Functional Characterization of SDF-1 Proximal Promoter

Abstract

Stromal-cell derived factor 1 (SDF1) is a CXC chemokine that binds and signals through the CXCR4 receptor, playing an essential role in embryonic B lymphopoiesis, myelopoiesis and organogenesis. The CXCR4/SDF1 pathway is associated with several pathologies. CXCR4 serves as a fusion cofactor for lymphotropic strains of human immunodeficiency virus type 1 and SDF1 inhibits viral entry. Moreover, recent works suggest an important role for SDF1 in metastasis progression and autoimmune diseases such as rheumatoid arthritis. To understand the molecular mechanisms that regulate SDF1 expression, we have cloned and functionally analysed its 5' flanking regulatory region. An SDF1-promoter luciferase construct showed high levels of reporter gene activity in transient transfection experiments. DNase I footprinting analysis revealed that the proximal promoter was occupied by six putative Sp1-binding motifs. Binding of Sp1 to the promoter was confirmed by electrophoretic mobility shift assay, and its importance in SDF1 gene expression verified by in vitro mutagenesis. Particularly, mutation of an Sp1 motif located between -57 and -39 upstream of the main transcription start-site resulted in a marked reduction in promoter activity. It has been shown that the SDF1 expression could be induced by mitogenic stimuli, X-ray radiation or treatment with IL1beta, depending on cell environment. We have analysed the effect of these stimuli on SDF1 promoter transactivation in three different cell lines. Phorbol myristated acetate plus ionomycin increased promoter activity in U373 and LC5 but repressed it in MS5 cells. On the contrary, gamma irradiation promoted SDF1 transcription in MS5 cells but not in the other cell lines. Interferon-gamma acted as a transcriptional repressor in U373 and LC5 but not in MS5 cells. Finally, IL1beta functions as mild activator only in U373 cells. The present study demonstrates that these stimuli mediate SDF1 production through promoter activation in a cell-specific manner.