Mechanism of prostaglandin E2-induced transcriptional up-regulation of Oncostatin-M by CREB and Sp1

Abstract

Oncostatin-M (OSM) is a pleotropic cytokine belonging to the interleukin-6 family. Differential expression of OSM in response to varying stimuli and exhibiting repertoire of functions in different cells renders it challenging to study the mechanism of its expression. Prostaglandin E2 (PGE2) transcriptionally increased osm levels. In silico studies of ∼1 kb upstream of osm promoter region yielded the presence of CRE (cyclic AMP response element)-like sites at the distal end (CREosm). Deletion and point mutation of CREosm clearly indicated that this region imparted an important role in PGE2-mediated transcription. Nuclear protein(s) from PGE2-treated U937 cells, bound to this region, was identified as CRE-binding protein (CREB). CREB was phosphorylated on treatment and was found to be directly associated with CREosm. The presence of cofactors p300 and CREB-binding protein in the complex was confirmed. A marked decrease in CREB phosphorylation, binding and transcriptional inhibition on treatment with PKA (protein kinase A) inhibitor, H89 (N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-soquinolinesulfonamide), revealed the role of phosphorylated CREB in osm transcription. Additionally, other nuclear protein(s) were specifically associated with the proximal GC region (GCosm) post PGE2 treatment, later confirmed to be specificity protein 1 (Sp1). Interestingly, Sp1 bound to the proximal osm promoter was found to be associated with phospho-CREB–p300 complex bound to the distal osm promoter. Knockdown of Sp1 abrogated the expression and functionality of OSM. Thus, the present study conclusively proves that these transcription factors, bound at the distal and proximal promoter elements are found to associate with each other in a DNA-dependent manner and both are responsible for the PGE2-mediated transcriptional up-regulation of Oncostatin-M.