Positive regulation of the Egr-1/osteopontin positive feedback loop in rat vascular smooth muscle cells by TGF-β, ERK, JNK, and p38 MAPK signaling

Previous studies identified a positive feedback loop in rat vascular smooth muscle cells (VSMCs) in which early growth response factor-1 (Egr-1) binds to the osteopontin (OPN) promoter and upregulates OPN expression, and OPN upregulates Egr-1 expression via the extracellular signal-regulated protein kinase (ERK) signaling pathway. The current study examined whether transforming growth factor-β (TGF-β) activity contributes to Egr-1 binding to the OPN promoter, and whether other signaling pathways act downstream of OPN to regulate Egr-1 expression. ChIP assays using an anti-Egr-1 antibody showed that amplification of the OPN promoter sequence decreased in TGF-β DNA enzyme-transfected VSMCs relative to control VSMCs. Treatment of VSMCs with PD98059 (ERK inhibitor), SP600125 (JNK inhibitor), or SB203580 (p38 MAPK inhibitor) significantly inhibited OPN-induced Egr-1 expression, and PD98059 treatment was associated with the most significant decrease in Egr-1 expression. OPN-stimulated VSMC cell migration was inhibited by SP600125 or SB203580, but not by PD98059. Furthermore, MTT assays showed that OPN-mediated cell proliferation was inhibited by PD98059, but not by SP600125 or SB203580. Taken together, the results of the current study show that Egr-1 binding to the OPN promoter is positively regulated by TGF-β, and that the p38 MAPK, JNK, and ERK pathways are involved in OPN-mediated Egr-1 upregulation.     

Osteopontin Is Expressed in the Mouse Uterus during Early Pregnancy and Promotes Mouse Blastocyst Attachment and Invasion In Vitro

Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion.     

V-sis induces Egr-1 expression by a pathway mediated by c-Ha-ras

The early growth response gene, Egr-1, is up-regulated transiently by mitogens and many other simuli in all cells tested. Using NIH3T3 cells conditionally expressing v-sis from a metallothionein promoter, we show that the addition of Zn²⁺ stimulates the production of PDGF-B(v-sis) and elicits the expression of Egr-1 in a dose-dependent and time-regulated manner. The signal is likely independent of protein kinase C, but depends on tyrosine kinase and other kinase activities and is mediated by c-Ha-Ras since the presence of dominant-negative mutants of Ras and Raf abrogates the induction of Egr-1 expression by Zn²⁺. Transiently activated Ras expression in NIH3T3 cells also stimulates the transient expression of Egr-1, but cells that constitutively express Rass do not have elevated levels of Egr-1. Transient assays also demonstrated that Zn²⁺ or activated Ras expression stimulated the activity of a 950 bp Egr-1 promoter-reporter gene construct and this is abrogated in the presence of mutant Ras and Raf. The accumulated data show that Egr-1 gene expression is regulated by multiple mechanisms, as would be needed for putative role in Cell proliferation, in suppression of transformation and in differentiation.     

Egr-1, a new downstream molecule of Epstein-Barr virus latent membrane protein 1

To investigate the effect of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) on human cancer cells, we sought to identify and analyze potential target genes that were differentially expressed in the presence and absence of LMP1. Our cDNA microarray analysis revealed that expression of early growth response gene-1 (Egr-1) was increased by LMP1 expression in MCF7 and Jurkat cells. An NFkappaB inhibitor (SN50) antagonized LMP1-induced enhancement of Egr-1 expression, indicating that LMP1 induced Egr-1 via NFkappaB. Furthermore, three lines of evidence indicated that Egr-1 was required for LMP1-induced cancer cell survival. First, Egr-1 expression enhanced the survival of doxorubicin-treated MCF7 cells. Second, inhibition of Egr-1 expression by siRNA (siEgr-1) effectively suppressed LMP-1-induced survival of MCF7 cells. Third, Egr-1 knockdown decreased LMP1-induced expression of Bfl-1. Similar relationships among EBV infection, Egr-1 and drug resistance were also observed in tissues of peripheral T-cell lymphoma-unspecified (PTCL-u) patients.     

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.