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Research Article

Glutamine Enhances Heat Shock Protein 70 Expression via Increased Hexosamine Biosynthetic Pathway Activity

¹University of Colorado, Denver ²University of Pittsburgh School of Medicine

Submitted 2 June 2009 ; revised 15 September 2009 ; accepted in final form 18 September 2009

Background and Aims: Glutamine (GLN) plays a key role in cellular protection following injury via enhancement of heat shock protein 70 (HSP70). The pathway by which GLN enhances HSP70 is unknown. GLN is a key substrate for the Hexosamine Biosynthetic Pathway (HBP), which has been shown to induce HSP70. We sought to explore the role of the HBP in GLN-mediated HSP70 expression. Methods: Both chemical inhibitors and siRNA knockdown of key HBP enzymes were used in Mouse Embryonic Fibroblast (MEF) cells to determine the effects of the HBP on HSP70 expression. The O-glycosylation, nuclear translocation, and transcriptional activation of Heat Shock Factor-1 (HSF-1) and Sp1 were evaluated using immunoprecipitation, western blotting, and luciferase assays. HSP70 expression levels were evaluated via ELISA and western blotting. Results: GLN augmented HBP activity prior to and post heat stress (HS). Chemical inhibition of HBP enzymes reduced GLN-mediated HSP70 expression. Specific siRNA targeting of the key HBP enzyme, UDP-GlcNAc: polypeptide-O-Beta-acetylglucosaminyltransferase (OGT), blocked GLN-mediated HSP70 expression and attenuated GLN-mediated cellular protection post-HS. Chemical and siRNA attenuation of the HBP blocked GLN-induced nuclear translocation of Sp1 and HSF-1, which are key to maximal HSP70 expression. Finally, immunoprecipitation revealed HSF-1 was O-glycosylated, and GLN enhanced this effect. Conclusions: These results suggest metabolism of GLN by the HBP enhances HSP70 expression. This effect appears to be mediated via O-glycosylation, nuclear translocation, and transcriptional activation of Sp1 and HSF-1. The HBP pathway appears to play a key role in GLN-mediated HSP70 expression.

O-GlcNAc; O-glycosylation; amino acid; heat shock protein; glutamine