Effect of estrogens on IL-1β promoter activity

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Abstract

It is well documented that steroid hormones modulate cytokine gene expression. In some tissues estrogens are known to suppress cytokine production while in other tissue types, cytokine expression is enhanced by the hormone. This study was conducted to investigate the regulatory mechanisms which underlie the modulation of the interleukin-1β (IL-1β) gene at the transcription level. To accomplish this, the macrophage cell line RAW264.7, which appeared insensitive to 17β-estradiol (E2) treatment, was stably transfected with the human estrogen receptor (ER) and an IL-1β promoter–CAT reporter construct. E2 markedly enhanced LPS-induced IL-1β promoter-driven CAT activity in an E2 dose dependent manner. This responsiveness was estrogen specific since no synergism was observed between LPS and the sex steroids testosterone or progesterone while the estrogen analogue 17α-estradiol stimulated only at 10 to 100 times the amount required for 17β-E2. Several antiestrogens, H1285, ICI 182 780, and tamoxifen inhibited the estrogen stimulated enhancement of IL-1β promoter activity in a dose-dependent manner, indicating that this effect was indeed mediated through the ER in a ligand dependent manner. The estrogenic effect appeared to be indirect and time dependent since the addition of E2 was required hours prior to LPS stimulation; addition of E2 and LPS at the same time resulted in a greatly reduced estrogenic effect. The estrogen metabolites 17-epiestriol and 16-keto-17β-E2 displayed an estrogenic response virtually indistinguishable from E2. 4-Hydroxyestradiol displayed activity only at 100-fold the concentration of E2 while 2-hydroxyestrone showed no activity at any of the concentrations tested. Overall the results demonstrate that E2 and some metabolites of E2 synergize with LPS to markedly enhance IL-1β promoter activity through ER mediated processes.

Introduction

Although modulation of cytokine gene expression is well established for the glucocorticoids1, 2, 3, increasing evidence indicates that sex steroids, particularly estrogens, also modulate cytokine production[4]. Much of this evidence indirectly comes from epidemiological studies which demonstrate that risk factors for both heart disease and osteoporosis in postmenopausal women are significantly reduced in those receiving estrogen replacement therapy5, 6. The proinflammatory cytokines, interleukin-one (IL-1), tumor necrosis factor-α (TNF-α), and interleukin-six (IL-6) appear to play an important role in the pathogenesis of these diseases[4]. Several reports in a number of different systems, both in vitro and in vivo, demonstrated that 17β-estradiol suppresses the production of these cytokines4, 7, 8, 9, 10. These findings are consistent with the observed increases in IL-1 and TNF in postmenopausal women concomitant with the risk of osteoporosis and heart disease and the amelioration of these diseases upon estrogen replacement therapy[4]. On the other hand, estrogen under certain conditions is known to enhance the production of cytokines such as IL-111, 12and γ interferon[13]. These conditions may in part be responsible for the increased frequency and severity of autoimmune disorders observed in women.

Interleukin-one (IL-1) is involved in a broad range of biological activities that effect immunological and inflammatory processes (see reviews,14, 15, 16, 17. Although the role of the IL-1 proteins in normal physiological responses in vivo remains incompletely defined, there is substantial evidence that excessive production of IL-1 contributes to the pathogenesis of many illnesses with autoimmune or inflammatory components, including rheumatoid18, 19, 20and osteoarthritis[21], atherosclerosis22, 23, and osteoporosis[4]. Despite the evidence that estrogen modulates IL-1 gene expression, little is known regarding the molecular mechanism which underlie this regulation.

Our laboratory has identified and characterized cis-acting elements and nuclear DNA-binding proteins that regulate murine IL-1β transcription in the murine macrophage cell line RAW264.724, 25, 26, 27, 28. The human estrogen receptor was permanently introduced into this otherwise E2 non-responsive cell line and was used as model system to study the role of estrogen and estrogen metabolites in the regulation of IL-1β transcription. The present study introduces this model transfected RAW264.7 cell line and, in contrast to the results of other cytokine studies, our results showed that estradiol markedly enhanced LPS-induced IL-1β promoter activity. This enhancement was mediated through the estrogen receptor in a time dependent manner.

Section snippets

Cell culture

RAW264.7[29]and MCF-7 cells (American Type Culture Collection, Rockville, MD) were maintained in Petri dishes in D5 (Dulbecco's modified Eagle medium containing 5% heat-inactivated fetal calf serum (HyClone, Logan, Utah), 2 mM l-glutamine, 100 U of penicillin per ml, and 100 μg of streptomycin per ml. Stable RAW264.7 transfected cell lines were maintained in D5 medium containing either the neomycin analog G418 (Gibco-BRL, Gaithersburg, MD) at 0.75 mg/ml or both G418 and Hygromycin B at 0.75 mg/ml and

Results

We first attempted to modulate IL-1β promoter activity in LPS-induced IL1β–CAT+ cells using a variety of concentrations and timed additions of E2. Regardless of the conditions used, E2 had no effect on IL-1β promoter activity as measured by CAT activity (data not shown). Estrogen receptor (ER) binding assays indicated that the parental RAW264.7 cells expressed very little binding activity (see Fig. 1) which may have been a reflection of very few non-functional ER per cell, or represent binding

Discussion

IL-1 production at the transcriptional level is modulated in several tissues by the presence of estradiol. The loss of the hormone can result in osteoporosis in postmenopausal women while the presence or inappropriate production of estradiol can potentiate and/or exacerbate autoimmune disease. In the studies reported here, we examined the regulation of the IL-1β promoter by E2 in a model murine macrophage cell line. Estrogen had no effect on LPS-induced CAT activity in the RAW264.7 IL1β-CAT+

Acknowledgements

The authors wish to thank Dr A. E. Wakeling, Zeneca Pharmaceuticals, for the gift of ICI 182780. The authors also wish to thank Ms. Susan Sulkey for her patient and expert secretarial assistance. This work was supported by NIH ES05968 and the National (94017520) and Missouri American Heart Associations.

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