacturer's instructions, and data were analyzed by Macrogen Japan (Kyoto, Japan). Output data were deposited in the DDBJ (DNA Data Bank of Japan) Sequence Read Archive (https://www.ddbj.nig.ac.jp/ dra/index-e.html (accessed on 1 November 2021) under accession numbers DRR304274 and DRR304275). Data were analyzed as described [35]. Briefly, trimmed sequences were analyzed using the TopHat/Cufflinks pipeline, the human genome (hg38), and reference annotations obtained from the UCSC genome browser (https://genome.ucsc.edu (accessed on 1 November 2021)). DEGs were categorized by gene-level FPKM (fragments per kilobase of exon per million mapped fragments) expression levels. Genes with an absolute expression level of >2 FPKM were selected. GO analyses were performed using the Enrichr tool (http://amp.pharm.mssm.edu/Enrichr/ (accessed on 1 November 2021)).

RNA Extraction and Quantitative RT-PCR
RNA was extracted from cells using RNeasy Mini Kits (Qiagen, Tokyo, Japan), according to the manufacturer's instructions, and the resulting mRNA was reverse transcribed using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Sequences in the resulting cDNA were subjected to qPCR amplification using specific primers (Table 1) and a PowerUP SYBR Green Master Mix (Thermo Fisher Scientific). Calibration curves were used to determine the level of expression of each target gene relative to that of a reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The mean crossing threshold (Ct) for each target was calculated using Sequence Detection System software v2.3 (Thermo Fisher Scientific) [35].

Cell Fusion Assay
BeWo cells were fixed with methanol and incubated with anti-E-cadherin antibody (1:200, #3195, CST) and AlexaFluor 594-conjugated goat anti-mouse antibody (Thermo Fisher Scientific) to distinguish cell surfaces. The nuclei were counterstained with 4 ,6diamino-2-phenylindole 2HCl (DAPI). The numbers of multinucleated cells in five randomly selected microscopic areas per sample were counted and evaluated in three independent experiments [34].

Statistical Analysis
Data are expressed as mean ± SEM and compared using Dunnett's test. All statistical analyses were performed using R software (ver.4.0.5; www.r-project.org, accessed on 1 November 2021), with p-values < 0.05 considered statistically significant.