Steroid receptor coactivator-2 expression in brain and physical associations with steroid receptors

Abstract

Estradiol and progesterone bind to their respective receptors in the hypothalamus and hippocampus to influence a variety of behavioral and physiological functions, including reproduction and cognition. Work from our lab and others has shown that the nuclear receptor coactivators, steroid receptor coactivator-1 (SRC-1) and SRC-2, are essential for efficient estrogen receptor (ER) and progestin receptor (PR) transcriptional activity in brain and for hormone-dependent behaviors. While the expression of SRC-1 in brain has been studied extensively, little is known about the expression of SRC-2 in brain. In the present studies, we found that SRC-2 was highly expressed throughout the hippocampus, amygdala and hypothalamus, including the medial preoptic area (MPOA), ventral medial nucleus (VMN), arcuate nucleus (ARC), bed nucleus of the stria terminalis, supraoptic nucleus and suprachiasmatic nucleus. In order for coactivators to function with steroid receptors, they must be expressed in the same cells. Indeed, SRC-2 and ER(alpha) were coexpressed in many cells in the MPOA, VMN and ARC, all brain regions known to be involved in female reproductive behavior and physiology. While in vitro studies indicate that SRC-2 physically associates with ER and PR, very little is known about receptor-coactivator interactions in brain. Therefore, we used pull-down assays to test the hypotheses that SRC-2 from hypothalamic and hippocampal tissue physically associate with ER and PR subtypes in a ligand-dependent manner. SRC-2 from both brain regions interacted with ER(alpha) bound to agonist, but not in the absence of ligand or in the presence of the selective ER modulator, tamoxifen. Analysis by mass spectrometry confirmed these ligand-dependent interactions between ER(alpha) and SRC-2 from brain. In dramatic contrast, SRC-2 from brain showed little to no interaction with ERbeta. Interestingly, SRC-2 from both brain regions interacted with PR-B, but not PR-A, in a ligand-dependent manner. Taken together, these findings reveal that SRC-2 is expressed in brain regions known to mediate a variety of steroid-dependent functions. Furthermore, SRC-2 is expressed in many ER(alpha) containing cells in the hypothalamus. Finally, SRC-2 from brain interacts with ER and PR in a subtype-specific manner, which may contribute to the functional differences of these steroid receptor subtypes in brain.

Figure 1

SRC-2 immunoreactive cells in the A) posterodorsal portion of the medial amygdala (MePD), B) supraoptic nucleus (SON), magnification bar = 500 μm, and C) ventromedial nucleus of the hypothalamus (VMN) and arcuate nucleus (ARC) of the female rat. Inset shows nuclear immunostaining of cells from the VMN, magnification bar = 10 μm. opt = optic tract; ox = optic chiasm; 3V = third ventricle.

Figure 2

Coexpression of SRC-2 and estrogen receptors (ER) in cells in the ventromedial nucleus of the hypothalamus (A–C) and arcuate nucleus (D–F). Sections were simultaneously immunostained for ERα (A and D) and SRC-2 (B and E). Overlaid images from the VMN (C) and ARC (F) show cells expressing both ERα and SRC-2. Open arrows point to cells containing ERα only (red), hatched arrows point to cells containing SRC-2 only (green) and solid arrows point to cells expressing both ERα and SRC-2 (orange/yellow). Magnification bar = 50 μm

Figure 3

SRC-2 from rat hypothalamic whole cell extracts associates with ERα, but not ERβ, in a ligand-dependent manner. SRC-2 from A) hippocampus or B) hypothalamus interacts with ERα in the presence of estradiol (lane 2), but not in the absence of ligand (lane 3) or in the presence of the SERM, tamoxifen (lane 4). SRC-2 interacts weakly with ERβ bound to estradiol (lane 5) with little to no interaction in the absence of ligand or tamoxifen (lanes 6 & 7). Inputs (2% of total) of SRC-2 from hippocampal or hypothalamic extracts are shown in lane 1. C) SRC-2 from hippocampus physically associates with ERα bound to estradiol, but not in the absence of ligand or in the presence of tamoxifen. In contrast, SRC-2 interacts weakly with ERβ. D) Hypothalamic SRC-2 interacts with ERα, and to a much lesser extent with ERβ, in a ligand dependent manner. *p < 0.05, n = 4–5 per treatment group.

Figure 4

Progestin receptor (PR) interactions with SRC-2 from brain. SRC-2 from A) hippocampal or B) hypothalamic whole cell extracts interacts with PR-B, and to a lesser extent PR-A, in the presence of the agonist R5020, but not in the absence of ligand or in the presence of the SPRM, RU486. Inputs (2% of total) of SRC-2 from hippocampal or hypothalamic extracts are shown in lane 1. SRC-2 from the C) hippocampus and D) hypothalamus interacts with PR-B in the presence of the agonist R5020, but not in the absence of ligand or in the presence of the SPRM, RU486. SRC-2 from each brain region interacts with PR-A to a lesser extent. *p < 0.05, n = 8 per treatment group.