Control of granulocyte-macrophage colony-stimulating factor production in normal endothelial cells by positive and negative regulatory elements

Abstract

Granulocyte-macrophage (GM)-CSF is an acidic glycoprotein involved in the hemopoietic response to inflammation and in the functional activation of mature blood cells. The protein is produced in response to a number of inflammatory mediators by mesenchymal cells present in the hemopoietic microenvironment and at peripheral sites of inflammation. To understand the molecular basis for the regulation of GM-CSF gene expression, nuclear run-on assays and a functional analysis of potential regulatory sequences were performed in normal human endothelial cells. These studies suggest that GM-CSF is regulated by both transcriptional and post-transcriptional mechanisms. By using hybrid constructs containing a reporter gene and varying lengths of the regions flanking the human GM-CSF gene, a 14-bp sequence was identified in the region upstream of the GM-CSF cap site which increased reporter expression in response to a number of inflammatory mediators. In addition, despite a failure to detect GM-CSF-specific RNA or protein in unstimulated cells, basal transcription from the GM-CSF promoter was readily detectable. To account for this apparent discrepancy, sequences present in the 3' untranslated region of the GM-CSF gene were found to substantially reduce the level of reporter gene expression in a number of cell types. Homologous sequences are found in other genes which share a similar pattern of expression, and may provide the molecular basis for the coordinate regulation of multiple inflammatory response genes.