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.

Ethanol stimulates the secretion of matrix metalloproteinases 2 and 9 in MCF-7 human breast cancer cells

“…Hence, in our study, in spite of the absence of serum and steroids in the medium, ethanol exposure could induce a PKA-mediated activation of ER· which could activate the transcription of a gene responsible for MMP-9 secretion, explaining that this effect is inhibited by the anti-estrogen. It is noteworthy that, in the case of ligand-independent activation of ER·, its degradation by the proteasome is inhibited in contrast to what occurs in the case of ligand-dependent activation (34). In our previous study of ethanol-treated MCF-7 cells, we observed by immunoblotting that the level of ER· was increased while no difference in the mRNA levels was observed by RT-PCR (14).…”

“…The phosphorylation of ER· by protein kinase A (PKA) has been well documented in this mechanism (33). Besides, forskolininduced ER activation, measured by the transcription of a reporter gene downstream a 2-ERE sequence, is inhibited by ICI 182,780 (34). Interestingly, ethanol has been shown to increase cAMP levels in MCF-7 cells (30).…”

Conformational dynamics of estrogen receptors α and β as revealed by intrinsic tryptophan fluorescence and circular dichroism

“…ERs mediate the function of the steroid hormone, estradiol (E 2 ), in both males and females. ERs and other nuclear receptors share a common modular structure consisting of five domains, named A/B, C, D, E and F, and some key functions have been assigned to each domain (Metzger et al 1995, Tsai et al 2004. The N-terminal A/B domain contains the ligand-independent transcription activation function 1 (AF-1; Metzger et al 1995, Tsai et al 2004).…”

“…ERs and other nuclear receptors share a common modular structure consisting of five domains, named A/B, C, D, E and F, and some key functions have been assigned to each domain (Metzger et al 1995, Tsai et al 2004. The N-terminal A/B domain contains the ligand-independent transcription activation function 1 (AF-1; Metzger et al 1995, Tsai et al 2004). The C domain has a characteristic zinc-finger structure responsible for the binding of estrogen-response elements (EREs; Danielian et al 1992, Krieg et al 2004.…”

The role of S6K1 in ER-positive breast cancer

“…19,39 Importantly, this site is associated with tamoxifen resistance. 40 PKA phosphorylates Ser236, which appears to promote the stability of ERα, 41,42 as well as Ser305. Ser305 appears to be important for dimerization and transactivation activity of ERα and is phosphorylated by PKA in the presence of estrogen 43 and by Akt through the IGF-I signaling pathway.…”