Progesterone receptor transactivation of the secretory leukocyte protease inhibitor gene in Ishikawa endometrial epithelial cells involves recruitment of Krüppel-like factor 9/basic transcription element binding protein-1.
ABSTRACT Progesterone receptor (PR), a ligand-inducible transcription factor, mediates the physiological actions of progesterone (P) through two distinct isoforms, PR-A and PR-B, and numerous nuclear coregulators. We previously demonstrated that basic transcription element binding protein-1 (BTEB1), a transcription factor of the Krüppel-like family, is a functional PR-interacting protein, based on the subfertility phenotype and reduced P sensitivity of uterine PR target genes, on BTEB1 null mutation. Here we examined the role of BTEB1 in PR-mediated signaling in endometrial epithelial cells using Ishikawa human endocarcinoma cells and the P-responsive secretory leukocyte protease inhibitor (SLPI) gene. Treatment of Ishikawa cells with P for 24 h increased SLPI and BTEB1 transcript levels without similar effects on PR expression. P induction was abolished by the PR antagonist RU486, whereas knockdown of BTEB1 with short interfering RNA reduced P-responsive BTEB1 but not SLPI expression to basal levels. Forced expression of BTEB1, either by stable or transient transfections of BTEB1 expression constructs in endometrial carcinoma cells, enhanced SLPI promoter activity. Chromatin immunoprecipitation with anti-BTEB1 antibody demonstrated BTEB1 recruitment to the proximal GC-rich containing SLPI promoter region (-97 to -86) in human endometrial carcinoma (Hec1A) cells overexpressing BTEB1. In Ishikawa cells, recruitment of BTEB1 to the proximal, GC-rich region and the distal, progesterone-responsive element-like containing region (-635 to -514) was P dependent and was accompanied by corecruitment of PR and the PR coactivator cAMP-response element binding protein-binding protein. PR-B, rather than PR-A isoform, preferentially associated with BTEB1 in the GC-rich region, whereas both PR isoforms were recruited to the progesterone-responsive element-like site along with BTEB1. Our findings define a novel pathway for BTEB1/PR cross-talk to facilitate P-dependent gene transcription in endometrial epithelial cells.
Article: Functional differentiation of uterine stromal cells involves cross-regulation between bone morphogenetic protein 2 and Kruppel-like factor (KLF) family members KLF9 and KLF13.[show abstract] [hide abstract]
ABSTRACT: The inability of the uterine epithelium to enter a state of receptivity for the embryo to implant is a significant underlying cause of early pregnancy loss. We previously showed that mice null for the progesterone receptor (PGR)-interacting protein Krüppel-like factor (KLF) 9 are subfertile and exhibit reduced uterine progesterone sensitivity. KLF9 expression is high in predecidual stroma, undetectable in decidua, and enhanced in uteri of mice with conditional ablation of bone morphogenetic protein 2 (BMP2). Given the individual importance of KLF9 and BMP2 for implantation success, we hypothesized that the establishment of uterine receptivity involves KLF9 and BMP2 functional cross-regulation. To address this, we used early pregnant wild-type and Klf9 null mice and KLF9 small interfering RNA-transfected human endometrial stromal cells (HESCs) induced to differentiate under standard conditions. Loss of KLF9 in mice and HESCs enhanced BMP2 expression, whereas recombinant BMP2 treatment of HESCs attenuated KLF9 mRNA levels. IGFBP1 and KLF9-related KLF13 expression were positively associated with BMP2 and inversely associated with KLF9. Prolonged, but not short-term, knockdown of KLF9 in HESCs reduced IGFBP1 expression. Mouse uterine Igfbp1 expression was similarly reduced with Klf9 ablation. PGR-A and PGR-B expression were positively associated with KLF9 in predecidual HESCs but not decidualizing HESCs. KLF13 knockdown attenuated BMP2 and PGR-B and abrogated BMP2-mediated inhibition of KLF9 expression. Results support cross-regulation among BMP2, KLF9, and KLF13 to maintain progesterone sensitivity in stromal cells undergoing differentiation and suggest that loss of this regulatory network compromises establishment of uterine receptivity and implantation success.Endocrinology 07/2010; 151(7):3396-406. · 4.46 Impact Factor
Article: Krüppel-like factors KLF2 and 6 and Ki-67 are direct targets of zoledronic acid in MCF-7 cells.[show abstract] [hide abstract]
ABSTRACT: Bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases due to breast and prostate cancer. Recent clinical studies indicated a benefit in survival and tumor relapse with the supportive treatment of breast cancer using zoledronic acid (ZA), thus stimulating the debate about its putative anti-tumor activity in vivo. MCF-7 breast cancer cells were treated for 3 h (pulse treatment) and 72 h (permanent treatment) with ZA, and apoptosis rates and cell viability, defined as ATP content, were determined after 72 h. Permanent and pulse stimulation with ZA inhibited the viability of MCF-7 cells, which could partly be rescued by atorvastatin (Ator) pre-treatment but not by geranylgeranyl pyrophosphate (GGPP) co-treatment. Microarray analysis of ZA treated MCF-7 cells identified genes of the mevalonate pathway as significantly upregulated, which was verified by qPCR. Additionally the putative tumor suppressors krüppel-like factor 2 and 6 (KLF2 and KLF6) were markedly upregulated, while the classical proliferation marker Ki-67 was clearly downregulated. The expression of all three genes was confirmed by qPCR on mRNA level and by immunocytochemistry or Western blot staining. Expression of target genes were also analyzed in other breast (MDA-MB-231, BT-20, ZR75-1, T47D) and prostate (LNCaP, PC3) cancer cell lines by qPCR. ZA responsiveness of KLF2, KLF6 and Ki-67 could be verified in PC3 and T47D cells, KLF6 responsiveness in LNCaP and KLF2 responsiveness in MDA-MB-231 and BT-20 cells. Here we demonstrate in the apoptosis insensitive MCF-7 cell line a remarkable impact of ZA exposure on cell viability and on the regulation of putative tumor suppressors of the KLF family. The molecular mechanism involved might be the accumulation of isopentenyl pyrophosphate (IPP) and ApppI, since we could partly rescue the ZA effect by Ator pre-treatment and GGPP co-treatment. These data should stimulate further research into both the role of the mevalonate pathway and the accumulation of pyrophosphate compounds like ApppI in tumorigenesis and differentiation and their potential apart from the inhibition of mitochondrial ADP/ATP translocase and apoptosis, since such effects might well be responsible for the adjuvant ZA treatment benefit of patients suffering from breast cancer.Bone 12/2011; 50(3):723-32. · 4.02 Impact Factor