Dual effects of 2-deoxyglucose on synthesis of the glycoprotein hormone common alpha-subunit in butyrate-treated HeLa cells.

Sodium butyrate (Btr) (3 mM) causes a 10-fold increase in production of the glycoprotein hormone alpha-subunit in HeLa cells. The following report demonstrates that this response could be inhibited about 95% by 5 mM 2-deoxy-D-glucose (dGlc), whereas alpha-subunit production in uninduced cells was affected little or not at all. Addition of D-mannose restored the Btr induction of Hela-alpha in cultures that had been treated with dGlc. When the alpha-subunits secreted by cells cultured in Btr plus dGlc or in Btr alone were compared by gel filtration (Sephadex G-75) and lectin affinity (concanavalin A and ricin) chromatography, differences were noted that probably reflect changes in their carbohydrate moieties. Immunoprecipitation of [35S]methionine-labeled HeLa-alpha and incubation with endoglycosidase H indicated that the subunit secreted from cells in the presence of dGlc contained oligosaccharide side chains that were not processed to the complex type. Cells that were simultaneously treated with Btr plus dGlc showed no increase in alpha-subunit production over cells receiving Btr only; in contrast, cells that were preincubated with Btr for either 16 or 36 h before dGlc was added exhibited high levels of subunit synthesis. Measurement of alpha-mRNA levels at various times after Btr and dGlc were added to cultures indicated that Btr brought about a dramatic increase in alpha-specific mRNA about 24 h after being added to cultures. This increase could be prevented by dGlc when added simultaneously with Btr but not when added after a 24-h preincubation. Although dGlc prevented the induction of alpha-subunit and alpha-mRNA in response to Btr, it had no effect on histone hyperacetylation, suggesting that if this chromatin modification is necessary for the induction process, it is not in itself sufficient. Together, the data demonstrate that dGlc inhibits the accumulation of alpha-subunit mRNA normally produced in response to Btr and that the subunit produced contains altered oligosaccharide constituents.     

Activation of interleukin-6 gene expression through the NF-kappa B transcription factor.

The promoter region of the interleukin-6 (IL-6) gene has a putative NF-kappa B-binding site. We found that a fragment of the IL-6 promoter containing the site specifically binds highly purified NF-kappa B protein and the NF-kappa B protein in nuclear extracts of phorbol ester-induced Jurkat cells. Mutations of the NF-kappa B site abolished complex formation with both purified NF-kappa B and the nuclear extract protein. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing the IL-6 promoter revealed very little activity of the promoter in U-937 monocytic cells and in HeLa cells before stimulation. However, stimulation of U-937 and HeLa cells by inducers of NF-kappa B led to a dramatic increase in CAT activity. Mutations in the NF-kappa B-binding site abolished inducibility of IL-6 promoter-cat constructs in U-937 cells by lipopolysaccharide, tumor necrosis factor alpha, the double-stranded RNA poly(IC), or phytohemagglutinin and in HeLa cells by tumor necrosis factor alpha and drastically reduced but did not completely eliminate inducibility in HeLa cells stimulated by double-stranded RNA poly(IC) or phorbol 12-myristate 13-acetate. These results suggest that NF-kappa B is an important mediator for activation of the IL-6 gene by a variety of IL-6 inducers in both U-937 and HeLa cells and that alternative inducible enhancer elements contribute in a cell-specific manner to IL-6 gene induction. Because NF-kappa B is involved in the control of a variety of genes activated upon inflammation, NF-kappa B may play a central role in the inflammatory response to infection and tissue injury.