Glutamine induces heat shock protein expression via O-glycosylation and phosphorylation of HSF-1 and Sp1.
BACKGROUND
Glutamine (GLN) improves outcome in experimental and clinical states of illness and injury. The authors hypothesized GLN-mediated enhancement of O-glycosylation and subsequent phosphorylation of key transcription factors in the HSP70 pathway would lead to increased HSP70 expression following experimental sepsis.


METHODS
Mice underwent cecal ligation and puncture (CLP)-induced sepsis and were treated with GLN (0.75 g/kg) or a saline placebo 30 minutes after CLP. A separate group of mice was treated with mithramycin, an Sp1 inhibitor. Lung tissue was harvested at 1, 2, 6, and 24 hours after CLP and was analyzed for HSF-1 and Sp1 O-GlcNAc modification, alpha-p-threonine modification, and HSP70.


RESULTS
GLN increased O-GlcNAc modification of HSF-1 and Sp1 at 1 and 2 hours after sepsis (P < .001 vs saline). Samples immunoprecipitated for Sp1 and probed for subsequent phosphorylation showed a significant increase in nuclear alpha-p-threonine-modified Sp1 at 2 and 6 hours after sepsis (P < .001 vs saline). GLN increased phosphorylated nuclear HSF-1 at 1 and 2 hours after CLP (P < .001). Finally, GLN treatment increased HSP70 4-fold (P < .01), but when treated with mithramycin, this increase was attenuated at 2, 6, and 24 hours (P < .001 vs no mithramycin treatment).


CONCLUSIONS
These results indicate that GLN induces HSF-1 and Sp1, which is known to lead to their nuclear translocation. The molecular mechanism of GLN-mediated HSP70 expression appears to be dependent on O-GlcNAc pathway activation and subsequent O-glycosylation and phosphorylation of key transcription factors required for HSP70 induction.
Singleton
Kristen D
KD
Department of Anesthesiology, University of Colorado Health Sciences Center, Denver, CO 80262, USA. Kristen.singleton@uchsc.edu
Wischmeyer
Paul E
PE
D000818:Animals; D002432:Cecum / Q000293:injuries; D005786:Gene Expression Regulation; D005973:Glutamine / Q000627:therapeutic use*; D006031:Glycosylation / Q000187:drug effects*; D018840:HSP70 Heat-Shock Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008026:Ligation; D008168:Lung / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D008926:Plicamycin / Q000627:therapeutic use; D011500:Protein Synthesis Inhibitors / Q000627:therapeutic use; D011897:Random Allocation; D018805:Sepsis / Q000188:drug therapy*
D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011500:Protein Synthesis Inhibitors; D005973:Glutamine; D008926:Plicamycin