Krüppel-like factor 9 and progesterone receptor coregulation of decidualizing endometrial stromal cells: implications for the pathogenesis of endometriosis

Abstract

Context: Endometriosis is characterized by progesterone resistance and associated with infertility. Krüppel-like factor 9 (KLF9) is a progesterone receptor (PGR)-interacting protein, and mice null for Klf9 are subfertile. Whether loss of KLF9 expression contributes to progesterone resistance of eutopic endometrium of women with endometriosis is unknown.

Objective: The aims were to investigate 1) KLF9 expression in eutopic endometrium of women with and without endometriosis, 2) effects of attenuated KLF9 expression on WNT-signaling component expression and on WNT inhibitor Dickkopf-1 promoter activity in human endometrial stromal cells (HESC), and 3) PGR and KLF9 coregulation of the stromal transcriptome network.

Methods: Transcript levels of KLF9, PGR, and WNT signaling components were measured in eutopic endometrium of women with and without endometriosis. Transcript and protein levels of WNT signaling components in HESC transfected with KLF9 and/or PGR small interfering RNA were analyzed by quantitative RT-PCR and Western blot. KLF9 and PGR coregulation of Dickkopf-1 promoter activity was evaluated using human Dickkopf-1-luciferase promoter/reporter constructs and by chromatin immunoprecipitation. KLF9 and PGR signaling networks were analyzed by gene expression array profiling.

Results: Eutopic endometrium from women with endometriosis had reduced expression of KLF9 mRNA together with those of PGR-B, WNT4, WNT2, and DKK1. KLF9 and PGR were recruited to the DKK1 promoter and modified each other's transactivity. In HESC, KLF9 and PGR coregulated components of the WNT, cytokine, and IGF gene networks that are implicated in endometriosis and infertility.

Conclusion: Loss of KLF9 coregulation of endometrial stromal PGR-responsive gene networks may underlie progesterone resistance in endometriosis.

Fig. 1.

Differentially expressed genes in eutopic endometrium of women with endometriosis. Transcript levels of PGR (panel A), PGR-B (panel B), WNT4 (panel C), WNT2 (panel D), DKK1 (panel E), and KLF9 (panel F) in eutopic endometrium of patients with (E) and without (N) endometriosis at different phases of the menstrual cycle were quantified by QPCR and normalized to that of 18S RNA. Each bar represents the mean ± sem of at least four samples per type per menstrual phase: PE, early secretory endometrium (ESE), MSE, and late-secretory endometrium (LSE). Significant differences (P < 0.05) between N and E were determined using two-way ANOVA followed by Tukey's test. *, P < 0.05 between N and E for each menstrual cycle phase by Student's t test.

Fig. 2.

Effects of KLF9 and PGR on expression of WNT signaling components in human endometrial stromal cells. A, Expression levels of genes in decidualizing HESC. Control (vehicle) and cAME-treated cells (2 d) were analyzed for gene expression by QPCR. Values (n = 6 wells per treatment group) were normalized to 18S and renormalized to vehicle-treated group. Transcript levels (mean ± sem) are expressed as fold change. *, P < 0.05 between vehicle- and cAME-treated groups using Student's t test. B, Representative Western blots of nuclear KLF9 and PGR protein levels from HESC treated with cAME for 2 d in the presence of nontargeting siRNA (NT), KLF9 siRNA (siKLF9) and PGR siRNA (siPGR). Lamin A served as loading control. C–E, Transcript levels (mean ± sem) of WNT4, WNT2, and DKK1 were analyzed by QPCR, normalized to 18S, and renormalized to the NT group. Values with different letters are significantly different at P < 0.05 by one-way ANOVA. F, Representative Western blots of cytoplasmic DKK1 protein from HESC treated with cAME for 2 d in the presence of nontargeting RNA (NT), KLF9 siRNA (siKLF9), PGR siRNA (siPGR), or si(KLF9+PGR) (siKP). β-Actin served as loading control.

Fig. 3.

KLF9 and PGR regulate DKK1 gene promoter activity and are recruited to the DKK1 promoter region. A, Schematic representation of human DKK1 promoter region showing the locations of the generated promoter-reporter constructs: −2238 to +112, −221 to +136, and −35 to +136; 1 refers to the translation initiation site. B, HESC cotransfected with 0.5 μg promoter-reporter plasmids or pGL3-basic (negative control) were treated with cAME. Luciferase activity of cell lysates (mean ± sem from two independent experiments, n = 4 per group per experiment) is expressed as relative luminescence units (RLU) normalized to the protein content. Significant differences among groups were identified by one-way ANOVA. Means with different letters differed at P < 0.05. C, Luciferase activity of the two promoter-reporter constructs (−2238/+112 and −221/+136) containing regions of the DKK1 promoter were cotransfected in HESC in the presence or absence of siRNA for KLF9, PGR, or siKLF9+PGR (siKP) and then treated with cAME. Luciferase activities were expressed as relative luminescence units (RLU) normalized to protein content. Significant differences were identified by one-way ANOVA, followed by Tukey's test. Means with different letters differed at P < 0.05. D, Map representation (not drawn to scale) of the DKK1 promoter region containing GC-rich (GGGAGG, CCTCCC), SP1 (GGGCGG, CACCC, GGGTG) and possible half-PRE/GRE (TGTTGT, TGTTTT, AGAACA) sites amplified by primer sets A and B (denoted by arrows, → ←). ChIP assays were performed with chromatin prepared from vehicle and cAME-treated HESC using anti-KLF9, anti-PGR, and anti-SP1 antibodies. Precipitated DNA was analyzed by PCR. Representative gels using primer set A (left) and primer set B (right) from one of three independent experiments using cells of similar passages are shown. Preimmune IgG was evaluated similar to the other antibodies and served as negative control.

Fig. 4.

Expression profiling of genes in HESC with PGR knockdown and with KLF9+PGR co-knockdowns. A, Venn diagram showing shared and unique genes in cAME-treated HESC between the pair-wise comparisons: 1) si(KLF9+PGR) (siKP) vs. nontargeting siRNA (NT) and 2) siPGR alone vs. NT. Only genes differing by at least 1.5-fold (P < 0.05) are included in the analyses. B, Heat map representation of transcripts perturbed by siKP that were previously reported to be deregulated in patients with endometriosis. Red indicates up-regulated; blue, down-regulated; and yellow, unchanged. C, QPCR analyses of representative genes differentially regulated by PGR (siPGR) and KLF9+PGR (siKP). D and E, Heat map representation of several transcripts associated with IL-6/IL-8/IL-17 (D) and IGF/ERK/MAPK (E) signaling pathways and whose expression levels are distinctly deregulated by siKP relative to siPGR. F, Molecular network from pathway analyses of dataset indicating perturbations in expression of molecules involved in cellular growth, proliferation, and apoptosis with loss of functional KLF9+PGR interactions. Red indicates up-regulated; blue, down-regulated; and white, other genes identified as part of the gene network.