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. 2011 May 12;25(7):1137–1144. doi: 10.1210/me.2010-0497

The Induction of KLF5 Transcription Factor by Progesterone Contributes to Progesterone-Induced Breast Cancer Cell Proliferation and Dedifferentiation

Rong Liu 1, Zhongmei Zhou 1, Dong Zhao 1, Ceshi Chen 1,✉
PMCID: PMC5417250  PMID: 21566082

Abstract

Progesterone (Pg) promotes normal breast development during pregnancy and lactation and increases the risk of developing basal-type invasive breast cancer. However, the mechanism of action of Pg has not been fully understood. In this study, we demonstrate that the mRNA and protein expression of Klf5, a pro-proliferation transcription factor in breast cancer, was dramatically up-regulated in mouse pregnant and lactating mammary glands. Pg, but not estrogen and prolactin, induced the expression of Krüpple-like factor 5 (KLF5) in multiple Pg receptor (PR)-positive breast cancer cell lines. Pg induced the KLF5 transcription through PR in the PR-positive T47D breast cancer cells. Pg-activated PR increased the KLF5 promoter activity likely through binding to a Pg response element at the KLF5 promoter. Importantly, Pg failed to promote T47D cell proliferation when the KLF5 induction was blocked by small interfering RNA. KLF5 is essential for Pg to up-regulate the expression of cell cycle genes, including CyclinA, Cdt1, and E2F3. In addition, KLF5 overexpression was sufficient to induce the cytokeratin 5 (CK5) expression, and the induction of CK5 by Pg was significantly reduced by KLF5 small interfering RNA. Consistently, the expression of KLF5 was positively correlated with that of CK5 in a panel of breast cancer cell lines. Taken together, we conclude that KLF5 is a Pg-induced gene that contributes to Pg-mediated breast epithelial cell proliferation and dedifferentiation.

Progesterone (Pg) is essential for normal postnatal breast development during pregnancy and lactation by stimulating ductal side branching and development of lobuloalveolar structures (1). Under the stimulation of Pg, the mammary gland epithelium undergoes extensive proliferation and remolding (1). Pg functions primarily through the ligand-activated nuclear receptor transcription factor Pg receptor (PR). PR knockout (KO) mice showed incomplete mammary gland ductal side branching and failure of lobuloalveolar structure development due to insufficient cell proliferation (2). However, the molecular mechanism of action of Pg and PR has not been completely elucidated.

Accumulated evidence suggests that Pg and PR promote mammary tumorigenesis (3, 4). Pg has been shown to stimulate breast cancer for menopausal women in several large-scale hormone-replacement therapy clinical studies (5–7). In these studies, Pg plus estrogen significantly increased the risk of invasive breast cancer compared with estrogen alone. Additionally, progestins have been shown to have proliferative effects in the PR-positive breast cancer cell lines in vitro (8) and in nude mice (9). Furthermore, progestins increased mammary tumor incidence in rats (10–12) and dogs (13).

Recently, progestins have been shown to reprogram a subset of estrogen receptor (ER)+PR+cytokeratin 5 (CK5)− differentiated cells into ER-PR-CK5+ basal-like progenitor cancer cells in vitro and in vivo (14–16). The ER-PR-CK5+ cells have progenitor potential and are less prone to drug-induced apoptosis during the endocrine therapy and chemotherapies (16). Interestingly, Pg increased the development of CK5+ basal type breast cancer in rats (17). However, the mechanism by which progestins reprogram ER+PR+CK5− cells into ER-PR-CK5+ cells is not clear.

Krüpple-like factor 5 (KLF5) is a transcription factor that regulates cell proliferation, survival, differentiation, and embryonic stem cell (ESC) self-renewal. Previously, we demonstrated that KLF5 overexpression promotes the G1/S cell cycle progression (18) and breast cell proliferation, survival, and tumor growth (19, 20). Furthermore, KLF5 has been reported to regulate smooth muscle cell and adipocyte differentiation (21, 22). Most recently, KLF5 was shown to promote the self-renewal of ESC and to maintain ESC in an undifferentiated state (23, 24). Interestingly, the gene expression signature of basal-like breast cancer cells is similar to that of ESC as shown through analyzing the microarray data from 1211 breast tumors (25). KLF5 and eight other genes are highly expressed in basal-type breast tumors (25). Indeed, positive KLF5 mRNA expression is an unfavorable prognostic marker correlated with shorter survival for breast cancer patients (26). However, the transcriptional regulation of KLF5 in breast cancer is largely unknown. We hypothesize that KLF5 is a Pg and PR downstream target gene and plays an important role in Pg-induced breast cancer cell proliferation and dedifferentiation.

Results

The expression of Klf5 is increased in mouse mammary glands during pregnancy and lactation stages

To explore the physiological regulation of Klf5 in the breast, we first examined the Klf5 protein expression by immunoblotting in the mouse mammary glands during four different stages (virgin, pregnancy, lactation, and involution). As shown in Fig. 1A, the protein expression level of Klf5 was undetectable in mammary glands during the stages of virgin and involution but increased dramatically in mammary glands during the stages of pregnancy and lactation. The expression up-regulation of Klf5 during the stages of pregnancy and lactation occurred at the mRNA level as assessed by RT-PCR (Fig. 1B). These results imply that the Klf5 transcription may be regulated by a specific hormone produced during the stages of pregnancy and lactation.

Fig. 1.

Fig. 1.

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The KLF5 expression is induced in mouse mammary glands during the stages of pregnancy and lactation and in PR-positive breast cancer cell lines by Pg. A, The protein expression levels of Klf5 are specifically up-regulated in mouse mammary glands during the stages of pregnancy and lactation. Mouse mammary glands were collected from female mice in different stages [V, virgin; P, pregnancy (13.5 d and 16.5 d); L, lactation; I, involution]. Two mice (B6) in each stage were examined. Mammary gland tissue lysates were used for immunoblotting. Gapdh was used as a loading control. B, The mRNA expression levels of Klf5 are specifically up-regulated in mouse mammary glands during pregnancy and lactation. The Klf5 mRNA levels were measured by RT-PCR. Gapdh was used as a loading control. C, Pg but not estrogen (E) or prolactin (Prl) induces the KLF5 protein expression. T47D cells were seeded in 12-well plates at 2.2 × 105 cells per well. The cells were serum-starved for 24 h and treated with Pg (100 nm), E (20 nm 17β-estradiol), Prl (100 ng/ml), or different combinations for 8 h. The KLF5 protein expression was measured by immunoblotting. GAPDH serves as a loading control. D, Pg induces the KLF5 protein expression in PR-positive but not in PR-negative breast cancer cell lines. All cells were treated with 100 nm Pg for 8 h or left untreated in serum-free media. The data shown in this figure are representative of two independent experiments.

Pg induces the KLF5 expression in ER+PR+ breast cancer cell lines

The postnatal development of normal mammary glands involves a coordinated effort of pituitary and ovarian hormones. Of those hormones, estrogen, Pg, and prolactin are the most significant regulators for duct growth and alveolar development. In an ER+PR+ luminal breast cancer cell line T47D in which the endogenous KLF5 protein level is very low, we demonstrated that Pg, but not estrogen and prolactin, induced the KLF5 protein expression (Fig. 1C). Estrogen and prolactin could not even increase the KLF5 expression when in combination with Pg (Fig. 1C). Pg also induced the KLF5 protein expression in several other ER+PR+ breast cancer cell lines, such as MCF7 and BT474, but not in ER−PR− breast cancer cell lines BT20 and MDA-MB-231 (Fig. 1D).

Pg activates the KLF5 transcription

Then, a time- and dosage-course experiment was conducted in T47D. As shown in Fig. 2, A and B, KLF5 was induced by Pg in time- and dosage-dependent manners at both the protein and mRNA levels. The induction of KLF5 by Pg could be detected after 1 h, suggesting that KLF5 is a Pg early-responsive gene and a possible PR direct-target gene. The maximum induction time for the KLF5 protein is about 6 h (Fig. 2A). The KLF5 expression can be significantly induced by Pg at physiological concentrations (1–100 nm). Similar results were observed in MCF7 (data not shown). These results suggest that Pg may increase the KLF5 transcription. To test whether Pg can activate the KLF5 promoter, we performed a luciferase reporter assay in T47D cells. As expected, the KLF5 promoter was significantly activated by synthetic Pg medroxyprogesterone 17-acetate (MPA) (Fig. 2C).

Fig. 2.

Fig. 2.

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Pg induces the KLF5 expression in time- and dosage-dependent manners and activates the KLF5 transcription in T47D. A, The KLF5 protein was induced by Pg in a time- and dosage-dependent manner. T47D cells were treated with Pg at 100 nm for the indicated time (left panel) or 8 h at the indicated concentration (right panel). B, Pg induced the KLF5 mRNA expression in a time- and dosage-dependent manner. The KLF5 mRNA expression levels were measured by qRT-PCR. Error bars show sd values from triplicates in a single representative experiment. C, Pg activated the KLF5 promoter. T47D cells were transfected with PR-B expressing plasmid and the KLF5 promoter luciferase reporter. One day after transfection, the cells were serum-starved overnight and treated with 20 μm MPA for 24 h. Cell lysates were collected for the dual-luciferase reporter assay. **, P < 0.001. D, PR specifically binds to the KLF5 promoter in vivo as determined by ChIP assays. The input DNA and water were used as the positive and negative controls. The coding sequence (CDS) of KLF5 was amplified to test the binding specificity. The Bcl-2 gene promoter region was amplified as a positive control for ChIP (28). All data shown in this figure have been confirmed by independent repeated experiments.

Because Pg activated the KLF5 gene promoter, we searched the KLF5 promoter sequence and identified a 6-bp potential Pg response element sequence (−548 TGTACA −543) (27). To determine whether PR binds to this Pg response element at the KLF5 promoter, we performed chromatin immunoprecipitation (ChIP) assays in T47D cells using the Bcl2 gene promoter as the positive control (28). As shown in Fig. 2D, the anti-PR antibody, but not the IgG control, specifically immunoprecipitated the KLF5 promoter rather than coding region. Furthermore, Pg dramatically enhanced the binding of PR to the KLF5 and Bcl2 promoters. These results suggest that KLF5 is a direct PR target gene.

Pg induces the KLF5 expression through PR

To test whether Pg induces the KLF5 expression through PR, we first blocked the PR activity using the PR antagonist RU-486 in T47D and found that the induction of KLF5 protein by Pg was completely abolished (Fig. 3A). Furthermore, two different PR small interfering (siRNA) almost completely blocked the KLF5 protein induction in T47D (Fig. 3B). These results clearly indicate that PR is required for the KLF5 induction by Pg.

Fig. 3.

Fig. 3.

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The induction of KLF5 by Pg is through PR and is essential for Pg to promote T47D cell proliferation and gene expression. A, The Pg antagonist RU-486 (100 nm) completely blocked the KLF5 induction by Pg (100 nm) or by E (20 nm)+Pg (100 nm). T47D cells were treated with hormones and RU-486 for 8 h. B, Two different PR siRNAs blocked the induction of KLF5 by Pg. T47D cells were transfected with control Luciferase siRNA (Lucsi) or two different PR siRNAs (PRsi#1 and PRsi#2). One day after transfection, the cells were serum starved for 24 h and then were treated with Pg (100 nm) overnight. There are two PR isoforms (B and A). C, The KLF5 and PR siRNA silenced the KLF5 and PR protein expression in T47D in the absence and presence of Pg. D, KLF5 or PR depletion by siRNA significantly blocked the DNA synthesis increase induced by Pg in T47D. The DNA synthesis was measured by [3H]thymidine incorporation assay. KLF5 siRNA itself decreased the DNA synthesis due to the depletion of endogenous KLF5 expression. **, P < 0.001, t test. E, KLF5 or PR depletion by siRNA significantly blocked the Pg-induced expression of CyclinA, Cdt1, and E2F3 in T47D. The mRNA levels were measured by qRT-PCR. GAPDH was used to normalize the input cDNA. Figures show representative data of three independent experiments.

The induction of KLF5 is essential for Pg to promote cell proliferation and gene expression

Because both Pg and KLF5 have been shown to promote cell proliferation, we wondered whether KLF5 is induced by Pg to promote cell proliferation. To test this, we blocked the Pg-induced KLF5 by a well-characterized KLF5 siRNA (18) and two PR siRNA in T47D and examined the DNA synthesis. As expected, Pg dramatically increased the DNA synthesis in Lucsi transfected cells (Fig. 3D). PR depletion not only blocked the KLF5 induction but also blocked the Pg-induced DNA synthesis increase (Fig. 3, C and D). When the KLF5 induction was depleted by KLF5 siRNA, Pg failed to increase the DNA synthesis in T47D (Fig. 3, C and D). To understand the mechanism by which KLF5 promoted cell proliferation, we examined the mRNA expression of several genes involved in cell cycle (29). As shown in Fig. 3E, the induction of CyclinA, Cdt1, and E2F3 by Pg was blocked by the depletion of KLF5 or PR. These results suggest that the KLF5 induction is essential for Pg to stimulate gene expression and cell proliferation in vitro.

KLF5 contributes to Pg-induced CK5 expression

Horwitz and co-workers (14, 15) showed that progestins could reprogram a small subset of ER+PR+CK5− T47D cells into ER-PR-CK5+ progenitor cells. As an ESC transcription factor, KLF5 may contribute to this dedifferentiation step. It has been shown that KLF5 is highly expressed in basal-type (CK5+) breast cancers (25). To test if KLF5 induces the CK5 expression, we infected T47D cells with green fluorescent protein (GFP) control and KLF5-IRES-GFP adenoviruses. Indeed, KLF5 induced the CK5 protein expression compared with the GFP control by immunofluorescence staining (IF) (Fig. 4A). However, only a small percentage (∼2–3%) of T47D cells infected with KLF5 adenoviruses expressed CK5. Furthermore, the CK5 expression was not only limited to GFP/KLF5-positive cells. The ER and PR levels were not steadily down-regulated by KLF5 by immunoblotting (data not shown). A 10-fold induction of CK5 mRNA expression by KLF5 was detected by quantitative RT-PCR (qRT-PCR) in T47D (Fig. 4B).

Fig. 4.

Fig. 4.

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KLF5 is sufficient to induce the CK5 expression and partially contributes to Pg-induced CK5 expression in T47D. A, KLF5 induces the CK5 protein expression in T47D, as determined by immunofluorescence. KLF5 was delivered into T47D by adenoviruses with the GFP marker (the GFP expression is independently regulated). The GFP adenoviruses were used as a negative control. B, KLF5 overexpression induced the CK5 mRNA expression. The KLF5 and CK5 mRNA levels were measured by qRT-PCR. C, KLF5 depletion significantly reduced the Pg-induced CK5 expression. T47D cells were transfected with Lucsi, KLF5si, and PRsi, respectively, and were treated with or without 100 nm Pg for 24 h. The KLF5, CK5, and SGK mRNA levels were measured by qRT-PCR. *, P < 0.01. Figures show representative data of three independent experiments. DAPI, 4′,6-Diamidino-2-phenylindole.

Because Pg has been shown to induce the CK5 expression in a small subset of T47D cells (14, 15), we tested whether Pg induces CK5 through KLF5. Indeed, when the induction of KLF5 was blocked by KLF5 siRNA, Pg-induced CK5 mRNA expression was significantly but not completely blocked (Fig. 4C). The induction of serum glucocorticoid regulated kinase (SGK), a PR direct target gene (30), was not affected by KLF5 siRNA at all. As expected, PR siRNA blocked the induction of KLF5, CK5, and SGK by Pg. These findings suggest that KLF5 may contribute to Pg-mediated cell dedifferentiation as indicated by inducing the CK5 expression.

The expression of KLF5 is negatively correlated with ER/PR but positively correlated with CK5 in breast cancer cell lines

Progestins have been shown to suppress ER and PR expression in addition to inducing CK5 expression (14, 15). To further test if KLF5 contributes to the reduction of ER and PR by progestins, we treated T47D cells with MPA for 6 and 24 h and examined the KLF5, ER, and PR expression. With the induction of KLF5, MPA also dramatically decreased the ER and PR expression (Fig. 5A). However, when the induction of KLF5 was blocked by KLF5 siRNA, the reduction of ER and PR was not affected at all. These results imply that the MPA-induced loss of ER and PR expression is not mediated by KLF5.

Fig. 5.

Fig. 5.

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The expression of KLF5 is negatively correlated with ER/PR but positively correlated with CK5 in breast cancer cell lines. A, The induction of KLF5 by MPA (20 μm) was in parallel with the reduction of ER and PR in T47D. KLF5 depletion did not block the Pg-induced loss of ER and PR expression. B, The protein expression of KLF5, CK5, ER, and PR in eight breast cancer cell lines was measured by immunoblotting. GAPDH was used as a loading control. Asterisk indicates a nonspecific band. Data show representative result of three independent experiments.

Finally, we examined the expression of KLF5, ER, PR, and CK5 by immunoblotting in a panel of eight breast cancer cell lines (Fig. 5B). In agreement with our previous report (31), KLF5 is lowly expressed in all ER+CK5− and/or PR+CK5− breast cancer cell lines (MCF7, HCC1500, MDA-MB-134, and T47D) but highly expressed in ER-PR-CK5+ breast cancer cell lines (SW527, HCC1937, HCC1806, and SUM149). This expression pattern suggests that the KLF5 expression may be a signature of ER-PR-CK5+ cells.

Discussion

Pg and its cognate receptor PR are essential for normal breast development during pregnancy and regulate breast carcinogenesis. However, the mechanisms of the action of Pg and PR have not been fully understood. For the first time, we demonstrate that the KLF5 transcription factor is induced by Pg and PR and contributes to Pg-induced cell proliferation and CK5 expression in PR-positive breast cancer cells. Our findings may reveal a novel mechanism for Pg and PR to regulate the development of normal breast and breast cancer.

KLF5 is an important Pg/PR downstream gene promoting cell cycle. In PR-positive breast cancer cell lines, Pg induces quiescent cells to enter the cell cycle through up-regulating several G1-S phase cell cycle proteins including Cyclin D1 (32). KLF5 has also been shown to induce Cyclin D1 (33). However, the induction of cyclin D1 by progestins is not mediated by KLF5 (data not shown). The KLF5 direct target gene, fibroblast growth factor-BP, was also not induced by Pg in T47D (data not shown). We found that KLF5 is essential for Pg to induce the expression of cell cycle genes including CyclinA, Cdt1, and E2F3 in T47D (Fig. 3E). These genes have been well implicated in Pg-induced breast cell cycle G1/S transition (29).

Pg has been shown to reprogram a small subset of ER+PR+CK5− differentiated luminal cells into ER-PR-CK5+ progenitor cancer cells (14, 15). Given the significant role of KLF5 in maintaining ESC self-renewal and preventing their differentiation, it is not surprising that KLF5 contributes to Pg-initiated cell reprogram. KLF5 contributes to Pg-induced cell dedifferentiation as suggested by inducing the CK5 expression (Fig. 4). Interestingly, the induction of CK5 by KLF5 in T47D showed a paracrine manner. The KLF5-negative cells next to KLF5-positive cells also expressed CK5. It is possible that KLF5 induced the expression of yet to be identified secreted proteins that indirectly induced the CK5 expression. Our result is consistent with a previous report that Pg acts by a paracrine mechanism in mammary epithelial cells (34). In T47D cells, Pg only induced CK5 in a very small percentage of cells by IF (data not shown; we could not steadily detect the induced CK5 by immunoblotting). When the induction of KLF5 is blocked, the Pg-induced CK5 mRNA levels were significantly reduced. Thus, KLF5 is not only sufficient to induce the CK5 expression but also essential for Pg to efficiently induce the CK5 expression.

The induction of KLF5 by progestins was also in parallel with the reduction of ER/PR in T47D. This explains the observation that KLF5 is highly expressed in ER-PR-CK5+ basal-like breast cancer cells although KLF5 is induced by Pg in ER+PR+CK5− luminal breast cancer cells. Thus, Pg can reprogram the ER+PR+KLF5-CK5− breast cancer cells into the ER-PR-KLF5+CK5+ cells by inducing the expression of KLF5, CK5, and other genes. How the KLF5 expression is maintained in ER-PR− breast cancer cells is still unknown. Recently, KLF5 has been reported to interact with ERα and suppresses its functions (35). However, KLF5 is not responsible for the loss of ER/PR after progestin stimulation. Whether KLF5 contributes to progestin-induced other differentiation gene expression is unknown. Because ER-PR-CK5+ cells from luminal breast cancers are less prone to drug-induced apoptosis and resistant to standard endocrine and chemotherapies (16), Pg/PR/KLF5 targeted therapy may prevent ER/PR-positive breast cancer progression and overcome the drug resistance.

Accumulated evidence suggests that KLF5 is a pro-proliferation and pro-survival oncogenic transcription factor specifically overexpressed in ER-PR-CK5+ breast cancers (19, 20, 31). The transcription of KLF5 has been shown to be up-regulated by several oncogenes including Ras (36), ErbB2 (37), and Wnt1 (38). We demonstrated that Pg induces the KLF5 transcription through PR (Fig. 3). Interestingly, KLF5 is also an androgen-induced gene in androgen receptor-positive prostate cancer cell lines (27, 39, 40). Dexamethasome, a glucocorticoid class hormone, can also induce the KLF5 expression in MCF7 (data not shown). It has been shown that the PR level is down-regulated in late pregnant stage and becomes undetectable during lactation (41). Thus the high levels of KLF5 protein and mRNA in the lactation stage are not likely due to Pg/PR. It is possible that Pg and PR only initiate the KLF5 expression induction during the early pregnancy stage. During the late pregnancy and lactation stages, the KLF5 expression level could be maintained by other mechanism, such as glucocorticoid receptor. Further investigation will be required to clarify the mechanism by which the KLF5 expression in mammary glands is maintained during the lactation stage.

The Klf5 expression is up-regulated after pregnancy in mice (Fig. 1). It is well known that Pg starts to increase at early pregnancy so that the induction of Klf5 in mice is likely caused by Pg and PR. The PR KO adult mice showed defects in normal breast development (34, 42). Because the klf5 homozygous KO mice are lethal, a breast-specific klf5 KO mouse model will be essential to evaluate the physiological role of Klf5 in normal breast development. We are currently characterizing the mouse mammary tumor virus-Cre; Klf5loxp/loxp mice (43). Given the essential role of KLF5 in the Pg/PR signaling pathway, klf5 may be indispensable for normal breast development.

In conclusion, the expression of KLF5 is induced by Pg through PR in PR-positive breast cancer cell lines and probably in mouse mammary glands. Importantly, the induction of KLF5 partially mediates the pro-proliferation and dedifferentiation functions of Pg in vitro. KLF5 contributed to Pg-induced CyclinA, Cdt1, E2F3, and CK5 expression. Consistently, KLF5 and CK5 are coexpressed in ER-PR− basal type breast cancer cell lines. These findings suggest that the induction of KLF5 transcription factor by Pg contributes to Pg-induced breast cancer cell proliferation and dedifferentiation.

Materials and Methods

Tissue culture, transfection, and adenovirus infection

T47D, MCF7, BT474, BT-20, MD-MB-231, and other breast cancer cell lines were cultured as described previously (19, 20). All plasmids and siRNA were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The KLF5 siRNA has been described in our previous study (19). The target sequences of PR siRNA are 5′-TTTTCGACCCTCCAAGGACC-3′ (no. 1) and 5′-TATGTAAGTTTCGAAAACC-3′, (no. 2) (Ambion, Austin, TX). The KLF5 and control GFP adenoviruses have been described previously (18).

Reagents

Pg and MPA were purchased from Sigma (St. Louis, MO). The anti-KLF5 antibody has been described in our previous study (44). The anti-PR and anti-ERα antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-CK5 antibody was from Leica Microsystems (Bannockburn, IL). The anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was from Cell Signaling Technology (Danvers, MA).

Quantitative RT-PCR

Total RNA were isolated from tissues or cultured cells using TRIzol reagent (Invitrogen). Reverse transcriptions were performed using the Iscript cDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA). Quantitative PCR was performed on the ABI-7300 system using ABsolute SYBR Green Fluorescein reagents (Thermo Fisher Scientific, Inc., Pittsburgh, PA). Primer sequences for KLF5, CK5, SGK, CyclinA, Cdt1, E2F3, and GAPDH will be provided upon request.

Dual luciferase assay

The 1.9-kb human KLF5 gene promoter has been described previously (27). T47D cells were seeded in 24-well plates at 1.2 × 105 cells per well. The cells were transfected 24 h later with the KLF5 promoter reporter plasmid, the PR-B expressing plasmid (a gift from Dr. Kathryn Horwitz, University of Colorado), and an internal control pRL-TK in triplicate. One day after transfection, the cells were serum starved for another day followed by 20 μm MPA treatment. Luciferase activities were measured 24 h later by using the dual luciferase reporter assay system (Promega Corp., Madison, WI).

CHIP assay

T47D cells were incubated with 100 nm Pg or vehicle for 1 h. The ChIP assay was performed using the T47D cells following a protocol provided by Abcam (Cambridge, MA). The diluted DNA-protein complex was incubated with an equal amount of either anti-PR antibody or rabbit IgG antibody (Santa Cruz, CA) overnight at 4 C in the presence of protein A/G beads. Chromosomal DNA was purified and analyzed by PCR for the presence of the KLF5 promoter. PCR primers used for amplifying the KLF5 promoter (−647 to −447) were: 5′-TCCGCGTCTCCACCCTAATT-3′ and 5′-ATGAGCAGGGAGAGAGGCAG-3′. Two primers (5′-ACACCAGACCGCAGCTCCA-3′ and 5′-TCCATTGCTGCTGTCTGATTTGTAG-3′) were used to amplify the KLF5 coding sequence (585–749). The Bcl-2 promoter region (−629 to −388) with PR binding site (28) was used as a positive control. Two primers used were 5′-CTGGAGAGTGCTGAAGATTG-3′ and 5′-ACACTACAAGTAACACGGC-3′.

Thymidine incorporation assay

T47D cells were seeded in 24-well plates and were transfected with siRNA on the following day. One day after transfection, the cells were serum-starved for 1 d followed by 100 nm Pg treatment for another day. The cells were incubated in complete medium with 1 mCi/ml [3H]thymidine (MP Biomedical, Solon, OH) for 4 h. The incorporated [3H]thymidine was measured by the Beckman LS-6500 scintillation counter.

Statistical analysis

The luciferase assay and the DNA synthesis assay were conducted in triplicate. When appropriate, the data were pooled to generate means ± sd and were analyzed by t test. P < 0.05 was considered to be significant.

Acknowledgments

We thank Dr. K.B. Horwitz from University of Colorado for kindly providing the PR constructs.

This work was supported by a grant from the American Cancer Society (RSG-08-199-01), a grant from the Department of Defense (W81XWH-07-1-0191), and a grant from Yunnan Province High-Profile Talents Program (2010CI114).

Disclosure Summary: The authors have no conflict of interest.

NURSA Molecule Pages†:

  • Nuclear Receptors: PR;

  • Ligands: Progesterone | RU486 | MPA.

Footnotes

Abbreviations:
ChIP
Chromatin immunoprecopitation
CK5
cytokeratin 5
ER
estrogen receptor
ESC
embryonic stem cell
GAPDH
glyceraldehyde 3-phosphate dehydrogenase
GFP
green fluorescent protein
KLF5
Krüpple-like factor 5
KO
knockout
MPA
medroxyprogesterone 17-acetate
Pg
progesterone
PR
Pg receptor
qRT-PCR
quantitative RT-PCR
siRNA
small interfering RNA.

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