Endocrine Abstracts

Oestrogens act via oestrogen receptors (ER) that are expressed in a wide range of tissues including the vasculature, bone and gonads. Two ER genes known as ERalpha and ERbeta have been identified. In the human splice variants of ERbeta have been identified. ERs form both homodimers and heterodimers upon ligand binding. The aim of the present experiments was to study the functional activity of ERbeta1 (wild type) and ERbeta variant (ERbeta2) in single and double transfection assays.
Full length ERbeta1 and ERbeta2 cDNAs were generated from human, macaque and marmoset testes and cloned into the TA cloning sites of pCDNA3.1/V5-His (Invitrogen). These constructs were transfected into Hek 293 cells in the presence of a 3xERE-luc reporter construct. In a second series of experiments the hERbeta1 and hERbeta2 constructs were cloned into the fluorescent EGFP and DsRed vectors (Clontech) and transfection results examined by confocal microscopy.
Transfection experiments with E₂ revealed slight differences in the observed positive dose response curve with the different cDNAs (human>macaque>marmoset). Cells transfected with ERbeta2 containing constructs failed to induce expression of the reporter gene in the presence of E₂. Co-transfection of ERbeta1 or ERalpha with ERbeta2 containing constructs and incubation with E₂ resulted in similar levels of reporter gene expression as those seen with ERbeta1 or ERalpha alone. On the confocal microscope incubation of GFP-ERbeta1 of GFP-ERalpha containing cells with E₂ resulted in a redistribution of nuclear receptors into a 'punctate' pattern. Redistribution of GFP-ERbeta2 was not observed.
In conclusion we have confirmed that the ERbeta2 variant does not act as an E₂ activated receptor. We have failed to find evidence for the dominant negative activity previously claimed for this 'receptor'.