Functional estrogen receptors in the nucleus and plasma membrane of TM3 Leydig cell line

Abstract

Estrogen receptor (ER) expression and function were examined using the TM3 Leydig cell line. Both ERα and ERβ were detected using Western blot analysis and immunocytochemistry. Fluorescence was detected in both the nucleus and non‐nuclear regions. To determine if the nuclear receptors were functional, cells were transfected with a reporter gene containing an estrogen response element. There was a significant decrease in luciferase activity in cells treated with estradiol (10, 100, and 1000 nM) compared to controls. To determine it the non‐nuclear fluorescence seen by immunocytochemistry was on the plasma membrane, cells were incubated with fluorescent labeled estradiol or non‐labeled estradiol at 4°C, rinsed with buffer, and levels of fluorescence measured by fluorometer. Significant levels of fluorescence were detected on the surface of these cells. To determine the function of these receptors, TM3 cells were treated with 17β‐estradiol for 5 minutes and protein kinase C activity measured using PepTag assay kit (Promega, Madison, WI). PKC activity was significantly decreased at 100 nM and 200 nM 17β‐estradiol compared to control. In summary, the TM3 Leydig cell line expresses both ERα and ERβ. These receptors may function as nuclear receptors that can down regulate transcription as well as putative plasma membrane receptors capable of decreasing protein kinase C activity.