Influence of oestradiol and tamoxifen on oestrogen receptors-α and -β protein degradation and non-genomic signalling pathways in uterine and breast carcinoma cells

Abstract

Tamoxifen acts as an oestrogen antagonist in the breast reducing cell proliferation, but in the uterus as an oestrogen agonist resulting in increased cell proliferation. Tamoxifen exerts its tissue-specific effects through the oestrogen receptors (ER or ER ). The levels and functions of the two ERs affect the response of the target tissue to oestrogen and tamoxifen. We examined the control of ER stability in breast and uterine cell lines using western blotting and RT-PCR. In MCF-7 breast-derived cells, ER and ER proteins were rapidly degraded via the proteasome pathway in response to oestradiol; conversely tamoxifen stabilised both receptors. In Ishikawa uterine-derived cells, oestradiol and tamoxifen stabilised ER but led to degradation of ER by the proteasome pathway. Further investigations showed that oestradiol induced activation of the non-genomic ER /Akt signalling pathway in MCF-7 cells. We have demonstrated that the alternative Erk signalling pathway is activated in Ishikawa cells following oestradiol treatment in the absence of an active proteasome pathway and therefore increased levels of ER . In conclusion, our data have demonstrated tamoxifen or oestradiol control of ER subtype stability and that non-genomic activation of transcription pathways is cell specific.

Ishikawa cells exhibit differential gene expression profiles in response to oestradiol or 4-hydroxytamoxifen

“…The negative cell cycle regulator cyclin G2 (CCNG2), which was recently identified as a primary ER target gene in MCF-7 breast cancer cells where it was robustly down-regulated by oestrogen but not OHT (Stossi et al 2006), was downregulated by both OHT and E 2 in Ishikawa cells demonstrating a further clear difference in response between breast and endometrial cells. We have previously shown that OHT is capable of stabilising ERa in Ishikawa cells, and inducing ligandmediated degradation of ERb protein, but not ERa (Horner-Glister et al 2005). This effect was shown to be cell-type specific, since in breast cancer cells, ERa protein is targeted for rapid degradation via the ubiquitin-proteasome pathway in response to E 2 (Dowsett & Ashworth 2003).…”

“…For all dosing experiments, the medium was replaced with RPMI 1640 containing 10% charcoal-stripped FCS (CSS, Hyclone) and 2 mM glutamine (Gmax) for 72 h prior to treatment with 10 K8 M 17b-oestradiol (E 2 ; Sigma), reported to significantly increase proliferation in MCF7 cells (Vanparys et al 2006), 10 K6 M (OHT; Sigma), which was shown to display both agonist and antagonist behaviour (Bramlett & Burris 2003) and induce proliferation (Horner-Glister et al 2005) …”

“…A standard curve was created by dilutions of BSA (200-1000 mg/ml), which were used as a reference. SDS-PAGE and blotting performed as previously described (Horner-Glister et al 2005). Human recombinant proteins for either ERa or ERb (10 ng; Calbiochem, Nottingham, UK) were used as positive controls and run alongside a standard molecular weight ladder (BioRad).…”

Estrogens and Progression of Diabetic Kidney Damage

“…In podocytes isolated from diabetic mice treated with E 2 , neither ER copy number nor protein expression was regulated by treatment. We did, however, find an increase in ER protein expression without a change in ER mRNA copy number, suggesting a posttranslational regulation, such as protein stabilization [70,71]. As ER expression regulates apoptosis and cell cycle in breast cancer cells [72], the increase in the ER protein expression in podocytes could lead to cell cycle changes and increased cell survival, an effect which could protect against podocyte depletion in diabetic kidney injury.…”

17β-Estradiol and tamoxifen stimulate rapid and transient ERK activation in MCF-7 cells via distinct signaling mechanisms

“…[18][19][20] This can be partially explained by differences in the expression levels of ER, EGFR, and the HER-2 receptor. [21][22][23][24] Furthermore, it has been suggested that multiple mechanisms exist by which estrogen stimulation can lead to ERK activation.…”