Transcription factor KLF11 integrates progesterone receptor signaling and proliferation in uterine leiomyoma cells

Abstract

Uterine leiomyoma is the most common tumor of the female genital tract and the leading cause of hysterectomy. Although progesterone stimulates the proliferation of uterine leiomyoma cells, the mechanism of progesterone action is not well understood. We used chromatin immunoprecipitation (ChIP)-cloning approach to identify progesterone receptor (PR) target genes in primary uterine leiomyoma smooth muscle cells. We identified 18 novel PR-binding sites, one of which was located 20.5 kb upstream of the transcriptional start site of the Krüppel-like transcription factor 11 (KLF11) gene. KLF11 mRNA levels were minimally downregulated by progesterone but robustly upregulated by the progesterone antagonist RU486. Luciferase reporter assays showed significant baseline and RU486-inducible promoter activity in the KLF11 basal promoter or distal PR-binding region, both of which contained multiple Sp1-binding sequences but lacked classic progesterone response elements. RU486 stimulated recruitment of Sp1, RNA polymerase II, PR, and the coactivators SRC-1 and SRC-2 to the distal region and basal promoter. siRNA knockdown of PR increased KLF11 expression, whereas knockdown of KLF11 increased leiomyoma cell proliferation and abolished the antiproliferative effect of RU486. In vivo, KLF11 expression was significantly lower in leiomyoma tissues compared with adjacent myometrial tissues. Taken together, using a ChIP-cloning approach, we uncovered KLF11 as an integrator of PR signaling and proliferation in uterine leiomyoma cells.

Figure 1

(A) Modulation of selected PR target gene expression by progesterone. LSM cells were treated with vehicle (ethanol) and progesterone (10⁻⁶ M) in the presence or absence of progesterone antagonist RU486 (10⁻⁴ M) for 2 and 4 hours. SLC7A8, SOX8, and KLF11 mRNA levels were measured by real-time PCR. The results are reported as fold change of vehicle-treated cells and represent the mean ± s.e. for cells from three subjects. (B) Regulation of KLF11 expression by progesterone or R5020. The cells were treated and mRNA levels were determined as described in Figure 1A. (C) In vivo recruitment of PR and RNA PolII to novel PR-binding modules. LSM cells were exposed to progesterone for 1 hour. ChIP was performed using nonspecific rabbit IgG, anti-PR, or anti-phosphorylated RNA PolII antibody, followed by 40 cycles of PCR. The results shown here are representative of three independent ChIP experiments.

Figure 2

Regulation of KLF11 mRNA and protein levels by RU486 and PR in LSM cells. (A) KLF11 mRNA levels were determined from LSM cells treated with vehicle or the indicated concentrations of RU486 for 6 hours. The asterisks on the columns (10⁻⁸, 10⁻⁷, and 10⁻⁶ M) were placed to compare each dose with the vehicle. (B) KLF11 protein levels were detected by immunoblotting from LSM cells treated with 10⁻⁶ M RU486 for 72 hours. (C) PR mRNA (upper panel) and PR protein (lower panel) levels were checked from control siRNA- or PR siRNA-transfected cells to determine the knockdown efficiency. (D) LSM cells were transfected with control siRNA or PR siRNA for 48 hours. KLF11 mRNA expression levels were detected using real-time PCR. The mean±s.e. for three independent experiments is reported.

Figure 3

Transcriptional activity of the distal PR-binding region and the basal promoter of the KLF11 gene. The distal PR-binding region was cloned into the pGL3 promoter vector and the basal promoter region was cloned into the pGL3 basic vector. T47D breast cancer cells were transiently transfected with each of these plasmids, exposed to vehicle (ethanol) or 10⁻⁶ M RU486 for 48 hours, and harvested for luciferase assay. Data shown are expressed as mean±s.e. for a representative triplicate experiment reproduced three times.

Figure 4

Association of transcription factors and steroid receptor coactivators with the distal PR-binding region and the basal promoter of the KLF11 gene. LSM cells were exposed to RU486 (10⁻⁶ M) or vehicle (ethanol) for 1 hour. DNA interaction with transcription factors and coactivators was detected by immunoprecipitation with an anti-phosphorylated RNA PolII, anti-PR, anti-Sp1 (A), anti-SRC-1, or anti-SRC-2 (B) antibody. Nonspecific rabbit IgG was used as a negative control. Real-time PCR was used to detect the fold-enrichment of these transcription factors and coactivators using primers flanking the distal PR-binding and proximal promoter regions. The enrichment was normalized by IgG and calculated as fold increase compared with vehicle-treated cells. The results are illustrated as mean±s.e. of triplicate replicates of cells from a representative experiment reproduced using cells from 4 subjects. (C) The effect of PR knockdown on SRC-2 association with the distal PR-binding region and the basal promoter of the KLF11 gene. LSM cells were transfected with control siRNA or PR siRNA for 72 hours, and then exposed to RU486 10⁻⁶ M for 1 hour. DNA interaction with SRC-2 was detected as described above. The results are illustrated as mean±s.e. of triplicate replicates of cells from a representative experiment reproduced using cells from 3 subjects.

Figure 5

Regulation of LSM cell proliferation by KLF11. (A) LSM cells were mock transfected (without siRNA) or transfected with control siRNA or KLF11 siRNA for 48 hours, then KLF11 mRNA expression levels were detected using real-time PCR and standardized to GAPDH mRNA. Data shown are the mean±s.e. from a representative experiment repeated in cells from 4 subjects. (B) and (C) The effects of KLF11 knockdown on proliferation of cells in the absence or presence of progesterone agonist R5020 or antagonist RU486. LSM cells were transfected with control siRNA or KLF11 siRNA for 24 hours, then cells were treated with vehicle (ethanol), R5020 (10⁻⁶ M), or RU486 (10⁻⁶ M) for 72 hours. The PCNA protein level was determined using anti-PCNA antibody. Immunoblot densities were quantified with ImageJ software. (D) LSM cells were treated as in (C) except that cells were treated with RU486 (10⁻⁶ M) or vehicle for 48 hours and BrdU (20 μM) was added for the final 4 hours. BrdU-labeled cells were detected by immunostaining. Data shown are mean±s.e. for the BrdU labeling index of LSM cells from 3 subjects.

Figure 6

mRNA and protein levels of KLF11 in human leiomyoma and matched myometrial tissues. (A) 36 samples from 18 subjects were analyzed for mRNA; 18 samples were obtained from leiomyomas and 18 from adjacent myometrial tissues. To allow comparisons of data obtained from samples from different subjects, mRNA levels in the myometrial tissues were normalized to 1. (B) 12 samples from 6 subjects were analyzed for KLF protein levels. Immunoblot densities were quantified with Image J software. M: myometrial tissue; L: leimyoma tissue.