title: The AhR is constitutively activated and affects granulosa cell features in the human cell line KGN.
abstract: A well-balanced activity of the aryl hydrocarbon receptor (AhR) is necessary for normal ovarian function. As known from murine AhR knock-out (KO) models, the AhR is involved in folliculogenesis, gonadotrophin receptor expression, proliferation of granulosa cells and intraovarian estrogen signalling. Highly potent, non-physiological ligands such as the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) lead to a blockade of ovulation, estrogen receptor degradation and reduction of estrogen levels. Estrogen synthesis is a typical function of granulosa cells and essential for normal cyclicity and fertility. We employed the human granulosa cell line KGN to further characterize AhR signalling and AhR function in granulosa cell physiology. Real-time PCR quantification of the target genes Cyp1a1 and Cyp1b1 and reporter gene assays after stimulation with TCDD or beta-naphthoflavone (BNF) or inhibition with alpha-naphthoflavone (ANF) or 3'-methoxy-4'-nitroflavone (3,4-MNF) of the AhR demonstrated constitutive activity and functionality of AhR pathway in KGN granulosa cells. In untreated KGN cells, AhR protein was exclusively detected in the nuclear fraction. TCDD stimulation affected the gonadotrophin receptor but not estrogen receptor β (ERβ) protein expression. Additionally, the constitutively activated AhR suppressed aromatase expression and estrogen synthesis (enzyme-linked immunoassay, ELISA) and enhanced proliferation [Bromodeoxyuridine (BrdU) ELISA] of KGN cells. Activation of the AhR by BNF did not override this inhibitory effect on estrogen synthesis or proliferation. In conclusion, the AhR pathway is constitutively activated and functional in human KGN granulosa cells. It is a potential target for endocrine disruption by exogenous ligands and subsequent dysfunction of granulosa cells.
authors.affiliation: Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Halle (Saale), Germany. k.horling@uke.uni-hamburg.de
authors.forename: Katja
authors.initials: K
authors.lastname: Horling
authors.forename: Anne Navarrete
authors.initials: AN
authors.lastname: Santos
authors.forename: Bernd
authors.initials: B
authors.lastname: Fischer
mesh_terms: D001141:Aromatase / Q000235:genetics; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D001571:Benzoflavones / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics; D065633:Cytochrome P-450 CYP1B1; D004797:Enzyme-Linked Immunosorbent Assay; D004967:Estrogens / Q000096:biosynthesis; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D010053:Ovary / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D018336:Receptors, Aryl Hydrocarbon / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D011967:Receptors, Gonadotropin / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019324:beta-Naphthoflavone / Q000494:pharmacology
chemical_list: C091768:3'-methoxy-4'-nitroflavone; D001571:Benzoflavones; D004967:Estrogens; D005419:Flavonoids; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011960:Receptors, Estrogen; D011967:Receptors, Gonadotropin; C011512:alpha-naphthoflavone; D019324:beta-Naphthoflavone; D001141:Aromatase; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1