Prolactin induces expression of the human prolactin receptor via transcriptional activation of its generic promoter by cross-talk between estrogen receptor α and Stat5

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 389-413-Signaling and Transcriptional Control in Endocrine Systems
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-400
Raghuveer Kavarthapu*1, Chon-Hwa Tsai-Morris2 and Maria L Dufau3
1National Institutes of Health, Bethesda, MD, 2NIH, Bethesda, MD, 3NIH-NICHD, Bethesda, MD
The prolactin receptor (PRLR) mediates the diverse cellular actions of prolactin (PRL) in target tissues and has been implicated in the development, tumoral growth and chemoresistance of breast cancer. Human PRLR expression is controlled at the transcriptional level by multiple promoters (a generic [hPIII] and five human specific [HPN1-5]) which direct transcription of specific non-coding exon 1, common exon 2 and coding exons 3-11. Our previous studies demonstrated hPRLR transcription via hPIII directed by estrogen receptor (ER) (basal) and estradiol (E2)/ERα through complex formation with Sp1 and C/EBPß that associate with their cognate elements (JBC 2006, MBC 2011). In this study, we investigate the regulation of PRLR transcription by PRL in MCF-7A2 breast cancer cells. Temporal and dose-related PRL stimulated hPIII promoter activity, PRLR mRNA and protein levels were prevented by the ER antagonist ICI. PRL-induced hPIII activity was abolished by Sp1or C/EBP site mutation. In E2 depleted cells, PRL caused rapid stimulation of ERα protein, its nuclear translocation and phosphorylation (ER118). Absence of changes in ER mRNA suggested a post-transcriptional effect of PRL on ER. ICI inhibition of PRLR-induced ER protein pointed to an E2 independent ER action.  Mutation of a GAS site in non-coding exon E13 abolished PRL induced hPIII promoter activity. Moreover, PRL stimulated hE13 mRNA expression was reduced upon Stat5a/b siRNA treatment. Since constitutive (basal) and E2 induced activation of ER regulates PRLR transcription by hPIII in MCF-7 via an ERE independent mechanism, loss of the hPIII activity by the mutated GAS suggested a cross-talk between Stat5 and ER for PRL-induced PRLR transcription. Knockdown of ER showed absence of PRL induced ER recruitment to Sp1/CEBPβ at the hPIII promoter by ChIP, but that of Stat5 to GAS was present, and knockdown of Stat5a/b abolished the recruitment of ER.  Re-ChIP analysis indicated complex formation of ER/Stat5 onto the endogenous PIII promoter and downstream GAS in exon1.  This study presents direct evidence for the effect of PRL independent of E2 on its receptor promoter activity by interaction between ER and Stat5 and complex formation of an activation loop via their binding to SP1/C/EBPß at their hPIII promoter sites and downstream GAS element in the hE13 exon. PRL produced in normal breast and mammary tumors is an important local regulator of PRLR transcription/expression.

Nothing to Disclose: RK, CHT, MLD

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