Methoxychlor stimulates the mouse lactoferrin gene promoter through a GC-rich element.

Abstract

The lactoferrin gene in the mouse uterus is a target gene for natural estrogens and xenoestrogens. One of the xenoestrogens is methyoxychlor, an insecticide that displays both estrogenic and antiandrogenic activities. Recently, methyoxychlor was found to stimulate lactoferrin gene expression in the uterus of an estrogen receptor null mouse. The present study is designed to uncover the methoxychlor response region in the mouse lactoferrin gene promoter. A series of different lengths of the mouse lactoferrin gene 5' flanking region were linked to a chloramphenicol acetyltransferase (CAT) reporter construct and transfected into human endometrial carcinoma HEC-1B cells, an estrogen receptor null cell line, in order to examine the methoxychlor response. The transfected cells were treated with methoxychlor or the metabolite of methoxychlor, HPTE, and the CAT reporter activities were measured. Constructs that contain a mouse lactoferrin 5' region longer than 100 bp were activated more than twofold by both methoxychlor and HPTE. The activation of the CAT reporter by the chemicals was dose dependent and reached saturation. Additional deletion mutants within the 100-bp region were tested, and a GC-rich sequence (GC-II) that we have previously characterized as an epidermal growth factor (EGF) response element was identified to be the region for the methoxychlor response. GC-II binds Sp1, Sp3, and IKLF transcription factors, collaborates with the AP1/CREB binding element, and confers the EGF response. Whether the effect of methoxychlor requires the AP1/CREB binding element has yet to be established; however, the present finding provides an alternative signaling pathway for the xenoestrogens.