GW1929 inhibits α7 nAChR expression through PPARγ-independent activation of p38 MAPK and inactivation of PI3-K/mTOR: The role of Egr-1

Studies demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) ligands reduce nicotine-induced non small cell lung carcinoma (NSCLC) cell growth through inhibition of nicotinic acetylcholine receptor (nAChR) mediated signaling pathways. However, the mechanisms by which PPARγ ligands inhibited nAChR expression remain elucidated. Here, we show that GW1929, a synthetic PPARγ ligand, not only inhibited but also antagonized the stimulatory effect of acetylcholine on NSCLC cell proliferation. Interestingly, GW1929 inhibited α7 nAChR expression, which was not blocked by GW9662, an antagonist of PPARγ, or by PPARγ siRNA, but was abrogated by the p38 MPAK inhibitor SB239063. GW1929 reduced the promoter activity of α7 nAChR and induced early growth response-1 (Egr-1) protein expression, which was overcame by SB239063, but enhanced by inhibitors of PI3-K and mTOR. Silencing of Egr-1 blocked, while overexpression of Egr-1 enhanced, the effect of GW1929 on α7 nAChR expression and promoter activity. Finally, GW1929 induced Egr-1 bound to specific DNA areas in the α7 nAChR gene promoter. Collectively, these results demonstrate that GW1929 not only inhibits but also antagonizes Ach-induced NSCLC cell growth by inhibition of α7 nAChR expression through PPARγ-independent signals that are associated with activation of p38 MPAK and inactivation of PI3-K/mTOR, followed by inducing Egr-1 protein and Egr-1 binding activity in the α7 nAChR gene promoter. By downregulation of the α7 nAchR, GW1929 blocks cholinergic signaling and inhibits NSCLC cell growth.     

Role of Egr-1 in Cholinergic Stimulation of Phenylethanolamine N-Methyltransferase Promoter

Abstract: The effects of the cholinergic agonist carbachol on phenylethanolamine N -methyltransferase promoter activity and Egr-1 mRNA expression in PC12-derived RS1 cells were examined to investigate the potential involvement of Egr-1 in the neural regulation of phenylethanolamine N -methyltransferase gene expression. Carbachol stimulated luciferase expression in cells transfected with a rat phenylethanolamine N -methyltransferase promoter-luciferase reporter gene construct and also elevated Egr-1 mRNA levels in untransfected cells. Maximum induction of Egr-1 mRNA by carbachol was rapid (0.5 h), whereas by comparison, peak luciferase activity was delayed (6 h). In addition, carbachol stimulation of both luciferase and Egr-1 mRNA expression could be completely inhibited by atropine but not hexamethonium. Furthermore, bethanechol but not nicotine could mimic the effects of carbachol, indicating that carbachol activation was medicated through muscarinic cholinergic receptors. Finally, carbachol failed to stimulate luciferase expression in cells transfected with a mutant construct, in which the Egr-1 binding element in the phenylethanolamine N -methyltransferase promoter was mutated. These results suggest that carbachol activates the phenylethanolamine N -methyltransferase promoter through stimulation of Egr-1 expression, and are consistent with the potential involvement of Egr-1 in the cholinergic activation of the phenylethanolamine N -methyltransferase gene.     

Egr-1 expression in surface Ig-mediated B cell activation. Kinetics and association with protein kinase C activation

We have studied the expression of an immediate/early type gene, Egr-1, in murine B lymphocyte responses to Ag receptor-generated signals. The Egr-1 gene encodes a zinc finger protein with sequence-specific DNA binding activity and is believed to act as an intracellular "third messenger," to couple receptor-generated signals to activation-associated changes in gene expression. We show here that Egr-1 mRNA expression is rapidly and transiently (returning to basal levels by 6 h) induced after receptor crosslinking with anti-receptor antibodies. Egr-1 protein expression is more prolonged, maintaining detectable levels through 12 h. The induction of Egr-1 is a primary response to Ag receptor signaling, as it is independent of new protein synthesis and is inhibited by actinomycin D. We have also examined the linkage of Egr-1 to known signaling pathways associated with G0 to G1 transition by these cells in response to signals generated through the B cell Ag receptor. Egr-1 mRNA was not induced after elevation of intracellular free Ca2+. In contrast, the pharmacologic agents PMA and SC-9, which directly activate protein kinase C, both cause marked increases in Egr-1 mRNA levels with the same kinetics as observed after anti-receptor antibody stimulation. Further, the protein kinase C inhibitors H7, sangivamycin, and staurosporin block anti-receptor antibody-induced expression of Egr-1, thus, B cell Ag receptor-linked Egr-1 expression is likely coupled to the protein kinase C component of transmembrane signaling. Preliminary promoter mapping studies are consistent with this conclusion, because both PMA and anti-receptor antibody act through the same or overlapping cis-regulatory elements.     

In vivo somatic delivery of plasmid DNA and retrograde transport to obtain cell-specific gene expression in the central nervous system

We utilised the retrograde transport machinery of neurones to deliver naked plasmid DNA into the central nervous system. A 5.4-kb fragment of the glycine receptor (GlyR) alpha1 subunit gene was cloned and used to drive the expression of a construct encoding for the enhanced green fluorescent protein (EGFP). Injections of the plasmid DNA in the tongue of mice resulted in the expression of the marker protein in hypoglossal motor neurones, showing that the GlyRalpha1 promoter sequence is sufficient to drive expression of the transgene. In order to determine the specificity of expression of the 5.4-kb fragment of the GlyR alpha1 subunit gene promoter, we subsequently injected the plasmid DNA into the mouse central nucleus of the amygdala. This nucleus receives projections from the parabrachial nucleus, a brainstem area that has a high density of GlyRs, and from the insular cortex, a forebrain structure devoid of GlyRs. We observed EGFP-labelled neurones in the parabrachial nucleus, but not in the insular cortex, indicating that the 5.4-kb GlyR alpha1 subunit gene promoter confers specificity of expression. This approach provides a simple and rapid way to identify, in vivo, promoter elements that mediate neurone-specific gene expression.     

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.