Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation

Abstract

Progestin-only long-acting reversible-contraceptive (pLARC)-exposed endometria displays decidualized human endometrial stromal cells (HESCs) and hyperdilated thin-walled fragile microvessels. The combination of fragile microvessels and enhanced tissue factor levels in decidualized HESCs generates excess thrombin, which contributes to abnormal uterine bleeding (AUB) by inducing inflammation, aberrant angiogenesis, and proteolysis. The- zinc finger and BTB domain containing 16 (ZBTB16) has been reported as an essential regulator of decidualization. Microarray studies have demonstrated that ZBTB16 levels are induced by medroxyprogesterone acetate (MPA) and etonogestrel (ETO) in cultured HESCs. We hypothesized that pLARC-induced ZBTB16 expression contributes to HESC decidualization, whereas prolonged enhancement of ZBTB16 levels triggers an inflammatory milieu by inducing pro-inflammatory gene expression and tissue-factor-mediated thrombin generation in decidualized HESCs. Thus, ZBTB16 immunostaining was performed in paired endometria from pre- and post-depo-MPA (DMPA)-administrated women and oophorectomized guinea pigs exposed to the vehicle, estradiol (E₂), MPA, or E₂ + MPA. The effect of progestins including MPA, ETO, and levonorgestrel (LNG) and estradiol + MPA + cyclic-AMP (E₂ + MPA + cAMP) on ZBTB16 levels were measured in HESC cultures by qPCR and immunoblotting. The regulation of ZBTB16 levels by MPA was evaluated in glucocorticoid-receptor-silenced HESC cultures. ZBTB16 was overexpressed in cultured HESCs for 72 h followed by a ± 1 IU/mL thrombin treatment for 6 h. DMPA administration in women and MPA treatment in guinea pigs enhanced ZBTB16 immunostaining in endometrial stromal and glandular epithelial cells. The in vitro findings indicated that: (1) ZBTB16 levels were significantly elevated by all progestin treatments; (2) MPA exerted the greatest effect on ZBTB16 levels; (3) MPA-induced ZBTB16 expression was inhibited in glucocorticoid-receptor-silenced HESCs. Moreover, ZBTB16 overexpression in HESCs significantly enhanced prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP1), and tissue factor (F3) levels. Thrombin-induced interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels in control-vector-transfected HESCs were further increased by ZBTB16 overexpression. In conclusion, these results supported that ZBTB16 is enhanced during decidualization, and long-term induction of ZBTB16 expression by pLARCs contributes to thrombin generation through enhancing tissue factor expression and inflammation by enhancing IL-8 and PTGS2 levels in decidualized HESCs.

Keywords: COX-2; IL-8; ZBTB16; decidualization; endometrial stromal cells; progestin-only contraceptives; tissue factor.

Figure 1

Endometrial stromal and glandular cells display enhanced ZBTB16 immunoreactivity in women administered depo-medroxyprogesterone acetate (DMPA). (A) Representative ZBTB16 immunostaining (brown) in paraffin sections from paired endometria obtained from pre- and 3 months post-DMPA administration. Original magnification ×40. Enhanced ZBTB16 immunoreactivity was observed in post-DMPA endometrial compared to pre-DMPA. A strong ZBTB16 immunoreactivity is seen in post-DPMA vascular endothelium (arrows) in the inset micrographs. Original magnification ×100. (B) HSCOREs for ZBTB16 immunoreactivity confirmed significantly higher ZBTB16 immunoreactivity in both endometrial stromal (185.4 ± 9.2 vs. 92.1 ± 13.8) and glandular cells (181.3 ± 20.4 vs. 47.7 ± 14.7), as well as endothelial cells (185.3 ± 19.9 vs. 45.4 ± 16.3; p < 0.001) in post- vs. pre-DMPA endometria. Bars represent the mean ± SEM; n = 7/each; *** p < 0.001 vs. pre-DMPA analyzed by the t-test.

Figure 2

MPA administration induces endometrial ZBTB16 expression in guinea pigs. (A) Representative images of ZBTB16 immunostaining in endometria obtained from ovariectomized guinea pigs treated 21 days with placebo as the control (Cont; n = 5), or estradiol (E₂; n = 4), or medroxyprogesterone acetate (MPA; n = 5), or E₂ + MPA (n = 6). Original magnification: ×20. The insert represents negative control staining. (B) HSCORE analysis of ZBTB16 immunoreactivity in stromal and glandular cells of endometria. Bars represent the mean ± SEM; * p < 0.05 vs. the control or E₂ treatment in stromal cells, and * p < 0.05 vs. the control or E₂ treatment in glandular epithelial cells analyzed by one-way ANOVA followed by the Holm–Sidak method.

Figure 3

Increased ZBTB16 expression during decidualization of cultured HESCs. (A) ZBTB16 mRNA levels in HESCs treated with 10⁻⁸ M E₂ + 10⁻⁷ MPA + 5 × 10⁻⁵ cyclic AMP (EMC) for 0, 3, or 6 days by qPCR. The data represent the fold change as the mean ± SEM; n = 5/each; *** p < 0.001 vs. Day 0 analyzed by one-way ANOVA followed by the Student–Newman–Keuls method. (B) MPA mediated upregulation of ZBTB16 mRNA levels in HESCs treated with vehicle (control; Cont) or E₂ or MPA or cAMP for 6 days. Bars represent mean ± SEM; n = 4/each; * p < 0.05 vs. Cont or E₂ or cAMP analyzed by one way ANOVA followed by Student-Newman-Keuls method.

Figure 4

pLARCs induce ZBTB16 mRNA and protein levels in cultured HESCs. ZBTB16 mRNA (A) and protein (B) levels were analyzed by qPCR and immunoblotting, respectively, in HESCs treated with 10⁻⁸ M E₂ ± 10⁻⁷ M ORG, ETO, LNG, MPA, or DEX for 7 days. Bars represent the mean ± SEM; (A) n = 4 for mRNA fold change; * p < 0.05 vs. E₂ alone; and (B) n = 3 for protein levels after normalization to β-actin; * p < 0.05 vs. E₂ alone analyzed by one-way ANOVA followed by the Student–Newman–Keuls method. E₂: estradiol, ORG: Organon 2058, ETO: etonogestrel, LNG: levonorgestrel, MPA: medroxyprogesterone acetate, DEX: dexamethasone. (C) NR3C1, ZBTB16, and FKBP5 mRNA levels in HESCs transfected with either the nonspecific control (Cont) or NR3C1-specific siRNA and treated with EMC for 3 days. Bars represent the mean ± SEM; n = 3; *** p < 0.001 vs. control siRNA by the t-test.

Figure 5

ZBTB16 overexpression induces decidualization markers, as well as F3, IL-8, and PTGS2 levels in HESCs. (A) Decidualization markers’, PRL and IGFBP1, mRNA levels in ZBTB16-vector (ZBTB16-v) or control-vector (Cont-v)-transfected HESCs treated with or without 10⁻⁸ M estradiol + 10⁻⁷ M medroxyprogesterone acetate + 5 × 10⁻⁵ M cAMP (EMC) for 3 days. Bars represent the mean ± SEM; n = 4; *** p < 0.05 vs. control-v-EMC; ⁺ p < 0.05 vs. ZBTB16-v + EMC. (B) Tissue factor (F3) mRNA levels in control- (Cont-v) or ZBTB16-vector (ZBTB16-v)-transfected HESCs treated with or without EMC for 3 days ± 1 U/mL of thrombin (THR) for 6 h. Bars represent the mean ± SEM; n = 4; * p < 0.05 vs. Cont-v or ZBTB16-v; ⁺ p < 0.05 vs. Cont-v; ^# p < 0.05 vs. Cont-v + THR. (C) Interleukin 8 (IL-8) and prostaglandin endoperoxide synthase 2 (PTGS2 aka cyclooxygenase 2; COX2) mRNA levels in the control- or ZBTB16-vector-transfected HESCs treated with EMC for 3 days ± 1 U/mL THR for 6 h. Bars represent the mean ± SEM; n = 4; * p < 0.05 vs. Cont-v or ZBTB16-v; ⁺ p < 0.05 vs. Cont-v; ^# p < 0.05 vs. Cont-v + THR. The data were analyzed by one-way ANOVA followed by the Student–Newman–Keuls method.

Figure 6

Schematic demonstration of the role of elevated ZBTB16 expression in the pathogenesis of pLARC-induced AUB. Administration of progestin-only, long-acting, reversible contraception (pLARCs) reduce uterine blood flow, which causes local hypoxia [8,9] and induces HESC decidualization, which increases tissue factor [15,23] and ZBTB16 levels. Increased ZBTB16 levels induce excess tissue factor expression, which generates thrombin. The resulting excess thrombin binds to protease-activated receptors (PARs), which increase the expression of several inflammatory factors and angiogenic factors, such as VEGF or IL-8 [19,20,48]. These factors promote excess angiogenesis, which results in abnormal uterine bleeding. pLARC-induced excess ZBTB16 exacerbates thrombin-induced endometrial angiogenesis and inflammation by increasing IL8 and PTGS2 levels in HESCs.