ABSTRACT

The effects of insulin-like growth factor-I (IGF-I) on aromatase P450 activity and steroid production in preimplantation pig conceptuses were evaluated in vitro. Conceptuses recovered from gilts on days 10 and 12 of pregnancy were incubated for 6 h in modified Eagle's Minimum Essential Medium (MEM) plus IGF-I (0·1 μg/ml) or insulin (8·5 μg/ml), and conceptuses were monitored for their ability to convert [1,2-³H]β-testosterone into oestrogens. Aromatase activity of day-10 conceptuses was low and unaffected by IGF-I or insulin. In contrast, basal aromatase activity in day-12 conceptuses was about threefold higher and was further increased by IGF-I (P < 0·02), but was unaffected by insulin. To determine whether higher aromatase P450 activity was associated with increased oestradiol production, concentrations of oestradiol were determined by radioimmunoassay in culture medium of day-11 and -12 conceptuses, after incubation in MEM alone or in the presence of dehydroepiandrosterone (DHA, 1 μg/ml) with or without IGF-I (0·1 μg/ml) or insulin (0·1 or 8·5 μg/ml) for 24 h. Conceptuses in MEM plus DHA produced more oestradiol (P < 0·01) than those in MEM alone. Addition of IGF-I or insulin did not increase the effect of DHA. Basal oestradiol production was dependent on conceptus size; however, IGF-I or insulin did not affect basal or DHA-stimulated oestradiol production regardless of conceptus size. These findings demonstrate that IGF-I can modulate aromatase activity in vitro, without affecting overall de-novo steroidogenesis. Thus, the developmental increase in conceptus oestradiol production observed during early pregnancy in the pig may reflect synergistic interactions between IGF-I and other regulatory factors present within the conceptus and/or uterine environment.

Journal of Endocrinology (1991) 130, 245–250

The over-expression of epidermal growth factor receptor (EGFR) and its ligands, epidermal growth factor (EGF) and transforming growth factor-α, is a common feature of epithelial carcinomas and correlates with neoplastic progression. Secretory leukocyte protease inhibitor (SLPI), a member of the Kazal superfamily of serine anti-proteases, induces proliferation and promotes malignancy of epithelial cells and is expressed at high levels in multiple tumor types. In the present study, we have demonstrated that EGF increases SLPI expression in the human endometrial epithelial cell line Ishikawa in a dose- and time-dependent manner. We have shown that this effect of EGF occurs, in part, at the level of the SLPI promoter and involves the MAP kinase signaling pathway. We have further shown that EGF promotion of cell proliferation, but not induction of cyclin D1 gene expression, involves SLPI. Our results suggest that the regulation of SLPI expression by EGFR ligand(s) may represent a ‘feed-forward’ mechanism by which the enhanced proliferative and migratory properties of EGFR over-expressing cancer cells are sustained. Increased SLPI expression is likely an important component of altered EGFR signaling in human tumors and may have significant therapeutic implications in cancer progression.

Inappropriate early exposure of the hormone-responsive uterus to estrogenic compounds is associated with increased risk for adult reproductive diseases including endometrial cancers. While the dysregulation of estrogen receptor-α (ESR1) signaling is well acknowledged to mediate early events in tumor initiation, mechanisms contributing to sustained ESR1 activity later in life and leading to induction of oncogenic pathways remain poorly understood. We had shown previously that the transcription factor Krüppel-like factor 9 (KLF9) represses ESR1 expression and activity in Ishikawa endometrial glandular epithelial cells. We hypothesized that KLF9 functions as a tumor suppressor, and that loss of its expression enhances ESR1 signaling. Here, we evaluated the contribution of KLF9 to early perturbations in uterine ESR1 signaling pathways elicited by the administration of synthetic estrogen diethylstilbestrol (DES) to wild-type (WT) and Klf9 null (KO) mice on postnatal days (PNDs) 1–5. Uterine tissues collected at PND84 were subjected to histological, immunological, and molecular analyses. Compared with WT mice, KO mice demonstrated larger endometrial glands and lower endometrial gland numbers; DES exposure exacerbated these differences. Loss of KLF9 expression resulted in increased glandular ESR1 immunoreactivity with DES, without effects on serum estradiol levels. Quantitative RT-PCR analyses indicated altered expression of uterine genes commonly dysregulated in endometrial cancers (Akt1, Mmp9, Slpi, and Tgf β 1) and of those involved in growth regulation (Fos, Myc, Tert, and Syk), with loss of Klf9, alone or in concert with DES. Our data support a molecular network between KLF9 and ESR1 in the uterus, and suggest that silencing of KLF9 may contribute to endometrial dysfunctions initiated by aberrant estrogen action.

Krüppel-like factors (KLFs), of which there are currently 17 known protein members, belong to the specificity protein (Sp) family of transcription factors and are characterized by the presence of Cys₂/His₂ zinc finger motifs in their carboxy-terminal domains that confer preferential binding to GC/GT-rich sequences in gene promoter and enhancer regions. While previously regarded to simply function as silencers of Sp1 transactivity, many KLFs are now shown to be relevant to human cancers by their newly identified abilities to mediate crosstalk with signaling pathways involved in the control of cell proliferation, apoptosis, migration, and differentiation. Several KLFs act as tumor suppressors and/or oncogenes under distinct cellular contexts, underscoring their prognostic potential for cancer survival and outcome. Recent studies suggest that a number of KLFs can influence steroid hormone signaling through transcriptional networks involving steroid hormone receptors and members of the nuclear receptor family of transcription factors. Since inappropriate sensitivity or resistance to steroid hormone actions underlies endocrine-related malignancies, we consider the intriguing possibility that dysregulation of expression and/or activity of KLF members is linked to the pathogenesis of endometrial and breast cancers. In this review, we focus on recently described mechanisms of actions of several KLFs (KLF4, KLF5, KLF6, and KLF9) in cancers of the mammary gland and uterus. We suggest that understanding the mode of actions of KLFs and their functional networks may lead to the development of novel therapeutics to improve current prospects for cancer prevention and cure.

Estrogen, acting through its cognate receptor estrogen receptor-α (ESR1), is a critical regulator of uterine endometrial epithelial proliferation. Although the dynamic communication between endometrial stromal (ST) and epithelial cells is considered to be an important component in this process, key molecular players in particular compartments remain poorly defined. Here, we used mice null for Krüppel-like factor 9 (KLF9) to evaluate the contribution of this nuclear protein in ST–epithelial interactions underlying proliferative effects of estrogen. We found that in ovariectomized mice administered estradiol-17β (E₂) for 24 h, Klf9 null mutation resulted in lack of E₂-induced proliferative response in all endometrial compartments. We demonstrated a negative association between Klf9 expression and nuclear levels of ESR1 transcriptional corepressor prohibitin (PHB) 2 in uterine ST and epithelial cells of E₂-treated wild-type (WT) and Klf9 null mice. In early pregnancy uteri of WT mice, the temporal pattern of Klf9 transcript levels was inversely associated with that of Phb2. Deletion of Klf9 up-regulated uterine Phb2 expression and increased PHB2 nuclear localization in endometrial ST and epithelial cells, with no effects on the expression of the related Phb1. In the human endometrial ST cell line treated with E₂ for 24 h, Klf9 siRNA targeting augmented PHB2 transcript and increased nuclear PHB2 protein levels, albeit this effect was not to the extent seen in vivo with Klf9 null mutants. Our findings suggest a novel mechanism for control of estrogen-induced luminal epithelial proliferation involving ST KLF9 regulation of paracrine factor(s) to repress epithelial expression of corepressor PHB2.