Cell- and Ligand-specific Regulation of Promoters Containing Activator Protein-1 and Sp1 Sites by Estrogen Receptors α and β

Jennifer R Schultz,Larry N Petz,Ann M Nardulli

doi:10.1074/jbc.m407879200

Jennifer R Schultz, Larry N Petz + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m407879200

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Abstract

Estrogen plays a critical role in development and maintenance of female reproductive and mammary tissues, but is also involved in maintenance of cardiovascular, skeletal, and neural function. Although it is widely accepted that the estrogen-occupied receptor mediates its effects by interacting with estrogen response elements (EREs) residing in target genes, a number of estrogen-responsive genes contain no identifiable ERE. To understand how estrogen-responsive genes lacking EREs but containing activator protein 1 (AP-1) and Sp1 sites respond to hormone treatment, we have identified four discrete regions of the human progesterone receptor gene that contain AP-1 or Sp1 sites and examined their abilities to modulate transcription in the presence of 17 beta-estradiol, ICI 182,780, tamoxifen, raloxifene, genistein, or daidzein. Transient cotransfection assays demonstrated that ER alpha was a more potent activator of transcription than ER beta in bone, uterine, and mammary cells. The Sp1-containing promoters were substantially more potent transcriptional enhancers than the AP-1-containing promoters, but a 1.5-kb region of the human progesterone receptor gene containing both AP-1 and Sp1 sites was the most hormone-responsive promoter tested. The ability of ligands to modulate transcription of AP-1- or Sp1-containing promoters was dependent on cell context, but the expression of AP-1 or Sp1 proteins was not necessarily related to transcriptional response. Taken together, these studies have helped to delineate the roles of ER alpha and ER beta in modulating transcription of genes containing AP-1 and Sp1 sites and define the effects of widely used, pharmacologic agents in target cells with distinct cellular environments.

Highlights

Menopausal symptoms, and decrease the incidence and recurrence of mammary tumors
To understand how estrogen-responsive genes lacking estrogen response elements (EREs) but containing activator protein 1 (AP-1) and Sp1 sites respond to hormone treatment, we have identified four discrete regions of the human progesterone receptor gene that contain AP-1 or Sp1 sites and examined their abilities to modulate transcription in the presence of 17␤-estradiol, ICI 182,780, tamoxifen, raloxifene, genistein, or daidzein
To better understand how estrogen receptor (ER)␣ and ER␤ regulate transcription of a complex promoter containing AP-1 and Sp1 sites in different cell environments, transient transfections were performed in bone (U2-OS), uterine (HEC-1), and mammary (SK-BR-3) cell lines using an ER␣ or ER␤ expression vector and a reporter plasmid containing 1.5 kb of the human progesterone receptor (PR) gene (Ϫ711/ϩ817 PR TATACAT), which contains the Ϫ61 Sp1, ϩ90 AP-1, ϩ571 ERE/Sp1, and ϩ745 AP-1 sites (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Cell Maintenance and Transient Transfections—Human osteosarcoma (U2-OS) cells were maintained in culture and transfected using Lipofectin (Invitrogen, Carlsbad, CA) as previously described [46]. The DNA/media mixture was removed and cells were maintained on phenol red-free improved minimal essential medium supplemented with 5% charcoal dextran-treated calf serum. Human breast cancer cells (SK-BR-3) were maintained in Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% heatinactivated fetal calf serum. Cells were plated in Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% charcoal/dextran-treated fetal calf serum 24 h prior to transfection and were transfected in serum-free Dulbecco’s modified Eagle’s medium/F-12. The DNA/media mixture was removed and cells were maintained on Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% charcoal/dextran-treated fetal calf serum. Cells were plated in McCoy’s 5A supplemented with 5% charcoal/dextran-treated calf serum 24 h prior to transfection and were transfected in serum-free McCoy’s 5A. The DNA/media mixture was removed and cells were maintained on McCoy’s 5A supplemented with 5% charcoal/dextran-treated fetal calf serum. Blots were probed with horseradish peroxidase-conjugated secondary antibody (Zymed Laboratories, Inc., South San Francisco, CA), and the SuperSignal West Femto Maximum Sensitivity Substrate chemiluminescent detection kit (Pierce Chemical Co.) was used to visualize the proteins as per the manufacturer’s instructions

RESULTS

DISCUSSION

TABLE I Compiled data from transient transfection studies