Heregulin-β1 regulates the estrogen receptor-α gene expression and activity via the ErbB2/PI 3-K/Akt pathway

This study examines whether the serine/threonine protein kinase, Akt, is involved in the crosstalk between the ErbB2 and estrogen receptor-α (ER-α) pathways. Treatment of MCF-7 cells with 10−9 M heregulin-β1 (HRG-β1) resulted in a rapid phosphorylation of Akt and a 15-fold increase in Akt activity. Akt phosphorylation was blocked by inhibitors of phosphatidylinositol 3-kinase (PI 3-K), by antiestrogens, the protein tyrosine kinase inhibitor, genistein, and by AG825, a selective ErbB2 inhibitor; but not by AG30, a selective EGFR inhibitor. Akt phosphorylation by HRG-β1 was abrogated by an arginine to cysteine mutation (R25C) in the pleckstrin homology (PH) domain of Akt, and HRG-β1 did not induce Akt phosphorylation in the ER-negative variant of MCF-7, MCF-7/ADR. Transient transfection of ER-α into these cells restored Akt phosphorylation by HRG-β1, suggesting the requirement of ER-α. HRG-β1 did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. Stable transfection of the cells with a dominant negative Akt or with the R25C-Akt mutant, as well as PI 3-K inhibitors, blocked the effect of HRG-β1 on ER-α expression and activity and on the growth of MCF-7 cells. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of HRG-β1. Experiments employing selective ErbB inhibitors demonstrate that the effect of HRG-β1 on ER-α expression and activity is also mediated by ErbB2 and not by EGFR, demonstrating that ErbB2 is the primary mediator of the effects of HRG-β1 on ER-α regulation. Taken together, our data suggest that HRG-β1, bound to the ErbB2 ErbB3 heterodimer, in the presence of membrane ER-α, interacts with and activates PI 3-K/Akt. Akt leads to nuclear ER-α phosphorylation, thereby altering its expression and transcriptional activity.