Protein Kinase A Activation of Estrogen Receptor α Transcription Does Not Require Proteasome Activity and Protects the Receptor from Ligand-Mediated Degradation

Abstract

17beta-Estradiol (E2)-stimulated estrogen receptor (ERalpha) transcription is accompanied by protein degradation via the 26S-proteasome pathway. Inhibition of proteasome activity stabilizes ERalpha protein and abolishes E2-activated transcription, suggesting functional linkages between transcription and degradation. It is not known whether ligand-independent ERalpha activation is coupled to proteolysis. In pituitary cells, forskolin (FSK) stimulates ERalpha transcription through the protein kinase A (PKA) pathway. This study examined interactions between E2-dependent and PKA-stimulated pathways in GH(3) cells by measuring transcription of a transfected reporter gene and endogenous ERalpha levels. E2 stimulated estrogen response element-mediated transcription 2- to 3-fold and decreased ERalpha protein levels to 40%. In contrast, FSK stimulated ERalpha transcription without decreasing ERalpha protein. Treatment with FSK plus E2 resulted in synergistic ERalpha transactivation, and FSK specifically prevented E2-induced ERalpha degradation. PKA is required for protection and was prevented by H89 (a PKA inhibitor), but not PD98059 (a MAPK kinase inhibitor). Propyl-pyrazole-triol and R,R-diethyl-tetrahydrochrysene, selective ERalpha agonists, reduced ERalpha protein by 50% while stimulating ERalpha transcriptional activity 4- to 8-fold. The antagonist ICI 182,780 similarly decreased ERalpha levels, but prevented ER activation. FSK prevented all ligand-induced ERalpha degradation. Lactacystin, a proteasome inhibitor, abolished E2-stimulated, but not FSK-stimulated, ERalpha transcription. Thus, stimulation of ERalpha transcription by the PKA-dependent pathway is dissociated from receptor degradation and proteasome activity. These data suggest a mechanism of ERalpha transcriptional activation by PKA that is distinct from E2 activation and that may contribute to the synergistic transcriptional activation of ERalpha by ligand-dependent and PKA-dependent pathways.