The gad2 Promoter Is a Transcriptional Target of Estrogen Receptor (ER)   and ER : A Unifying Hypothesis to Explain Diverse Effects of Estradiol

Abstract

Estradiol (E 2 ) regulates a wide range of neural functions, many of which require activation of estrogen receptor ␣ (ER␣) and/or ER␤, ligand-gated transcriptional regulators. Surprisingly, very few neural gene targets of ERs have been identified, and these cannot easily explain the myriad effects of E 2 . GABA regulates most of the same neural functions as E 2 , and GABAergic neurons throughout the brain contain ER. Therefore, we examined whether E 2 directly regulates expression of glutamic acid decarboxylase 2 (gad2), the enzyme primarily responsible for GABA synthesis for synaptic release. Using dual luciferase assays, we found that E 2 , but not other gonadal steroids, stimulated the activity of a 2691 bp rat gad2 promoter reporter construct. Activation required either ER␣ or ER␤, and ER␤ did not repress ER␣-mediated transactivation. Site-directed mutagenesis studies identified three estrogen response elements (EREs) with cell-specific functions. An ERE at Ϫ711 upstream of the gad2 translational start site was essential for transactivation in both MCF-7 breast cancer cells and SN56.B5.G4 neural cells, but an ERE at Ϫ546 enhanced transcription only in neural cells. A third ERE at Ϫ1958 was inactive in neural cells but exerted potent transcriptional repression in E 2 -treated MCF-7 cells. Chromatin immunoprecipitation assays in mouse GABAergic N42 cells confirmed that E 2 induced ER␣ binding to a DNA fragment containing sequences corresponding to the Ϫ546 and Ϫ711 EREs of the rat promoter. Based on these data, we propose that direct transcriptional regulation of gad2 may explain, at least in part, the ability of E 2 to impact such a diverse array of neural functions.

GABAergic influence on temporomandibular joint-responsive spinomedullary neurons depends on estrogen status

“…The gad2 promoter is a target of ER-positive neurons (Hudgens et al, 2009), while in PAG (McCarthy et al 1995) and hippocampus estrogen status regulates glutamate decarboxylase (GAD) mRNA and protein expression (Nakamura et al, 2004) and dendritic spine density through a GABA-dependent mechanism (Murphy et al, 1998). A mechanism for E2-mediated disinhibition in hippocampus (Rudick and Woolley 2001; Ledoux and Woolley 2005) and forebrain (Rudick et al, 2003) may involve a decrease in release probability at GABAergic synapses.…”

“…GABA acts through ionotropic GABA A receptors as well as G protein-coupled GABA B receptors to alter sensory processing at spinal levels (Malcangio and Bowery, 1996; Hammond, 1997). Estrogen status influences GABA biosynthesis by targeting the gad2 promoter (Hudgens et al 2009) and, more indirectly, by altering the expression of GABA A receptor subunits in the trigeminal ganglion (Puri et al 2011) and elsewhere in the brain (McCarthy et al 1995; Nakamura et al 2004; Lovick 2008). To determine if estrogen status alters GABAergic influences on TMJ nociception, we recorded from Vc/C 1–2 neurons under high (HE) and low (LE) estrogen conditions in ovariectomized female rats and tested the effects of locally applied antagonists and agonists for GABA A receptors.…”

Cadmium-induced cell death of basal forebrain cholinergic neurons mediated by muscarinic M1 receptor blockade, increase in GSK-3β enzyme, β-amyloid and tau protein levels

“…The cells were maintained at 37 °C and 5 % CO 2 in DMEM supplemented with 10 % FBS, penicillin/streptomycin, 2 mM l-glutamine (Sigma, Madrid, Spain) and 1 mM sodium pyruvate. Medium was changed every 48 h (Hudgens et al 2009). In order to determine cadmium binding to M1R, the cellular Aβ 1-40, Aβ 1-42, total and phosphorylated tau content, M1R, GSK-3β and AChE splice variants gene expression, and single and simultaneous knockdown effects of AChE, M1R, GSK-3β and βAPP genes, cells were seeded in 6-well plates at a density of 10 6 cells/well.…”

Acute and long-term exposure to chlorpyrifos induces cell death of basal forebrain cholinergic neurons through AChE variants alteration

“…The cells were maintained at 37 C and 5% CO 2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, 2 mM L-glutamine (Sigma, Madrid, Spain), and 1 mM sodium pyruvate. The medium was changed every 48 h (Hudgens et al, 2009). Differentiation of the cells was achieved by culturing for 3 days with 1 mM dibutyryl-cAMP and 1 mM retinoic acid as described (Bielarczyk et al, 2003;Szutowicz et al, 2006), which produced morphological maturation and 3-4-fold increase of ChAT activity and acetylcholine level in the cells.…”