Estrogen receptor b agonistdiarylpropionitrile inhibitslipopolysaccharide-induced regulatedon activation normal T cell expressedand secreted (RANTES) production inmacrophages by repressing nuclearfactor kB activation

Shi-ying Huang, M.S.,a,c Hong Xin, M.D.,a Jing Sun, M.D.,b Rui Li, M.S.,a Xue-mei Zhang, Ph.D.,a

and Dong Zhao, M.D.b

a Department of Pharmacology, School of Pharmacy, Fudan University, b Department of Minimally Invasive GynecologicSurgery, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, and c Department ofPharmacy, Shanghai Children's Medical Center, Shanghai Jiaotong University, Shanghai, People's Republic of China

Objective: To investigate the effect of the estrogen receptor-b (ERb) agonist diarylpropionitrile (DPN) on lipopolysaccharide (LPS)-in-duced regulated on activation normal T cell expressed and secreted (RANTES) production in macrophages and the possible mechanisms.Design: Cellular and molecular biology experimental study.Setting: University-based research laboratory.Patient(s): None.Intervention(s): ERb mRNA and protein expression determined in murine macrophage cell line RAW264.7 cells using reverse-transcription polymerase chain reaction and Western blot analysis; RANTES production detected by ELISA in LPS-stimulatedRAW264.7 cells and ERb knockdown RAW264.7 cells after the addition of DPN, phosphorylation of p65 and IkB degradationdetected by Western blot analysis; and nuclear accumulation of p65 visualized using immunofluorescence.Main Outcome Measure(s): LPS-induced RANTES production and phosphorylation of p65 and IkB.Result(s): ERbwasexpressed inRAW264.7 cells, andDPNstatistically significantlydecreasedLPS-inducedRANTESproduction inRAW264.7cells. Small interfering RNA targeting the ERb gene inhibited the effect of DPN on RANTES production. In addition, DPN inhibited nucleartranslocation and phosphorylation of p65 by inhibiting IkB degradation and thus prohibited the activation of nuclear factor kB (NF-kB).

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Conclusion(s): Diarylpropionitrile down-regulates LPS-induced RANTES production via ERb.This effect of DPN is likely due to repression of nuclear factor kB activation. (Fertil Steril�2013;100:234–40. �2013 by American Society for Reproductive Medicine.)Key Words: DPN, estrogen receptor beta, macrophage, RANTES, NF-kB

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Received January 3, 2012; revised and accepted February 28, 2013; published online April 1, 2013.S.-y.H. has nothing to disclose. H.X. has nothing to disclose. J.S. has nothing to disclose. R.L. has

nothing to disclose. X.-m.Z. has nothing to disclose. D.Z. has nothing to disclose.S.-y.H. and H.X. contributed equally to this work.Supported by the Shanghai Rising Star program (grant number 08QA1407000) and Shanghai Science

and Technology Innovation Grant (grant number 12140903000).Reprint requests: Dong Zhao, M.D., Minimally Invasive Gynecologic Surgery, Shanghai First Maternity

and Infant Hospital, Tongji University School of Medicine, Chang Le Road 180 Hao, Shanghai200032, People's Republic of China (E-mail: hendryz@gmail.com); and Xue-mei Zhang, Ph.D.,Department of Pharmacology, School of Pharmacy, Fudan University, 826 Zhang Heng Road,Shanghai 201203, People's Republic of China (E-mail: xuemzhang@fudan.edu.cn).

Fertility and Sterility® Vol. 100, No. 1, July 2013 0015-0282/$36.00Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc.http://dx.doi.org/10.1016/j.fertnstert.2013.02.052

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ndometriosis is a common dis-

E order often associated withpelvic pain, dysmenorrhea, and

infertility. The development andprogression of chronic inflammatoryendometriosis is often accompaniedby macrophage aggregation andinfiltration, resulting in the secretionof large amounts of inflammatorycytokines and chemokines. Increased

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regulated on activation normal T cell expressed and secreted(RANTES) expression is one pathologic change that is ob-served (1, 2). RANTES is primarily secreted by endometrialstromal cells and peritoneal macrophages (PMF). Inendometriosis, RANTES is secreted by ectopic endometrialcells and activated T cells, causing the aggregation ofmore macrophages and T cells, and thus the secretion ofmore cytokines and chemokines. This forms a positivefeedback loop and locally mediates the pathogenesis ofendometriosis.

Because occurrence spontaneous endometriosis is depen-dent on menstruation, the development of this diseasedepends on hormones. Estrogen is a key regulator of growthand differentiation and is also involved in the immune systemand elicits complex immune functions by acting on variousimmune cells. The estrogen receptors (ERs), ERa and ERb,have both overlapping and unique biologic functions. TheERa agonists are thought tomediate most of the classic effectsof estrogen. In contrast, ERb-selective agonists have beendemonstrated to possess anti-inflammatory properties ininflammatory disease models (3). Some studies found thatthe commercial ERb agonist diarylpropionitrile (DPN) inhibitsproinflammatory cytokine secretion from microglia (4, 5), thecentral nervous system inflammatory response, andastrocytes stimulated by lipopolysaccharide (LPS). Theinhibitory effect of DPN was also observed in a ratischemia-reperfusion (I/R) model (6, 7). Studies have alsodetected decreased levels of serum inflammatory cytokinesin peripheral blood mononuclear cells, natural killer (NK)cells, Kupffer cells, and other immune cells (6, 8–11) in theDPN-pretreated trauma-hemorrhage rat model. The highlyselective ERb agonist ERB-041 was found to be therapeuticin animal models of chronic inflammatory conditions suchas colitis (12), rheumatoid arthritis (12, 13), andendometriosis (14). These data imply that ERb-selectiveagonists may exert their inhibitory effects by acting directlyon immune cells.

Macrophages play a pivotal role in cell infiltration andchemokine production in endometriosis, and the ERbanti-inflammatory mechanism may target these cells. Inthis study, we investigated the effects of DPN in LPS-induced RANTES production in macrophages and exploredthe influence of DPN on inflammatory pathways.

FIGURE 1

ERb expression in RAW264.7 cells. (A) RAW264.7 and mouse ovarylysates were analyzed by RT-PCR for the presence of the mRNAencoding ERb. (B) ERb protein expression in mouse ovary, murinePMF, and RAW264.7 (RAW) lysates using Western blot analysis.Huang. ERb agonist inhibits LPS-induced RANTES production. Fertil Steril 2013.

MATERIALS AND METHODSCell Culture

The murine macrophage cell line RAW264.7 was purchasedfrom the Type Culture Collection of the Chinese Academy ofSciences (Shanghai, People's Republic of China). The cellswere seeded in 24-well plates (2.5 � 105 cells/well), culturedfor at least 24 hours in phenol red-free Dulbecco's modifiedEagle's medium (DMEM; Invitrogen) with 10% charcoal-treated fetal bovine serum (Hyclone), and then pretreatedfor 2 hours with dexamethasone (10�6 and 10�5 mol/L) orwith increasing concentrations (from 10�10 to 10�6 mol/L)of DPN (Tocris Bioscience) before the addition of 1 mg/mLLPS (Sigma-Aldrich). After 24 hours, the supernatants werecollected for cytokine production assays, and the cells were

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lysed for polymerase chain reaction (PCR) or immunoblotexperiments.

Cytokine Measurements

The RANTES concentrations were determined from cell-freesupernatants using an ELISA kit (R&D Systems) accordingto the manufacturer's instructions. The detection range forRANTES is from 15.6 to 1,000 pg/mL, and the both interassayand intra-assay variability is <10%.

RNA Extraction and RT-PCR Analysis

The murine ovary homogenate was generously providedby Dr. Yi-Zhun Zhu (School of Pharmacy, Fudan University),and the RAW264.7 cells were lysed in Tri-Reagent (Sigma-Al-drich), with the total RNA harvested as described by the man-ufacturer's protocol. Total RNA (5 mg) from each sample wasused as template for the reverse transcription reaction usingrandom hexamers and Improm-II Reverse Transcriptase(Promega). Reactions were performed according to the manu-facturer's protocol. The following primers were used: for ERb,50-CAG TAA CAA GGG CAT GGA AC and 50-GTA CAT GTCCCA CTT CTG AC; for b-actin, 50-CTA TTG GCA ACG AGCGGT TCC and 50-CAG CAC TGT GTT GGC ATA GAG G.

RNA Interference

The RNA oligonucleotides that were directed against ERb anda mismatch negative control small interfering RNA (siRNA)were purchased from Bioneer. We cultured RAW264.7 inmedium as previously described, but without the gentami-cin/amphotericin B. The cells were plated at a density of2.0 � 105 cells per well in 24-well plate for 1 day beforetransfection to achieve approximately 30% to 50% conflu-ence at the time of transfection. On the day of transfection,the Lipofectamine 2000 (Invitrogen) was combined with 200nmol/L siRNA duplexes that were diluted in DMEM andapplied to the cells. Six hours after the start of transfection,complete growth medium without antibiotics was added.The controls included cells that were exposed to onlyDMEM and cells undergoing mock transfection with exposureto Lipofectamine 2000 in the absence of RNAi oligos. The cells

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FIGURE 2

Effect of DPN on RANTES production in murine RAW264.7 and RAW264.7 after ERb siRNA transfection. (A) RANTES production was determinedafter a 2-hour pretreatment with saline, DPN, or dexamethasone (positive control), followed by treatment for 24 hours with LPS. (B) RANTES levelafter siERb transfection. Nontargeting siRNA [RNAi(�)] was set as the negative control. RAW264.7 cells were pretreated with 10�7 mol/L DPN orsaline, and after 24 hours LPS stimulation. The data reported are the mean� SEM of three independent experiments, each performed in duplicate.*P<.05 as compared with the control group. (C) ERb protein level in RAW264.7 after ERb siRNA transfection.Huang. ERb agonist inhibits LPS-induced RANTES production. Fertil Steril 2013.

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were starved overnight, then treated with vehicle or LPS(1 mg/mL) for 24 hours. This was timed to allow for proteinisolation at 48 hours from the start of transfection.

Immunoblotting

The murine PMFs were obtained as kind gift from Dr. Hong Li(School of Pharmacy, Fudan University). The lysates of PMF

and RAW264.7 cells were collected. Equal amounts of protein(50 mg) in the lysates were resolved by 10% SDS-PAGE andtransferred to polyvinylidene difluoride membranes. Themembranes were incubated with the following antibodies(diluted 1:1000 in 5% bovine serum albumin): p65,phospho-p65, IkB (Cell Signaling Technology) and ERb(Abcam). The blots were incubated with goat anti-rabbithorseradish peroxidase and were visualized with enhanced

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chemiluminescence detection reagents (Applygen). Digitalimages were captured and quantified using AlphaEaseFCimaging system (Alpha Innotech).

Immunofluorescence and Quantification ofNuclear and Cytoplasmic Fluorescence

The RAW264.7 cells were cultured on glass coverslips,pretreated for 2 hours with DPN, and then treated for 15 min-utes with 1 mg/mL LPS. The cells were fixed with 4% parafor-maldehyde in phosphate-buffered saline, permeabilized for5 minutes in 0.1% Triton X-100, and blocked for 2 hourswith 1% bovine serum albumin. The cells were incubatedwith the nuclear factor kB (NF-kB)/p65 antibody and stainedwith CY3-conjugated AffiniPure goat anti-rabbit IgG(Jackson ImmunoResearch) and DAPI (Wolsen). The resulting

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FIGURE 3

DPN suppresses p65 phosphorylation and IkB degradation in RAW264.7. (A) Effects of NF-kB signal on RANTES accumulation. Cells were pretreatedfor 2 hours with DPN (10�7 mol/L) or PDTC (10�5 mol/L) and then were stimulated with LPS for 24 hours. *P<.05 as compared with the controlgroup. #P<.05 as compared with the LPS group. (B) Effect of DPN on LPS-induced NF-kB activation and p65 phosphorylation in time course.(C) Effect of DPN on IkB degradation in time course. (D, E) Quantitative data depicting the phospho-p65/total p65 ratio (D) and the IkB/b-actinratio (E). Cells were pretreated for 2 hours with or without DPN (10�7 mol/L) and then were stimulated with LPS (1 mg/mL) for 0, 5, 10, 15, 30,and 60 minutes. The protein levels were quantitated by mean density value. The results are expressed as the mean � SEM of four independentexperiments, each performed in triplicate. *P<.05 compared with the LPS group at each respective time point.Huang. ERb agonist inhibits LPS-induced RANTES production. Fertil Steril 2013.

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immunofluorescence was visualized with Axiovert 40 CFL(Carl Zeiss). Fluorescence of p65 in the nucleus or cytoplasmwas quantified using the image analysis software Image-ProPlus (Media Cybernetics). The intensity readings were takenfrom either the nucleus or the cytoplasm of over 80 cells ineach group.

Statistical Analyses

We used SPSS 18.0 software (IBM) for statistical analyses.Each parameter is presented as the mean � standard errorof the mean (SEM) and was compared using one-way analysisof variance (ANOVA) followed by Student-Newman-Keulsmultiple comparisons tests. P< .05 was considered statisti-cally significant.

RESULTSERb Protein Is Expressed in Murine MacrophageCell Line RAW264.7

Although it has been demonstrated that ERb mRNA isexpressed in the murine PMF and the murine macrophagecell line RAW264.7 (15), ERb protein expression had not yetbeen observed in these cells. We used mouse ovary lysate as

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a positive control and determined the expression of ERb inRAW264.7 cells. The ERb mRNA and protein expression inRAW264.7 cells was detected using RT-PCR (Fig. 1A) andWestern blot analysis (see Fig. 1B). The RAW264.7 cellsexpressed ERb mRNA and protein at a lower level comparedwith mouse ovary homogenate.

DPN Decreases the Secretion of RANTES fromRAW264.7 Cells

To investigate the effect of DPN on LPS-induced RANTESproduction, we used dexamethasone as a positive control.Stimulation of macrophages with LPS (1 mg/mL) evoked therelease of a statistically significant amount of RANTES inRAW264.7 cells (Fig. 2A). Preincubation with DPN (10�10 to10�6 mol/L) or dexamethasone (10�5 to 10�6 mol/L)decreased the level of RANTES. We performed ERb siRNAtransfection to determine whether DPN decreases the RANTESproduction via ERb. The results showed that treatment of 10�7

mol/L DPN did not decrease the LPS-stimulated RANTES levelin cells after siERb transfection compared with the nontarget-ing siRNA control (see Fig. 2B). This suggests that the effect ofDPN on RANTES production may be exerted via ERb. Theefficiency of ERb siRNA was evaluated by Western blot

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FIGURE 4

DPN suppressed NF-kB nuclear translocation in RAW264.7. (A) RAW264.7 cells were grown on glass coverslips and [1] left untreated, [2] treatedwith LPS for 15 minutes, or [3] pretreated with DPN for 2 hours, followed by 15minutes of LPS stimulation. Cells were visualized using a fluorescentmicroscope (scale bar¼ 20 mm). (B) The relative intensity (nucleus/cytoplasm) of the p65 fluorescence in panel A was quantified. Over 80 cells wereanalyzed in each case. The results are expressed as the mean � SEM of four independent experiments. *P<.05 compared with the control group.#P<.05 as compared with the LPS group.Huang. ERb agonist inhibits LPS-induced RANTES production. Fertil Steril 2013.

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analysis, and the ERb protein expression was lower in thesiERb cells than in the control group (see Fig. 2C).

DPN Inhibits NF-kB Activation in LPS-StimulatedRAW264.7 Cells

The genes encoding RANTES and other proinflammatoryeffectors are controlled by the transcription factor NF-kB,so we investigated whether DPN treatment affects NF-kBsignaling. First, NF-kB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) was employed to assess effects of NF-kBsignal on RANTES accumulation (Fig. 3A). The accumulationof RANTES evoked by LPS stimulation was markedlyinhibited in the presence of PDTC and DPN, respectively.This suggests that DPN may play a role similar to PDTC,which exerts its effect by suppressing NF-kB activation. Theeffect of DPN on NF-kB activation was also demonstratedin time course experiments. Pretreating the macrophageswith DPN inhibited p65 phosphorylation as comparedwith the LPS-stimulated group (see Fig. 3B and D). Inaddition, there was a decrease in LPS-induced IkBdegradation (see Fig. 3C and E), which may result in decreasedp65 phosphorylation.

Because NF-kB activation also requires nuclear translo-cation of the RelA/p65 subunit of NF-kB, we examined theeffect of DPN on LPS-stimulated p65 translocation usingimmunostaining (Fig. 4A). Before LPS treatment, p65 was

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localized mainly in the cytoplasm. After treatment for 15minutes with 1 mg/mL LPS, there was extensive nuclear stain-ing of the p65 protein, indicating an accumulation of p65 inthe nuclei. However, pretreatment with 10�7 mol/L DPNblocked LPS-stimulated translocation of p65 from the cyto-plasm to the nucleus in RAW264.7 cells (see Fig. 4A and B).Taken together, these observations reconfirmed that DPNlimits NF-kB activation in macrophages in response to LPSand suggest that this may contribute to the observedinhibition of migration in vivo.

DISCUSSIONThe expression of ERs in macrophages determines whetherthe ERb agonist DPN can affect these cells. Although somestudies have reported that peripheral mononuclear cellsexpress ERb (16, 17), it remained controversial whether ERbis expressed in macrophage strain RAW264.7. In the presentstudy, we found that ERb mRNA and protein wereexpressed on RAW264.7 cells. This finding is consistentwith the study of Lambert et al. (15) that reportedexpression of both ERa and ERb mRNA in PMF stimulatedby sodium thioglycolate. Conversely, a previous studyindicated that there was no detectable ERb mRNAexpression in RAW264.7 cells (18). The expression of ERb isdown-regulated in cells with low estrogen levels (19), whichmay explain this discrepancy. The previous study involved

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long-term culture of cells in media free of endogenous steroidhormones, which may have resulted in a decline in ERbexpression.

The effects of ERb in the modulation of inflammatorydisease are well known (12–14). The current potentialtherapeutic use of selective ERb modulators ininflammatory disease states such as endometriosis addressesthe need to define the ERb role in immune cells (14). Thepresent study focused on inflammatory chemokine RANTESsecretion in macrophages in response to DPN. We foundthat DPN markedly inhibited RANTES secretion inRAW264.7. Although DPN is 70 times more selective forERb than ERa (20), it remains unclear whether ERa or ERbmediate the effects of DPN in macrophages. To determinewhether DPN decreases RANTES production via ERb, weperformed ERb siRNA transfection. The result confirmedthat the effect of DPN may mainly be exerted via ERb.Furthermore, an antichemotactic role of DPN was observed(data not shown). It shows a potential therapeutic role ofDPN in endometriosis by suppressing both the productionof chemokines and the response to chemotactic stimuli.

Because the RANTES promoter contains two NF-kBregulatory sites (21), it is suggested that RANTES expressionis regulated by NF-kB. In our study, RANTES accumulationevoked by LPS stimulation was markedly inhibited in thepresence of PDTC and DPN, respectively. This suggests thatDPN may affect RANTES production via blocking theactivation of NF-kB, which is similar to PDTC. Nuclear factorkB (heterodimer of p65 and p50) is mainly located in thecytosol, complexed with the inhibitory protein IkB in itsinactivated state. A variety of extracellular signals such asLPS can eventually result in the degradation of IkB and therelease of NF-kB. The activated NF-kB is then translocatedinto the nucleus, where it binds to specific sequences ofDNA and finally regulates the transcription. To test ourhypothesis that DPN could also inhibit the NF-kB pathway,we examined the influence of DPN on p65 phosphorylationand nuclear translocation as well as its effect on IkB inRAW264.7 cells. The results indicate that DPN inhibitedLPS-induced IkB hydrolysis in macrophages and inhibitedp65 phosphorylation and nuclear translocation.

Estradiol has been shown to influence NF-kB signaling bysuppressing IkB kinase (IKK) phosphorylation and IkBdegradation (22). Estrogen receptors, predominantly ERa,have been shown to repress NF-kB signaling on a numberof levels: they inhibit IKK activity and degradation of IkBproteins, can block DNA binding by NF-kB, can competewith NF-kB for coactivators, and can bind to DNA-boundNF-kB and inhibit transcription. An interaction betweenERb and the NF-kB pathway also has been reported, but theoutcome varies among cell lines. The NF-kB pathway is in-hibited in ERb-transfected human osteoblast cells in a li-gand-dependent manner (23). Administration of estrogeninhibited NF-kB activity but did not affect IkB phosphoryla-tion and DNA binding. However, in ovarian granulosa cellstransfected with ERb, transcriptional inhibition of NF-kBwas also observed (24). In addition, it has been reported thatin inflammatory breast cancer NF-kB activation is associatedwith ER down-regulation, secondary to EGFR and/or ErB2

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overexpression and mitogen-activated protein kinase(MAPK) hyperactivation (25).

The estrogen receptor is a nuclear receptor that regulatesgene transcription and synthesis by directly binding estrogenresponse elements (ERE) in specific gene promoters. It alsoaffects transcription through its interaction with othertranscription factors such as activator protein-1 (AP-1)(26, 27) and specificity protein 1 (SP1) (28) in a nongenomicmanner. This mechanism is referred as crosstalk. Similarly,the estrogen receptor may interact with NF-kB (via protein-protein interactions) (23, 29), resulting in modulation of thebinding of NF-kB to NF-kB binding sites on the DNA (NF-kB response elements), thus regulating NF-kB dependentgene transcription. Recent data also have suggested a directlink between the ERs and the fast and transient activationof the MAPK signaling cascade (30). This concept is alsosupported by a study that DPN and estrogen pretreatmentnormalized the inducible nitric oxide synthase (iNOS) andcyclooxygenase-2 (COX-2) protein levels in LPS stimulatedmicroglia by extracellular signal-regulated protein kinases1 and 2 (ERK1/2) activation but not p38 (6). As it is knownthat transcriptional activation of RANTES promoter by LPSis also dependent on the specific AP-1 response element(31), the question as to whether the DPN affects RANTESthrough other signaling mechanism needs further study.

Taken together, our results indicate that DPNdown-regulates LPS-induced RANTES production mainlyvia the ERb, which might alleviate inflammation. In addition,our data suggest the NF-kB signaling is repressed in thepresence of DPN.

Acknowledgments: The authors thank Dr. Hong LI inFudan University for helpful suggestions prompting thisstudy, and Qi-jing Chen for the assistance with cell culturing.

REFERENCES1. Hornung D, Ryan IP, Chao VA, Vigne JL, Schriock ED, Taylor RN.

Immunolocalization and regulation of the chemokine RANTES in humanendometrial and endometriosis tissues and cells. J Clin Endocr Metab1997;82:1621–8.

2. Khorram O, Taylor RN, Ryan IP, Schall TJ, Landers DV. Peritoneal fluidconcentrations of the cytokine RANTES correlate with the severity ofendometriosis. Am J Obstet Gynecol 1993;169:1545–9.

3. Harris HH. The unexpected science of estrogen receptor-b selective agonists:a new class of anti-inflammatory agents? Nucl Recept Signal 2006;4:e012.

4. Lewis DK, Johnson AB, Stohlgren S, Harms A, Sohrabji F. Effects of estrogenreceptor agonists on regulation of the inflammatory response in astrocytesfrom young adult and middle-aged female rats. J Neuroimmunol 2008;195:47–59.

5. Baker AE, Brautigam VM, Watters JJ. Estrogen modulates microglialinflammatory mediator production via interactions with estrogen receptorbeta. Endocrinology 2004;145:5021–32.

6. Suzuki T, Shimizu T, Yu HP, Hsieh YC, Choudhry MA, Schwacha MG, et al.Tissue compartment-specific role of estrogen receptor subtypes in immunecell cytokine production following trauma-hemorrhage. J Appl Physiol 2007;102:163–8.

7. Vornehm ND,WangM, Abarbanell A, Herrmann J, Weil B, Tan J, et al. Acutepostischemic treatment with estrogen receptor-alpha agonist or estrogenreceptor-beta agonist improves myocardial recovery. Surgery 2009;146:145–54.

8. Suzuki T, Shimizu T, Yu HP, Hsieh YC, Choudhry MA, Chaudry IH. Salutaryeffects of 17b-estradiol on T-cell signaling and cytokine production after

239

ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE

trauma-hemorrhage are mediated primarily via estrogen receptor-alpha.Am J Physiol Cell Physiol 2007;292:C2103–11.

9. Kawasaki T, Choudhry MA, Suzuki T, Schwacha MG, Bland KI, Chaudry IH.17b-Estradiol's salutary effects on splenic dendritic cell functions followingtrauma-hemorrhage are mediated via estrogen receptor-alpha. MolImmunol 2008;45:376–85.

10. Moeinpour F,ChoudhryMA,deFigueiredoLF, BlandKI,Chaudry IH.Estradiol'ssalutary effects on keratinocytes following trauma-hemorrhage are mediatedby estrogen receptor (ER)-alpha and ER-beta. Mol Med 2008;14:689–96.

11. Shimizu T, Suzuki T, Yu H-P, Yokoyama Y, Choudhry MA, Bland KI, et al. Therole of estrogen receptor subtypes on hepatic neutrophil accumulationfollowing trauma-hemorrhage: direct modulation of CINC-1 productionby Kupffer cells. Cytokine 2008;43:88–92.

12. Harris HA, Albert LM, Leathurby Y, Malamas MS, Mewshaw RE, Miller CP,et al. Evaluation of an estrogen receptor-beta agonist in animal models ofhuman disease. Endocrinology 2003;144:4241–9.

13. Follettie MT, Pinard M, Keith JC Jr, Wang L, Chelsky D, Hayward C, et al.Organ messenger ribonucleic acid and plasma proteome changes in theadjuvant-induced arthritis model: responses to disease induction andtherapy with the estrogen receptor-beta selective agonist ERB-041.Endocrinology 2006;147:714–23.

14. Harris HA, Bruner-Tran KL, Zhang X, Osteen KG, Lyttle CR. A selectiveestrogen receptor-beta agonist causes lesion regression in an experimentallyinduced model of endometriosis. Hum Reprod 2005;20:936–41.

15. Lambert KC, Curran EM, Judy BM, Lubahn DB, Estes DM. Estrogenreceptor-alpha deficiency promotes increased TNF-alpha secretion andbacterial killing by murine macrophages in response to microbial stimuliin vitro. J Leukocyte Biol 2004;75:1166–72.

16. Stygar D, Masironi B, Eriksson H, Sahlin L. Studies on estrogen receptor (ER)alpha and beta responses on gene regulation in peripheral blood leukocytesin vivo using selective ER agonists. J Endocrinol 2007;194:101–19.

17. Stygar D, Westlund P, Eriksson H, Sahlin L. Identification of wild type andvariants of oestrogen receptors in polymorphonuclear and mononuclearleucocytes. Clin Endocrinol (Oxf) 2006;64:74–81.

18. Palacios VG, Robinson LJ, Borysenko CW, Lehmann T, Kalla SE, Blair HC.Negative regulation of RANKL-induced osteoclastic differentiation inRAW264.7 cells by estrogen and phytoestrogens. J Biol Chem 2005;280:13720–7.

19. Trukhacheva E, Lin Z, Reierstad S, Cheng YH, Milad M, Bulun SE. Estrogenreceptor (ER) b regulates ERa expression in stromal cells derived from ovarianendometriosis. J Clin Endocrinol Metab 2009;94:615–22.

20. Meyers MJ, Sun J, Carlson KE, Marriner GA, Katzenellenbogen BS,Katzenellenbogen JA. Estrogen receptor-beta potency-selective ligands:

240

structure-activity relationship studies of diarylpropionitriles and theiracetylene and polar analogues. J Med Chem 2001;44:4230–51.

21. Hirano F, Kobayashi A, Hirano Y, Nomura Y, Fukawa E, Makino I. Nuclearfactor-kB regulates RANTES chemokine expression in response to tumornecrosis factor-a in fibroblast-like synoviocytes. Mod Rheumatol 2002;12:37–43.

22. Stice JP, Knowlton AA. Estrogen, NFkB, and the heat shock response. MolMed 2008;14:517–27.

23. Quaedackers ME, Van den Brink CE, Wissink S, Schreurs RHMM,Gustafsson JA, Van der Saag PT, et al. 4-Hydroxytamoxifen trans-repressesnuclear factor-k B activity in human osteoblastic U2-OS cells throughestrogen receptor (ER)a, and not through ER b. Endocrinology 2001;142:1156–66.

24. Chu S. Transrepression of estrogen receptor signaling by nuclear factor-kB inovarian granulosa cells. Mol Endocrinol 2004;18:1919–28.

25. Van Laere SJ, Van der Auwera I, Van den Eynden GG, van Dam P, VanMarck EA, Vermeulen PB, et al. NF-kB activation in inflammatory breastcancer is associated with oestrogen receptor downregulation, secondaryto EGFR and//or ErbB2 overexpression and MAPK hyperactivation. Br J Can-cer 2007;97:659–69.

26. Liu MM, Albanese C, Anderson CM, Hilty K, Webb P, Uht RM, et al.Opposing action of estrogen receptors alpha and beta on cyclin D1 geneexpression. J Biol Chem 2002;277:24353–60.

27. Zhao C, Gao H, Liu Y, Papoutsi Z, Jaffrey S, Gustafsson JA, et al. Genome-wide mapping of estrogen receptor-beta-binding regions reveals extensivecross-talk with transcription factor activator protein-1. Cancer Res 2010;70:5174–83.

28. Salvatori L, Pallante P, Ravenna L, Chinzari P, Frati L, Russo MA, et al.Oestrogens and selective oestrogen receptor (ER) modulators regulate EGFreceptor gene expression through human ER alpha and beta subtypes viaan Sp1 site. Oncogene 2003;22:4875–81.

29. Ghisletti S, Meda C, Maggi A, Vegeto E. 17 beta-estradiol inhibitsinflammatory gene expression by controlling NF-kappa B intracellularlocalization. Mol Cell Biol 2005;25:2957–68.

30. Wong CW,McNally C, Nickbarg E, Komm BS, Cheskis BJ. Estrogen receptor-interacting protein that modulates its nongenomic activity-crosstalkwith Src/Erk phosphorylation cascade. Proc Natl Acad Sci USA 2002;99:14783–8.

31. Hiura TS, Kempiak SJ, Nel AE. Activation of the human RANTES genepromoter in a macrophage cell line by lipopolysaccharide is dependent onstress-activated protein kinases and the IkB kinase cascade: implicationsfor exacerbation of allergic inflammation by environmental pollutants.Clin Immunol 1999;90:287–301.

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