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. 2022 May 13;6(7):bvac078.
doi: 10.1210/jendso/bvac078. eCollection 2022 Jul 1.

The PCOS GWAS Candidate Gene ZNF217 Influences Theca Cell Expression of DENND1A.V2, CYP17A1, and Androgen Production

Jamaia S Waterbury  1 , Maria E Teves  2 , Alison Gaynor  3 , Angela X Han  1 , Grace Mavodza  4 , Jordan Newell  1 , Jerome F Strauss 3rd  2 , Jan M McAllister  1
Affiliations

The PCOS GWAS Candidate Gene ZNF217 Influences Theca Cell Expression of DENND1A.V2, CYP17A1, and Androgen Production

Jamaia S Waterbury et al. J Endocr Soc. .

Abstract

Polycystic ovary syndrome (PCOS), a common endocrine disorder of women, is characterized by increased ovarian androgen production and anovulatory infertility. Genome-wide association studies (GWAS) have identified more than 20 PCOS candidate loci. One GWAS candidate locus encompasses ZNF217, a zinc finger transcription factor. Immunohistochemical staining of ovarian tissue demonstrated significantly lower staining intensity for ZNF217 protein in PCOS theca interna compared to ovarian tissue from normal ovulatory women. Immunofluorescence staining of normal and PCOS theca cells demonstrated nuclear localization of ZNF217, with lower intensity in PCOS cells. Western blotting showed reduced ZNF217 protein in PCOS theca cells compared to normal theca cells, and that treatment with forskolin, which mimics the action of luteinizing hormone (LH), reduces ZNF217 expression. Lower ZNF217 expression in PCOS theca cells was confirmed by quantitative reverse transcription polymerase chain reaction. Notably, there was an inverse relationship between ZNF217 messenger RNA (mRNA) levels and theca cell androgen (dehydroepiandrosterone; DHEA) synthesis. The abundance of mRNA encoding a splice variant of DENND1A (DENND1A.V2), a PCOS candidate gene that positively regulates androgen biosynthesis, was also inversely related to ZNF217 mRNA levels. This relationship may be driven by increased miR-130b-3p, which targets DENND1A.V2 transcripts and is directly correlated with ZNF217 expression. Forced expression of ZNF217 in PCOS theca cells reduced androgen production, CYP17A1 and DENND1A.V2 mRNA, while increasing mIR-130b-3p. Conversely, knockdown of ZNF217 in normal theca cells with short hairpin RNA-expressing lentivirus particles increased DENND1A.V2 and CYP17A1 mRNA. These observations suggest that ZNF217 is part of a network of PCOS candidate genes regulating thecal cell androgen production involving DENND1A.V2 and miR-130b-3p.

Keywords: CYP17A1; DENND1A.V2; ZNF217; miR-130b-3p; polycystic ovary syndrome; theca cells.

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Figures

Figure 1.
Figure 1.
A, ZNF217 immunohistochemical staining of normal ovarian tissue (right) and polycystic ovary syndrome (PCOS) ovarian tissue (left). Images were taken at 40× magnification. In comparison to the theca cells of the normal cycling ovary, ZNF217 protein expression was decreased in theca layer of follicles of PCOS ovarian specimens. Antibody neutralized with the ZNF217 immunogenic peptide was used in the bottom 2 images to confirm specificity of the immunoperoxidase signal. B, Comparison of ZNF217 staining intensity in the designated region of interest (ROI, theca interna) revealed a significant decrease in the theca cell layer of PCOS ovarian tissue (N = 4) compared to normal ovarian tissue (N = 4); *P < .001.
Figure 2.
Figure 2.
ZNF217 expression in normal and polycystic ovary syndrome (PCOS) theca cells. A, ZNF217 (green signal) is localized in punctate nuclear structures in human theca cells by immunofluorescence. The ZNF217 signal is reduced in PCOS theca cells compared to normal theca cells cultured without forskolin, and in normal theca cells treated with forskolin (20 μM, 24 hours). B, Representative Western blots comparing ZNF217 and DENND1A.V2 protein expression in 2 normal and 2 PCOS theca cell preparations cultured in the C, absence, or F, presence of forskolin (20 μM) for 24 hours. C, Quantitative Western blot data from 5 normal and 5 PCOS preparations demonstrated that ZNF217 protein was decreased by forskolin treatment in normal theca cells (αP < .001). ZNF217 protein in PCOS theca cells was decreased compared to normal theca cells (***P < .001) under control culture conditions. D, Relative DENND1A.V2 protein normalized by mammalian target of rapamycin (mTOR) was elevated both in forskolin-treated (***P < .005) and untreated (***P < .005) PCOS cells.
Figure 3.
Figure 3.
ZNF217 abundance in normal and polycystic ovary syndrome (PCOS) theca cells and the relationship between ZNF217 messenger RNA (mRNA) to dehydroepiandrosterone (DHEA) accumulation, DENND1A.V2, CYP17A1, and miR-130b-3p expression. Individual theca cell preparations from normal (n = 5) and PCOS (n = 5) women were cultured in the absence or presence of forskolin (20 μM). For quantitative reverse transcription polymerase chain reaction, mRNA was harvested from the cells 16 hours following treatment and ZNF217, CYP17A1, DENND1A.V2, miR-130b-3p expression was quantified. In parallel cultures of normal and PCOS theca cells, conditioned medium was collected at 48 hours and DHEA biosynthesis was assayed. A, PCOS theca cells showed decreased expression of ZNF217 mRNA compared to normal theca cells both in forskolin-treated (***P < .001) and untreated cultures (**P < .001. In normal theca cells, the addition of forskolin significantly decreased ZNF217 mRNA (*P < .01). B, ZNF217 mRNA abundance compared to DHEA production. PCOS theca cells cultured in the absence or presence of forskolin showed increased DHEA production and decreased ZNF217 mRNA expression compared to normal theca cells. There was an inverse negative correlation between ZNF217 mRNA expression and DHEA production (Spearman ρ = –0.7353; P < .001). C, CYP17A1 gene expression in PCOS theca cells, required for androgen biosynthesis in PCOS, was negatively correlated with decreased ZNF217 mRNA abundance (Spearman ρ = –0.67890; P = .001). D, Comparison of ZNF217 mRNA expression to DENND1A.V2 mRNA abundance in PCOS and normal theca cells treated without or with forskolin revealed an inverse negative correlation (Spearman ρ = –0.6992; P < .001). E, miR-130b-3p, which targets DENND1A.V2 transcripts, was positively correlated with ZNF217 expression (Spearman ρ = 0.7815; P < .001).
Figure 4.
Figure 4.
Forced expression of ZNF217 in polycystic ovary syndrome (PCOS) theca cells converts PCOS theca cells to a normal phenotype of reduced DENND1A.V2, CYP17A1, and dehydroepiandrosterone (DHEA) biosynthesis using a ZNF217 adenovirus (Adv). PCOS theca cells (N=5) were infected with either 3 pfu/cell of Null (empty) vector adenovirus (Null Adv) or ZNF217 adenovirus (ZNF217 Adv) overnight, then treated in the absence (Control) and presence of forskolin (20 μM) for 36 hours for RNA studies or 72 hours for DHEA studies. A, ZNF217 Adv infection augmented ZNF217 messenger RNA (mRNA) expression under unstimulated (control, ***P < .0001) and forskolin-stimulated conditions (**P < .001). ZNF217 Adv infection following forskolin stimulation resulted in decreased ZNF217 mRNA compared to control untreated cells (αP < .01). B, Following ZNF217 Adv infection, PCOS theca cells were converted to a normal phenotype of diminished DENND1A.V2 mRNA accumulation, under both control (**P < .001) and forskolin-stimulated conditions (**P < .001), compared to Null Adv (control) infected cells. C, CYP17A1 mRNA accumulation was increased following forskolin-treatment (αP < .01) in Null Adv–infected cells. In addition, ZNF217 Adv infection dramatically decreased CYP17A1 mRNA accumulation under both control (**P < .001) and forskolin-stimulated conditions (**P < .001). D, At 72 hours following infection with Null Adv, DHEA accumulation was observed to be increased by forskolin treatment (αP < .01). ZNF217 Adv infection resulted in decreased DHEA biosynthesis under both control (**P < .001) and forskolin-stimulated cells (**P < .001). E, Forced ZNF217 overexpression with ZNF217 Adv, augmented miR-130b-3p expression under unstimulated (control, **P < .001) and forskolin-stimulated conditions (*P < .1). ZNF217 Adv infection following forskolin stimulation resulted in decreased ZNF217 mRNA compared to control untreated cells (αP < .01).
Figure 5.
Figure 5.
ZNF217 knockdown in normal theca cells results in augmented DENND1A.V2 and CYP17A1 messenger RNA (mRNA) expression. Normal theca cells (N=5) were infected with equivalent amounts of control short hairpin RNA (shRNA) or ZNF217 shRNA lentiviral particles (3 pmol/106 cells) for 24 hours, then treated for 36 hours in the absence (control) or presence of 20 μM forskolin. A, In the absence of forskolin, ZNF217 expression was decreased following infection with ZNF217 lentiviral shRNA (*P < .05) as compared control shRNA. Forskolin treatment inhibited ZNF217 mRNA in control shRNA-infected cells (αP < .05). B, DENND1A.V2 expression was increased following ZNF217 shRNA lentivirus infection under both control (***P < .001) and forskolin-stimulated (***P < .001) conditions. C, CYP17 A1 mRNA was increased by forskolin-stimulation in normal theca cells infected with control shRNA (αP < .01). CYP17 A1 mRNA was increased following ZNF217 shRNA lentivirus infection under both control (*P < .01) and forskolin-stimulated (**P < .001) conditions, compared to control shRNA-infected cells.
Figure 6.
Figure 6.
A polycystic ovary syndrome (PCOS) genome-wide association study (GWAS) candidate gene network encompassing ZNF217 and DENND1A.V2. PCOS is a manifestation of altered expression of a network of PCOS GWAS candidate genes including LHCGR, ZNF217, DENND1A, and RAB5B. Decreased ZNF217 expression in PCOS theca cells is associated with increased DENND1A.V2 and CYP17A1 expression, and downstream, androgen biosynthesis. Alternatively, ZNF217 could indirectly influence DENND1A.V2, RAB5B, and subsequently CYP17A1 expression, through the intermediacy of miR-130b-3p.
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