Human cell-based estrogen receptor beta dimerization assay

Estrogen is not only responsible for important functions in the human body, such as cell growth, reproduction, differentiation, and development [1], but it is also deeply related to pathological processes, such as cancer, metabolic and cardiovascular diseases, and neurodegeneration [[1], [2], [3]]. Estrogen, which is mainly synthesized and secreted from the ovaries, binds to the estrogen receptor (ER) present in the nucleus of the target cell. This binding force is due to the structural diversity that determines receptor affinity [3,4].

The estrogen-bound ER regulates the transcription of target genes [3,4]. The ER has two subtypes, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), which enable various physiological regulations and are due to the diverse expression patterns in each tissue [1]. ERα is mainly expressed in the uterus, ovary, breast, kidney, bone, white adipose tissue, and liver, whereas ERβ is mainly expressed in the ovary, central nervous system (CNS), cardiovascular system, lung, male reproductive system, prostate, colon, kidney, and hyperimmune system [1]. More specifically, although both receptors can be expressed simultaneously in the same tissue, the expression ratio of ERα and ERβ in individual tissues is different, thus making it possible to regulate their functions [5,6]. Therefore, it is very important to determine the expression balance of ERα and ERβ, as these two receptors have specific roles that differentiate them from each other [5,7].

The ER signaling pathway can be subdivided into multiple steps, including transcriptional activity through ligand-receptor binding, ligand-bound receptor dimerization, and receptor-estrogen-responsive element (ERE) binding. Currently, methods for identifying compounds with ERα binding activity in vitro are the ERα binding and transactivation assays, which are Organization for Economic Co-operation and Development (OECD)-approved tests, and the ERα dimerization assay developed in the previous study. Also, an ERβ binding assay using HEK293 cells has been reported [8]. However, currently no methods to identify compounds with ERβ dimerization activity is available.

In this study, we developed an ERβ dimerization assay useful for identifying estrogenic compounds with ERβ binding affinity using the human embryonic kidney (HEK293) cell line. The human ERβ gene fused to Nanoluciferase (NL) or Halotag (HT) was expressed in a human cell line to induce ER dimerization, which was determined by measuring the BRET signal. The ERβ dimerization assay can provide information on the ERβ dimerization potential of a compound, which can potentially identify the comprehensive ER signaling mechanism, which includes ERα and ERβ.

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