Glucocorticoid Receptor Signaling Contributes to

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Dec 13, 2012 - Anh Nguyen, and Todd Rosen. Department of Obstetrics, Gynecology, and Reproductive Sciences, Division of Maternal-Fetal Medicine,.
ORIGINAL

RESEARCH

Glucocorticoid Receptor Signaling Contributes to Constitutive Activation of the Noncanonical NF-␬B Pathway in Term Human Placenta Bingbing Wang, Kristy Palomares, Nataliya Parobchak, John Cece, Max Rosen, Anh Nguyen, and Todd Rosen Department of Obstetrics, Gynecology, and Reproductive Sciences, Division of Maternal-Fetal Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901

Our recent study demonstrated that constitutively activated RelB/NF-␬B2 positively regulates the CRH in the human placenta. In the current study, we explored the role of the glucocorticoid receptor (GR) signaling in constitutive activation of the noncanonical NF-␬B pathway. A glucocorticoid response element (GRE) motif search suggests that both NF-␬B inducing kinase (NIK) and RelB genes, which are key regulators of the noncanonical NF-␬B pathway, have a putative GRE within their promoter, approximately 1 kb upstream from the transcription start site. By using chromatin immunoprecipitation assay we identified that the GR and phosphorylated GR at Ser211 were associated with the GREs of both NIK and RelB. Dexamethasone stimulated expression of NIK, RelB, NF-␬B2 as well as CRH and cyclooxygenase-2 (COX-2). Repression of GR by short interfering RNA resulted in inhibition of NIK, RelB, NF-␬B2, CRH, and COX-2. In addition, depletion of GR attenuated glucocorticoid-mediated up-regulation of NIK, RelB, NF-␬B2, CRH, and COX-2. Furthermore, siRNA specifically targeting NIK down-regulated CRH and COX-2. Taken together, these results suggest that constitutive activation of the noncanonical NF-␬B pathway in term human placenta is driven by the GR signaling, which in turn up-regulates placental CRH and other NF-␬B-responsive genes. (Molecular Endocrinology 27: 203–211, 2013)

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RH is a 41-amino acid peptide hormone and neurotransmitter that was originally identified in the hypothalamus (1). CRH is widely distributed in areas of central nerve system; however, the parvocellullar paraventricullar nucleus neurons of the hypothalamus have the highest concentration (2). In addition to being secreted from the hypothalamus, CRH is detected in a variety of peripheral tissues, including lymphocytes and the reproductive system (3). CRH is highly expressed in the human placenta and believed to be part of the placental clock determining the length of gestation (4, 5). Despite a product of the same gene, CRH is repressed by glucocorticoids in the hypothalamus but up-regulated in the placenta (6, 7). We believe that elucidating how CRH is regulated in the placenta will

assist in understanding of the mechanisms that lead to parturition and delivery. The glucocorticoid receptor (GR) is a ligand-activated and ubiquitously expressed transcription factor that regulates expression of numerous genes. There are two isoforms of GR, GR␣ and GR␤ with GR␤ functioning as a dominant-negative inhibitor (8). Cross-linked hormone and GR are associated with a specific genomic site, known as the glucocorticoid response element (GRE), to regulate target genes. Elevated levels of GR␤ in neurons may account, in part, for hypothalamic CRH repression by glucocorticoids (9). However, nested deletions of CRH gene promoter coupled with luciferase reporter assay suggest an alternative mechanism, also known as the composite mechanism because of existence of a negative GRE

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2013 by The Endocrine Society doi: 10.1210/me.2012-1309 Received September 11, 2012. Accepted November 12, 2012. First Published Online December 13, 2012

Abbreviations: ChIP, Chromatin immunoprecipitation; COX-2, cyclooxygenase-2; CT, cycle threshold; DEX, dexamethasone; FL, firefly luciferase; GR, glucocorticoid receptor; GRE, glucocorticoid response element; 11␤-HSD2, 11␤-hydroxysteroid dehydrogenase 2; NF-␬B, nuclear factor-␬B; nGRE, negative GRE; NIK, NF-␬B-inducing kinase; PG, prostaglandin; siRNA, short interfering RNA; TSS, transcriptional start site.

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(nGRE) within the CRH gene promoter (10, 11). With respect to positive regulation of CRH by glucocorticoid in the placenta, our recent study has shown that it is the noncanonical nuclear factor-␬B (NF-␬B) signaling (RelB/ NF-␬B2) that plays a critical role in regulation of placental CRH (12). Indeed, we showed that constitutively nuclear RelB/NF-␬B2 (p52) interacts with an NF-␬B enhancer within the CRH gene promoter to regulate CRH in primary cultures of cytotrophoblasts. Progesterone represses CRH by blocking processing of the active form, NF-␬B2 (p52), from the precursor form, NF-␬B2 (p100), and also blocking nuclear translocation of NF-␬B2 (p52). Moreover, glucocorticoid up-regulates CRH by increasing expression of RelB/NF-␬B2 and their subsequent nuclear translocation. In this study, we test the hypothesis that the GR signaling regulates genes involved in the noncanonical NF-␬B pathway, NF-␬B-inducing kinase (NIK), RelB, and NF-␬B2 in the human placenta. We examined the putative GRE(s) within the promoter of NIK, NF-␬B2, and RelB genes, occupancy of GR at these GRE(s), and the effects of GR signaling on NIK, NF-␬B2, and RelB and NF-␬B-responsive genes including CRH as well as cyclooxygenase-2 (COX-2) in cell culture of primary term cytotrophoblast.

facturers. The staining was detected with a 3,3⬘-diaminobenzidine detection kit (Ventana, Roche, AZ).

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Materials and Methods Study approval Placentas were collected after the patients signed the written informed consent in protocols approved by the Institutional Review Board of The University of Medicine and Dentistry of New Jersey.

Purification of primary cytotrophoblasts Purification of term cytotrophoblasts was performed as recently detailed with purity of greater than 98% (12, 13). Cytotrophoblasts were cultured in DMEM-F12 and 10% fetal bovine serum and maintained at 37 C and 5% CO2 for 24 h before further analysis. Early trimester trophoblast, HTR-8/SVneo (HTR-8) cell line (14), was obtained from Dr. Seth Guller at Yale University (New Haven, CT) in an agreement of material transfer with Dr. Charles Graham at Queen’s University, Canada.

Immunohistochemical staining Immunohistochemical staining was performed at Anatomical Tissue Services, Cancer Institute of New Jersey. Tissues from term human placentas were fixed in buffered formalin for 24 h, dehydrated in 70% ethanol, paraffin embedded, and sectioned. Paraffin slides were incubated with primary antibodies to, GR at 1:50 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), CRH at 1:50 (Abnova, Walnut, CA), and COX-2 at 1:100 (Cell Signaling Technology, Danvers, MA) as recommended by the manu-

Chromatin immunoprecipitation (ChIP) Formaldehyde cross-linking ChIP was performed in cultured primary cytotrophoblasts as recently detailed with use of ChIP grade antibodies to GR (Cell Signaling Technology), phospho-GR Ser211 (Cell Signaling Technology), RelB (Santa Cruz Biotechnology), and NF-␬B2 (Abcam, Cambridge, MA) (12). Normal rabbit IgG antibody (Cell Signaling Technology) was used as the nonspecific control antibody. Real-time PCR was performed triplicates by use of SYBR green dye (QIAGEN, Valencia, CA) on an Mx3000 real-time PCR instrument (Agilent Technologies, Santa Clara, CA). Primers used for CRH promoter were: forward, 5⬘-GGCCTTTCATAGTAAGAGGTCAA3⬘; reverse, 5⬘-TCTCACATCCAATTATATCAACAGA-3⬘; COX-2 promoter: forward, 5⬘-AACATAAAACATGTCAGCCTTTCTTAACC-3⬘; reverse, 5⬘-GTTTCTTTCCGCCTTTTCGTACC-3⬘; RelB promoter: forward, 5⬘-CTCCACTTCCTGGGTTCAAG3⬘; reverse, 5⬘-GTGGCTCATGCCTGTAATCC-3⬘; NIK: forward, 5⬘-CCTCTAGCTGGGACTGCAAG-3⬘; reverse, 5⬘-CGGTGGCTCACTCCTGTAAT-3⬘; NF-␬B2: forward, 5⬘AGCAATCCTCCCACCTTAGC-3⬘; reverse, 5⬘-TTTTCACTTTGGGAGGCTGA-3⬘. Endogenous control primers for a human DNA ␣ satellite to which no transcription factors should bind were: forward, 5⬘-TCTCAGAATCTTCCTTTTGATGTG-3⬘; reverse, 5⬘-CCAGTTGCAGATCCTACAAAGA-3⬘. The occupancy of each transcription factor at target gene promoter was presented as the fold enrichment that was derived by the method of cycle threshold (CT) in the formula of 2⫺⌬⌬CT. ⌬⌬CT ⫽ (mean CT of triplicates of test primers and test antibody ⫺ mean CT of triplicates of endogenous control primers and test antibody) ⫺ (mean CT of triplicates of test primers and nonspecific control antibody ⫺ mean CT of triplicates of endogenous control primers and nonspecific control antibody). The error bars represent standard deviation of fold enrichment from experiments performed in three independent term placentas.

Gene silencing assay Flextube short interfering RNAs (siRNAs) were purchased from QIAGEN. siRNA transfection in primary cytotrophoblasts was performed as recently detailed with use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and siRNA at a final concentration of 50 nM (12). A nontargeting scramble RNA was used as a negative control with the targeting sequence: 5⬘-AACAGUCGCGUUGUCGACUGGUU-3⬘. After 48 h, cells were harvested for further analyses.

RT-PCR Total RNAs were extracted by the Trizol method (Invitrogen). Total cDNA synthesis was prepared by a mixture of oligo-dT and random primer method with use of the GoScript reverse transcription system (Promega Corp., Madison, WI). The following primers were used for quantitative analysis with SYBR green dye (QIAGEN) in real-time PCR: NIK, forward, 5⬘-CAAGCCTCTGAAGGAACCAG-3⬘; reverse, 5⬘-AGGGATGAGGCAGTCTGCTA-3⬘; COX-2: forward, 5⬘-TGAGCATCTACGGTTTGCTG-3⬘; reverse, 5⬘-TGCTTGTCTGGAACAACTGC-3⬘; RelB: forward, 5⬘-TCCCAACCAGGATGTCTAGC-3⬘; reverse, 5⬘-AGCCATGTCCCTTTTCCTCT-3⬘; NF-␬B2: forward, 5⬘-GAACAGCCTTGCATCTAGCC-3⬘;

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Mol Endocrinol, February 2013, 27(2):203–211

reverse, 5⬘-TCCCAGTCGCTATCAGAGG-3⬘; GR: forward, 5⬘-AGGGAGCAATTCCAGTTTCA-3⬘; reverse, 5⬘-TGACCAGCCAAGATGGAAAT-3⬘; CRH: forward, 5⬘-GCAGTTAGCACAGCAAGCTCAC-3⬘, reverse: 5⬘-CAAATGGCATAAGAGCAGCG-3⬘; and glyceraldehyde-3-phosphate dehydrogenase: forward, 5⬘-CTCCCGCTTCGCTCTCTG3⬘; reverse, 5⬘-CTGGCGACGCAAAAGAAG-3⬘.

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ciferase assays were performed on Victor 3V (PerkinElmer, Waltham, MA) according to the manufacturer’s protocol (Promega).

Statistical analysis We performed one-way ANOVA analysis to test significant differences among groups and Dunnett’s test to compare the reference with each test group.

Western blotting analysis Whole-cell lysates were resuspended in 1⫻ Laemmli sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA) supplemented with 5% 2-mercaptoethanl and boiled at 95 C for 5 min. Samples were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad). Membranes were incubated with antibodies to GR, phospho-GR Ser 211, RelB, NF-␬B2, CRH, COX-2 at 1:500 and ␤-actin at 1:1000 (Sigma-Aldrich, St. Louis, MO) at 4 C overnight. The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies and developed by Immun-Star HRP Substrate (Bio-Rad).

Results

Immunohistochemical localization of GR in term human placentas GR is a transcription factor that is expressed in almost every cell in human body and predominantly cytoplasmic. Upon ligand binding, this hormone-receptor complex translocates into the nucleus where it interacts with GRE(s) to modulate target gene transcription. Paraffin tissue slides from term placentas were used for determinDNA constructs and dual-luciferase assay ing spatial patterns of GR and CRH. As shown in Fig. 1, To create DNA constructs of firefly luciferase (FL) reporter, expression of which is regulated by the GRE, we linearized positive cytoplasmic and nuclear staining for GR was obpGL4.32 (Promega) with KpnI and HindIII. Three sets of DNA served in villous syncytiotrophoblasts in the syncytium oligonucleotides, NIK-GRE (5⬘-CATATTGCCCAGGATand syncytiol knot in term human placenta. Villous cyGTTCTCCAACTA-3⬘ and 5⬘-AGCTTAGTTGGAGTTCATCtotrophoblast cells are sparse in term placenta. Cells beCTGGGCAATATGGTAC-3⬘), RelB-GRE (5⬘-CGAGCTTTTneath the syncytium that resemble cytotrophoblasts by GAGCTCAAAGTGGTCTGGGCTATTTAA-3⬘, and 5⬘-AGCTTTAAATAGCCCAGACCACTTTGAGCTCAAAAGCTCGmorphology and other intravillous cells also demonGTAC-3⬘), NF-␬B2-GRE (5⬘-CTCCCTATGTTGCCCAGstrated positive staining in the cytoplasm and nucleus. GCTGTTCTCAAACTCCTGA-3⬘ and 5⬘-AGCTTCAGGAGPositive staining for CRH was only observed in the cytoTTTGAGAACAGCCTGGGCAACATAGGGAGGTAC-3⬘) were plasm of villous syncytiotrophoblasts in the syncytium purchased (Integrated DNA Technologies, Coralville, IA) and anand syncytiol knot, but not in cytotrophoblast and other nealed each other to generate three double-stranded DNA fragintravillous cells, consistent with previous findings (15). ments that contained cohesive ends that were compatible with sites of KpnI and HindIII at 5⬘- and 3⬘-ends, respectively. These three There are two isoforms of cyclooxygenase (COX), double-stranded DNA fragments were ligated to KpnI/HindIII-linCOX-1 and COX-2. COX-1, which is constitutively exearized pGL4.32 backbone to generate pGL4.32-NIK-GRE, pressed in a variety of human tissues, is responsible for pGL4.32-RelB-GRE, and pGL4.32-NF-␬B2-GRE, respectively. baseline levels of prostaglandins (PGs). COX-2, the inThe individual FL reporter construct was cotransfected with a Reducible form, produces PGs that are closely associated nilla luciferase reporter construct, pRL-CMV with use of Lipofectamine 2000 (Invitrogen) as recently described (12). Dual luwith initiation and progression of human labor by a mean of stimulation (16). COX-2 has been previously detected in human term placenta (17). Importantly, COX-2 constitutes an NF-␬B target gene, and upregulation of COX-2 in placental trophoblasts is also stimulated by glucocorticoids (16, 18). Thus, we hypothesized that COX-2 is regulated similarly to CRH in the placenta. As shown in Supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://mend.enGR CRH dojournals.org, the term placenta FIG. 1. Representative immunohistochemical staining of GR and CRH performed in three showed an abundant expression of independent term human placentas. Paraffin slides were stained with antibody to GR (left COX-2 in syncytiotrophoblasts in the panel) or CRH (right panel) as indicated. Original magnification, ⫻400.

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T

GR GRα GRβ p-GR CRH β−actin

FIG. 2. Representative gel pattern and analysis of GR isoforms and phosphorylation of in HTR-8 cell (H) and culture of primary cytotrophoblast from three independent term placentas (T) by Western blotting. p-GR, phospho-GR at Ser211.

syncytium and the syncytial knot, which is in accordance with our previous observation that the nuclei of villous trophoblasts of term placentas contained robust activity of RelB and NF-␬B2(12). Taken together, the nuclear localization of GR and robust expression of CRH and COX-2 in villous trophoblasts suggests that GR signaling may participate in regulation of CRH and COX-2 in term human placenta.

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junction with ligand binding. Ligand binding-induced phosphorylation of GR at Ser211 facilitates cofactor interaction, which in turn recruits the RNA polymerase II and the basal transcriptional machinery to promote transcription of many genes (20). To determine isoform composition and phosphorylation of GR in human placentas, we performed Western blotting analysis in early-trimester trophoblast HTR-8 cells (14), in which CRH is not produced, and primary cytotrophoblasts from term placentas. As shown in Fig. 2, GR␣ and GR␤ were almost equally detected in HTR-8 cells as previously described (21); however, the expression of GR␣ dominated in term cytotrophoblast cells. In addition, phosphorylation of GR␣ at Ser211 increased in the term placenta compared with HTR-8 cells. Similar to patterns of gene expression with CRH, COX-2 was barely detectable in HTR-8 cells, but noticeably expressed in term placentas (Supplemental Fig. 2). These results suggest that the expression of CRH and COX-2 may be associated with a more active form of GR and phosphorylation at Ser211.

Fold of RelB

Fold of NIK

GR associates with genes involved in the noncanonical NF-␬B signaling in term human placenta Isoforms and phosphorylation of GR in term In unstimulated cells, the noncanonical NF-␬B pathhuman placenta way is inactivated due to low abundance of NIK. IntraThere are two GR isoforms, GR␣ and GR␤, with GR␤ cellular accumulation of NIK is necessary and sufficient acting as the dominant-negative inhibitor of the active for activation of the noncanonical NF-␬B heterodimer form, GR␣ (8). Like many other regulatory proteins, GR (RelB/NF-␬B2) (22). GR stimulates gene expression by is a phosphoprotein (19). GR can be phosphorylated in binding to GRE and inhibits target genes by binding to absence of ligand, which can be enhanced further in con- nGRE. Although nGRE contained within 1 kb upstream of the transcriptional start site (TSS) is necessary and sufficient for GR-mediA -1092 1=TSS GRE ated repression of target gene (23), NIK TGCCCAGGCTGTTCT GREs that have been proven to be suf-1093 ficient for gene stimulation are local1=TSS GRE ized as far as 7 kb upstream of TSS RelB AGCTCAAAGTGGTCT (24). To test the hypothesis that the glucocorticoid-GR complex stimulates * * B * CRH by up-regulating gene expression * of the noncanonical NF-␬B pathway, we started by performing a GRE motif search within 7 kb upstream TSS of NIK, RelB, and NF-␬B2 genes based on position weight matrix of the conIgG p-GR GR IgG p-GR GR sensus GRE sequence: 5⬘-AGAAFIG. 3. Both human NIK and RelB genes contain a putative GRE. A, GRE motif search CANNNTGTTCT-3⬘ (24). Both RelB indentified that a GRE is located at ⫺1092 and ⫺1093 within NIK and RelB promoter, and NIK contain a putative GRE respectively. TSS, transcription start site. B, ChIP was performed to determine occupancy of GR or phospho-GR at Ser 211 (p-GR) at NIK (left panel) and RelB (right panel). The bars within approximately 1 kb upstream of indicate the average of fold enrichment (y-axis) against each member (x-axis) with error bars TSS (Fig. 3A), which was not observed representing the SD from experiments performed in three independent term placentas. The for NF-␬B2 (data not shown). When fold enrichment with IgG was defined as 1. *, P ⬍ 0.01, as determined by one-way ANOVA we performed assay of ChIP, we found with Dunnett’s test.

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that GR and phosphorylated GR at Ser211 (pGR-Ser211) occupied both NIK and RelB genes at these putative GREs, approximately 1.5-fold over the baseline control (Fig. 3B). Collectively, these results further suggest that GR and pGR-Ser211 may play a role in regulation of NIK and RelB in term human placenta.

GR

NF-κB2 (p100)

Relative Level

sion of NIK at transcriptional level, and repressed NF␬B2, RelB, and CRH at both the translational and transcriptional levels. Interaction between GR or pGRSer211 and NIK or RelB was also inhibited. Moreover, repressed GR prevented DEX-mediated up-regulation of NIK, NF-␬B2, CRH and association of pGR␣-S211 and GR with the NIK and RelB genes. GR signaling regulates NIK, RelB, and NF-␬B2 in We also examined the effects of GR signaling on term human placenta COX-2 of placental origin. As shown in Supplemental To further assess the interaction between GR and the Fig. 3, A–C, DEX treatment increased COX-2 by approxnoncanonical NF-␬B pathway, primary term cytotropho- imately 1.8-fold at both transcriptional and translational blast cells were transfected with the siRNAs specifically levels. In accordance with this increase, RelB and NF-␬B2 targeting the GR. As shown in Fig. 4, although not chang- were associated with the COX-2 gene at ␬B1 site in the ing total mRNA or protein level of GR␣, dexamethasone human placenta, which was further promoted by DEX (DEX) treatment promoted phosphorylation of GR␣ at treatment. Reduction in GR expression decreased glucoSer211, and proteins levels of RelB, NF-␬B2, and CRH. corticoid-mediated up-regulation of COX-2. InterestThe quantitative RT-PCR further showed that DEX stim- ingly, neither RelB nor NF-␬B2 occupied COX-2 at the ulated transcriptional activity of NIK, RelB, NF-␬B2, and ␬B2 site and DEX did not enhance such association (SupCRH by approximately 2-fold. The association of GR or plemental Fig. 4D), because the binding of RelB/p52 hetpGR-Ser211 with NIK or RelB was enhanced by up to erodimer is dependent on a middle segment of a consec2.5-fold when primary cytotrophoblasts were treated utive A:T in NF-␬B enhancer (25). These results suggest with DEX. GR knockdown resulted in significant repres- that glucocorticoid-stimulated expression of genes such as CRH and COX-2 is dependent on ligand binding-activated phosphorylaA SS B SS GR siRNA GR siRNA GR siRNA/DEX tion of GR␣ that, in turn, up-regulates SS/DEX − + − + DEX (1 µM) * the noncanonical NF-␬B pathway. * p-GR

C

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NIK

β-actin

Fold Enrichment

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GR siRNA GR siRNA/DEX

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p-GR

FIG. 4. The effects of GR silencing on RelB/NF-␬B2 and CRH and their interaction in term cytotrophoblasts. Cell cultures of primary term cytotrophoblast were transfected with the scramble siRNA (SS) or siRNA targeting GR for 24 h and then treated with DEX for additional 24 h. A, Representative gel patterns and analysis in whole-cell lysates from three independent term placentas by Western blotting with use of antibody as indicated. p-GR, phospho-GR at Ser 211. B, Total RNAs were extracted, and quantitative RT-PCR was performed with normalization to glyceradehyde-3-phosphate dehydrogenase mRNA level. The bars represent average of relative mRNA levels and error bars represent SD from experiments performed in three independent term placentas. The mRNA level with SS but without DEX was defined as 1. C, ChIP was performed to determine occupancy of GR or phospho-GR Ser 211 (p-GR) at NIK or RelB. The bars indicate the average of fold enrichment (y-axis) against each member (x-axis) with error bars representing the SD from experiments performed in three independent experiments. The fold enrichment with SS but without DEX was defined as 1. *, P ⬍ 0.01; ***, P ⬎ 0.05.

The GREs of NIK and RelB regulate transcriptional activity of a heterologous promoter NIK and RelB regulatory elements were placed upstream of a minimal promoter as recently described (12), to make firefly luciferase reporter (FL) constructs. As shown in Fig. 5, DEX was able to stimulate FL activity by more than 2-fold for both NIK and RelB GRE-regulated FL reporter expression. Knockdown of the GR reduced or prevented DEX-mediated increase of FL activity. These results suggest that the GRE of NIK or RelB is sufficient to regulate transcription to a heterologous promoter. Constitutive activation of RelB/NF␬B2 correlates with intracellular abundance of NIK in term human placenta In addition to serving the precursor for the active form, NF-␬B2 (p52), NF␬B2 (p100) functions as an I␬B-like

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A

TGCCCAGGCTGTTCT

TATA

FL

pGL-NIK-GRE1X

AGCTCAAAGTGGTCT

TATA

FL

pGL-RelB-GRE1X

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ing of NF-␬B2 (p100) to generate NF-␬B2 (p52), which induces nuclear translocation of these transcription factors. To test the role of NIK in constitutive activation of RelB/NF-␬B2, primary term cytotrophoblasts were transfected with siRNA targeting NIK, and processing of p100 and CRH production was assayed. As shown in Fig. 6, NIK inhibition resulted in reduced p100 processing and CRH synthesis, but no significant change in expression of RelB or NF-␬B2. In matched ChIP assays, the occupancy of RelB or NF-␬B2 (p52) at the CRH gene was also significantly inhibited. In both cases, DEX mediated up-regulation of CRH was abolished. p100 processing and association of RelB or NF␬B2 (p52) with CRH gene were also decreased. Similarly, COX-2 activity and association of RelB or NF␬B2 with COX-2 gene were significantly reduced when NIK was repressed (Supplemental Fig. 4). These results suggest that the glucocorticoid-GR complex acts as an intracellular signal to induce a cascade of events including activation of NIK, nuclear translocation of the RelB/NF-␬B2 (p52) heterodimer, and its association with NF-␬B enhancer to regulate transcription of NF-␬B target genes including CRH and COX-2.

* pGL-NIK-GRE1X

* FL activity

pGL-RelB-GRE1X

* * −

* *



DEX

DEX

SS

GR siRNA

FIG. 5. The putative GRE of NIK or RelB regulates transcription of a heterologous promoter. A, Schematic presentation of luciferase reporter constructs based on pGL4.32 (Promega). B, Cell cultures of primary term cytotrophoblast were cotransfected with pCMV-RL (200 ng) and FL reporter construct (1 ␮g) as indicated for 24 h. Then the cells were transfected with scramble siRNA (SS) or siRNA targeting GR mRNA for 24 h and then treated with DEX for an additional 24 h. The bars represent average of fold change of FL with normalization to Renilla luciferase, and error bars represent the SD experiment performed in three independent term placentas. *, P ⬍ 0.01.

molecule that prevents RelB nuclear translocation. Signal-induced intracellular accumulation of NIK is essential for triggering of the downstream signaling events in the noncanonical NF-␬B pathway by initiating process-

Discussion −

NIK siRNA −

+

DEX (1 µM)

B Relative Level

SS

RelB NF-κB2 (p100)

NF-κB2 (p52) CRH β-actin

SS NIK siRNA

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* ** ** NIK

* ** **

RelB

NF-κB2 CRH mRNA

C RelB NF-κB2

CRH Enrichment

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FIG. 6. The effects of NIK silencing on RelB/NF-␬B2 and CRH and their interaction in term cytotrophoblasts. Cell cultures of primary cytotrophoblast were transfected with scramble siRNA (SS) or siRNA targeting NIK mRNA for 24 h and then treated with DEX for an additional 24 h. A, Representative gel patterns and analysis of whole-cell lysates from three independent term placentas by Western blotting with use of antibody as indicated. B, In matched assays with panel A, total RNAs were extracted from and quantitative RT-PCR was performed with normalization to glyceraldehyde-3phosphate dehydrogenase mRNA. The bars represent relative level in mRNA levels, and error bars represent SD performed in three independent term placentas. The mRNA level with SS but without DEX was defined as 1. C, ChIP was performed to determine occupancy of RelB or NF-␬B2 at the CRH gene. The bars indicate the average of fold enrichment (y-axis) against each member (x-axis) with error bars representing the SD experiments performed in three independent term placentas. The fold enrichment with SS but without DEX was defined as 1. *, P ⬍ 0.01; **, P ⬎ 0.05.

Glucocorticoids inhibit hypothalamic CRH but paradoxically stimulate placental CRH production. Understanding how CRH is regulated could increase understanding of how a placental clock that governs the length of human gestation functions. Human labor is heralded by functional withdrawal of progesterone. Progesterone competes with action of cortisol at GR to down-regulate CRH but maintain PG dehydrogenase that inactivates PGs for maintenance of pregnancy. At term, increased cortisol antagonizes progesterone in binding to GR to increase CRH but inhibit PG dehydrogenase (6, 26). These studies provide significant insights into the role of GR in gene regulation in human placenta. Our group recently found that the noncanonical NF-␬B pathway drives CRH production in the placenta. Glucocorticoid up-regulated CRH by stimulating expression and nuclear transloac-

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tion of RelB and NF-␬B2 (p52), and progesterone repressed CRH by inhibiting nuclear translocation of RelB and NF-␬B2 (p52) (12). In the current study, we have explored mechanisms of constitutive activation of NF-␬B in term human placenta. We determined that GR signaling drives NIK, NF-␬B2, and RelB transcription, and liberation of the NF-␬B complex. Consequently, liberated RelB/NF-␬B2 (p52) heterodimer proceeds into the nucleus, regulating genes involved in human parturition, such as CRH and COX-2 that contain NF-␬B enhancer(s) within their promoter. In this study, we have shown, for the first time, that transcriptional activity of NIK is promoted by glucocorticoid in primary cytotrophoblasts, complementing the previous findings that glucocorticoid induced synthesis and nuclear translocation of RelB in lymphoblastic cells (27) and synthesis and nuclear translocation of RelB and NF-␬B2 in primary cytotrotrophoblast (12). NIK is a signal integrator as well as a central component of the noncanonical NF-␬B pathway, intracellular accumulation of which is necessary and sufficient for p100 phosphorylation (22). Under normal condition, NIK is inactivated because of being bound by a component of a multi-subunit NIK ubiquitin ligase, which mediates constant ubiquitination followed by proteasomal degradation of NIK. Activation of NIK is mediated by ligand cross-linking to a subset of TNF receptor superfamily, which triggers degradation of this ubiquitin ligase to stabilize NIK level. Constitutive activation of NIK has been noted in B-lymphocytes because of persistent degradation of the NIK-ubiquitin ligase (28), and cancer such as multiple myeloma because of increased gene dosage of NIK (29). In term human placenta, expression of TNF receptors, like B-cell activating factor receptor coupled with production of B-cell activating factor in villous mesenchymal cells may contribute, in part, to activation of NIK in a paracrine manner (30, 31). However, our data that glucocorticoid stimulated expression of RelB, NF-␬B2, and NIK strongly suggest that the GR signaling plays an essential role in constitutive activation of the noncanonical NF-␬B pathway. It is possible that cortisol secreted by the fetus enters the placenta to induce expression of NIK. During pregnancy, this process may be tightly regulated by 11␤-hydroxysteroid dehydrogenase 2 (11␤-HSD2), which is detected in abundance in the placenta (32). Placental 11␤-HSD2 converts active cortisol to inactive cortisone, thereby preventing passage of maternal glucocorticoids into fetal circulation. With advancing pregnancy, cortisol production exceeds oxidative ability of 11␤-HSD2. Thus, the expression of NIK along with RelB and NF-␬B2 accumulates, thereby

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activating NF-␬B-responsive genes including CRH and COX-2 in the placenta. The expression of GR-responsive genes is classified as phosphorylation-dependent and -independent. Phosphorylation of GR can occur at multiple sites that include S203, S211, and S226 in the N terminus (20). Phosphorylation of GR at different sites exerts differential effects on expression of target genes, which is exemplified by the fact that GR trans-activation reaches a peak when Ser211 is phosphorylated (33). Furthermore, phosphorylated GR binds to cofactors in a gene-specific manner and, in turn, recruits RNA polymerase II and the basal transcriptional machinery to promote target gene expression (20, 34). In this study, the expression of the inhibitory isoform of GR, GR␤, was significantly inhibited in term human placenta. The observation that Ser211 phosphorylation was absent in early-trimester trophoblasts but increased with advancing of gestation is consistent with the central role played by GR␣ phosphorylation at Ser211 in NIK, RelB, and NF-␬B2 expression in the placenta. Hypothalamic CRH repression by glucocorticoids is mediated by direct interaction between GR and the nGRE of CRH gene. In fact, this composite mechanism involves competitive DNA binding of GR and AP-1 nucleoproteins at discrete adjacent sites within the CRH promoter (10, 11). Whereas the mechanism underlying this differential regulation by glucocorticoid needs further investigation, differences in the noncanonical NF-␬B signaling between the placenta and the brain may account for this phenomenon. The studies about spatial pattern of expression of NF-␬B components in rat brain may have provided insight into this differential regulation (35–37). NF-␬B target genes are widely expressed in multiple types of cell. GR signaling-induced activation of the noncanonical NF-␬B pathway may target any gene containing NF-␬B enhancer within the promoter. As a result, the noncanonical NF-␬B signaling may play a fundamental role in regulating not only CRH, but also other genes involved in the onset of parturition such as COX-2. PGs are closely associated with the initiation and progression of human labor (16, 38). COX-2, the inducible form of COX, dramatically increases in the decidua and myometrium at term and preterm labor. Our finding that glucocorticoids activate the noncanonical NF-␬B pathway, and that RelB/p52 positively regulates COX-2, suggest that GR signaling may play a central integrative role in regulating length of pregnancy in humans, in part through its effects on the noncanonical NF-␬B pathway. Assessing the role of the GR and the noncanonical NF-␬B on other NF-␬B-re-

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GR Activates the Noncanonical NF-␬B Pathway

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sponsive genes may yield additional insight into this basic biological process.

Khoo N, Lala PK 1993 Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res 206:204 –211 Riley SC, Walton JC, Herlick JM, Challis JR 1991 The localization and distribution of corticotropin-releasing hormone in the human placenta and fetal membranes throughout gestation. J Clin Endocrinol Metab 72:1001–1007 Challis JR, Sloboda DM, Alfaidy N, Lye SJ, Gibb W, Patel FA, Whittle WL, Newnham JP 2002 Prostaglandins and mechanisms of preterm birth. Reproduction 124:1–17 Xu Y, Knipp GT, Cook TJ 2005 Expression of cyclooxygenase isoforms in developing rat placenta, human term placenta, and BeWo human trophoblast model. Mol Pharm 2:481– 490 Deng WG, Zhu Y, Wu KK 2003 Up-regulation of p300 binding and p50 acetylation in tumor necrosis factor-␣-induced cyclooxygenase-2 promoter activation. J Biol Chem 278:4770 – 4777 Kumar R, Calhoun WJ 2008 Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation. Biologics 2:845– 854 Chen W, Dang T, Blind RD, Wang Z, Cavasotto CN, Hittelman AB, Rogatsky I, Logan SK, Garabedian MJ 2008 Glucocorticoid receptor phosphorylation differentially affects target gene expression. Mol Endocrinol 22:1754 –1766 Cervellati F, Pavan B, Lunghi L, Manni E, Fabbri E, Mascoli C, Biondi C, Patella A, Vesce F 2011 Betamethasone, progesterone and RU-486 (mifepristone) exert similar effects on connexin expression in trophoblast-derived HTR-8/SVneo cells. Reprod Fertil Dev 23:319 –328 Sun SC 2011 Non-canonical NF-␬B signaling pathway. Cell Res 21:71– 85 Van LP, Spengler DH, Holsboer F 1990 Glucocorticoid repression of 3⬘,5⬘-cyclic-adenosine monophosphate-dependent human corticotropin-releasing-hormone gene promoter activity in a transfected mouse anterior pituitary cell line. Endocrinology 127:1412–1418 Datson NA, Polman JA, de Jonge RT, van Boheemen PT, van Maanen EM, Welten J, McEwen BS, Meiland HC, Meijer OC 2011 Specific regulatory motifs predict glucocorticoid responsiveness of hippocampal gene expression. Endocrinology 152:3749 –3757 Fusco AJ, Huang DB, Miller D, Wang VY, Vu D, Ghosh G 2009 NF-␬B p52:RelB heterodimer recognizes two classes of ␬B sites with two distinct modes. EMBO Rep 10:152–159 Patel FA, Funder JW, Challis JR 2003 Mechanism of cortisol/progesterone antagonism in the regulation of 15-hydroxyprostaglandin dehydrogenase activity and messenger ribonucleic acid levels in human chorion and placental trophoblast cells at term. J Clin Endocrinol Metab 88:2922–2933 Castro-Caldas M, Mendes AF, Carvalho AP, Duarte CB, Lopes MC 2003 Dexamethasone prevents interleukin-1␤-induced nuclear factor-␬B activation by upregulating I␬B-␣ synthesis, in lymphoblastic cells. Mediators Inflamm 12:37– 46 Liao G, Zhang M, Harhaj EW, Sun SC 2004 Regulation of the NF-␬B-inducing kinase by tumor necrosis factor receptor-associated factor 3-induced degradation. J Biol Chem 279:26243–26250 Annunziata CM, Davis RE, Demchenko Y, Bellamy W, Gabrea A, Zhan F, Lenz G, Hanamura I, Wright G, Xiao W, Dave S, Hurt EM, Tan B, Zhao H, Stephens O, Santra M, Williams DR, Dang L, Barlogie B, Shaughnessy Jr JD, Kuehl WM, Staudt LM 2007 Frequent engagement of the classical and alternative NF-␬B pathways by diverse genetic abnormalities in multiple myeloma. Cancer Cell 12:115–130 Gill RM, Ni J, Hunt JS 2002 Differential expression of LIGHT and its receptors in human placental villi and amniochorion membranes. Am J Pathol 161:2011–2017 Langat DL, Wheaton DA, Platt JS, Sifers T, Hunt JS 2008 Signaling pathways for B cell-activating factor (BAFF) and a proliferationinducing ligand (APRIL) in human placenta. Am J Pathol 172: 1303–1311

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Acknowledgments We thank Lei Cong at Cancer Institute of New Jersey for immunohistochemical staining. Address all correspondence and requests for reprints to: Bingbing Wang, Clinical Academic Building, Room 7056, 125 Paterson Street, New Brunswick, New Jersey 08901. E-mail: [email protected] or Todd Rosen, Clinical Academic Building, Room 2140, 125 Paterson Street, New Brunswick, New Jersey 08901. E-mail: [email protected]. Disclosure Summary: The authors have nothing to disclose.

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