Ginsenoside Rg1 exerts estrogen-like activities via ligand-independent activation of ERalpha pathway

Ginsenoside Rg1, an active ingredient commonly found in ginseng root, was previously demonstrated to be a phytoestrogen that exerted estrogen-like activity without direct interaction with estrogen receptors (ERs) in human breast cancer (MCF-7) cells. The present study was designed to determine the molecular mechanism by which Rg1 exerted estrogenic effects. Co-incubation of MCF-7 cells with 1 microM of ER antagonist ICI182780 abolished the inductive effects of Rg1 on pS2 expression as well as ERE-luciferase activity, suggesting that the estrogenic effects of Rg1 were mediated through the endogenous ERs. To evaluate the relative involvement of ERalpha and ERbeta in mediating the actions of Rg1, ER-negative human embryonic kidney (HEK293) cells were co-transfected with the ERE-luciferase reporter construct and either ERalpha or ERbeta construct. The results showed that Rg1 could activate ERE-luciferase activity via the ERalpha-mediated pathway in a dose-dependent manner (10(-14) to 10(-6)M); whereas, the activation of ERbeta-mediated ERE-luciferase activity by Rg1 only occur at high concentration (10(-6)M). Furthermore, the results showed that 1pM Rg1 could rapidly induce phosphorylation of the AF-1 domain of ERalpha at serine 118 residue within the first 5 min of incubation, suggesting that Rg1 activates ERalpha in a ligand-independent manner. Taken together, our results indicate that Rg1 preferentially activates ERalpha via phosphorylation of AF-1 domain in the absence of receptor binding. This study is the first to provide evidence that ginsenoside Rg1 exerts estrogen-like actions via ligand-independent activation of ERalpha pathway.     

Cobalt chloride-induced estrogen receptor alpha down-regulation involves hypoxia-inducible factor-1alpha in MCF-7 human breast cancer cells

The estrogen receptor (ER) is down-regulated under hypoxia via a proteasome-dependent pathway. We studied the mechanism of ERalpha degradation under hypoxic mimetic conditions. Cobalt chloride-induced ERalpha down-regulation was dependent on the expression of newly synthesized protein(s), one possibility of which was hypoxia-inducible factor-1alpha (HIF-1alpha). To examine the role of HIF-1alpha expression in ERalpha down-regulation under hypoxic-mimetic conditions, we used a constitutively active form of HIF-1alpha, HIF-1alpha/herpes simplex viral protein 16 (VP16), constructed by replacing the transactivation domain of HIF-1alpha with that of VP16. Western blot analysis revealed that HIF-1alpha/VP16 down-regulated ERalpha in a dose-dependent manner via a proteasome-dependent pathway. The kinase pathway inhibitors PD98059, U0126, wortmannin, and SB203580 did not affect the down-regulation. A mammalian two-hybrid screen and immunoprecipitation assays indicated that ERalpha interacted with HIF-1alpha physically. These results suggest that ERalpha down-regulation under hypoxia involves protein-protein interactions between the ERalpha and HIF-1alpha.     

Nitric oxide reverses desferrioxamine- and hypoxia-evoked HIF-1alpha accumulation--implications for prolyl hydroxylase activity and iron

Hypoxia inducible factor 1 (HIF-1) senses and coordinates cellular responses towards hypoxia. HIF-1 activity is primarily determined by stability regulation of its alpha subunit that is degraded by the 26S proteasome under normoxia due to hydroxylation by prolyl hydroxylases (PHDs) but is stabilized under hypoxia. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha and promotes hypoxia-responsive target gene expression under normoxia. However, in hypoxia, NO attenuates HIF-1alpha stabilization and gene activation. It was our intention to explain the contrasting behavior of NO under hypoxia. We used the iron chelator desferrioxamine (DFX) or hypoxia to accumulate HIF-1alpha in HEK293 cells. Once the protein accumulated, we supplied NO donors and followed HIF-1alpha disappearance. NO-evoked HIF-1alpha destabilization was reversed by proteasomal inhibition or by blocking PHD activity. By using the von Hippel Lindau (pVHL)-HIF-1alpha capture assay, we went on to demonstrate binding of pVHL to HIF-1alpha under DFX/NO but not DFX alone. Showing increased intracellular free iron under conditions of hypoxia/NO compared to hypoxia alone, we assume that increased free iron contributes to regain PHD activity. Variables that allow efficient PHD activation such as oxygen availability, iron content, or cofactor accessibility at that end allow NO to modulate HIF-1alpha accumulation.     

ERalpha and STAT5a cross-talk: interaction through C-terminal portions of the proteins decreases STAT5a phosphorylation, nuclear translocation and DNA-binding

Cross-talk between ERalpha and STAT5a was demonstrated to mediate through a direct physical association between the two proteins. By GST pull-down assays and functional assays with various constructs of ERalpha and STAT5a, it was shown that the C-termini of these two proteins were mainly responsible for this interaction. Furthermore, the interaction between ERalpha and STAT5a was demonstrated to give rise to functional changes in their signaling events. In cell transfection studies, it was shown that ERalpha activation could attenuate PRLR signaling through STAT5a. This ERalpha-mediated attenuation of PRLR signaling was substantiated by observed decreases in the phosphorylation of JAK2 and STAT5a, reduced translocation of STAT5a into the nucleus, and reduced binding of STAT5a onto a GAS-containing nucleotide. Apart from transfected cells, the interaction between ERalpha and STAT5a could also be observed in established breast cancer cell lines of MCF-7 and T-47D in co-immunoprecipitation studies. However, the functional consequence of the interaction in these cancer cells was very different from the transfected HEK293 cells. ER activation could lead to potentiation of PRLR signaling in MCF-7 cells but not in T-47D cells. Conversely, in both MCF-7 and T-47D cells, PRLR activation could lead to attenuation of ER signaling. These data serve to elucidate the mechanisms underlying the ERalpha-STAT5a cross-talk and in demonstrating that the functional consequence of this cross-talk depends on the precise milieus of the intracellular environment.