HOXA10 inhibits Kruppel-like factor 9 expression in the human endometrial epithelium

Abstract

Kruppel-like factor 9 (KLF9) is a zinc finger transcription factor that regulates estrogen and progesterone action by modulating the activity of progesterone receptor (PGR). The transition from proliferative to secretory endometrial epithelium involves loss of estrogen receptor/PGR expression and loss of direct response to sex steroids. HOXA10 partially mediates progesterone responsiveness in the endometrium. Here, we demonstrate that HOXA10 directly regulates KLF9 in endometrial epithelial cells and not in stromal cells. Immunohistochemistry performed on endometrial tissue obtained from normal, reproductive-age women revealed that KLF9 expression was decreased in the secretory phase of the menstrual cycle compared to the proliferative phase. In vitro, HOXA10 transfection of human endometrial epithelial cells (Ishikawa), but not stromal cells (HESC), resulted in a greater than 50% decrease in KLF9 mRNA and protein expression. Reporter constructs driven by the KLF9 promoter were repressed by cotransfection with HOXA10. Electrophoretic mobility shift assay was used to demonstrate direct binding of HOXA10 to the KLF9 promoter. Targeted mutation of the HOXA10-binding site in the KLF9 promoter resulted in loss of HOXA10 binding and loss of repression by HOXA10 in reporter assays. HOXA10 directly and selectively repressed KLF9 expression in endometrial epithelial cells. HOXA10 repression of KLF9 likely contributes to the loss of sex steroid responsiveness in secretory-phase endometrial epithelium.

FIG. 1.

KLF9 protein expression in human cycling endometrium tissue. KLF9 protein was expressed in nuclei of both stromal and glandular cells in human endometrium. A) Immunohistochemical results show KLF9 expression was relatively high in the proliferative phase in glandular epithelial cells. B) KLF9 expression was lower in the secretory phase in glandular epithelial cells. Original magnification ×600; bar = 50 μm.

FIG. 2.

HOXA10 mRNA expression in human endometrial cell lines. A) Real-time PCR results show that HOXA10 expression was increased more than 1000-fold (P < 0.05) after transfection with pcDNA3.1/HOXA10 vector in Ishikawa cells. B) HOXA10 expression was decreased to 27% (P < 0.05) of pretreatment level after transfection with HOXA10 siRNA in Ishikawa cells. C) Similarly, real-time PCR results show HOXA10 expression was increased more than 1000-fold (P < 0.05) after transfection with pcDNA3.1/HOXA10 vector in HESC cells. D) HOXA10 expression was decreased to approximately 70% (P < 0.05) of pretreatment level after transfection with HOXA10 siRNA in HESC cells. *P < 0.05.

FIG. 3.

HOXA10 regulated KLF9 mRNA expression in human endometrium cell line. A) Real-time PCR results show that pcDNA3.1/HOXA10 transfection decreased KLF9 mRNA expression to 44% of pretreatment level (P < 0.05) in Ishikawa cells. B) No change was found in KLF9 mRNA expression after transfection with HOXA10 siRNA in Ishikawa cells. C) Real-time PCR results show that pcDNA3.1/HOXA10 transfection decreased KLF9 mRNA expression to 52% of pretreatment level in HESC cells; however, this did not reach statistical significance. D) No increase in KLF9 mRNA expression was found after transfection of HOXA10 siRNA in HESC cells. *P < 0.05.

FIG. 4.

HOXA10 protein expression in human endometrial cell lines. A) Western blot results demonstrated that HOXA10 gene expression increased after transfection with the pcDNA-HOXA10 vector and decreased after transfection with HOXA10 siRNA in Ishikawa cells. B) Western blot results demonstrated that HOXA10 gene expression increased after transfection with the pcDNA-HOXA10 vector and decreased after transfection HOXA10 siRNA in the HESC cell line as well.

FIG. 5.

HOXA10 regulated KLF9 protein expression in human endometrial cell lines. A) Western blot results show that pcDNA/HOXA10 transfection decreased KLF9 protein expression in Ishikawa cells. No change was observed in KLF9 protein expression after HOXA10 siRNA transfection. B) Western blot results show no change in KLF9 protein expression in HESC cells after the pcDNA3.1/HOXA10 transfection or HOXA10 siRNA transfection. The results are each representative of three separate experiments using each cell type.

FIG. 6.

Putative HOXA10 binding sites drove luciferase activity in response to HOXA10. A) A schematic representation of 5′ region in KLF9 gene. B) In transactivation assays, the 692-bp pGL3-control-KLF9 sequence resulted in a 43% decrease in luciferase expression induced by HOXA10 in Ishikawa cells. The smaller, 131-bp segment containing the HOXA10-binding sites cloned into pGL3-control resulted in a 54.7% decrease in luciferase expression induced by HOXA10 in Ishikawa cells. No decrease in luciferase activity driven by HOXA10 was observed using the 131-bp mutant pGL3-control-KLF9 sequence in Ishikawa cells that lack the HOXA10-binding sites. *P < 0.05.

FIG. 7.

EMSA showing the HOXA10-binding to the KLF9 promoter. Nuclear extract was obtained from Ishikawa cells for use in EMSA. Lanes 1 and 2 demonstrate the ³²P-labeled, 131-bp, wild-type and mutated probes, respectively. Lane 3 demonstrates the interaction of ³²P-labeled, wild-type probe with nuclear extract from Ishikawa cells; significant binding is observed. Lane 4 demonstrates loss of binding to the mutated probe. Lane 5 demonstrates the cold unlabeled competing probe and HOXA10 polyclonal antibody added into the reaction of labeled wild probe with nuclear extract from Ishikawa cells. Lane 6 demonstrated further retarded mobility of the shifted complex (supershift) using 131-bp wild-type probe, HOXA10 polyclonal antibody, and Ishikawa nuclear extract. The arrow indicates the supershifted complex. Results are representative of seven independent experiments.