A distal estrogen responsive element upstream the cap site of human transthyretin gene is an enhancer-like element upon ERα and/or ERβ transactivation

摘要

Previous studies reported that 17 beta-estradiol (E2) is responsible for the up-regulation of transthyretin (TTR) expression via an estrogen receptor (ER)-dependent pathway in rat choroid plexus (CP) and liver. A computer-assisted homology search identified a putative estrogen-responsive element (ERE) in the 5' flanking region of the human TTR (hTTR) gene (ERETTR), with the sequence aAGTCAAAGTGACCa, between - 3406 and - 3392. bp. Luciferase reporter assays and electrophoretic mobility shift (EMSA) and supershift analysis were carried out to investigate if E2 regulates TTR transcription via this putative ERE. Luciferase reporter assays in COS-7 cells were carried out with a plasmid construction where the TTR fragment containing the putative ERETTR was cloned in pGL2-promoter vector (pGL2-P) (pGL2-P/TTR), co-transfected with estrogen receptor α (ERα) and/or estrogen receptor β (ERβ) expression vectors. These assays demonstrated that, upon incubation with E2, one or both ERs (α and/or β) transactivate the reporter gene. The pGL2-P/TTR showed a significant transactivation of up to 6.8-fold, by E2, when co-transfected with ERβ, and up to 4-fold with ERα. Specific binding of ER (α and/or β) to ERETTR was demonstrated by EMSA and supershift assays confirmed the binding to ERα and/or ERβ. Our findings further suggest a mechanism underlying the regulation of TTR expression through the identification of a novel ERE in the TTR gene, which functions as an E2-dependent enhancer-like element.