12-O-tetradecanoylphorbol-13-acetate activation of the MDR1 promoter is mediated by EGR1.

P-glycoprotein, the product of the MDR1 gene (multidrug resistance gene 1), is an energy-dependent efflux pump associated with treatment failure in some hematopoietic malignancies. Its expression is regulated during normal hematopoietic differentiation, although its function in normal hematopoietic cells is unknown. To identify cellular factors that regulate the expression of MDR1 in hematopoietic cells, we characterized the cis- and trans-acting factors mediating 12-O-tetradecanoylphorbol-13-acetate (TPA) activation of the MDR1 promoter in K562 cells. Transient-transfection assays demonstrated that an MDR1 promoter construct containing nucleotides -69 to +20 conferred a TPA response equal to that of a construct containing nucleotides -434 to +105. TPA induced EGR1 binding to the -69/+20 promoter sequences over a time course which correlated with increased MDR1 promoter activity and increased steady-state MDR1 RNA levels. The -69/+20 promoter region contains an overlapping SP1/EGR site. The TPA-responsive element was localized to the overlapping SP1/EGR site by using a synthetic reporter construct. A mutation in this site that inhibited EGR protein binding blocked the -69/+20 MDR1 promoter response to TPA. The expression of a dominant negative EGR protein also blocked the TPA response of the -69/+20 promoter construct. Finally, the expression of EGR1 was sufficient to activate a construct containing tandem MDR1 promoter SP1/EGR sites. These data suggest a role for EGR1 in modulating MDR1 promoter activity in hematopoietic cells.     

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.     

Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.     

High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3'' untranslated region.

The high-level expression of the rat whey acidic protein (WAP) gene in transgenic mice depends on the interaction of 5'-flanking promoter sequences and intragenic sequences. Constructs containing 949 bp of promoter sequences and only 70 bp of 3'-flanking DNA were expressed at uniformly high levels, comparable to or higher than that of the endogenous gene. Although this WAP transgene was developmentally regulated, it was expressed earlier during pregnancy than was the endogenous WAP gene. Replacement of 3' sequences, including the WAP poly(A) addition site, with simian virus 40 late poly(A) sequences resulted in an approximately 20-fold reduction in the expression of WAP mRNA in the mammary gland during lactation. Nevertheless, position-independent expression of the transgene was still observed. Further deletion of 91 bp of conserved WAP 3' untranslated region (UTR) led to integration site-dependent expression. Position independence was restored following reinsertion of the WAP 3' UTR into the deleted construct at the same location, but only when the insertion was in the sense orientation. The marked differences observed between the expression levels of the 3'-end deletion constructs in transgenic mice were not seen in transfected CID 9 mammary epithelial cells. In these cells, expression of the endogenous WAP gene was dependent on the interaction of these cells with a complex extracellular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in cells cultured on plastic and the precocious developmental expression of WAP in transgenic mice may reflect the absence of a negative control element(s) within these recombinant constructs.