Molecular characterization of two estrogen receptor (ER) alpha subtype cDNAs from goldfish (Carassius auratus) and cross-talk between ERalpha and prolactin-activated signal transducers and activators of transcription (STAT) 5a

Existence of two estrogen receptor (ER) subtypes, α and β, has been demonstrated in various species. Because goldfish is a tetraploid species, its genome would contain a double dose of each ER subtype. Consistent with this, two ERβ cDNA sequences have been isolated from the goldfish liver and brain respectively. In this work, two ER cDNA sequences were obtained from goldfish. One of them was found to contain 1985 base pairs (bp), including an open reading frame that encodes 606 amino acids (aa). While the other, 1695 by in length, encodes a 564 as protein. Comparison of the deduced as sequences of these two ER sequences with other cloned vertebrate ERs revealed that both of them belong to the α subtype of ER. These two ERα sequences in goldfish were thus named as ERα1 representing the 606 as receptor and ERα2 representing the 564 as receptor. The two goldfish ERα sequences share a high level of homology in their nucleotide sequences and aa sequences throughout the whole protein. The DNA-binding domain and the ligand-binding domain were particularly conversed, not only in the two goldfish ERα sequences, but also in the goldfish ERβ sequences as well as in the other ER sequences. Despite of this similarity in sequence, ERα1 and ERα2 are expressed differentially among different tissues of the fish. Semi-quantitative RT-PCR analysis revealed that ERα2 is strongly expressed in the goldfish pituitary, brain and all the peripheral tissues examined including gill, heart, intestine, kidney, liver and male/female gonad but not in skeletal muscle. On the other hand, ERα1 is expressed in a similar level in the kidney and liver, but at a much lower level in other peripheral tissues. Skeletal muscle expresses ERα1 exclusively. ERα1 is barely detectable in the fish brain and pituitary. Transient transfection experiments revealed that both ERα1 and ERα2 are able to mediate estrogen response element-containing reporter gene transcription upon 17β-estradiol stimulation in a dose dependent manner. ERα2 has stronger transactivation ability than ERα1.; Estrogen exerts its biological effect on the target tissues in conjunction with other hormones such as prolactin (PRL). Cross-talk among the different hormone receptors further fine-tune the actions of these hormones on their target tissues. Cross-talk between ERα and PRL activated signal transducers and activators of transcription (STAT) 5a was identified and the mechanism was investigated in the present study. 17β-Estradiol activated ERα was found to inhibit the PRL induced STAT5a mediated transcription activation. Further experiments indicated that the negative regulation of ERα on STAT5a transactivation might be through a direct and hormone independent protein-protein interaction, resulting in a decrease in tyrosine phosphorylation of the STAT5a and Janus kinase (JAK) 2 protein, STAT5a DNA binding ability and STAT5a nuclear translocation. Binding sites in two proteins were mapped to the C-F domains in ERα and the SH2-TAD regions in STAT5a. ERα-STAT5a physiological association and functional interaction in two human breast cancer cell lines, MCF-7 and T-47D, were also identified.