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Molecular Endocrinology, doi:10.1210/me.2007-0242
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Molecular Endocrinology 21 (12): 2988-3001
Copyright © 2007 by The Endocrine Society

Krüppel-Like Factor 9 Is a Negative Regulator of Ligand-Dependent Estrogen Receptor {alpha} Signaling in Ishikawa Endometrial Adenocarcinoma Cells

Michael C. Velarde1, Zhaoyang Zeng1, Jennelle R. McQuown, Frank A. Simmen and Rosalia C. M. Simmen

Department of Physiology and Biophysics, University of Arkansas for Medical Sciences and Arkansas Children’s Nutrition Center, Little Rock, Arkansas 72202

Address all correspondence and requests for reprints to: Rosalia C.M. Simmen, Ph.D., Department of Physiology and Biophysics, University of Arkansas for Medical Sciences and Arkansas Children’s Nutrition Center, 1120 Marshall Street, Little Rock, Arkansas 72202. E-mail: simmenrosalia{at}uams.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen and progesterone, acting through their respective receptors and other nuclear proteins, exhibit opposing activities in target cells. We previously reported that Krüppel-like factor 9 (KLF9) cooperates with progesterone receptor (PR) to facilitate P-dependent gene transcription in uterine epithelial cells. Here we evaluated whether KLF9 may further support PR function by directly opposing estrogen receptor (ER) signaling. Using human Ishikawa endometrial epithelial cells, we showed that 17β-estradiol (E2)-dependent down-regulation of ER{alpha} expression was reversed by a small interfering RNA to KLF9. Transcription assays with the E2-sensitive 4x estrogen-responsive element-thymidine kinase-promoter-luciferase reporter gene demonstrated inhibition of ligand-dependent ER{alpha} transactivation with ectopic KLF9 expression. E2 induced PR-A/B and PR-B isoform expression in the absence of effects on KLF9 levels. Addition of KLF9 small interfering RNA augmented E2 induction of PR-A/B while abrogating that of PR-B, indicating selective E2-mediated inhibition of PR-A by KLF9. Chromatin immunoprecipitation of the ER{alpha} minimal promoter demonstrated KLF9 promotion of E2-dependent ER{alpha} association to a region containing functional GC-rich motifs. KLF9 inhibited the recruitment of the ER{alpha} coactivator specificity protein 1 (Sp1) to the PR proximal promoter region containing a half-estrogen responsive element and GC-rich sites, but had no effect on Sp1 association to the PR distal promoter region containing GC-rich sequences. In vivo association of KLF9 and Sp1, but not of ER{alpha} with KLF9 or Sp1, was observed in control and E2-treated cells. Our data identify KLF9 as a transcriptional repressor of ER{alpha} signaling and suggest that it may function at the node of PR and ER genomic pathways to influence cell proliferation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE STEROID HORMONE estrogen (E) classically acts through two estrogen receptor (ER) isoforms, ER{alpha} and ERβ, to regulate transcriptional processes in target cells (1, 2). Both isoforms, although encoded by distinct genes, exhibit similar structural features that include the presence of activation function domains 1 and 2; a centrally located and highly conserved DNA-binding domain; and a carboxy terminally located ligand-binding domain (3, 4, 5, 6). Upon binding E, these receptors undergo conformational changes, form dimers, and selectively bind to consensus estrogen response elements (EREs) or half-EREs within promoter and enhancer/silencer regions of target genes (7). The interaction of ER with DNA facilitates the subsequent recruitment of different coactivators and chromatin remodeling enzymes, leading to the assembly of a functional transcriptional complex with components of the RNA machinery (8). Whether ERβ forms the same receptor coregulator complexes as ER{alpha} remains unclear, given that it can counter ER{alpha}-mediated E-induced gene transcription. More recently, the existence of alternative (nonclassical) pathways for the genomic actions of ligand-bound ER{alpha} was suggested, given the lack of ERE-like sequences in many E-responsive genes (9, 10). Studies now indicate that the mechanisms for the genomic actions of ER{alpha} also include: 1) the direct binding of ER{alpha} to other DNA-binding proteins such as specificity protein 1 (Sp1), activator protein-1, and nuclear factor-{kappa}B (11, 12, 13); 2) the simultaneous occupation of ER{alpha} binding sites and proximal transcription factor binding sites for maximal E response (14, 15); and 3) the direct binding of ER{alpha} to nucleotide recognition sequences for other nonrelated nuclear receptors (e.g. the orphan nuclear hormone receptor steroidogenic factor-1) (16). Less well known is whether ER{alpha} selectively partners with distinct steroid receptor coactivators to modulate transcription under these different genomic contexts.

Progesterone (P) opposes ER{alpha} actions in the uterine endometrium by binding to its cognate nuclear receptor progesterone receptor (PR) (1, 17). The mechanisms underlying the counterregulation of E signaling by P and vice versa on growth regulation, differentiation, and transcriptional activities remain poorly understood despite the many studies undertaken to elucidate this biologically important regulatory process (18). Selective regulation of ER{alpha} signaling to facilitate PR signaling has been posited to underlie opposing P and E actions in vivo. In support of this, ligand-bound PR repression of ER{alpha} expression levels (19); competition between ligand-activated ER{alpha} and PR for nuclear coactivators, which are normally present in limiting quantities in target cells (20); and differential cellular expression of ER{alpha} and PR, leading to distinct target gene sensitivity and susceptibility to E or P effects (21, 22), have been demonstrated. Given that endogenous E excess and loss of PR, specifically of PR-B, expression are associated with high risks for and poor outcome of endometrial carcinoma (23, 24), the most common gynecological malignancy in the Western world, understanding the major contributors and their respective roles in the regulation of ER{alpha} and PR signaling may have important consequences in the etiology and prognosis of this deadly disease of women.

Our laboratory has identified Krüppel-like factor 9 (KLF9; previously designated basic transcription element binding protein 1, BTEB1) as a PR coactivator in distinct cell types of the uterine endometrium (25, 26). KLF9 is one of 25 currently known members of the Sp/KLF family of transcription factors (27). Members of this family are characterized by the presence of a highly conserved C-terminal region, which contains three zinc finger domains that bind GC-rich sequence motifs in DNA, and extremely divergent amino-terminal regions that confer specificity and selectivity to their transcriptional activities and protein-protein interactions (27, 28). Gene targeting studies demonstrated that KLF9 is functionally involved in early pregnancy events, consistent with its role as a PR interacting protein (29). Female mice null for the KLF9 gene are subfertile due to decreased numbers of implanting embryos and partial uterine insensitivity to P. Subsequent studies showed that this phenotype is partly a consequence of aberrant expression of paracrine-acting uterine stromal PR, resulting in delayed proliferation of luminal epithelial cells and leading to developmental asynchrony of the uterine endometrium and implantation-ready embryo (30). In vitro studies suggest that KLF9 is also a PR target gene and cooperates with both PR-A and PR-B isoforms to transactivate P-responsive genes in uterine glandular epithelial cells (31).

The role of KLF9 in ER{alpha} signaling has not been previously examined. Because KLF9 is a positive regulator of PR-dependent gene transcription in endometrial epithelial cells (25, 26, 31), we hypothesized that one mechanism by which KLF9 may further support PR function in this cell type is by opposing ER{alpha} signaling. In this report, we show that KLF9, in a background of high 17β-estradiol (E2) mimicking the condition of unopposed E found in endometrial carcinoma (32), facilitates E2 inhibition of ER{alpha} expression and alters ligand-activated ER{alpha} transactivation. We further show that KLF9 contributes to ligand-dependent ER{alpha}-selective regulation of PR isoform expression. We demonstrate that KLF9 promotes E2-dependent association of ER{alpha} to the ER{alpha} promoter and disrupts the interaction of the ER{alpha}-interacting protein Sp1 to the E2-responsive PR-proximal but not to the PR-distal, promoter. Together, these findings suggest that KLF9, by mediating epithelial sensitivity to E2 and ligand-dependent ER{alpha} transactivation of gene expression, may function in E- and P-mediated control of endometrial epithelial cell proliferation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E2 Does Not Regulate KLF9 Gene Expression in Ishikawa Cells
Previous studies have shown that E2 increases PR-A/B expression in a number of tissues and cell types, including the well-differentiated ER- and PR-positive human endometrial adenocarcinoma cell line Ishikawa, which exhibits a glandular epithelial phenotype (33, 34). To confirm functional ER signaling in the Ishikawa cells used in the present studies, we measured PR-A/B transcripts in E2-treated cells (for 24 h) by quantitative real-time PCR (QPCR). E2 at 100 nM was used for the treatments to mimic the hyperestrogenic condition in an endometrial carcinoma environment (35). E2 increased PR-A/B mRNA levels in Ishikawa cells, and this induction was abolished by the E antagonist ICI when the latter was added 30 min before E2 treatment (Fig. 1Go). KLF9 transcript levels were unaffected by E2 ± ICI (Fig. 1Go). Because ER signaling in normal human endometrium and well-differentiated human endometrial cells (e.g. Ishikawa) occurs predominantly through ER{alpha} (33, 36), E2 induction of PR-A/B expression is likely ER{alpha} mediated and occurs in the absence of E2 effects on KLF9 expression.


Figure 1
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  		Fig. 1. E2 Regulates PR-A/B But Not KLF9 Gene Expression

Ishikawa cells were incubated in phenol red-free medium containing 10% CSS ± E2 (100 nM) in the presence or absence of ER antagonist ICI for 24 h and analyzed for KLF9 and PR mRNA abundance by QPCR. Results (least-square means ± SEM) are from four independent experiments and are expressed as fold-change over the control value (vehicle, -ICI). Differences were identified by two-way ANOVA, followed by Tukey’s test. Means with different superscripts differed at P < 0.05.

 
KLF9 Regulates E2-Dependent ER{alpha} Gene Expression
To determine whether KLF9 affects ER signaling at the level of ER{alpha} expression, we measured ER{alpha} transcript levels in control and E2-treated cells with and without the addition of KLF9 small interfering RNA (KLF9siRNA) duplex at 50 nM concentration (Fig. 2Go). We have previously shown that use of scrambled siRNA did not mimic the effect of KLF9 siRNA duplex on KLF9 expression (31). Control and E2-treated cells had comparable KLF9 mRNA levels, which were diminished by greater than 70% upon transfection of KLF9 siRNA (Fig. 2AGo). By contrast, KLF9 siRNA did not alter the expression of the closely related family member KLF13 (27) in these cells. Consistent with a previously published study (37), E2 decreased ER{alpha} expression at the levels of mRNA (Fig. 2AGo) and protein (Fig. 2BGo). Knockdown of KLF9, although having no effect on basal ER{alpha} mRNA levels, abrogated E2-inhibition of ER{alpha} expression (Fig. 2AGo). To confirm the latter at the protein level, we examined ER{alpha} expression by immunofluorescence (Fig. 2CGo) and Western blot analysis (Fig. 2EGo). ER{alpha} protein (shown as red staining) was found at comparable levels in unstimulated (control) cells in the absence or presence of KLF9 siRNA (Fig. 2Go, C and D). E2 treatment decreased ER{alpha} protein levels, relative to control cells (Fig. 2Go, C and D). KLF9 knockdown reversed the ligand-induced decrease in ER{alpha} protein (Fig. 2Go, C and D). Western blot analysis also showed decreased receptor levels in nuclear extracts with E2 treatment, which were increased to the level of control cells upon KLF9siRNA addition (Fig. 2EGo). Although there were some variations in the magnitude of changes in ER{alpha} protein levels with the two protocols, our results are consistent with loss of E2-mediated ER{alpha} down-regulation with attenuated KLF9 expression.


Figure 2
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  		Fig. 2. KLF9 Enhances E2-Dependent Down-Regulation of ER{alpha} Expression

Ishikawa cells were mock transfected or transfected with siRNA to KLF9 (50 nM) and incubated in phenol red-free medium containing 10% CSS ± E2 (100 nM) for 24 h. Harvested cells were analyzed for (A) KLF9, ER{alpha}, and KLF13 mRNA and (B–E) ER{alpha} protein levels. A, KLF9, ER{alpha}, and KLF13 mRNA expression were quantified by QPCR and normalized to that of 18S RNA. Transcript levels (least-square means ± SEM) were determined from four independent experiments and the value of control (vehicle, –siKLF9) was set to 1. Differences among treatment groups were determined by two-way ANOVA, followed by Tukey’s test. Means with different superscripts differed at P < 0.05. B, Whole-cell extracts from control and E2-treated Ishikawa cells from two independent experiments were subjected to immunoblot analysis using anti-ER{alpha} and anti-{alpha}-tubulin antibodies. C, Cells were immunostained for ER{alpha} (red) and counterstained for DAPI (blue). Immunopositive cells were visualized using fluorescent antibodies. Overlay of anti-ER{alpha}- and DAPI-stained cells showed nuclear localization of ER{alpha} (purple). Representative immunofluorescent cells for each treatment group are shown. D, Staining intensities of immunopositive cells were expressed on a per cell basis. The bar graphs represent the average staining intensity per cell normalized to those of control (vehicle, –siKLF9) cells, from three independent experiments. Differences were identified by two-way ANOVA, followed by Tukey’s test. Means with different superscripts differed at P < 0.05. E, Western blot of nuclear extracts prepared from Ishikawa cells of control (vehicle) and E2-treated cells without and with KLF9 siRNA transfection. The blot is representative of two independent experiments; Lamin B1 immunostaining was used as control for protein loading.

 
KLF9 Promotes E2-Dependent Association of ER{alpha} to ER{alpha} Promoter Region
Previous studies have shown that GC/CA-rich motifs that bind Sp1 transcription factor family members mediate ER{alpha} transcription (38), and that interfering with the interaction of Sp1 family members to these sites results in disruption of ER{alpha} promoter activity (39). Because KLF9 is a member of the Sp/KLF family and exhibits the characteristic zinc-finger domains that bind GC-rich regions (27, 28), we evaluated whether increased levels of ER{alpha} mRNA and protein due to loss of KLF9 expression, in E2-treated relative to control cells, may be due to altered recruitment of Sp1, KLF9, or ER{alpha} to GC-rich regions within the ER{alpha} minimal promoter. Based on published studies using deletion promoter-reporter constructs that defined GC-rich regions functionally contributing to ER{alpha} promoter activity (38, 40), we examined the human ER{alpha} minimal promoter region (–259 to –30 nt), which contains two Sp1 binding motifs (Fig. 3AGo). By performing chromatin immunoprecipitation (ChIP) analyses on control and E2-treated cells in the absence and presence of KLF9 siRNA, we found that whereas Sp1 was recruited to this ER{alpha} promoter region at comparable levels under all treatment conditions, KLF9 was not detected in this region under basal (vehicle) and E2-dominated conditions (Fig. 3Go, B and C). Interestingly, KLF9 facilitated the recruitment of ER{alpha} to this promoter region (by ~2-fold) under an environment of high E2, because this recruitment was lost with the addition of KLF9 siRNA and was undetectable (not different from vehicle only) in non-E2-treated cells (Fig. 3BGo). Our results suggest that KLF9 contributes to ER{alpha} autoregulation (41) by enhancing ER{alpha} association to this region of the promoter in an E2-dependent manner.


Figure 3
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  		Fig. 3. KLF9 Promotes Association of ER{alpha} with the ER{alpha} Promoter Region

A, Schematic representation of human ER{alpha} minimal promoter region containing two Sp1 (GC-rich) binding sites originally described in Ref. 38 . The bracketed area (–259 to –30 nt) represents the promoter region amplified by the PCR primers. B, ChIP assays were performed with chromatin prepared from control and E2-treated Ishikawa cells, in the absence and presence of siRNA to KLF9 (50 nM), using anti-ER{alpha} and anti-Sp1 antibodies. Precipitated DNA was analyzed by PCR, using primers spanning the Sp1 binding sites. Representative ChIP PCR gels (stained with ethidium bromide) are shown. The fold-change in PCR signals among the treatment groups was calculated from densitometric scans of PCR bands obtained from three independent experiments and was expressed relative to vehicle-treated cells without KLF9 siRNA added. C, ChIP assays were performed with chromatin prepared from control and E2-treated Ishikawa cells, using anti-KLF9 antibody or control (preimmune) goat IgG. Results of PCR amplification from three different experiments are expressed as fold-change relative to vehicle-treated cells immunoprecipitated with preimmune IgG. Differences among treatment groups were determined by two-way ANOVA, followed by Tukey’s test. Means with different superscripts differed at P < 0.05.

 
KLF9 Attenuates ER{alpha} Transactivity
One mechanism by which KLF9 can also inhibit ER{alpha} signaling is by repression of this receptor’s transcriptional activity. To address this possibility, we used the E2-responsive 4xERE-thymidine kinase (TK)-luciferase construct as a reporter for ER{alpha} transactivation (Fig. 4AGo). Ishikawa cells were cotransfected with the reporter construct in the presence of a human ER{alpha} expression construct (hER{alpha}-pCMV), a KLF9 expression construct (pCDNA3-BTEB/KLF9) containing the full-length rat KLF9 cDNA, which shares 96% homology with human KLF9 (42), or both. Preliminary studies with these various constructs indicated comparable transfection efficiencies between control and E2-treated cells as measured by cotransfection of the reporter plasmid pCMV-β-galactosidase; hence, the latter was not used in subsequent transfection studies (data not shown). Without E2 treatment, cells transfected with empty vectors (pCMV, pCDNA3) and expression constructs for ER{alpha} and KLF9, alone and together, showed comparable luciferase reporter activities (Fig. 4BGo). Treatment with E2 resulted in approximately 4-fold increase in 4xERE-TK-Luc promoter activity in ER{alpha}-transfected cells; this was significantly reduced upon cotransfection with KLF9 expression construct (Fig. 4BGo). The absence of E2 induction in Ishikawa cells not transfected with ER{alpha} expression construct, despite endogenous ER{alpha} expression, may be attributed to limiting amounts of ER{alpha} relative to exogenously added template DNA. Results demonstrate the inhibitory effect of KLF9 on ER{alpha} transactivation of an estrogen-responsive promoter.


Figure 4
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  		Fig. 4. KLF9 Attenuates E2-Induced ER{alpha} Transcription Activity

Ishikawa cells were transfected with 4xERE-TK-Luc promoter, followed by cotransfection of KLF9 and ER{alpha} expression constructs or empty vectors pCDNA and pCMV, respectively. Cells were incubated in phenol red-free medium containing 10% CSS ± E2 (100 nM) for 24 h. A, Schematic representation of promoter-reporter construct used in cotransfection experiments. B, Cell lysates were analyzed for Luc activity. Results (least-square means ± SEM) are from three independent experiments and are expressed as fold-change over the control value (–E2, pCMV+pcDNA). Differences among treatment groups were identified by two-way ANOVA, followed by Tukey’s test. Means with different superscripts differed at P < 0.05.

 
KLF9 Differentially Regulates PR Isoform Expression
We considered the possibility that KLF9 may contribute to ligand-dependent ER{alpha} inhibition of PR signaling by affecting ER{alpha} regulation of specific PR isoform expression. PR exists as two major isoforms, PR-A and PR-B, which are encoded by a single gene with two different transcription start sites (43, 44). The longer PR-B isoform exhibits distinct transcriptional activities from the truncated PR-A protein (45, 46), and loss of its expression, resulting in altered ratios of PR-B to PR-A, has been associated with E-dependent progression of endometrial carcinoma and poor prognosis of the disease (23, 24). We performed siRNA knockdown of KLF9 in control and E2-treated (100 nM) Ishikawa cells and measured the expression levels of PR-A and PR-B. To distinguish PR-B from PR-A mRNA by QPCR, we used one primer set that amplifies a region common to both PR isoform mRNAs (PR-A/B) and a second primer set that recognizes a region unique to PR-B mRNA (31). PR-A mRNA levels were inferred from PR-A/B and PR-B mRNA values. E2-treated cells showed a 2-fold increase in both PR-B and PR-A/B transcript levels, relative to control (untreated) cells (Fig. 5AGo), suggesting that E2 predominantly induced PR-B isoform expression, consistent with published reports (23). Interestingly, whereas knockdown of KLF9 diminished E2-induced PR-B expression to basal levels, PR-A/B transcript levels were significantly enhanced; this suggests E2 induction of PR-A mRNA with loss of KLF9 expression. Specific antibodies that recognize only PR-B or PR-A by immunofluorescence (Ref. 21 and Clarke, C. L., personal communication) were used to evaluate changes in the levels of these proteins (shown as red staining) in control and E2-treated cells, with and without KLF9 siRNA transfection. KLF9 knockdown did not affect basal levels of PR-B protein (Fig. 5Go, B and D). Consistent with the differences noted in PR-B transcript levels, E2 increased the abundance of PR-B protein; this effect of E2 was abrogated by the presence of KLF9 siRNA. By contrast, PR-A protein levels did not differ for untreated and E2-treated cells in the absence of KLF9 siRNA. However, KLF9 knockdown significantly increased PR-A levels in E2-treated cells (Fig. 5Go, C and D). Results suggest that KLF9 cooperates with ligand-bound ER{alpha} to induce PR-B expression while inhibiting expression of PR-A.


Figure 5
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  		Fig. 5. KLF9 Up-Regulates PR-B but Attenuates PR-A Expression

Ishikawa cells were mock transfected or transfected with siRNA to KLF9 (50 nM) and incubated in phenol red-free medium containing 10% CSS ± E2 (100 nM) for 24 h. Harvested cells were analyzed for (A) transcript and (B–D) protein levels. A, PR-B and PR-A/B mRNA levels were quantified by QPCR. Results (least-square means ± SEM; n = 4 independent experiments) represent normalized values to 18S RNA and are expressed as fold-change over the control value (vehicle, –siKLF9). B–D, Control and E2-treated cells in the presence or absence of siRNA to KLF9 were immunostained with (B) anti-PR-B and (C) anti-PR-A antibodies, followed by incubation with biotinylated secondary antibody and rhodamine-conjugated streptavidin to visualize PR immunopositive cells (red staining). Cells were counterstained with DAPI to identify nuclei (blue). Overlay of DAPI and antibody-stained cells showed nuclear localization of proteins (purple). Representative data for each treatment group are shown. D, Staining intensities of immunopositive cells were expressed on a per cell basis. The bar graphs represent the average staining intensity per cell normalized to those of control (vehicle, –siKLF9) cells, from three independent experiments. Differences were identified by two-way ANOVA, followed by Tukey’s test. Means with different superscripts differed at P < 0.05.

 
KLF9 Disrupts Recruitment of Sp1 to the PR Proximal Promoter
The proximal and distal promoters of the PR gene are thought to regulate PR-A and PR-B mRNA synthesis, respectively (4). Both promoters, albeit E responsive, do not contain canonical ERE sequences; however, the PR proximal promoter contains an ERE-half site [+571 to +575 nucleotides (nt)] adjacent to two GC boxes (at +580 to +585 nt and +590 to +595 nt) (Fig. 6AGo) that bind Sp1 and were found to be critical to E induction of PR-A (47, 48). To evaluate the molecular basis for KLF9 inhibition of PR-A expression, we examined a role for KLF9 in the recruitment of ER{alpha} and Sp1 to the PR proximal promoter, in response to E2. For these studies, we analyzed control and E2-treated cells in the absence and presence of KLF9 siRNA, in conjunction with specific antibodies to ER{alpha}, Sp1, and KLF9. Preimmune sera did not precipitate the PR proximal promoter region in control and E2-treated cells (data not shown). DNA-bound ER{alpha} was detected with anti-ER{alpha} in E2-treated but not in control cells (Fig. 6BGo). Addition of KLF9 siRNA to E2-treated cells did not affect chromatin recruitment of ER{alpha}. Anti-Sp1 antibody precipitated the PR proximal promoter region irrespective of E2 treatment, but only in the presence of KLF9 siRNA (Fig. 6BGo). No significant amounts of KLF9 were recruited to this promoter region in both untreated and E2-treated cells, relative to preimmune antibody (Fig. 6CGo). The levels of Sp1 associated with the promoter region were comparable in non-E2 and E2-treated cells with and without KLF9 siRNA, consistent with the absence of E2 and KLF9 effects on Sp1 mRNA levels (Fig. 6DGo). There was no recruitment of ER{alpha}, KLF9, or Sp1 to a sequence within exon 7 of PR (negative control) in the presence of specific antibodies to these proteins, in control and treated cells (data not shown). Altogether, results indicate that Sp1 binding to the PR proximal promoter, which occurs under basal (no E2) and E2 conditions, is inhibited by the presence of KLF9, albeit KLF9 by itself is not recruited to this region of the promoter. Moreover, corecruitment of ER{alpha} and Sp1 to the PR proximal promoter, in the absence of KLF9 (Fig. 6BGo) likely underlies the observed increase in PR-A isoform expression in E2-treated cells with KLF9 knockdown (Fig. 5Go).


Figure 6
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  		Fig. 6. KLF9 Interferes with Recruitment of Sp1, But Not of ER{alpha}, to the PR Proximal Promoter

A, Schematic representation of the location of ERE half-site at +571 to +575 nt, followed by two Sp1 sites at +580 to +585 nt and +590 to +595 nt of the PR proximal (PR-A) promoter, as originally reported by Kastner et al. (43 ). The bracketed area (+514 to +663 nt) indicates the PR promoter region amplified by the PCR primers. B and C, Ishikawa cells were incubated in phenol red-free medium containing 10% CSS ± E2 (100 nM) ± KLF9 siRNA (50 nM) for 24 h. Cross-linked, sheared chromatin was immunoprecipitated with antibodies against ER{alpha}, Sp1, and KLF9. DNA was analyzed by PCR using primers described in panel A. B, Representative ChIP PCR gels showing recruitment of Sp1 and ER{alpha} to the PR proximal promoter. ChIP PCR results (least-square means ± SEM) from three independent experiments are expressed as the fold-change relative to control (vehicle, –siKLF9) cells. C, ChIP PCR results for recruitment of KLF9 to PR proximal promoter. Results (least-square means ± SEM; n = 3 independent experiments) are expressed as the fold-change relative to vehicle-treated cells immunoprecipitated with preimmune IgG. D, Ishikawa cells treated as above were analyzed for levels of Sp1 mRNA by QPCR. Results (least-square means ± SEM) from four independent experiments are expressed as the fold-change over the control (vehicle, –siKLF9) cells. E, ChIP PCR for PR distal (PR-B) promoter (described in Ref. 38 ), using anti-KLF9, -Sp1, and -ER{alpha} antibodies. Representative ChIP PCR gels demonstrating recruitment of Sp1 but not ER{alpha} to the PR distal promoter are shown. Results (least-square means ± SEM) from four independent experiments are expressed as the fold-change relative to control (for KLF9: vehicle, preimmune IgG; for Sp1 and ER{alpha}: vehicle, –siKLF9) cells. Differences among treatment groups were identified by one-way ANOVA, followed by Tukey’s test. Means with different superscript differed at P < 0.05.

 
Parallel analysis was carried out on a region of the PR-B (distal) promoter containing GC-rich (Sp1 binding) sites previously identified to partly drive PR-B transcription (Fig. 6EGo and Ref. 15). As similarly shown for the PR-A promoter, robust recruitment of Sp1, but not KLF9, to this region was observed (Fig. 6EGo). However, unlike that for PR-A promoter, ER{alpha} was not localized to this promoter region, and the recruitment of Sp1 did not vary with KLF9 siRNA addition under basal or E2 conditions (Fig. 6EGo).

KLF9 Physically Associates with Sp1 But Not ER{alpha}
To further elucidate how KLF9 participates in the ER{alpha}-mediated transcriptional regulation of ER{alpha}, PR-B, and PR-A in Ishikawa cells, we determined whether KLF9 interacts with ER{alpha} and/or Sp1 protein in vivo. For these studies, whole-cell lysates from Ishikawa cells treated with vehicle or E2 for 24 h were subjected to immunoprecipitation with antibodies specific to KLF9, Sp1, ER{alpha}, or with control IgG. Immunoprecipitates were then analyzed by immunoblots using these antibodies. Anti-KLF9 antibody precipitated Sp1 protein (Fig. 7AGo) and conversely, anti-Sp1 precipitated KLF9 (Fig. 7BGo) in both control and E2-treated cells. Control IgG did not precipitate either protein (Fig. 7AGo for Sp1; data not shown for KLF9). Moreover, no association between ER{alpha} and KLF9 (Fig. 7Go, A and C) nor ER{alpha} and Sp1 (Fig. 7CGo) was detected when coimmunoprecipitation was carried out with anti-KLF9 or anti-ER{alpha}. Western blots of cell lysates used for coimmunoprecipitation (designated Input) indicated comparable levels of Sp1 (Fig. 7Go, A and C) and KLF9 (Fig. 7Go, B and C) proteins in control and E2-treated cells relative to the loading control {alpha}-tubulin, consistent with the lack of changes for their respective transcripts (KLF9 in Fig. 1Go; Sp1 in Fig. 6Go) with E2. By comparison, E2-treated cells had lower ER{alpha} protein levels than control cells (Fig. 7AGo).


Figure 7
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  		Fig. 7. KLF9 Physically Associates with Sp1 But Not with ER{alpha}

Cell lysates isolated from Ishikawa cells treated with vehicle or E2 (100 nM) for 24 h were immunoprecipitated (IP) with anti-KLF9, -Sp1, or ER{alpha} antibodies or control IgG. Precipitates were subsequently analyzed by immunoblotting with indicated antibodies. A, CoIP was carried out with anti-KLF9 antibody or rabbit IgG. Protein blots of resultant precipitates and starting cell lysates used for IP were analyzed with anti-Sp1 (top panel) or anti-ER{alpha} (bottom panel) antibodies. B, CoIP was carried out with anti-Sp1 antibody. Protein blots of resultant precipitates and starting cell lysates used for IP were analyzed with anti-KLF9 antibody (top panel). Blot was also probed with anti-tubulin antibody to demonstrate the specificity of the CoIP procedure. C, CoIP was carried out with anti-ER{alpha} antibody. Protein blots of resultant precipitates and input lysates for IP were analyzed with anti-KLF9 (top panel) or anti-Sp1 (bottom panel) antibodies. Results shown are representative of three independent experiments, with each experiment carried out in duplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The present study provides evidence for a negative regulatory role of the PR coactivator KLF9 in ligand-dependent ER{alpha} signaling in Ishikawa endometrial glandular epithelial cells. We show that in an E2-dominated environment, KLF9 facilitated E2 down-regulation of ER{alpha} expression at the levels of mRNA and protein and altered agonist-bound ER{alpha} transactivation of E2-responsive reporter and endogenous promoters. The underlying mechanism by which KLF9 negatively influenced ER{alpha} expression appears to involve the ability of KLF9 to facilitate the association of ER{alpha} to its own promoter, specifically to a region containing GC-rich motifs that are essential for Sp1 regulation of ER{alpha} transcription (38). Using PR as paradigm for an endogenous ER{alpha}-regulated gene, we further show that KLF9 interfered with the formation of an ER{alpha}-Sp1 complex that is essential for ligand-dependent ER{alpha}-mediated expression of PR-A isoform. Specifically, KLF9 excluded Sp1 from binding to GC-rich motifs located in close vicinity to a half-ERE within the PR proximal promoter. Intriguingly, this KLF9 effect was selective and promoter context dependent, because expression of PR-B was positively regulated in contrast to PR-A. The actions of KLF9 on ER{alpha}, PR-B, and PR-A promoters occurred in the absence of its recruitment to the respective promoter regions, suggesting that albeit KLF9 and Sp1 can physically associate, the mechanism for the observed effects of KLF9 did not involve the formation of an ER{alpha}/Sp1/KLF9 complex. To our knowledge this study represents the first report of KLF9 as a negative mediator of ER{alpha} action in E2-responsive cells. More importantly, given the association between loss of PR-B expression, resulting in higher expression ratios of PR-A to PR-B isoforms, and E2-dependent progression of endometrial carcinoma and poor prognosis of the disease (23, 24) and that PR-B isoform mediates the P-dependent reversal of highly proliferative endometrial cells to the differentiated, secretory phenotype (49), our findings lay the groundwork for the possible manipulation of KLF9 expression and/or activity as a targeted approach for control of abnormal endometrial epithelial proliferation resulting from loss of PR-B expression and underlying carcinoma.

Previously, we demonstrated that P-dependent transactivation of the secretory leukocyte protease inhibitor (SLPI) gene in Ishikawa cells occurred, in part, through P/PR induction of KLF9 gene expression and P-mediated corecruitment of KLF9, PR-B, and the PR coactivator cAMP responsive binding protein binding protein to the SLPI promoter (31). The role of KLF9 as a mediator of P-induced gene expression (SLPI) contrasts with its repression (ER{alpha}, PR-A) of E-mediated transcription in these same cells as reported here. Although the dual functions of KLF9 as a trans-activator and trans-repressor of gene promoter activities are not entirely surprising, given previous reports of such activities for various other target genes (50, 51), our findings are noteworthy because they suggest that E and P actions, which have opposite consequences on growth control in endometrial epithelial cells, may converge at the level of KLF9. Interestingly, the participation of KLF9 in the molecular regulation of ER{alpha} (negative), PR-A (negative), and PR-B (positive) expression under an E2-dominated cellular environment, leading to higher PR-B than PR-A expression, is consistent with our previous reports of PR-B as a favored KLF9-interacting protein, and the absence of KLF9 effects on ligand-dependent PR-A transactivity (26). Thus, we suggest that KLF9 may serve as a molecular rheostat of ER{alpha}/PR cross talk by influencing ER{alpha} regulation of specific PR isoform expression, and consequently, the direction of P/PR signaling (49).

The present study used ChIP, RNA interference, and coimmunoprecipitation coupled with immunoblotting to delineate the contribution of KLF9 to E2-dependent PR-A transcriptional regulation in Ishikawa cells. Our results showing lack of recruitment of KLF9 to the PR-A promoter, despite physical association of endogenous Sp1 and KLF9, are consistent with the mechanism of direct sequestration by KLF9 of Sp1, reducing the latter’s availability for binding to the promoter region where it acts as PR-A transactivator. In the breast cancer cell line MCF-7, E2-dependent up-regulation of PR-A involves binding of both ER{alpha} and Sp1, respectively, to the half-ERE and two Sp1 sites located at the PR proximal promoter (47, 48). Indeed, occupation of the GC-rich motifs by Sp1 appeared to be critical for PR proximal promoter activation because mutations of these sites completely abolished E-mediated induction of the PR gene (48). Moreover, mutation of the half-ERE induced transcription, suggesting that binding of ER{alpha} to this region, in the absence of Sp1 binding, is inhibitory to PR transcription. Our findings that KLF9 knockdown allowed Sp1 recruitment to the PR proximal promoter, in concert with ER{alpha}, and coincident with induction of PR-A expression, support the importance of these GC motifs in PR-A transcription and the role of KLF9 in inhibiting this process. Furthermore, the observed recruitment of ER{alpha} to the ERE/Sp1 site in the presence of KLF9, coincident with the observed lack of E2 induction of PR-A expression, is consistent with the suggested nonfunctionality of the ERE-bound ER{alpha} in the absence of Sp1 (48). Mutational mapping of KLF9 and Sp1 in future studies should address the functional domains within each protein that support their physical association leading to functional repression.

The mechanism by which KLF9 participates in E/ER{alpha} induction of PR-B transcription is presently unknown. Unlike the PR-A promoter, the PR distal promoter does not contain ERE or half-ERE, albeit two GC boxes are located within this region (15). The robust recruitment of Sp1, independent of KLF9, and the lack thereof of ER{alpha} to this region in E2-treated cells, contrasted with the KLF9-dependent (Sp1) and -independent (ER{alpha}) recruitment of both proteins to the PR-A proximal promoter and suggest the distinct involvement of KLF9 in PR-A and PR-B transcriptional mechanisms. The nature and context of the Sp1 motifs within the PR-B promoter region, the lack of a neighboring ERE, and the potential proximity of cis-elements for other promoter-specific factors, which can influence the active recruitment of coactivators or sequestration of corepressors, may account for these findings. More definitive characterization of the PR-B promoter and associated regulatory regions is requisite for addressing the mechanisms of E2 induction of PR-B expression involving KLF9.

Our findings highlight a role for KLF9 in facilitating E2 induction of ER{alpha} autoregulation of its transcription. We found that attenuated KLF9 expression in E2-treated cells resulted in loss of ER{alpha} recruitment to the ER{alpha} minimal promoter, leading to maintenance of ER{alpha} mRNA and protein levels. The specific mechanism(s) by which KLF9 mediates this effect is not clear, given 1) the absence of physical association between ER{alpha} and KLF9, 2) the constitutive recruitment of Sp1 to the ER{alpha} promoter, 3) the lack of KLF9 recruitment to the same promoter, and 4) the noted physical association between Sp1 and KLF9. Albeit speculative at the present time, these observations raise the interesting possibility of KLF9 involvement in ER{alpha} degradation upon ligand binding. Given that inhibition of the ubiquitin-proteasome pathway, specifically involving nuclear-expressed, neural precursor cell expressed developmentally down-regulated 8 (NEDD8) protein leads to maintenance of ER{alpha} transcriptional activity (52, 53), we suggest that interaction of KLF9 (directly or indirectly) with nuclear components of this pathway, resulting in inhibition of ER{alpha} proteolysis, could underlie KLF9’s promotion of ER{alpha} binding to its promoter and subsequent decreased ER{alpha} gene expression. Increased ER{alpha} down-regulation in the presence of KLF9 may also account for KLF9 repression of ligand-activated ER{alpha} induction of the ERE-driven reporter gene promoter activity. By contrast, the induction of PR-B activity in the presence of KLF9 and the loss thereof with KLF9 knockdown suggest that the above mechanism involving KLF9 may not apply to all E2-responsive genes. In this regard, it has been reported that E2-induced transcription of the PR gene is not directly dependent on ER{alpha} occupancy of its promoter (54) and that the relationship between ER{alpha} transcriptional activation and proteasome inhibition may not be applicable to E2-induced genes with complex (i.e. non-ERE) promoters (53). Future studies will address regulation by KLF9 of E2-induced ER{alpha} proteolysis involving the ubiquitin-proteasome system as a novel mechanism by which KLF9 may control ER{alpha} signaling.

The results presented here provide some insights into the potential functional relevance of KLF9 in the control of epithelial proliferation, dysregulation of which underlies the development of endometrial carcinoma. The selective inhibition by KLF9 of ER{alpha} expression at high E2 concentration (100 nM) implies that ER{alpha}-dependent proliferation in a background of unopposed E action can be potentially inhibited by overexpression of KLF9. Analysis of tissue arrays of adenocarcinoma ranging from well-differentiated to poorly differentiated phenotypes, for KLF9 expression and correlation with ER{alpha} expression, could potentially address this question.

In summary, KLF9 repression of ligand-activated ER{alpha} signaling can occur through its inhibition of ER{alpha} expression and selective regulation of ER{alpha} transactivity in E2-responsive promoters. Our results suggest that KLF9 may prime epithelial cells for increased sensitivity to PR-B action by increasing the relative ratios of PR-B to PR-A, findings which may have clinical ramifications given the distinct roles of PR-B and PR-A on cellular differentiation and in the development and progression of endometrial carcinoma (55, 56). Because KLF9 is also a PR interacting protein, preferentially of PR-B in glandular epithelial cells (25, 26, 31), our data tentatively place KLF9 at the node of PR and ER{alpha} signaling pathways and suggest its potential to coordinately influence the expression of multiple genes regulated by E and P. Whether KLF9 functions as brake for ER{alpha} actions in all epithelial cells and underlies deregulated estrogen signaling in those lacking its expression are important questions that require further elucidation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Cell culture media, antibiotic/antimycotic solution, and TriZol reagent were purchased from Invitrogen (Carlsbad, CA). Oligonucleotides were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). All other reagents, except where noted, were purchased from Fisher Scientific (Pittsburgh, PA).

Cell Culture and Treatments
Ishikawa cells were routinely grown at 37 C in MEM supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution in an atmosphere of 5% CO2 /95% air (31). For E2 treatment, cells were seeded in 60-mm plates at a density of 6 x 105 cells per well. Subconfluent cells (~50%) were serum starved in phenol red-free MEM containing 0.5% charcoal-stripped fetal bovine serum (CSS) for 24 h, and then treated with vehicle (PBS), or 17β-estradiol (E2; 100 nM final concentration) (Sigma Chemical Co., St. Louis, MO) in the presence or absence of the ER antagonist, ICI 182,780 (ICI; 1 µM; Tocris Bioscience, Ellisville, MO). Treatments were performed in phenol red-free MEM containing 10% CSS for 24 h. For KLF9 knockdown, double-stranded siRNA targeting human KLF9 mRNA (Catalog no. 16708, identification no. 3082; Ambion, Austin, TX) was used at 50 nM final concentration. The siRNA was introduced into cells by reverse transfection using siPORT NeoFX Lipid-Based Agent (Ambion) in OPTI-MEM I reduced serum medium (Invitrogen), as described previously (31). Cells were then treated with vehicle or E2 (100 nM) for 24 h. Experiments were performed in quadruplicate on three separate occasions.

RNA Isolation and QPCR
Total RNA was isolated from cells using TriZol reagent (Invitrogen) and analyzed for integrity using the Agilent 2100 Bioanalyzer and RNA 6000 NanoLabChip kit (Agilent Biotechnologies, Palo Alto, CA). RNA samples were reverse transcribed using random primers and a cDNA synthesis kit (Applied Biosystems, Foster City, CA). mRNA levels were evaluated by real-time QPCR, using primer pairs spanning an intron to eliminate genomic DNA amplification (PrimerExpress; Applied Biosystems). Primers used to detect KLF9, PR-A/B, PR-B, and 18S rRNA, and amplification protocols were described in a previous study (31). The forward and reverse primers for human ER{alpha}, Sp1, and KLF13, respectively, and resultant PCR product sizes (in parentheses) are: ER{alpha}: 5'-CGG CAT TCT ACA GGC CAA ATT-3' and 5'-AGC GAG TCT CCT TGG CAG ATT-3' (111 bp); Sp1: 5'-TGG TGG TGG TGC CTT TTC A-3' and 5'-GCT GTT CTC ATT GGG TGA CTC A-3' (145 bp); and KLF13: 5'-TGC GAG AAA GTT TAC GGG AAA-3' and 5'-CCC GTG TGT GTG CGG TAG T-3' (149 bp). For each sample, relative gene expression was calculated with 18S rRNA as internal reference.

Transient Transfection and Luciferase Assays
Plasmid DNAs for transfection studies were prepared using the Maxiprep system (QIAGEN, Valencia, CA). The sources of expression vectors and reporter DNA constructs were as follows: 1) 4xERE-TK-Luc reporter construct, human ER{alpha} in pCMV5 (hER{alpha}-pCMV5), and corresponding empty vector (pCMV5) was supplied by Dr. Benita S. Katzenellenbogen [University of Illinois at Urbana-Champaign, IL (57, 58)] and 2) rat KLF9/BTEB1 cDNA cloned into pCDNA3 vector (pCDNA3-BTEB/Klf9) and corresponding empty vector (pCDNA3) supplied by Dr. Hiroaki Imataka [McGill University, Quebec, Canada (59)]. Ishikawa cells were transfected using lipofectamine with 4xERE-TK-Luc reporter plasmid (5 µg/well) and various combinations of expression (hER{alpha}-pCMV5; pCDNA3-BTEB/KLF9) and empty (pCDNA3; pCMV5) vectors (0.5 µg/well), following previously described protocols (25, 26). Treatment of cells with E2 (100 nM) or vehicle was for 48 h. Cells were lysed in 1x lysis buffer (Promega Corp., Madison, WI), and quantitative determination of luciferase activity (measured as relative light units) in cell lysates was carried out using an Autolumat Luminometer (EG&G Berthold, Bad Wildbad, Germany). Luciferase activity was normalized to total cellular extract protein, determined by the Bradford dye-binding procedure (60). Data are presented as least-square means ± SEM from three independent experiments, with each experiment performed in triplicate.

Immunofluorescence
Ishikawa cells were seeded on sterile 22-mm glass cover slides at a density of 3.0 x 105 cells per well and grown overnight. Cells were transfected with KLF9 siRNA (50 nM) and treated with vehicle or E2 (100 nM) for 24 h. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized in 0.25% Triton-PBS for 30 min, with 1x PBS washes after each step. Cells were incubated in 1% goat serum blocking solution (Vectastain elite ABC kit, Vector Laboratories, Inc., Burlingame, CA) for 30 min and then overnight at 4 C with anti-ER{alpha} rabbit polyclonal antibody (sc-542; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (1:500 dilution), or antibodies (1:500 dilution) that recognize only PR-B (hPRa6; LabVision Corp., Fremont, CA) or PR-A (sc-538; Santa Cruz Biotechnology) by immunofluorescence in human cells (21). Cells were then incubated with the biotinylated secondary antibody (Vectastain elite ABC kit) for 30 min at room temperature and rhodamine (tetramethylrhodamine isothiocyanate) conjugated-streptavidin (1:250 dilution) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min at room temperature, with 1x PBS washes before and after each step. Cells were mounted using Vectashield Mounting Medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Fluorescence was visualized under a Carl Zeiss Axiovision microscope (Carl Zeiss AG, Oberkochen, Germany) and quantified based on densitometric mean per cell using the Axiovision software program. Results (least square means ± SEM) are from nine different areas of each of three independent experiments and are expressed as fold-induction over the control (vehicle; –siKLF9) cells.

ChIP
Ishikawa cells (2.5 x 106) were seeded in 10-cm plates in MEM containing 10% FBS overnight, and were then serum starved in 0.5% CSS phenol red-free MEM for 24 h. Cells were treated with vehicle or E2 (100 nM) in phenol red-free MEM containing 10% CSS for 24 h. Treatment with KLF9 siRNA followed protocols described above. Cells were processed for ChIP using EZ-Chip kit following the manufacturer’s recommendations (Upstate Biotechnology, Inc., Lake Placid, NY). Briefly, cells were cross-linked with 10% formaldehyde for 10 min, and the reaction was stopped with 1x Glycine solution. Cellular DNA was sheared to about 400 bp in length by sonication (Sonic Dismembrator 50; Fisher) in ice for 15 pulses in 10-sec cycles. Aliquots of the lysate were set aside to quantify genomic DNA present (DNA input); the remainder was diluted (1:10) in ChIP dilution buffer. Antibodies (1 µg; Santa Cruz Biotechnology) for immunoprecipitation of cell lysates were: 1) anti-BTEB1/KLF9 goat polyclonal antibody (sc-12994); 2) anti-ER{alpha} rabbit polyclonal antibody (sc-542); and 3) anti-Sp1 goat polyclonal antibody (sc-59). For the preimmune control, an equal amount (1 µg) of normal goat IgG (sc-2028; Santa Cruz Biotechnology) was substituted for the test antibody. Immune complexes were recovered by incubation with salmon sperm DNA/Protein A (rabbit antibody) or Protein G (goat antibody) for 1 h at 4 C, successively washed with a series of salt solutions, and then eluted with 1% sodium dodecyl sulfate/0.1 M NaHCO3. Cross-links were reversed by treatments with 0.2 M NaCl for 4 h at 65 C, RNAse (50 µg/ml) for 30 min at 37 C, and Proteinase K (50 µg/µl in 10 mM Tris-HCl/10 mM EDTA) for 1.5 h at 45 C. The ChIP-captured DNA was purified for PCR by affinity columns included in the kit. RT-PCR using primers spanning the human ER{alpha} minimal promoter region (hER{alpha}prom2: forward, 5'-GTC TTC CCT GGG CCA CCT T-3' and reverse, 5'-TTT GGA GCG ATC CCA AAG AG-3') containing binding sites for Sp1 transcription factor family members (38); to the human PR proximal promoter containing the Sp1/ERE-half-site (5'-GGG ACA AAC GAC AGC CAC AGT TC-3' and 5'-GGG GCA GAG GGA GGA GAA AGT G-3'; 150 bp) (47, 48), and to the human PR distal promoter containing GC-rich regions binding Sp1 (5'-ACG GGT GGA AAT GCC AAC T-3' and 5'-AGG CTT ACC CCG ATT AGT GAC A-3'; 145 bp) were carried out under the following conditions: 1) hot start at 94 C for 5 min; 2) addition of Taq polymerase and then 94 C for 5 min; 3) 35 cycles of 94 C for 45 sec, 60 C for 1 min, and 72 C for 30 sec; and 4) final extension of 72 C for 7 min. PCR products were resolved on 2% agarose gels containing ethidium bromide and visualized under UV light. Band intensities were quantified using the Bio-Rad molecular analyst detection system (Bio-Rad Laboratories, Inc., Hercules, CA) and Quantity One Software. Three independent experiments were performed using cells of similar passages.

Immunoprecipitation and Immunoblotting
For immunoprecipitation experiments, Ishikawa cells were plated on 100-mm dishes at a density of 2.5 x 106 cells per dish in MEM containing 10% FBS overnight. Cells, when 70% confluent, were serum starved in 0.5% CSS phenol red-free MEM for 24 h. Cells were treated with vehicle or E2 (100 nM) in phenol red-free MEM containing 10% CSS. Twenty four hours after treatment, cells were chilled on ice, washed with ice-cold PBS, and lysed in RIPA Lysis Buffer (sc-24948; Santa Cruz Biotechnology). Cell lysates were isolated after sonication (10 sec on ice) and centrifugation. The protein concentrations were determined by the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). Coimmunoprecipitation (CoIP) assays were done with the Catch and Release Immunoprecipitation System following the manufacturer’s instructions (Upstate Biotechnology, Lake Placid, NY). Briefly, lysates (800 µg protein) were diluted in 1xCatch and Release Lysis/Wash Buffer and incubated with 4 µg of rabbit anti-rat KLF9 (25), anti-Sp1 (sc-59), or anti-ER{alpha} (sc-542; Santa Cruz Biotechnology, Inc.) antibodies and 1 µg of Antibody Capture Affinity Ligand. The mixtures were placed on a rocking platform overnight at 4 C. Protein complexes were purified by binding to Protein A-affinity beads in a spin column provided in the immunoprecipitation (IP) system, followed by several washings in 1x Catch and Release lysis/wash buffer and brief centrifugation. Bound complexes were eluted in buffer containing 0.5% β-mercaptoethanol, and analyzed by Western blots following previously described procedures (25). Membranes were probed with rabbit anti-KLF9 (diluted 1:500), anti-Sp1 (sc59; diluted 1:600), or anti-ER{alpha} (sc-542; diluted 1:1000), using Tris-buffered saline containing 0.05% Tween 20 as dilution buffer. Immunoreactive bands were detected by incubation with antirabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology). Anti-{alpha}-tubulin (sc-5546; Santa Cruz Biotechnology) or anti-LaminB1 (ab16048; Abcam, Inc., Cambridge, MA) antibodies at 1:1000 dilutions were used to reprobe blots as internal loading controls. Nuclear extracts and cell lysates were prepared as previously described (25).

Data Analysis
Data, presented as least-square means ± SEM, were subjected to analyses by Student’s t test, one-way ANOVA, or two-way ANOVA. Differences between means in two-way ANOVA were further analyzed by Tukey’s test. P values < 0.05 were considered statistically significant.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Christine Clarke (University of Sydney, New South Wales, Australia) for helpful advice on PR antibodies used for immunofluorescence, and Renea Easton for technical assistance.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant HD-21961.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 23, 2007

1 M.C.V. and Z.Z. contributed equally to this work and should be considered as first co-authors. Back

Abbreviations: BTEB1, Basic transcription element binding protein-1; ChIP, chromatin immunoprecipitation; CSS, charcoal-stripped fetal bovine serum; DAPI, 4',6-diamidino-2-phenylindole; E, estrogen; E2, 17β-estradiol; ER, estrogen receptor; ERE, estrogen-responsive element; FBS, fetal bovine serum; IP, immunoprecipitation; KLF, Krüppel-like factor; nt, nucleotide; P, progesterone; PR, progesterone receptor; QPCR, quantitative real-time PCR; siKLF9, small interfering KLF9; siRNA, small interfering RNA; Sp1, specificity protein 1; tk, thymidine kinase.

Received for publication May 9, 2007. Accepted for publication August 9, 2007.


   REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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