Nuclear receptor coactivator 6 promotes HTR‐8/SVneo cell invasion and migration by activating NF‐κB‐mediated MMP9 transcription

Abstract Objectives NCOA6 is a transcription coactivator; its deletion in mice results in growth retardation and lethality between 8.5 and 12.5 dpc with defects in the placenta. However, the transcription factor(s) and the mechanism(s) involved in the function of NCOA6 in placentation have not been elucidated. Here, the roles of NCOA6 in human cytotrophoblast invasion and migration were studied. Materials and Methods Human placenta tissues were collected from normal pregnancies and pregnancies complicated by early‐onset severe preeclampsia (sPE). Immunofluorescence, RT‐qPCR and Western blotting were used to determine NCOA6 expression. Transwell invasion/migration assays were performed to explore whether NCOA6 knockdown affected human placenta‐derived HTR‐8/SVneo cell invasion/migration. Gelatin zymography was performed to examine the change in the gelatinolytic activities of secreted MMP2 and MMP9. Luciferase reporter assays were used to explore whether NCOA6 coactivated NF‐κB‐mediated MMP9 transcription. Results NCOA6 is mainly expressed in the human placental trophoblast column, as well as in the EVTs. HTR‐8/SVneo cell invasion and migration were significantly attenuated after NCOA6 knockdown, and the secretion of MMP9 was decreased due to transcriptional suppression. NCOA6 was further found to coactivate NF‐κB‐mediated MMP9 transcription. Moreover, expression of NCOA6 was impaired in placentas of patients complicated by early‐onset sPE. Conclusions Thus, we demonstrated that NCOA6 is important for cytotrophoblast invasion/migration, at least partially, by activating NF‐κB‐mediated MMP9 transcription; the downregulation of NCOA6 may contribute to the pathogenesis of early‐onset sPE.


| INTRODUC TI ON
Normal placentation is indispensable for foetal development. Two major types of differentiation exist in trophoblasts during human placentation, which include trophoblast invasion and syncytialization. During invasion, cytotrophoblasts (CTBs) proliferate to form the trophoblast column (TC), from which they differentiate into the invasive extravillous trophoblasts (EVTs) that either invade into the maternal decidua/myometrium to anchor the placenta (as interstitial EVTs) or invade and remodel maternal spiral arteries to improve maternal blood flow as required for pregnancy (as endovascular EVTs).
During syncytialization, mononucleated CTBs fuse to become multinucleated syncytiotrophoblasts (STBs). 1 Abnormal trophoblast differentiation and subsequent functional impairment result in pregnancy-related diseases, including preeclampsia (PE) due to shallow interstitial EVT invasion into the maternal decidua/myometrium and incomplete remodelling of maternal vessels caused by impaired endovascular EVT invasion into blood vessels. [2][3][4] Nuclear receptor coactivator 6 (NCOA6) was first identified in human breast tumours and was originally named Amplified in breast 3 (AIB3). 5,6 In addition to gene amplification in breast cancer, NCOA6 was also found to be amplified and overexpressed in colon cancers and lung cancers. 7 As a transcription coactivator, NCOA6 plays multiple roles by coactivating specific transcription factors, including PPARα, 8 PPARγ, 9 AP-1, CRE and NF-κB. 10 Mice with Ncoa6 deletion showed growth retardation and lethality between 8.5-12.5 days post-conception (dpc), most likely due to developmental defects in the placenta. 9,11,12 Since both the labyrinth and spongiotrophoblast layers of the placenta in Ncoa6 −/− mice were severely affected, the essential roles of NCOA6 might be the coactivation of key transcription factors in placentation. 9,11 However, the transcription factor(s) and the mechanism(s) involved in the function of NCOA6 in placentation have not been elucidated.
In this study, we explored the functions of NCOA6 in human trophoblast invasion and migration. NCOA6 was mainly expressed in the TC and EVT of human placentas. NCOA6 knockdown significantly suppressed the invasion and migration of human placenta-derived HTR-8/SVneo cells with reduced matrix metalloproteinase 9 (MMP9) secretion. Luciferase reporter assays were further used to test whether NCOA6 contributed to NF-κB-mediated MMP9 transcription. In addition, the transcription of NCOA6 in placentas from patients with early-onset sPE, showing inadequate trophoblast invasion and subsequent incomplete remodelling of maternal spiral arteries, was found to be impaired. Thus, we demonstrated that NCOA6 promotes the invasion and migration of HTR-8/SVneo cells, at least partially, by coactivating NF-κB-mediated MMP9 transcription.

| Human placenta collection
Placental villi from the first trimester (6-8 weeks of gestation, n = 3) were sampled from normal pregnancies after legal abortion; placental tissues from the second trimester (17-21 weeks of gestation, n = 3) were collected after inevitable abortions that had been accidentally caused by external hurt; the third-trimester placenta samples were obtained from normal pregnancies (normal group, between 36 and 40 weeks of gestation; n = 15) and pregnancies complicated by early-onset sPE (early-onset sPE group, between 33 and 37 weeks of gestation; n = 13). All tissues were sampled with informed consent at the Department of Gynecology and Obstetrics in the First Affiliated Hospital of Zhengzhou University.
Individuals with early-onset sPE were recruited as previously reported, without any other maternal complications. 13

| Paraffin-embedded section preparation and immunofluorescence
Paraffin-embedded sections of placental tissue were prepared as previously reported. 15 After antigen retrieval and blocking, the serial sections were treated with the following primary antibod-

| Primary CTB isolation and subsequent in vitro induction of EVT
Primary CTBs were isolated as previously reported. 17 For the in vitro differentiation of primary CTBs into EVTs, 2.5 × 10 6 CTBs were

| Transwell invasion/migration assays
Transwell invasion/migration assays were carried out as previously reported. 14

| Cell counting kit-8 (CCK-8) assay
CCK-8 incubations were carried out with CCK-8 reagent (Dojindo Laboratories) 0, 24, 48 and 72 hours after cell-seeding as previously reported. 14 The cell medium was renewed every day to ensure sufficient growth nutrition for plated cells. The absorbance at 450 nm of CCK-8 incubation medium was measured by a Varioskan ™ Flash Microplate Reader (Thermo Scientific).

| Gelatin zymography
Gelatin zymography was carried out by using an MMP zymography assay kit (Applygen). Before the cells that invaded through the insert membrane in the invasion assays were fixed, 20 μL cell medium inside the insert was mixed with 2× SDS-PAGE non-reducing buffer, loaded on a gel (20 μL each) and separated via 10% SDS-PAGE containing 1.0 mg/mL gelatin. After Buffer A rinsing, Buffer B incubation and R-250 staining, bands representing the gelatinolytic activities of secreted MMP2 and MMP9 were recorded with a ChemiDoc MP System (Bio-Rad).

| RT-qPCR
RT-qPCR assays were carried out as previously reported. 14

| Statistical analysis
All data are represented as the mean ± SD (standard deviation) following three and more independent experiments, and an independent-samples t test in SPSS Statistics 17.0 was used to determine the significance of the differences between treatments and corresponding controls. Statistical significance is indicated mainly by one asterisk (*) for P < .05 or two asterisks (**) for P < .01.

| NCOA6 is expressed in human placental TC and invasive EVT cells
The expression of NCOA6 in the first-and second-trimester human

| NCOA6 knockdown blocks the activation of NF-κB-mediated MMP9 transcription
Trophoblasts degrade the extracellular matrix (ECM) when they invade and migrate into the maternal decidua and myometrium.

Among the various proteinases secreted by trophoblasts, MMP2
and MMP9, belonging to the gelatinase group of the MMP family and being mainly expressed in trophoblasts of early gestation, play essential roles in the invasion and migration of trophoblasts. 25,26 To investigate whether MMP2 and MMP9 are involved in suppressing the invasion and migration of NCOA6-knockdown HTR-8/SVneo cells, gelatin zymography assays were performed to examine the changes in the gelatinolytic activities of MMP2 and MMP9. As a result, MMP9 activity was significantly decreased after NCOA6 was knocked down; however, no change in MMP2 activity was found ( Figure 4A). Tissue inhibitors of metalloproteinases (TIMPs) inhibit most MMP activities in tissues with a certain degree of specificity. 26 Interestingly, in addition to the reduced level of MMP9 transcription, the transcription of TIMP1, which has been reported to preferentially bind MMP9, 27,28 was increased, while the transcription of TIMP2, which preferentially binds MMP2, 28,29 was not changed, in accordance with the MMP2 results ( Figure 4B). These data suggested the important role of MMP9 but not MMP2 in regulating the invasion and migration mediated by NCOA6 in HTR-8/SVneo cells.
Since NCOA6 is a transcription coactivator of NF-κB, 10 which has been reported to regulate MMP9 transcription via the binding of its subunit RELA/p65 to MMP9 promoter, 20,30,31 we further explored whether NCOA6 coactivates NF-κB-mediated MMP9 transcription using luciferase reporter assays. Knockdown of NCOA6 not only significantly decreased the basic reporter activity of the MMP9 promoter but also blocked increased reporter activity of the MMP9 promoter following RELA/p65 overexpression ( Figure 4C). However, compared with RELA/p65 overexpression alone, the co-overexpression of NCOA6 and RELA/p65 did not synergistically enhance the increased reporter activity of the MMP9 promoter ( Figure 4D). These results further demonstrate that the coactivating role of NCOA6 is essential for NF-κB-mediated MMP9 transcription.

| Transcript levels of NCOA6 were decreased in early-onset sPE placentas
Since inadequate EVT invasion and migration primarily contribute to early-onset PE, the transcript levels of NCOA6 in the placentas of pregnancies with early-onset sPE were examined to evaluate whether placental transcription of NCOA6 is also altered in this diseased state. Of interest, transcript levels for NCOA6 in the early-onset sPE placentas were significantly impaired compared with those in the paired placentas from normal pregnancies ( Figure 6).

**
NCOA6 is a transcription coactivator that executes a variety of functions with specific transcription factors including PPARα, PPARγ, AP-1, CRE and NF-κB. [8][9][10] The deletion of Ncoa6 in mice resulted in developmental defects in the placenta and lethality between 8.5 and 12.5 dpc 9,11,12 ; however, the mechanisms involved remain largely unknown. In the present work, NCOA6 was found to be expressed in the TC and invasive EVTs of human placenta. In addition, the RNA level of NCOA6 was also increased as those of the specific EVT markers HLA-G and ITGA5 during the in vitro induction of isolated primary CTBs into EVTs. These results suggested that Mmp9-null embryos. 38 These results demonstrated the crucial roles of MMP9 in trophoblast invasion during human placentation. We found in this study that the gelatinase activity of secreted MMP9, but not that of secreted MMP2, was significantly impaired after NCOA6 siRNA treatment.
NF-κB, one of the transcription factors that can be coactivated by NCOA6, has been reported to regulate MMP9 transcription via the binding of its subunit RELA/p65 to the promoter. 20,30,31 We found in this study that NCOA6 knockdown not only significantly decreased the basic reporter activity of the MMP9 promoter but also prevented the increased reporter activity of the MMP9 promoter induced by RELA/p65 overexpression, suggesting an essential role of NCOA6 in the coactivation of NF-κB-mediated MMP9 transcription.
However, co-overexpression of NCOA6 and RELA/p65 did not promote increased reporter activity of the MMP9 promoter induced by RELA/p65 overexpression alone.
Two binding sites of NF-κB on the MMP9 proximal promoter, including one (at −619 to −609 bp) reported previously, 20 were bioinformatically predicted, and both sites were validated by site mutagenesis. However, the activity of the reporter with both binding sites mutated remained significantly decreased after NCOA6 knockdown, suggested the presence of other NCOA6-coactivated transcription factors, for example AP-1, 10,20 that bind to the proximal promoter of MMP9 in HTR-8/SVneo cells.
Since NCOA6 has essential roles in trophoblast invasion and migration and inadequate trophoblast invasion/migration and subsequent incomplete reconstruction of maternal spiral arteries are the main contributors to the early-onset PE, 2-4,39 the transcript levels of NCOA6 were compared in placentas from pregnancies with early-onset sPE and normal pregnancies. Notably, early-onset sPE placentas showed significantly fewer NCOA6 transcripts than normal placentas. The transcriptomics of CTBs have been found to be dysregulated in sPE due to its diseased in vivo environment. 40 As addressed above, NCOA6 is a transcription coactivator that executes a variety of functions with many specific transcription factors. These results imply transcription coactivators including NCOA6 might play critical roles in the pathogenesis of sPE. However, one limitation exists in this section: placental tissues from normal pregnancies and pregnancies complicated by early-onset sPE were not completely "term-matched" ( Table 1).
Significant difference in genstational age might partly contribute to the decreased NCOA6 transcript levels observed in early-onset sPE placentas.
In conclusion, our results demonstrated that NCOA6, a transcription coactivator, promotes the invasion and migration of trophoblasts, at least partially, by activating NF-κB-mediated MMP9 transcription. The "in vivo" functions of NCOA6 in placentation and the roles of downregulated NCOA6 in sPE development need to be investigated in the future; for example, placenta-specific Ncoa6 knockout mice should be constructed and analysed to study the "in vivo" function of NCOA6 in placental development. Furthermore, additional researches are also needed to verify other transcription factors that are coactivated by NCOA6 during placentation and sPE development.

ACK N OWLED G EM ENTS
We honestly thank all patients enrolled in this study. We ap-

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

AUTH O R CO NTR I B UTI O N S
The contributions each author made to the study are specified as follows: YPS and LW designed the study; LW, KQZ, WW, LNC and JS performed the experiments and collected the data; YPS, LW, KQZ, WW, LLH and XXJ analysed the data; LW and KQZ prepared the manuscript; YPS and LW revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the prediction of RELA/p65 binding sites in the MMP9 proximal promoter in this study are available from LASAGNA-Search 2.0 (https://biogr id-lasag na.engr.uconn.edu/lasag na_searc h/), 22 Tfsitescan (http://www.ifti.org/cgi-bin/ifti/Tfsit escan. pl) and Gene2Promoter (http://www.genom atix.de), with the default parameters.