Basal transcription is regulated by lipopolysaccharide and glucosamine via the regulation of DNA binding of RNA polymerase II in RAW264.7 cells

Abstract

Aims: The objective of this study is to investigate glucosamine (GlcN) as a transcriptional regulator of iNOS and other genes in association with the dynamic O-GlcNAcylation of RNA polymerase II (RNAPII).

Main methods: The LPS- and/or GlcN-stimulated transcriptional activities of various Gal4-binding site/TATA-box-containing reporter constructs were measured.

Key findings: Basal transcriptional activities of nuclear factor-κB (NF-κB) and nitric oxide synthase (iNOS) reporter plasmids are inhibited by GlcN in RAW264.7 cells. Furthermore, GlcN suppressed whereas lipopolysaccharide (LPS) stimulated the basal activity of Gal4-binding site/TATA-box-containing reporter constructs. LPS reduced the O-linked N-acetylglucosamine modification (O-GlcNAcylation) of RNAPII, but enhanced the binding of this enzyme to the iNOS promoter. In contrast, GlcN enhanced RNAPII O-GlcNAcylation, but inhibited iNOS promoter binding. Furthermore, the basal activities of reporter plasmids containing activator protein 1 (AP1), E2F, or cyclic AMP response element (CRE) binding sites were consistently inhibited by GlcN in a dose-dependent manner. However, GlcN did not inhibit the phorbol 12-myristate 13-acetate- (PMA-) or forskolin-induced transcriptional activities of AP1 and CRE. The transcriptional activity of transforming growth factor alpha (TGF-α) was slightly increased by both LPS and GlcN.

Significance: In conclusion, our data demonstrate that LPS activates, whereas GlcN suppresses, basal activities of transcription through the regulation of RNAPII O-GlcNAcylation and DNA binding.

Keywords: Glucosamine; O-GlcNAc; RNAPII.