Egr-1 is activated by 17beta-estradiol in MCF-7 cells by mitogen-activated protein kinase-dependent phosphorylation of ELK-1

Early growth response-1 (Egr-1) is an immediate-early gene induced by E2 in the rodent uterus and breast cancer cells. E2 induces Egr-1 mRNA and protein levels in MCF-7 human breast cancer cells and reporter gene activity in cells transfected with pEgr-1A, a construct containing the -600 to +12 region of the Egr-1 promoter linked to the firefly luciferase gene. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region of the promoter that contained serum response element (SRE)3 (-376 to -350) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a mitogen-activated protein kinase kinase inhibitor, but not by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3-K). These results contrasted with hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002. Differences in activation of the SREs in Egr-1 and c-fos were related to promoter sequence, which defines the affinities of Elk-1 and SRF to their respective binding sites. Thus, Egr-1, like c-fos, is activated through non-genomic (extranuclear) pathways of estrogen action in breast cancer cells.     

Mechanisms of estrogen receptor action in the myocardium. Rapid gene activation via the ERK1/2 pathway and serum response elements

We have previously shown that the myocardium is a target tissue for estrogen. Here, we have identified rapid non-nuclear estrogen effects on the expression of the early growth response gene-1 (Egr-1) in cardiomyocytes. Egr-1 mRNA and protein were rapidly and strongly induced by estrogen in an estrogen receptor-dependent manner via the extracellular signal-regulated kinase, ERK1/2. A promoter analysis study of a 1.2-kilobase Egr-1 promoter fragment revealed that the serum response elements (SREs) but not the estrogen response elements or AP-1 sites are responsible for Egr-1 induction by estrogen, identifying a novel mechanism of estrogen receptor-dependent gene activation in the myocardium. Both estrogen receptor-alpha and -beta induced the Egr-1 promoter via the SREs as well as an artificial promoter consisting of only five SREs in cardiomyocytes. Electrophoretic mobility shift assays showed that a protein complex containing serum response factor or an antigenically related protein was recruited to the SREs by estrogen treatment of primary cardiomyocytes. The recruitment of the protein complex was inhibited by the specific estrogen receptor antagonist ICI 182,780 as well as the MEK inhibitor PD 98059. Taken together, these results identify SREs as important promoter control elements for an estrogen receptor-dependent mechanism of gene activation in the myocardium.     

M1 muscarinic acetylcholine receptors activate zif268 gene expression via small G-protein Rho-dependent and lambda-independent pathways in PC12D cells.

We have previously shown that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) in neuronal PC12D cells rapidly induces the immediate-early gene zif 268 [Ebihara, T. & Saffen, D. (1997) J. Neurochem. 68, 1001-1010]. Here we show that stimulation of M1 mAChRs in these cells activates four distal serum response elements (SREs) in the zif 268 promoter, and that this activation is strongly inhibited by Clostridium botulinum C3 exoenzyme (C3), which specifically inactivates the small G-protein Rho. Even with high doses of C3, however, a portion of the activation remains intact, indicating that stimulation of M1 mAChRs activates zif 268 SREs via Rho-dependent and Rho-independent pathways. Moreover, the Rho-independent activation of zif 268 SREs is inhibited by the dominant-negative form of the small G-protein Ras, suggesting that Rho-independent activation of zif 268 SREs is mediated by Ras. To determine if muscarinic agonists activate RhoA, we also measured the translocation of RhoA from the cytosolic fraction to the particulate fraction. Translocation of RhoA to the particulate fraction was observed within 15 min following stimulation of M1 mAChRs, indicating that RhoA is activated with sufficient rapidity to participate in the induction of zif 268 mRNA. Together, these results suggest that RhoA is activated following stimulation of M1 mAChRs and functions in SRE-dependent induction of the zif 268 gene within a Ras-independent pathway.     

Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway

E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.