Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype

Abstract

Polycystic ovary syndrome (PCOS), characterized by increased ovarian androgen biosynthesis, anovulation, and infertility, affects 5-7% of reproductive-age women. Genome-wide association studies identified PCOS candidate loci that were replicated in subsequent reports, including DENND1A, which encodes a protein associated with clathrin-coated pits where cell-surface receptors reside. However, these studies provided no information about functional roles for DENND1A in the pathogenesis of PCOS. DENND1A protein was located in the cytoplasm as well as nuclei of theca cells, suggesting a possible role in gene regulation. DENND1A immunostaining was more intense in the theca of PCOS ovaries. Using theca cells isolated and propagated from normal cycling and PCOS women, we found that DENND1A variant 2 (DENND1A.V2) protein and mRNA levels are increased in PCOS theca cells. Exosomal DENND1A.V2 RNA was significantly elevated in urine from PCOS women compared with normal cycling women. Forced overexpression of DENND1A.V2 in normal theca cells resulted in a PCOS phenotype of augmented CYP17A1 and CYP11A1 gene transcription, mRNA abundance, and androgen biosynthesis. Knock-down of DENND1A.V2 in PCOS theca cells reduced androgen biosynthesis and CYP17A1 and CYP11A1 gene transcription. An IgG specific to DENND1A.V2 also reduced androgen biosynthesis and CYP17 and CYP11A1 mRNA when added to the medium of cultured PCOS theca cells. We conclude that the PCOS candidate gene, DENND1A, plays a key role in the hyperandrogenemia associated with PCOS. These observations have both diagnostic and therapeutic implications for this common disorder.

Fig. 1.

Western analyses of DENND1A.V2 in normal and PCOS theca cells. (A) Representative Western analysis of ∼62-kDa DENND1A.V2 and ∼112-kDa DENND1A.V1 in whole-cell extracts from normal and PCOS theca cells treated in the absence (−) and presence (+) of 20 μM forskolin. Total mTOR was used for protein normalization. (B) Quantitative Western data from theca cells isolated from five normal cycling and five PCOS subjects, presented as the mean ± SEM, demonstrated that DENND1A.V2 protein was increased in both basal and forskolin-stimulated (*P < 0.01) PCOS theca cells compared with normal theca cells. (C) DENND1A.V1 was increased by forskolin treatment in normal theca cells (*P < 0.01). Forskolin-stimulated DENND1A.V1 was decreased in PCOS theca cells as compared normal cells (**P < 0.01). (D) The ratio of DENND1A.V2/V1 was (*P < 0.01) increased in PCOS theca cells under control and forskolin-stimulated conditions.

Fig. 2.

Imunnohistochemical localization of DENND1A protein in normal cycling and PCOS ovary. (A) DENND1A protein was localized in the theca interna of the ovarian follicles, and was increased in PCOS theca (Lower) compared with normal theca (Upper). (B) DENND1A staining in PCOS theca and granulosa cells (Left, 40× magnification). Staining, primarily in the PCOS theca cell nuclei, cytoplasm, and cell membrane (Right, 100× magnification, under oil).

Fig. 3.

DENND1A.V2 mRNA accumulation is greater in PCOS theca cells and is correlated with DHEA production. (A) DENND1A.V2 mRNA abundance was compared in theca cells propagated from six individual normal and six individual PCOS women that were treated in the absence (C) and presence (F) of 20 μM forskolin. DENND1A.V2 mRNA was increased under basal (*P < 0.05) and forskolin (**P < 0.01)-stimulated conditions in PCOS theca cells, compared with normal cells. (B) Control (C) and forskolin (F)-stimulated DHEA accumulation in the six normal and six PCOS women’s theca cell preparations were compared with DENND1A.V2 mRNA under the same conditions.

Fig. 4.

Exosomal DENND1A.V2 RNA in urine from normal cycling and PCOS women. Comparison of DENND1A.V2 RNA accumulation in exosomal mRNA purified and isolated from midday urine obtained from five normal cycling and six PCOS women (*P < 0.001) using qRT-PCR analysis.

Fig. 5.

Forced expression of DENND1A.V2 in normal theca cells results in augmented androgen and progestin production. (A) DHEA production following infection of normal theca cells, with 0.3, 1.0, 3.0, and 10 pfu per cell of either empty (Null) or DENND1A.V2 (DENN.V2) adenovirus, treated in the absence (C) or presence (F) of 20 μM forskolin for 72 h. (B) Quantitative Western analysis following infection of normal theca cells with 3 pfu Null or DENND1A.V2 adenovirus to confirm DENND1A.V2 protein expression. (C) DHEA, (D) 17OHP4, (E) T, and (F) Progesterone biosynthesis in normal theca cells infected with either 3 pfu per cell of DENND1A.V2 or control, Null adenovirus and treated in the absence (C) or presence of (F) of 20 μM forskolin for 72 h. DENND1A.V2 infection increased basal 17OHP4 (*P < 0.01), T (*P < 0.05), and P4 (*P < 0.05) accumulation compared with control Null adenovirus. DENND1A.V2 infection also increased forskolin-stimulated DHEA (*P < 0.001), 17OHP4 (**P < 0.001), and P4 (**P < 0.001) compared with control Null adenovirus.

Fig. 6.

Forced expression of DENND1A.V2 in normal theca cells results in augmented CYP17A1 and CYP11A1 expression. To examine the effects of DENND1A.V2 on (A) CYP17 and (B) CYP11A1 mRNA accumulation, normal theca cells were infected either 3 pfu per cell of DENND1A.V2, or control, Null adenovirus and treated in the absence (C) or presence of (F) of 20 μM forskolin. DENND1A.V2 infection increased forskolin-stimulated (A) CYP17 mRNA (*P < 0.01) and (B) CYP11A1 mRNA (*P < 0.05) accumulation. (C) To examine the effects of DENND1A.V2 on CYP17A1 transcription, normal theca cells were transfected with a CYP17A1 promoter gene plasmid (−770 CYP17A1/LUC), and infected with DENND1A V2 or Null adenovirus. DENND1A.V2 infection increased both basal (*P < 0.05) and forskolin-stimulated (**P < 0.05) −770 CYP17A1 promoter activity, compared with Null control adenovirus. (D) The CYP11A1 promoter construct (−160/−90 CYP11A1/LUC) was used to examine the effects of DENND1A.V2 on CYP11A1 transcription in theca cells. Normal theca cells were transfected with the −160/−90 CYP11A1/LUC plasmid and a DENND1A.V2/pCMV-XL4 or control pCMV-XL4 plasmid. DENND1A.V2 increased forskolin-stimulated (*P < 0.001) −160/−90 CYP11A1/LUC promoter activity compared with empty plasmid.

Fig. 7.

Knock-down of DENND1A.V2 in PCOS theca cells results in a reduction in CYP17A1 and CYP11A1 expression, and decreased androgen and progestin biosynthesis. (A) Transfection of PCOS theca cells with silencing DENND1A.V2 (DENN.V2) shRNA1 and shRNA2 plasmids inhibited both basal (P < 0.05) and 20 μM forskolin (*P < 0.01) -stimulated CYP17 mRNA accumulation, compared with scrambled plasmid. (B) The DENND1A.V2 shRNA plasmids also inhibited forskolin-stimulated (*P < 0.05) CYP11A1 mRNA accumulation in PCOS theca cells. (C) Cotransfection of −235/+44 of the CYP17A1 promoter fused to the luciferase gene in a pGL3 plasmid (−235 CYP17A1/LUC) with DENND1A.V2 shRNA1 and shRNA2 plasmids resulted in an inhibition of forskolin-dependent CYP17A1 reporter activity in PCOS theca cells, compared with scrambled shRNA (*P < 0.05). (D–F) Infection with silencing shRNA DENND1A.V2 lentivirus particles inhibited forskolin-stimulated (D) 17OHP4 (*P < 0.001), (E) DHEA (*P < 0.001), and (F) Progesterone biosynthesis (*P < 0.001), compared with control nonsilencing lentivirus.

Fig. 8.

Anti-DENND1A.V2 IgG reduces androgen biosynthesis and CYP17 and CYP11A1 mRNA PCOS theca cells. (A) PCOS theca cells were treated with increasing concentrations (0.1–3.0 μg/mL) of anti-DENND1A.V2 IgG or nonspecific IgG, in the absence (C) or presence of 20 μM forskolin (F). Anti-DENND1A.V2 IgG inhibited forskolin-stimulated DHEA (*P < 0.01) with an approximate ID₅₀ of 0.25 μg/mL, compared with nonspecific IgG. (B and C) Experiments were performed to examine the effects of 0.5 μg/mL of anti-DENND1A.V2 IgG (DENNN.V2) or 0.5 μg/mL nonspecific IgG on (B) CYP17 and (C) CYP11A1 mRNA (Fig. 7C) accumulation in the absence (C) or presence of 20 μM forskolin (F), demonstrated that anti-DENND1A.V2 IgG inhibits CYP17 mRNA accumulation under control (*P < 0.01) and CYP17 mRNA and CYP11A1 mRNA under forskolin-stimulated (**P < 0.01) conditions in PCOS theca cells, but had no effect in normal theca cells. (D–F) Parallel experiments to examine the effects of 0.5 μg/mL of anti-DENND1A.V2 IgG or control IgG on basal and forskolin-stimulated (D) DHEA (E) 17OHP4, and (F) P4 biosynthesis similarly demonstrated that anti-DENND1A.V2 IgG-inhibited forskolin-stimulated DHEA (*P < 0.001) and 17OHP4 (*P < 0.001), in PCOS theca cells by 50% compared with control IgG, without affecting normal theca cells.