Differential subcellular distribution and transcriptional activity of Sigma E3, Sigma E4, and Sigma E3-4 isoforms of the rat estrogen receptor-alpha

Sigma E3, Sigma E4, and Sigma E3-4 are naturally occurring estrogen recepto r (ER) isoforms, generated through differential splicing of the ER alpha pr imary transcript and abundantly expressed in embryonic rat pituitary. Studi es in COS cells transfected with full-length ER alpha or its three splice v ariants fused to green fluorescent protein (GFP), revealed a different subc ellular localization for each isoform. In the absence of estradiol, full-le ngth ER alpha -GFP was predominantly nuclear, and Sigma E3-GFP and Sigma E4 -GFP were present both in cytoplasm and nucleus, whereas Sigma E3-4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistributi on of full-length ER alpha -GFP and Sigma E3-GFP, from a diffuse to punctat e pattern, occurred within the nucleus. In contrast, the distribution of Si gma E4-GFP and Sigma E3-4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-alpha and Sigma E3 displayed the same hormone-i nduced ability to tether to nuclear matrix, whereas nuclear Sigma E4 appear ed to remain loosely associated to functional nuclear constituents. When co transfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER- negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, Sigma E3-4 exhi bited a very weak estrogen-dependent transactivation activity, whereas Sigm a E3 had an inhibitory effect on full-length ER action. Conversely, Sigma E 4 displayed estrogen-independent transcriptional activity in ER-negative ce lls, and in ER-positive cells, enhanced the estrogen-induced gene expressio n as efficiently as full-length ER alpha. In a gel mobility shift assay, ph osphorylated Sigma E4 was able to form a specific complex with a consensus ERE, while Sigma E3 and Sigma E3-4 never did bind by themselves. The observ ed inhibitory action of Sigma E3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodime rs with full-length ER alpha, as suggested by immunoprecipitation followed by Western blotting. These data suggest that Sigma E3 and Sigma E4 may play a physiologically relevant role as negative or constitutively positive mod ulators of transcription, in the developing rat pituitary.