CCAAT-binding factor regulates expression of the β1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line

Abstract

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of α and β subunits. We investigated human β1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5′ upstream region of the β1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the β1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the β1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of β1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human β1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in β1 sGC expression.