Estrogen-induced phosphorylation of P53 at Ser15 in aging HMEC in the absence of estrogen receptor

Abstract

615 Background: A variety of studies have established primary human mammary epithelial cells (HMEC) as an ex vivo model. It was of interest therefore, to investigate aging-related functional alterations in response to estrogen treatment as part of a hormone replacement therapy (HRT). Method: Primary HMEC were cultured for 5d, 19d, and 50d, respectively. During this aging process, functional changes including cell cycle distribution and estrogen receptor expression were determined by flow cytometry, respectively. Expression of P53 and phosphorylated P53 were detected by Western blot analysis and quantified by a P53 phospho ELISA. Results: Primary HMEC cultures underwent significant morphological and functional changes during the process of aging. Cells in S phase significantly declined after 19d and accumulated in G0/G1 and G2/M phase after 50d. Simultaneously, the initial estrogen receptor expression of 32,3% dropped to less than 1% in 19d and 50d aged HMEC, respectively. Short term treatment of HMEC with 1μM β-estradiol demonstrated unaltered levels of P53 and phosphorylated P53 after 60min. Following 24h of estrogen exposure, however, Western blot analysis revealed a significantly enhanced phosphorylation of P53 at Ser15 in 5d and 19d HMEC populations, respectively. A phospho-P53 ELISA confirmed a more than 2-fold increased Ser15-phosphorylation of P53 in these two HMEC populations following 24h of estrogen stimulation, but no significant changes after 60min, respectively. Conclusions: According to the estrogen receptor depletion in aged HMEC, these data indicated a receptor-independent signaling and thus suggest a dual estrogen mechanism: 1) a receptor-dependent probably short-term transcriptional effect, and 2) a receptor-independent long-term effect associated with P53 phosphorylation and subsequent repair. Whereas these dual estrogen signaling pathways are accompanied by aging-related functional alterations of the breast tissue, this would also complicate HRT to balance protective and damaging effects of estrogen. No significant financial relationships to disclose.