Molecular assays to profile 10 estrogen receptor beta isoform mRNA copy numbers in ovary, breast, uterus, and bone tissues

Abstract

Estrogens regulate various biological processes in a diverse range of reproductive and nonreproductive tissues through two genetically distinct but structurally related high affinity nuclear receptors, the estrogen receptor alpha and beta (ERα and ERβ). The physiological significance of the presence of two ERs that have redundant functions is not known. Several unique properties of ERβ together with its distinct expression patterns are considered to be, in part, the basis for diverse functional actions of estrogens and opposing actions of selective estrogen receptor modulators (SERMs) in different tissues. To understand how relative expression levels of two ERs correlate to seemingly dissimilar actions of estrogens and SERMs, quantitative methods are required that can precisely measure the levels of every isoform. Previously, methods to quantify eight ERα isoforms have been described [Poola I. (2003) Anal. Biochem. 314, 217–226]. In this article, real-time PCR-based molecular assays are described that can distinguish and quantify as low as 100 copies of 10 ERβ isoform mRNAs, the ERβ1, ERβ2, ERβ4, ERβ5, and ERβ exon 2Δ, exon 3Δ, exon 4Δ, exon 5Δ, exon 6Δ, and exons 5–6Δ. Each isoform mRNA is quantified using a specific primer pair and a 5′FAM (carboxy-fluorescein)- and 3′TAMARA (6-carboxy tetraethyl-rhodamine)-labeled probe and in comparison with a standard curve constructed with known copy numbers of its respective reverse transcribed cRNA. The devised assays were applied to profile 10 ERβ isoforms in four estrogen-sensitive tissues—ovary, breast, uterus, and bone. The sensitivity of detection of each isoform in these tissues varied from picograms to nanograms of reverse-transcribed total RNA depending on the isoform and the tissue. The results presented also show that each tissue has a distinct profile of 10 isoform mRNAs. Interestingly, ERα-negative breast cancer cell lines and tumors expressed significant amounts of ERβ isoforms suggesting that mitogenic stimulation by estrogen exists in these tissues. Bone tissues expressed several isoforms, although wild type was not present. In addition to the assay development, evidence is presented to demonstrate for the first time that ERβ4 and ERβ5 are full length receptors, contrary to previous reports that they are short receptors of exon 7–8. It is expected that the methods described here will significantly contribute to delineating the functional roles of various ERβ isoforms and in conjunction with ERα isoform profiling, will highly facilitate designing of individualized tissue specific therapies to treat estrogen-related pathologies.