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.

Endometriosis is an estrogen-dependent disorder commonly associated with infertility in reproductive-aged women (1). Of the 6–10% of women affected with endometriosis, 35–50% are found to be infertile (2). Defective implantation is considered to be an underlying cause of endometriosis-related infertility in women (3) and mouse models (4). Patients undergoing in vitro fertilization and diagnosed with endometriosis have poor pregnancy outcomes (5, 6). Furthermore, patients with endometriosis showed higher rates of pregnancy loss and pregnancy-associated complications (7, 8). Nonetheless, a definitive mechanistic association between endometriosis and infertility remains lacking.

Progesterone (P) resistance is considered to underlie endometriosis (1). Genes affected by P are dysregulated during the window of uterine receptivity for embryo implantation in eutopic endometrium of women with endometriosis (9, 10). Regulators of P receptor (PGR) expression and transactivation constitute major determinants of successful implantation and pregnancy (11). Thus, the loss of PGR activity due to reduced PGR (12, 13) and/or inappropriate PGR coactivator (14, 15) expression, cumulatively leading to deregulated downstream effector signaling (9, 10, 14, 16), may link endometriosis with endometriosis-associated infertility.

Dissecting the mechanisms by which PGR regulates gene networks for establishment of a successful pregnancy is complicated by the presence of a large repertoire of PGR coregulator proteins that function under distinct contexts (17). Accordingly, a systematic evaluation of the functional consequence of individual PGR coregulators under physiological and pathophysiological conditions is warranted to delineate their coordinate, opposing, and compensatory functions in PGR-mediated responses. In the present study, we examined the contribution of the transcription factor Krüppel-like factor 9 (KLF9) to the PGR network in human endometrial stromal cells and how it may be associated with the pathological condition of endometriosis. KLF9 is likely participatory to PGR function linking endometriosis and endometriosis-associated infertility, given our previous findings that KLF9 is a PGR-B-interacting protein (18); loss of KLF9 expression in mice leads to subfertility, P-resistance, and uterine hypoplasia (19); and in human endometrial stromal cells, premature expression of a key decidualizing factor, bone morphogenetic protein 2, leading to compromised stromal function is a consequence of deregulated KLF9 activity (20).

Materials and Methods

Tissues

Endometrial tissue samples were obtained from women without (control) and with diagnosed endometriosis undergoing endometrial biopsy, following protocols approved by the University of California San Francisco Committee on Human Research (14, 15). Participants (Supplemental Table 1, published on The Endocrine Society's Journals Online web site at http://jcem.endojournals.org) were documented to be nonpregnant and not to have undergone hormone treatments for at least 3 months before surgery.

Cell culture and treatments

The human endometrial stromal cell (HESC) line was treated with a cocktail of 8-bromo-cAMP (0.5 mm), 1 μm progestin [medroxyprogesterone acetate (MPA)] and 10 nm estradiol-17β (E₂) (Sigma-Aldrich, St. Louis, MO), henceforth designated 8-bromo-cAMP+MPA+E₂ (cAME) (20).

RNA isolation and analyses

Total RNA, isolated using RNeasy Plus minikit (QIAGEN, Valencia, CA), was reverse transcribed (iScript; Bio-Rad Laboratories, Hercules, CA) and used for SYBR green-based real-time quantitative PCR (QPCR) (20). Primer sequences are provided in Supplemental Table 2.

Western blot analyses

Nuclear and cytoplasmic proteins were resolved by SDS-PAGE. Proteins were incubated with rabbit antirat KLF9 (18), monoclonal goat antihuman Dickkopf-1 (DKK1) (R&D Systems, Minneapolis, MN), and mouse antihuman PGR (Pgr-1294; Dako, Carpinteria, CA). Protein-antibody complexes were detected as described (20). Lamin A and β-actin were used as normalizing controls (20).

RNA interference

Transfection was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in OPTI-MEM reduced serum-containing medium (20). Small interfering RNA (siRNA) targeting human KLF9 and PGR (siGeNOME SMART pool) and nontargeting (siCONTROL) siRNA (Dharmacon, Waltham, MA) were used at 50 nm concentration. When appropriate, final siRNA concentrations were made up to 100 nm with the addition of nontargeting siRNA. The transfected groups and treatments were 1) nontargeting siRNA plus vehicle alone (vehicle), 2) nontargeting siRNA plus cAME (NT); 3) KLF9 siRNA plus 0.5cAME (siKLF9), 4) PGR siRNA plus 0.5cAME (siPGR), and 5) KLF9 plus PGR siRNA plus 0.5cAME (siKP). Six hours after transfection, cells were washed, incubated for 24 h in phenol red-free DMEM-F12 containing 2% charcoal-stripped bovine calf serum, and then exposed for 48 h in medium containing 0.5cAME or vehicle. Collected cells were subjected to Western blot or RNA expression analyses.

DKK1 promoter-driven luciferase assay

The DKK1 promoter and 5′-flanking regions were amplified (by PCR) from human genomic DNA and subcloned into the NheI and Xho sites of pGL3-basic vector containing the luciferase reporter gene (Promega, Madison, WI). Reporter constructs and pGL3-basic (0.5 μg per well) were transiently transfected with appropriate siRNA using Lipofectamine 2000 in OPTI-MEM. Six hours after transfection, cells were washed, transferred to phenol red-free DMEM-F12 medium containing 2% charcoal-stripped bovine calf serum and after 24 h, treated with 0.5cAME. Cells harvested after 48 h were processed for luciferase activity (luciferase reagent kit; Promega).

Chromatin immunoprecipitation (ChIP)

Cells were processed for ChIP using the ChIP-IT Express Kit (Activ Motif, Carlsbad, CA). Chromatin was immunoprecipitated with goat antihuman KLF9 polyclonal antibody (sc-12994; Santa Cruz Biotechnology, Santa Cruz, CA), mouse antihuman PGR clone 636 monoclonal antibody (M3569; Dako), or goat antirat specificity protein 1 (SP1) polyclonal antibody (sc-59G; Santa Cruz). Preimmune control antibodies were normal goat IgG (sc-2028; Santa Cruz) or normal mouse IgG (X0931; Dako). Antibody-bound protein-DNA complexes were recovered using protein G-coated magnetic beads, and the final DNA were analyzed by PCR. The oligonucleotide primers used for amplification of the proximal [−190 to −12 nucleotides (nt)] and distal (−1850 to −1250 nt) regions of the DKK1 promoter were as follows: proximal, 5′-CCAGCC GAGCGACTAAGCAA-3′ and 5′-ACCGCGGCTGCCTTTATACC-3′ (forward and reverse, 179 bp), and distal, 5′-TGGAATTTGGGATGGGAAGGACAC-3′ and 5′-CTGCCCTCTGGGTTGTTACCTTAT-3′ (forward and reverse, 601 bp).

Gene expression profiling with microarrays

Gene expression analyses used Human Genome U133 Plus version 2.0 high-density oligonucleotide arrays (Affymetrix Inc., Santa Clara, CA), with 54,120 probe sets to interrogate 38,500 well-characterized human transcripts. cRNA preparation, hybridization, washes, and detection followed the manufacturer's recommendations. Samples were HESC from the five treatment groups described under RNA interference. RNA were prepared using GeneChip 3′ IVT Express Kit (Affymetrix). RNA pooled from triplicate samples per treatment group constituted one biological replicate and were hybridized to an array; two independent replicates were evaluated per treatment group. Expression data were analyzed using GeneSpring GX version 11 software (Agilent Technologies, Santa Clara, CA). The .CEL files containing probe level intensities were processed using the GeneChip robust multiarray analysis (GC-RMA) with quantile normalization. The normalized data were subjected to pairwise comparisons: 1) NT vs. vehicle, 2) siKLF9 vs. NT, 3) siPGR vs. NT, and 4) siKP vs. NT. Gene sets were subjected to genome set enrichment analyses (21) and biological function and ontology analyses using Affymetrix NetAffx and GeneSpring, and genes with at least 1.5-fold change and P ≤ 0.05 were analyzed for functional gene annotations using Ingenuity pathway analysis (IPA; Ingenuity Systems, Redwood City, CA). Data discussed herein were deposited in Gene Expression Omnibus (GSE31683).

Statistical analysis

Data (mean ± sem) were analyzed by one-way ANOVA or two-tailed Student's t test using SigmaStat version 3.5 software (SPSS, Chicago, IL). P ≤ 0.05 was considered significant.

Results

Aberrant WNT signaling and deregulated KLF9 expression in eutopic endometrium of women with endometriosis

Eutopic endometria from women with (E) and without (N) endometriosis at different menstrual cycle phases [proliferative endometrium (PE), early secretory endometrium, midsecretory endometrium (MSE), and late secretory endometrium] were evaluated for expression of KLF9 and other genes functionally relevant to endometriosis and endometriosis-associated infertility (1, 9, 14), by QPCR. Levels of total PGR, PGR-B, WNT4, WNT2, DKK1, and KLF9 transcripts were reduced in eutopic endometria of women with vs. without endometriosis (Fig. 1). Reductions in PGR, PGR-B, and WNT4 transcripts for PE (Fig. 1, A–C) and of DKK1 and KLF9 transcripts for MSE (Fig. 1, E and F) were significant for women with vs. without endometriosis. Similarly, women with endometriosis had lower (P < 0.05) WNT2 expression in PE and MSE than women without disease (Fig. 1D).

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.

KLF9 participates in PGR regulation of WNT signaling components in endometrial stromal cells

Because KLF9 is predominantly expressed in endometrial stromal cells (19), we used HESC treated with cAME for 48 h to investigate KLF9 and PGR regulation of WNT signaling under conditions that mimicked the uterine receptive phase (MSE). cAME-treated cells showed increased transcript levels for IGFBP1 (130-fold), PRL (116-fold), and BMP2 (4-fold) relative to untreated cells. The WNT components WNT4, WNT2, and DKK1 were highly up-regulated by cAME, with WNT4 showing the greatest response (Fig. 2A).

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.

To evaluate whether loss of KLF9 in the background of P resistance, conditions found in eutopic endometrium of women with endometriosis (Fig. 1), contributes to deregulated WNT signaling associated with endometriosis and infertility (9, 22), we used siRNA to decrease KLF9 and PGR expression before cAME treatment. siRNA to KLF9 and PGR reduced their respective transcript levels by more than 75%, relative to control siRNA (data not shown). Nuclear KLF9 protein levels were reduced to a comparable extent with siKLF9 (Fig. 2B) without affecting PGR isoform levels. With PGR siRNA, nuclear PGR-A and PGR-B levels were drastically decreased, whereas KLF9 levels showed a modest reduction (Fig. 2B).

Knockdowns of KLF9, PGR, and KLF9+PGR (KP) progressively reduced WNT4 transcript levels in cAME-treated HESC (Fig. 2C). WNT2 transcript levels were unchanged and increased, respectively, with knockdown of KLF9 and PGR, whereas coaddition of both siRNA augmented the increase above that of siPGR (Fig. 2D). DKK1 transcript levels were increased and decreased, respectively, with siRNA to KLF9 and to PGR (Fig. 2E); these changes were mirrored by cytoplasmic DKK1 protein (Fig. 2F). KP co-knockdown led to total loss of DKK1 protein, similar to that found with siPGR (Fig. 2, E and F).

KLF9 and PGR regulate DKK1 promoter activity in endometrial stromal cells

Because DKK1 and KLF9 transcript levels are coincidentally reduced in MSE of women with vs. without endometriosis, when PGR levels are already low (Fig. 1) and because HESC had drastically reduced DKK1 expression in KP vs. KLF9 knockdowns, we examined whether loss of KLF9+PGR regulation of DKK1 promoter activity underlies attenuated DKK1 expression in endometriosis. A region of the human DKK1 promoter (−2238/+136 nt) containing multiple GC-rich, SP1/KLF binding sites (23) and several half-P response element/glucocorticoid response element (PRE/GRE) sites (24) (Fig. 3A) was evaluated for activity by transfection of promoter-reporter constructs containing the entire (−2238/+112 nt) or truncated (−221/+136 nt; −35/+136 nt) regions (translation initiation site ATG, +1). The −2238/+112 and −221/+136 DKK1-Luc constructs demonstrated comparable luciferase activities, indicating DKK1 promoter activity to mostly reside between −221 to +112 nt (Fig. 3B). To determine which sequences within the DKK1 promoter region are KLF9 and/or PGR responsive, HESC cotransfected with KLF9, PGR, or KP siRNA and the longer and shorter DKK1-Luc constructs were evaluated for reporter activity. KLF9 siRNA increased, whereas PGR siRNA decreased the activities of both promoter/reporter constructs to similar extents (Fig. 3C).

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.

KLF9 and PGR are recruited to the DKK1 promoter

To evaluate the molecular basis for KLF9 and PGR regulation of DKK1, we examined the recruitment of both transcription factors to the DKK1 promoter region using ChIP assay. Chromatin isolated from vehicle and cAME-treated HESC was precipitated using KLF9 or PGR antibodies, and precipitated DNA was amplified using primers flanking two regions of the DKK1 promoter. Primer set A (−1850/−1250 nt) amplified a region containing two GC/SP1 and three possible half-PRE/GRE sites, whereas primer set B (−190/−12 nt) amplified a region containing seven GC/SP1 sites and one half-PRE/GRE site (Fig. 3D). Amplification of the precipitated DNA yielded the expected product sizes of 601 and 179 bp, respectively (data not shown). The region amplified by primer set A showed KLF9 but undetectable PGR binding in vehicle- and cAME-treated HESC (Fig. 3D, left). The region amplified by primer set B bound both KLF9 and PGR in cAME- but not in vehicle-treated cells (Fig. 3D, right). SP1 ubiquitously bound to the latter site, irrespective of cAME treatment. Preimmune sera did not precipitate the DKK1 promoter regions containing KLF9 or PGR binding sites.

Microarray analyses

Gene expression profiling was conducted in decidualizing HESC, perturbed by KLF9 and PGR knockdowns, to identify additional KLF9 and PGR coregulated genes. Samples for the microarray analyses were those used for the QPCR analyses (Fig. 2, C–E), and non-cAME-treated HESC transfected with NT siRNA, added as a control group. Unsupervised hierarchical clustering of expression profiles indicated that cAME-treated HESC grouped together and separately from the non-cAME-treated group (data not shown). A total of 1740 annotated genes were significantly regulated by at least 1.5-fold with cAME treatment; the top 100 genes (50 up- and 50 down-regulated) are presented in Supplemental Table 3. The list included genes reportedly regulated in decidualizing stromal cells (25), such as somatostatin (SST), IGF-binding protein 1 (IGFBP1), IGF1, WNT4, prolactin (PRL), and forkhead box O1 (FOXO1). Of the cAME-regulated genes, expression of 34 (2.0%), including IGFBP1, was perturbed with KLF9 knockdown (Supplemental Table 3). With siPGR, expression of 109 (6.3%) genes were modified; these included IGF1, IL8, monoamine oxidase (MAOA), and RAR-related orphan receptor B (RORB).

Analyses of common and unique genes perturbed by siPGR and by siKP in decidualizing HESC (Fig. 4A) indicated that more genes were uniquely regulated by siKP than by siPGR alone (309 vs. 97). Table 1 lists siKP up- and down-regulated genes whose expression were either unaffected by siPGR or affected by greater than 25% over that of siPGR alone. Genes unaffected by siPGR but that were either up- or down-regulated with siKP included epiregulin (EREG), IL-1β (IL1B), dual-specificity phosphatase 10 (DUSP10), matrix metallopeptidase I (MMP1), caspase 1 (CASP1), KLF4, and tissue inhibitor of metalloproteinase 3 (TIMP3). Genes whose expression was affected by siPGR and additionally affected by siKP included early growth response (EGR1), stanniocalcin 1 (STC1), amphiregulin (AREG), and DUSP6.

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.

Table 1.

List of selected up- and down-regulated genes in cAME-treated HESC with knockdown of PGR ± KLF9

Gene symbol	Gene name	Fold change		hESFendo vs. hESFnonendo
		siKP vs. NT	siPGR vs. NT	EPa,c	cAMPb,c
Up-regulated
INHBE	Inhibin, β E	13.01	18.59
CNIH3	Cornichon homolog 3 (Drosophila)	9.40	5.89	↑
KIAA1199	KIAA1199	8.52	3.31
EGR1	Early growth response 1	7.85	3.55
C2orf88	Chromosome 2 open reading frame 88	6.27	3.05
TMEM100	Transmembrane protein 100	6.20	2.05
STC1	Stanniocalcin 1	5.97	2.09	↑
PTHLH	PTH-like hormone	5.49	3.70		↓
RXFP1	Relaxin/insulin-like family peptide receptor 1	4.74	3.86	↓	↓
DUSP6	Dual-specificity phosphatase 6	4.56	1.56	↑
TNFRSF11B	TNF receptor superfamily, member 11b	4.04	2.49
TNFSF13B	TNF (ligand) superfamily, member 13b	4.03	2.44
IER3	Immediate early response 3	3.84	2.08	↑
LOC100131897	Uncharacterized protein LOC100131897	3.76	2.05
AREG	Amphiregulin	3.71	2.31	↑
RGS18	Regulator of G-protein signaling 18	3.65	2.40
FAM84A	Family with sequence similarity 84, member A	3.56	2.08
ETV1	ETS variant 1	3.45	2.10
FJX1	Four jointed box 1 (Drosophila)	3.37		↑
HK2	Hexokinase 2	3.27	2.06	↑
FAM20A	Family with sequence similarity 20, member A	3.14	2.27	↓
IL8	IL-8	3.11	2.11		↓
SERPINB2	Serpin peptidase inhibitor, clade B (ovalbumin), member 2	3.08	1.62
SHC4	SHC (Src homology 2 domain containing) family, member 4	3.02	2.28	↑
TMSB15A	Thymosin β 15a	3.01	2.28
TNFSF10	TNF (ligand) superfamily, member 10	2.98	2.26	↓
FABP5	Fatty acid binding protein 5 (psoriasis-associated)	2.95	1.93	↓
GEM	GTP binding protein overexpressed in skeletal muscle	2.90	2.24
ETV5	ETS variant 5	2.79	1.58	↑
KYNU	Kynureninase (l-kynurenine hydrolase)	2.77			↓
SPRY1	Sprouty homolog 1, antagonist of FGF signaling (Drosophila)	2.73
OAS1	2′,5′-oligoadenylate synthetase 1, 40/46 kDa	2.73	1.92	↓
KCNE4	Potassium voltage-gated channel, Isk-related family, member 4	2.69	2.00	↓
VWA5A	von Willebrand factor A domain containing 5A	2.67
IFIT2	Interferon-induced protein with tetratricopeptide repeats 2	2.60	2.08	↓
CCL7	Chemokine (C-C motif) ligand 7	2.59	1.60
G0S2	G0/G1switch 2	2.59
ARSG	Arylsulfatase G	2.58	1.70
KRTAP1–5	Keratin-associated protein 1–5	2.55		↑
HMGA2	High-mobility group AT-hook 2	2.53	1.61
GDF15	Growth differentiation factor 15	2.52	1.77	↑
RGL1	RAL guanine nucleotide dissociation stimulator-like 1	2.48	1.99	↑
TMEM158	Transmembrane protein 158	2.45
EREG	Epiregulin	2.44
KCND2	Potassium voltage-gated channel, Shal-related subfamily, member 2	2.41	1.60
IL1B	IL-1, β	2.33			↓
PPFIBP2	PTPRF interacting protein, binding protein 2 (liprin β 2)	2.31	1.50
NR4A2	Nuclear receptor subfamily 4, group A, member 2	2.29	1.55		↓
SLC22A4	Solute carrier family 22 (organic cation/ergothioneine transporter), member 4	2.29	1.60
TSPAN13	Tetraspanin 13	2.26	1.68
LOC729345	Hypothetical LOC729345	2.19	1.17
TMEM106C	Transmembrane protein 106C	2.18	1.67
C21orf7	Chromosome 21 open reading frame 7	2.18	1.54	↑
AHR	Aryl hydrocarbon receptor	2.16	1.50
LOC154761	Hypothetical LOC154761	2.09
SLC45A4	Solute carrier family 45, member 4	2.01
MYLIP	Myosin regulatory light chain interacting protein	2.01	1.61	↓
ITPRIP	Inositol 1,4,5-triphosphate receptor interacting protein	1.99
TNFAIP6	TNF, α-induced protein 6	1.99			↓
DHCR7	7-Dehydrocholesterol reductase	1.99
TPBG	Trophoblast glycoprotein	1.98
TNFRSF12A	TNF receptor superfamily, member 12A	1.96
ARRDC3	Arrestin domain containing 3	1.95
IGF2BP3	IGF-II mRNA binding protein 3	1.93
EWSR1/ FLI1	Ewing sarcoma breakpoint region 1/friend leukemia virus integration 1	1.92
ZFP36L2	Zinc finger protein 36, C3H type-like 2	1.92
MAFF	v-MAF musculoaponeurotic fibrosarcoma oncogene homolog F (avian)	1.91
PDE4B	Phosphodiesterase 4B, cAMP-specific (phosphodiesterase E4 dunce homolog, Drosophila)	1.90		↑
NPC1	Niemann-Pick disease, type C1	1.90
LOC387763	Hypothetical protein LOC387763	1.90	1.51
FAM102B	Family with sequence similarity 102, member B	1.86
SGPL1	Sphingosine-1-phosphate lyase 1	1.86
NRM	Nurim (nuclear envelope membrane protein)	1.85
SP4	Sp4 transcription factor	1.84
RGMB	RGM domain family, member B	1.83		↑
CD97	CD97 molecule	1.83
DIRAS3	DIRAS family, GTP-binding RAS-like 3	1.83
RNF144B	Ring finger protein 144B	1.82
vTSTA3	Tissue-specific transplantation antigen P35B	1.81
HEY1	Hairy/enhancer-of-split related with YRPW motif 1	1.78			↑
ALG1	Asparagine-linked glycosylation 1, β-1,4-mannosyltransferase homolog (Saccharomyces cerevisiae)	1.78
C14orf109	Chromosome 14 open reading frame 109	1.78
PITPNC1	Phosphatidylinositol transfer protein, cytoplasmic 1	1.78
RNF24	Ring finger protein 24	1.78
ADAM17	ADAM metallopeptidase domain 17	1.78
RAB3D	RAB3D, member RAS oncogene family	1.77
ZNF75D	Zinc finger protein 75D	1.76
HSD17B6	Hydroxysteroid (17-β) dehydrogenase 6 homolog (mouse)	1.75
PNPLA4	Patatin-like phospholipase domain containing 4	1.75
PPP1R3C	Protein phosphatase 1, regulatory (inhibitor) subunit 3C	1.75
SCD	Stearoyl-CoA desaturase (δ-9-desaturase)	1.74
LPCAT2	Lysophosphatidylcholine acyltransferase 2	1.74		↑
INHBA	Inhibin, β A	1.73
S1PR2	Sphingosine-1-phosphate receptor 2	1.73
CSRNP1	Cysteine-serine-rich nuclear protein 1	1.72
SDC1	Syndecan 1	1.72
PUSL1	Pseudouridylate synthase-like 1	1.70
ZC3HAV1L	Zinc finger CCCH-type, antiviral 1-like	1.70
ZNRF1	Zinc and ring finger 1	1.70
GPR89B/GPR89C/GPR89A	G protein-coupled receptor 89B/G protein-coupled receptor 89A/G protein-coupled receptor 89C	1.69
ILVBL	ILVB (bacterial acetolactate synthase)-like	1.68
CRABP2	Cellular retinoic acid binding protein 2	1.68
PLCL2	Phospholipase C-like 2	1.67
ALG5	Asparagine-linked glycosylation 5, dolichyl-phosphate β-glucosyltransferase homolog (S. cerevisiae)	1.67
DUSP10	Dual-specificity phosphatase 10	1.66		↑
FAM158A	Family with sequence similarity 158, member A	1.66
EPOR	Erythropoietin receptor	1.66
STEAP1	Six transmembrane epithelial antigen of the prostate 1	1.65
PAICS	Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase	1.64
GK/GK3P	Glycerol kinase/glycerol kinase 3 pseudogene	1.64
HERC6	Hect domain and RLD 6	1.64
RIN2	Ras and Rab interactor 2	1.64
MXI1	MAX interactor 1	1.64
CD47	CD47 molecule	1.63
SLC41A3	Solute carrier family 41, member 3	1.63
UCHL3	Ubiquitin carboxyl-terminal esterase L3 (ubiquitin thiolesterase)	1.63
IFI35	Interferon-induced protein 35	1.63
RNF145	Ring finger protein 145	1.63
GOLPH3L	Golgi phosphoprotein 3-like	1.62
TBL2	Transducin (β)-like 2	1.61
IER2	Immediate early response 2	1.61
PKM2	Pyruvate kinase, muscle	1.61
LAP3	Leucine aminopeptidase 3	1.61
ADFP	Adipose differentiation-related protein	1.61		↓
CASP1	Caspase 1, apoptosis-related cysteine peptidase (IL-1, β, convertase)	1.60		↑
TM4SF1	Transmembrane 4L six family member 1	1.60		↑
KIAA1715	KIAA1715	1.59
UAP1L1	UDP-N-acteylglucosamine pyrophosphorylase 1-like 1	1.59
POLD3	Polymerase (DNA-directed), δ 3, accessory subunit	1.57
GAS2L3	Growth arrest-specific 2 like 3	1.57
LOC100129973	Hypothetical protein LOC100129973	1.57
MAD1L1	MAD1 mitotic arrest deficient-like 1 (yeast)	1.57
EXTL3	Exostoses (multiple)-like 3	1.56
YPEL2	Yippee-like 2 (Drosophila)	1.55
GMDS	GDP-mannose 4,6-dehydratase	1.55
DTX3L	Deltex 3-like (Drosophila)	1.55
SP110	SP110 nuclear body protein	1.54
PHF1	PHD finger protein 1	1.53
ATP13A3	ATPase type 13A3	1.53
XYLT2	Xylosyltransferase II	1.53
SOAT1	Sterol O-acyltransferase 1	1.53
SAC3D1	SAC3 domain containing 1	1.53
FAM46A	Family with sequence similarity 46, member A	1.53
FAM57A	Family with sequence similarity 57, member A	1.53
FAM162A	Family with sequence similarity 162, member A	1.53
REL	v-Rel reticuloendotheliosis viral oncogene homolog (avian)	1.52
JUNB	Jun B protooncogene	1.52
AKAP2	A kinase (PRKA) anchor protein 2	1.52
NTN4	Netrin 4	1.52
PDXK	Pyridoxal (pyridoxine, vitamin B6) kinase	1.52
IFI44L	Interferon-induced protein 44-like	1.52		↓
TMEM60	Transmembrane protein 60	1.52
C19orf66	Chromosome 19 open reading frame 66	1.52
NEU1	Sialidase 1 (lysosomal sialidase)	1.51
DOCK10	Dedicator of cytokinesis 10	1.51		↓
SLC15A3	Solute carrier family 15, member 3	1.51
SRPK2	SFRS protein kinase 2	1.51
SUSD3	Sushi domain containing 3	1.51
DHRS7	Dehydrogenase/reductase (SDR family) member 7	1.51
SLC31A1	Solute carrier family 31 (copper transporters), member 1	1.51
RQCD1	RCD1 required for cell differentiation1 homolog (S. pombe)	1.50
TAF1A	TATA box binding protein (TBP)-associated factor, RNA polymerase I, A, 48 kDa	1.50
Down-regulated
RORB	RAR-related orphan receptor B	9.19	5.67	↓
ALDH1A3	Aldehyde dehydrogenase 1 family, member A3	7.69	4.23
IGF1	IGF-I (somatomedin C)	7.68	3.78	↓
KLF9	Kruppel-like factor 9	5.97
RASD1	RAS, dexamethasone-induced 1	5.91	4.64
PGR	Progesterone receptor	5.36	5.88	↓
NPR3	Natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide receptor C)	4.98	2.61	↓
CNTN3	Contactin 3 (plasmacytoma associated)	4.56	3.59	↓
DHRS3	Dehydrogenase/reductase (SDR family) member 3	4.53	2.48	↓	↑
C5orf23	Chromosome 5 open reading frame 23	4.30	2.85	↓
PRKAB2	Protein kinase, AMP-activated, β 2 non-catalytic subunit	4.16	1.68
SDPR	Serum deprivation response (phosphatidylserine binding protein)	3.51	2.31
FERMT2	Fermitin family homolog 2 (Drosophila)	3.27	2.58	↓
PLCXD3	Phosphatidylinositol-specific phospholipase C, X domain containing 3	3.18	2.16
FAM116A	Family with sequence similarity 116, member A	3.09	1.73
ITGBL1	Integrin, β-like 1 (with EGF-like repeat domains)	2.78	2.24
CCDC69	Coiled-coil domain containing 69	2.74	1.57
MMP1	Matrix metallopeptidase 1 (interstitial collagenase)	2.72
ODC1	Ornithine decarboxylase 1	2.63
GLT8D3	Glycosyltransferase 8 domain containing 3	2.62	1.97
ST6GALNAC5	ST6 (α-N-acetyl-neuraminyl-2, 3-β-galactosyl-1, 3)-N-acetylgalactosaminide α-2, 6-sialyltransferase 5	2.55	1.98
ERRFI1	ERBB receptor feedback inhibitor 1	2.54	1.78	↓
ADAM19	ADAM metallopeptidase domain 19 (meltrin β)	2.45
ZBTB47	Zinc finger and BTB domain containing 47	2.43	1.60
QDPR	Quinoid dihydropteridine reductase	2.42
CDKN1C	Cyclin-dependent kinase inhibitor 1C (p57, Kip2)	2.42
NEXN	Nexilin (F actin-binding protein)	2.40
PDK4	pyruvate dehydrogenase kinase, isozyme 4	2.39	1.88
TGFBR3	TGF, β receptor III	2.33	1.71
NBN	Nibrin	2.31
RHOU	Ras homolog gene family, member U	2.26		↓
MESDC2	Mesoderm development candidate 2	2.26
C1orf9	Chromosome 1 open reading frame 9	2.23	1.52
ZNF675	Zinc finger protein 675	2.22	1.56
KIAA0317	KIAA0317	2.22
SLC11A2	Solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2	2.21
NBLA00301	Nbla00301	2.18	1.59	↓
PLBD1	Phospholipase B domain containing 1	2.16
UST	Uronyl-2-sulfotransferase	2.16		↓
C17orf80	Chromosome 17 open reading frame 80	2.16
SUCLA2	Succinate-CoA ligase, ADP-forming, β subunit	2.14
SATB1	SATB homeobox 1	2.12
SLC40A1	Solute carrier family 40 (iron-regulated transporter), member 1	2.10		↓
MRVI1	Murine retrovirus integration site 1 homolog	2.06	1.56
LPHN3	Latrophilin 3	2.04
MAOA	Monoamine oxidase A	2.03	1.64
RASL11B	RAS-like, family 11, member B	2.03	1.53
ZNF91	Zinc finger protein 91	1.98
ARNT	Aryl hydrocarbon receptor nuclear translocator	1.97
TIMP3	TIMP metallopeptidase inhibitor 3	1.95		↓
TGOLN2	Trans-golgi network protein 2	1.95
INPP5A	Inositol polyphosphate-5-phosphatase, 40 kDa	1.95		↓
HSP90B1	Heat-shock protein 90 kDa β (Grp94), member 1	1.94
TBCEL	Tubulin folding cofactor E-like	1.93
STAG3L4	Stromal antigen 3-like 4	1.91
TSHZ1	Teashirt zinc finger homeobox 1	1.91
CD55	CD55 molecule, decay accelerating factor for complement (Cromer blood group)	1.91
CFL2	Cofilin 2 (muscle)	1.89
PIK3R3	Phosphoinositide-3-kinase, regulatory subunit 3 (γ)	1.88
FAM167A	Family with sequence similarity 167, member A	1.88
GAS1	Growth arrest-specific 1	1.88		↓
HIGD1A	HIG1 hypoxia inducible domain family, member 1A	1.86
EXOSC3	Exosome component 3	1.85
MASP1	Mannan-binding lectin serine peptidase 1 (C4/C2 activating component of Ra-reactive factor)	1.83
SYNJ2BP	Synaptojanin 2 binding protein	1.82
IGFBP1	IGF-binding protein 1	1.81			↓
KIAA0146	KIAA0146	1.81
MCEE	Methylmalonyl CoA epimerase	1.79
ACP5	Acid phosphatase 5, tartrate resistant	1.78
TCEAL1	Transcription elongation factor A (SII)-like 1	1.78
ACER3	Alkaline ceramidase 3	1.78
ZADH2	Zinc binding alcohol dehydrogenase domain containing 2	1.78
AKAP7	A kinase (PRKA) anchor protein 7	1.78
RAP1B	RAP1B, member of RAS oncogene family	1.77
ATP1B1	ATPase, Na+/K+ transporting, β 1 polypeptide	1.77			↑
AGPS	Alkylglycerone phosphate synthase	1.76
LYRM7	Lyrm7 homolog (mouse)	1.75
ANKRD13C	Ankyrin repeat domain 13C	1.75
LOC253039	Hypothetical LOC253039	1.74
DNMBP	Dynamin binding protein	1.73		↑
TP53INP1	Tumor protein p53 inducible nuclear protein 1	1.71
RUNX1T1	Runt-related transcription factor 1; translocated to, 1 (cyclin d-related)	1.71
KLF4	Kruppel-like factor 4 (gut)	1.71
PTN	Pleiotrophin	1.70
VAPA	VAMP (vesicle-associated membrane protein)-associated protein A, 33 kDa	1.70
PRDM2	PR domain containing 2, with ZNF domain	1.69
EARS2	Glutamyl-tRNA synthetase 2, mitochondrial (putative)	1.68
LOC100129387	Hypothetical LOC100129387	1.68
DIABLO	Diablo homolog (Drosophila)	1.67
FBN1	Fibrillin 1	1.66
JAZF1	JAZF zinc finger 1	1.66
PLA2G16	Phospholipase A2, group XVI	1.66
CGA	Glycoprotein hormones, α polypeptide	1.64			↓
TMED8	Transmembrane emp24 protein transport domain containing 8	1.64
C15orf17	Chromosome 15 open reading frame 17	1.64
C1orf124	Chromosome 1 open reading frame 124	1.63
ZCCHC6	Zinc finger, CCHC domain containing 6	1.63
PCYOX1	Prenylcysteine oxidase 1	1.63
OBFC2A	Oligonucleotide/oligosaccharide-binding fold containing 2A	1.63
KCNMA1	Potassium large conductance calcium-activated channel, subfamily M, α member 1	1.63
COX15	COX15 homolog, cytochrome c oxidase assembly protein (yeast)	1.62
C1orf2	Chromosome 1 open reading frame 2	1.62
BCAT1	Branched chain aminotransferase 1, cytosolic	1.62		↓
COL8A1	Collagen, type VIII, α 1	1.62
TPM1	Tropomyosin 1 (α)	1.62
TES	Testis derived transcript (3 LIM domains)	1.61
ABHD14B	Abhydrolase domain containing 14B	1.60
CDC14B/CDC14C	CDC14 cell division cycle 14 homolog B (S. cerevisiae)/CDC14 cell division cycle 14 homolog C (S. cerevisiae)	1.59
RPGR	Retinitis pigmentosa GTPase regulator	1.59
GADD45B	Growth arrest and DNA-damage-inducible, β	1.59
PPP1R12A	Protein phosphatase 1, regulatory (inhibitor) subunit 12A	1.59
PCDH19	Protocadherin 19	1.58
CDC37L1	Cell division cycle 37 homolog (S. cerevisiae)-like 1	1.57
MDFIC	MyoD family inhibitor domain containing	1.57
KCTD12	Potassium channel tetramerization domain containing 12	1.57
TNPO1	Transportin 1	1.56
RHOBTB2	ρ-Related BTB domain containing 2	1.55
SRPR	Signal recognition particle receptor (docking protein)	1.54
TRMT11	tRNA methyltransferase 11 homolog (S. cerevisiae)	1.53
RAB6A/RAB6C	RAB6A, member RAS oncogene family/RAB6C, member RAS oncogene family	1.53
C11orf70	Chromosome 11 open reading frame 70	1.53
TCEAL4	Transcription elongation factor A (SII)-like 4	1.53
JUN	Jun oncogene	1.53
MAP7D3	MAP7 domain containing 3	1.53
CKS2	CDC28 protein kinase regulatory subunit 2	1.53
SEMA5A	SEMA domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A	1.52
SAT1	Spermidine/spermine N1-acetyltransferase 1	1.52
GLUL	Glutamate-ammonia ligase (glutamine synthetase)	1.51
MST150	MSTP150	1.51
CHN1	Chimerin (chimaerin) 1	1.51
LGMN	Legumain	1.51

Results are expressed as fold change vs. nontargeting (NT) siRNA (≥1.5-fold change cutoff). ↑, Up-regulated; ↓, down-regulated.

^aGenes significantly regulated in hESF_endoEP vs. hESF_nonendoEP (27); E₂+MPA (EP).

^bGenes significantly regulated in hESF_endocAMP vs. hESF_nonendocAMP (26).

^cSimilar entities (overlaps) were determined using Genespring software based on the hypergeometric distribution with P < 0.001 (siKP vs. NT and hESF_endoEP vs. hESF_nonendoEP) and P < 0.001 (siKP vs. NT and hESF_endocAMP vs. hESF_nonendocAMP), respectively.

KLF9 and PGR coregulate endometriosis-associated genes

We compared our transcriptome data with previously reported expression profiles for human endometrial stromal fibroblasts (hESF) isolated from women with endometriosis and showing deregulated P or cAMP responsiveness (26, 27). Of the P-resistant genes in hESF of women with endometriosis, 48 (e.g. STC1, DUSP6, AREG, CASP1, IGF1, ERRFI1, and TIMP3) were also found in our list of up- or down-regulated genes with KP knockdown [Table 1; hESF_endo vs. hESF_nonendo (EP)]. Of these, 36 showed deregulated expression in the same direction as those for disrupted KLF9+PGR expression. A number of cAMP-responsive genes (e.g. IL8, IL1B, and IGFBP1) whose expression was deregulated in hESF of women with endometriosis, also showed overlap with genes altered by KP knockdown [Table 1; hESF_endo vs. hESF_nonendo (cAMP)]. Dysregulated genes in the siKP-HESC dataset and those with deregulated P or cAMP responses were highly correlated (P < 0.001).

Analyses using genome set enrichment analyses upon KLF9, PGR, or KLF9+PGR knockdowns indicated that KLF9 can function independently of PGR (Table 2). The gene functions regulated by KLF9+PGR (enriched or diminished with siKP) did not overlap with those identified for KLF9 alone (siKLF9). Although there was overlap in the gene functions regulated by siKP and siPGR (compare Diminished in siKP vs. Diminished in siPGR), KLF9+PGR loss significantly exacerbated the effect of siPGR alone, as indicated by the false discovery rate values (Table 2).

Table 2.

Summary of GSEA with FDR no more than 0.25

Gene set	FDR
Enriched in siKP
Chemokine activity	0.154
G-protein coupled receptor binding	0.158
Cytokine activity	0.209
Defense response	0.235
Response to biotic stimulus	0.237
Negative regulation of MAP kinase activity	0.249
Diminished in siKP
Proteinaceous extracellular matrix	<0.001
Extracellular matrix	<0.001
Collagen proteins	<0.001
Basement membrane components	<0.001
Actin cytoskeleton	0.025
Structural constituents of muscle	0.026
Basal lamina	0.044
Peptidase activity	0.120
Integrin binding	0.125
Intramolecular oxidoreductase activity	0.190
Metallopeptidase activity	0.197
Enriched in siKLF9
Microtubule cytoskeleton organization and biogenesis	0.008
Transcription factors	0.223
Mitotic cell cycle	0.238
DNA polymerase activity	0.243
Cell cycle process	0.248
Diminished in siKLF9
Protein phosphorylation	0.043
Jak-stat cascade	0.171
Diminished in siPGR
Proteinaceous extracellular matrix	0.002
Extracellular matrix	0.006
Collagen proteins	0.051
Structural constituents of muscle	0.111
Peptidase activity	0.210
Basement membrane components	0.250
Intramolecular oxidoreductase activity	0.250

Refer to Supplemental Table 8 for the complete list that includes gene ontology, functional processes, and genes involved. FDR, False discovery rate.

We further analyzed by Ingenuity pathway analysis the 417 annotated genes deregulated by siKP (≥1.5-fold change). The top gene networks (Supplemental Table 4), functional processes (Supplemental Table 5), and canonical pathways (Supplemental Table 6) deregulated with KLF9+PGR over PGR knockdown include cell proliferation, angiogenesis, apoptosis, and immune signaling. Predictably, uterine cancer (34 of 417) and other reproductive system diseases (57 of 417), notably endometriosis, were associated with disruption of KLF9+PGR expression (Supplemental Table 7).

The heat map representation of identified genes affected by siKP relative to siPGR that were previously reported to be deregulated in patients with endometriosis (9, 22, 26, 27) is shown in Fig. 4B. Selected genes were confirmed for differential expression by QPCR (Fig. 4C). EGR1 and IL8 levels were increased by siPGR alone and further increased by siKP. IGFBP1 levels were significantly decreased by siKP but not by siPGR. PGR mRNA levels were not additionally affected by siKP.

With respect to physiological functions, immune cell trafficking was highly affected by KP loss (Supplemental Table 5). Correlation-based hierarchical clustering showed enrichment of selected cytokine (Fig. 4D) and IGF/MAPK (Fig. 4E) signaling component genes with siKP, relative to siPGR alone. Figure 4F illustrates representative overlapping gene networks impacted by loss of functional KLF9+PGR interactions.

Discussion

This study addressed whether endometriosis, a uterine pathology characterized by attenuated PGR expression and transactivity, involves the coordinate loss of KLF9 function. Here, we report that eutopic endometrium of women with vs. without endometriosis, had significantly reduced KLF9 expression, coincident with reductions in PGR, PGR-B, WNT4, WNT2, and DKK1 levels. We demonstrate a causal relationship between loss of KLF9+PGR and reduced expression of WNT4 and DKK1 using the siRNA approach in cAME-treated HESC. Furthermore, we show that KLF9/PGR coregulation of DKK1 expression involves their corecruitment to the DKK1 proximal promoter region to regulate DKK1 gene transcription. Finally, we establish the global aspects of KLF9/PGR coregulation of human endometrial stromal genes by gene expression array and identify unique stromal KLF9/PGR gene targets that previously showed resistance to P action in hESF of women with endometriosis (27). Together, our results implicate KLF9 loss of expression as a participatory event in the pathogenesis of endometriosis.

Our study revealed for the first time that although KLF9 and PGR can function independently of each other, their interaction modifies PGR transactivity such that their coincident loss of expression elicits biological responses distinct from those caused by their individual losses. This was supported by our candidate gene approach for DKK1, WNT2, and WNT4 and, on a more global scale, by our identification of genes and biological functions distinctly deregulated by siKP vs. siPGR alone. KLF9+PGR-regulated genes include predominant candidate contributors to the pathology of endometriosis such as IGF1, IL8, AREG, MMP1, STC1, DUSP6, MAOA, EGR1, and CASP1 (10, 13, 22, 26, 27, 28). Importantly, we showed the potential validity of identified KLF9+ PGR-regulated genes as clinically relevant to endometriosis by demonstrating significant overlaps in expression with those exhibiting abnormal response to P in hESF isolated from women with endometriosis (27). Although our studies were carried out using total PGR rather than specific PGR-B isoform knockdown, raising the potential contribution of PGR-A to the effects shown here, this is not likely because KLF9 preferentially interacts with PGR-B (18, 29) and endometriosis is largely associated with decreased PGR-B expression (12, 13).

Our studies also established a mechanistic underpinning for how KLF9 and PGR cooperate, using DKK1 as a paradigm target in stromal cells. DKK1 is a potent inhibitor of the canonical WNT signaling pathway (30), and disruption of its expression leads to abnormal uterine cellular proliferation, differentiation, and death (31). In the decidualizing stroma, KLF9 and PGR are recruited to the DKK1 proximal promoter, and although KLF family member SP1 can bind to these same promoter sequences, only KLF9 interacted with PGR under the proper decidual stimulus. Interestingly, loss of KLF9 alone in HESC enhanced DKK1 promoter activity in contrast to the noted reduction with the coincident loss of KLF9+PGR. Data suggest that KLF9 may provide specificity to and/or fine-tune the promoter response to P/PGR and that KLF9 effects may involve gene networks distinct from those perturbed by KLF9/PGR together. The latter possibility is consistent with the universal loss of DKK1 expression in eutopic endometrium of women with endometriosis (14, 26, 27) and the distinct enrichment of functional gene sets with KLF9 vs. KLF9+PGR loss of expression (this study).

Our results identified the WNT signaling pathway as a major target of KLF9/PGR interactions. Another signaling pathway potentially influenced by KLF9+PGR in stromal cells is the IGF system, with IGF-I and IGF-binding protein 1 as major candidate targets of KLF9/PGR coregulation. The immune system component IL-8, whose increased expression in eutopic endometria of women with endometriosis has been suggested to facilitate disease progression (32), is also distinctly regulated by KLF9+PGR. Furthermore, the pronounced up-regulated expression of EGR1, STC1, AREG, and EREG, all of which are hallmarks of escape from normal growth regulation, with loss of KLF9+PGR expression, provides additional proof to the biological importance of their interactions. Nevertheless, it remains unclear whether these genes are direct targets of KLF9/PGR, as we have shown here for DKK1 or whether other PGR coregulators may also contribute. Given that the Klf9-null mouse exhibits a subfertile phenotype (19) and the recent data suggesting deregulated expression of several KLF family members in endometriosis (22), it is possible that perturbations of the KLF family regulatory network (20, 33–35) contributes to the pathogenesis of this disease.

An important question is what links endometriosis and infertility. Our work confirms other previously published studies (9, 22) that disruption of WNT signaling contributes to this association. Mutant mice lacking Wnt4 are subfertile, due partly to defects in endometrial cell survival, differentiation, and P sensitivity (36). Targeted Wnt2 ablation in mice also resulted in placentation defects (37). Thus, studies on how appropriate expression of WNT signaling components is maintained by PGR and its coregulatory proteins, one of which is KLF9, may present viable options for the treatment of these disorders. An equally important question is the underlying mechanism for decreased KLF9 expression in eutopic endometrium of women with endometriosis. Although PGR may contribute to KLF9 regulation, because PGR knockdown in HESC modestly reduced KLF9 protein levels (this study), it is likely that other, more predominant mechanisms for the dramatic loss of KLF9 expression in endometriosis exist. Given that PGR hypermethylation leading to reduced PGR expression is found in both endometrial cancer (38) and in endometriosis (39) and the recent report that KLF9 is subject to epigenetic regulation (40), it is tempting to speculate that aberrant epigenetic mechanisms may cause loss of KLF9 and PGR expression in endometriosis, thereby initiating a cascade of deregulated transcriptional events.

In conclusion, our study demonstrates that KLF9, by modifying PGR action, plays a functional role in uterine PGR sensitivity, the loss of which may underlie the pathogenesis of endometriosis. The breadth of the KLF9+PGR network shown here suggests that further investigations into these linkages may provide therapeutic targets for endometriosis and endometriosis-associated infertility.