skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: TLR4 activates NF-{kappa}B in human ovarian granulosa tumor cells

Abstract

Highlights: {yields} TLR4 is expressed in human ovarian granulosa tumor cells. {yields} Acting through TLR4, LPS and HSP60 induce a NF{kappa}B signaling cascade in human ovarian granulosa tumor cells. {yields} NF{kappa}B activation or inhibition did not alter chemosensitivity to TRAIL or cisplatin. -- Abstract: Previous studies have demonstrated expression of Toll-like receptors (TLRs) in the surface epithelium of normal ovaries (OSE) and in epithelial ovarian tumors. Most notably, OSE-derived cancers express TLR4, which activates the nuclear factor-kappa B (NF-{kappa}B) signaling cascade as a mediator of inflammatory response. Currently, there is considerable interest in elucidating the role of TLR-mediated signaling in cancers. Nevertheless, the expression of TLRs in granulosa cell tumors (GCTs) of the ovary, and the extent to which GCT expression of TLRs may influence cell-signaling pathways and/or modulate the efficacy of chemotherapeutics, has yet to be determined. In the present study, human GCT lines (COV434 and KGN) were utilized to evaluate expression of functional TLR4. TLR4 is expressed in GCT cell lines and ligation of TLR4 with bacterial lipopolysaccharide (LPS) led to I{kappa}B degradation and activation of NF-{kappa}B. NF-{kappa}B activation was confirmed by nuclear localization of NF-{kappa}B p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably,more » immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-{kappa}B signaling attenuated LPS-induced TNF{alpha} plus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-{kappa}B signaling failed to sensitize GCT lines to TRAIL or cisplatin. In summary, findings herein are the first to demonstrate a functional TLR-signaling pathway specifically in GCTs, and indicate that in contrast to OSE-derived cancers, inhibition of NF-{kappa}B does not sensitize GCTs to TRAIL or cisplatin.« less

Authors:
 [1];  [1];  [2];  [3]
  1. Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114 (United States)
  2. University of California, San Francisco, School of Dentistry, San Francisco, CA 94143 (United States)
  3. Center for Reproductive Biology and Health, The Pennsylvania State University, University Park, PA 16802 (United States)
Publication Date:
OSTI Identifier:
22204961
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 409; Journal Issue: 4; Other Information: Copyright (c) 2011 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; APOPTOSIS; EPITHELIUM; INFLAMMATION; INHIBITION; LIGANDS; NEOPLASMS; OVARIES; RECEPTORS; TUMOR CELLS

Citation Formats

Woods, Dori C., E-mail: dwoods2@partners.org, White, Yvonne A.R., Dau, Caroline, and Johnson, A.L. TLR4 activates NF-{kappa}B in human ovarian granulosa tumor cells. United States: N. p., 2011. Web. doi:10.1016/J.BBRC.2011.05.063.
Woods, Dori C., E-mail: dwoods2@partners.org, White, Yvonne A.R., Dau, Caroline, & Johnson, A.L. TLR4 activates NF-{kappa}B in human ovarian granulosa tumor cells. United States. doi:10.1016/J.BBRC.2011.05.063.
Woods, Dori C., E-mail: dwoods2@partners.org, White, Yvonne A.R., Dau, Caroline, and Johnson, A.L. 2011. "TLR4 activates NF-{kappa}B in human ovarian granulosa tumor cells". United States. doi:10.1016/J.BBRC.2011.05.063.
@article{osti_22204961,
title = {TLR4 activates NF-{kappa}B in human ovarian granulosa tumor cells},
author = {Woods, Dori C., E-mail: dwoods2@partners.org and White, Yvonne A.R. and Dau, Caroline and Johnson, A.L.},
abstractNote = {Highlights: {yields} TLR4 is expressed in human ovarian granulosa tumor cells. {yields} Acting through TLR4, LPS and HSP60 induce a NF{kappa}B signaling cascade in human ovarian granulosa tumor cells. {yields} NF{kappa}B activation or inhibition did not alter chemosensitivity to TRAIL or cisplatin. -- Abstract: Previous studies have demonstrated expression of Toll-like receptors (TLRs) in the surface epithelium of normal ovaries (OSE) and in epithelial ovarian tumors. Most notably, OSE-derived cancers express TLR4, which activates the nuclear factor-kappa B (NF-{kappa}B) signaling cascade as a mediator of inflammatory response. Currently, there is considerable interest in elucidating the role of TLR-mediated signaling in cancers. Nevertheless, the expression of TLRs in granulosa cell tumors (GCTs) of the ovary, and the extent to which GCT expression of TLRs may influence cell-signaling pathways and/or modulate the efficacy of chemotherapeutics, has yet to be determined. In the present study, human GCT lines (COV434 and KGN) were utilized to evaluate expression of functional TLR4. TLR4 is expressed in GCT cell lines and ligation of TLR4 with bacterial lipopolysaccharide (LPS) led to I{kappa}B degradation and activation of NF-{kappa}B. NF-{kappa}B activation was confirmed by nuclear localization of NF-{kappa}B p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably, immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-{kappa}B signaling attenuated LPS-induced TNF{alpha} plus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-{kappa}B signaling failed to sensitize GCT lines to TRAIL or cisplatin. In summary, findings herein are the first to demonstrate a functional TLR-signaling pathway specifically in GCTs, and indicate that in contrast to OSE-derived cancers, inhibition of NF-{kappa}B does not sensitize GCTs to TRAIL or cisplatin.},
doi = {10.1016/J.BBRC.2011.05.063},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 409,
place = {United States},
year = 2011,
month = 6
}
  • Aromatase is the only enzyme in vertebrates to catalyze the biosynthesis of estrogens. Although inhibitors of aromatase have been developed for the treatment of estrogen-dependent breast cancer, the whole-body inhibition of aromatase causes severe adverse effects. Thus, tissue-selective aromatase inhibitors are important for the treatment of estrogen-dependent cancers. In this study, 63 natural products with diverse structures were examined for their effects on estrogen biosynthesis in human ovarian granulosa-like KGN cells. Two compounds—trans-phytol (SA-20) and (22E)-ergosta-6,9,22-triene-3β,5α,8α-triol (SA-48)—were found to potently inhibit estrogen biosynthesis (IC{sub 50}: 1 μM and 0.5 μM, respectively). Both compounds decreased aromatase mRNA and protein expression levelsmore » in KGN cells, but had no effect on the aromatase catalytic activity in aromatase-overexpressing HEK293A cells and recombinant expressed aromatase. The two compounds decreased the expression of aromatase promoter I.3/II. Neither compound affected intracellular cyclic AMP (cAMP) levels, but they inhibited the phosphorylation or protein expression of cAMP response element-binding protein (CREB). The effects of these two compounds on extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs), and AKT/phosphoinositide 3-kinase (PI3K) pathway were examined. Inhibition of p38 MAPK could be the mechanism underpinning the actions of these compounds. Our results suggests that natural products structurally similar to SA-20 and SA-48 may be a new source of tissue-selective aromatase modulators, and that p38 MAPK is important in the basal control of aromatase in ovarian granulosa cells. SA-20 and SA-48 warrant further investigation as new pharmaceutical tools for the prevention and treatment of estrogen-dependent cancers. - Highlights: • Two natural products inhibited estrogen biosynthesis in human ovarian granulosa cells. • They inhibited aromatase transcription without affecting its catalytic activity. • They decreased the transcription or protein expression of CREB. • They inhibited p38 MAPK to exert their inhibitory effects on aromatase expression.« less
  • AngRem104 [angiotensin II (Ang II)-related genes in human mesangial cells (MCs), clone104], a novel gene in human MCs induced by Ang II, was previously identified in human MCs and found to interact with several proteins. The current study used a yeast two-hybrid system and co-immunoprecipitation to investigate the interaction between AngRem104 and glucocorticoid receptor (GR) AF-1-specific elongation factor (GR-EF). GR expression was downregulated and the number of MCs positive for activated nuclear factor {kappa}B (NF-{kappa}B) was increased when AngRem104 was overexpressed. Transfection with antisense AngRem104 vector resulted in the upregulation of GR protein and reduced numbers of MCs with activatedmore » NF-{kappa}B. These results indicate that the novel gene AngRem104 is involved in the in vivo regulation of GR expression and the activation of NF-{kappa}B through interaction with GR-EF in human MCs.« less
  • The distribution and movement of cholesterol were studied in granulosa cells from the ovaries of estrogen-stimulated hypophysectomized immature rats cultured in serum-free medium. Plasma membrane cholesterol was distinguished from intracellular cholesterol with cholesterol oxidase, an enzyme that converts cell surface cholesterol to cholestenone, leaving intracellular cholesterol untouched. Using this approach we showed that 82% of unesterified cholesterol was associated with the plasma membrane in granulosa cells cultured for 48 h in serum-free medium in both the presence and absence of added androstenedione and FSH. FSH and androstenedione stimulated a marked increase in steroid hormone (progestin) production. The movement of newlymore » synthesized cholesterol to the plasma membrane also was followed using cholesterol oxidase. Newly synthesized cholesterol reached the plasma membrane too rapidly to be measured in unstimulated cells (t1/2 less than 20 min); however, in cells stimulated by FSH and androstenedione, this rate was considerably slower (t1/2 approximately 2h). Therefore, cholesterol movement to the plasma membrane appears to be regulated by gonadotropins in these cells. We tested whether steroid biosynthesis used all cell cholesterol pools equally. To this end we administered (3H)acetate and (14C)acetate at different times and determined their relative specific contents in various steroids after defined intervals. The relative ages of the steroids (youngest to oldest) were: lanosterol, progestins, intracellular cholesterol, and plasma membrane cholesterol. This finding suggests that progestins use newly synthesized intracellular cholesterol in preference to preexisting intracellular or cell surface cholesterol.« less
  • Highlights: {yields} Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression. {yields}Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom. {yields} Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. {yields}Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. {yields} The expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. -- Abstract: Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number ofmore » granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.« less
  • Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6more » μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.« less