Myeloid ELF1-like Factor Is a Potent Activator of Interleukin-8 Expression in Hematopoietic Cells

Cyrus V Hedvat,Jinjuan Yao,Robert A Sokolic,Stephen D Nimer

doi:10.1074/jbc.m307524200

Abstract

Myeloid ELF1-like factor (MEF), also known as ELF4, is a member of the ETS family of transcription factors which is expressed in hematopoietic cells. MEF-deficient mice have defects in natural killer cell and natural killer T cell development, suggesting a role for MEF in regulating innate immunity. MEF also functions in myeloid cells, where it can transactivate target genes. To identify MEF target genes in a "myeloid" environment, we created an inducible expression system and used oligonucleotide microarrays to examine the transcript profile of HEL cells after induction of MEF expression. Sixteen genes were reproducibly turned on or off more than 2-fold, 8 h after induction of MEF expression, and we examined one of the genes, interleukin-8 (IL-8), in greater detail. IL-8 is a CXC chemokine involved in neutrophil chemoattraction, angiogenesis, and stem cell mobilization. It is expressed by several tumor types, and its expression is regulated primarily transcriptionally. The IL-8 promoter contains three ETS binding sites, and we identified the specific site that binds MEF and is required for MEF responsiveness. MEF, but not the closely related ETS factors PEA3, ETS1, ETS2, ELF1, or PU.1, strongly activates the IL-8 promoter. MEF overexpression is sufficient to induce IL-8 protein expression, and reduction in MEF expression (using RNA interference) results in decreased IL-8 levels. These data demonstrates that MEF is an important regulator of IL-8 expression.

Highlights

Myeloid ELF1-like factor (MEF),1 known as ELF4, is a member of the ETS family of transcriptional regulators and was originally isolated from a myeloid hematopoietic cell line [1]
We have shown that MEF regulates cytokine gene expression, including GM-CSF and IL-3 [1]
The identification of true ETS target genes is complicated by the simplicity of their consensus recognition elements (GGAA), which are present at high frequency throughout genomic DNA and by the great number of related ETS proteins, which are often coexpressed in several different cell types

Summary

EXPERIMENTAL PROCEDURES

Generation of Cells with Inducible MEF Expression—HEL cells and NB4 –306 (a subclone of NB4, which is ATRA-resistant) cells were maintained in RPMI 1640 medium and COS-7 cells in Dulbecco’s modified Eagle’s medium, all supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine. Oligonucleotide Array-based Expression Profiling—Total RNA was isolated from HEL/IND-MEF cells after an 8-h incubation with 5 ␮M ponasterone A, using TrIzol (Invitrogen) followed by RNeasy purification (Qiagen). Luciferase Reporter Assays—5 ␮g of either the pCMV5 cytomegalovirus promoter-based expression vector alone or pCMV5 containing the MEF, ETS1, ETS2, PU., ELF1, or PEA3 cDNA [1] was used to cotransfect COS-7 cells using the calcium phosphate precipitation method together with 1 ␮g of pGL3-Basic-IL8 and 10 ng of pRL-CMV control plasmid, used to control for transfection efficiency. HEL cells were electroporated in RPMI with 10% fetal calf serum without antibiotics at 250 mV, 960 microfarads with 10 ␮g of reporter plasmid, 10 ␮g of expression vector, and 100 ng of pRL-CMV. MEF and IL-8 mRNA levels were determined by real time PCR

RESULTS

Chromatin binding

DISCUSSION