Isoform-Specific Degradation of PR-B by E6-AP Is Critical for Normal Mammary Gland Development

Abstract

E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a coactivator of estrogen (ER-α) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that either overexpress wild-type (WT) human E6-AP (E6-AP(WT)) or ubiquitin-protein ligase-defective E6-AP (E6-AP(C833S)) in the mammary gland. Here we show that overexpression of E6-AP(WT) results in impaired mammary gland development. In contrast, overexpression of E6-AP(C833S) or loss of E6-AP (E6-AP(KO)) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are due, in large part, to the alteration of PR-B protein levels. We also observed alteration in ER-α protein level, which might contribute to the observed mammary phenotype by regulating PR expression. Furthermore, E6-AP regulates PR-B protein levels via the ubiquitin-proteasome pathway. Additionally, we also show that E6-AP impairs progesterone-induced Wnt-4 expression by decreasing the steady state level of PR-B in both mice and in human breast cancer cells. In conclusion, we present the novel observation that E6-AP controls mammary gland development by regulating PR-B protein turnover via the ubiquitin proteasome pathway. For the first time, we show that the E3-ligase activity rather than the coactivation function of E6-AP plays an important role in the mammary gland development, and the ubiquitin-dependent PR-B degradation is not required for its transactivation functions. This mechanism appears to regulate normal mammogenesis, and dysregulation of this process may be an important contributor to mammary cancer development and progression.

Progesterone Decreases Levels of the Adhesion Protein E-Cadherin and Promotes Invasiveness of Steroid Receptor Positive Breast Cancers

“…Both RU486 and Wnt inhibitors blocked this effect of R5020, indicating that both the genomic action of PRB and the activation of the Wnt pathway were required to decrease the E-cadherin protein. These results are consistent with previous reports that P acting through PRB induces Wnt-1 and Wnt-4 mRNA in YB cells, and loss of Wnt1 blocks soft agar growth of YB T47D cells [32,33]. However, other Wnt proteins may be induced by PS to regulate E-cadherin.…”

Polycomb subunit BMI1 determines uterine progesterone responsiveness essential for normal embryo implantation

“…In fact, BMI1 is essential for normal functional interaction of the PR with E6AP. A previous study has shown that E6AP is involved in human PRB ubiquitination for its timely turnover via the proteasome pathway during mammary gland development (32). In this regard, we observed a comparable expression level of PR proteins upon the loss of Bmi1, pointing toward a disassociation of PR ubiquitination by the BMI1-PR-E6AP complex from the process of PR protein turnover.…”

Role of the ubiquitin ligase E6AP/UBE3A in controlling levels of the synaptic protein Arc

“…increased in brain lysates derived from Ube3a-null mice (26)? E6AP was reported to affect nuclear hormone receptor-mediated transcription by E3-independent and E3-dependent mechanisms (41)(42)(43). Furthermore, expression of the human Arc gene is increased by estradiol signaling (44).…”