title: Anti-apoptotic effects of L-glutamine-mediated transcriptional modulation of the heat shock protein 72 during heat shock.
abstract: OBJECTIVE
  During physiologic stress, L-glutamine becomes conditionally essential. Its deficiency results in altered epithelial barrier competence, bacterial translocation, and decreased survival. L-glutamine may attenuate these effects by modulating heat shock protein expression, a well-described effect in vitro. We sought to characterize L-glutamine-dependent transcriptional regulation in heat-shocked intestinal cells and to determine its physiologic relevance.
  
  
  METHODS
  IEC-18 and H4 intestinal cells were used. Heat shock protein 72 (Hsp72) gene expression was determined by Northern blotting and luciferase assays. Heat shock factor-1 (HSF-1) activation was assessed by electromobility shift assay, Western blotting, and HSF-1 minimal promoters. Phosphorylation and trimerization of HSF-1 were determined by immunoprecipitation and native nonreducing gradient polyacrylamide gel electrophoresis (PAGE). Camptothecin-induced apoptosis was monitored using caspase-3 and poly (ADP-ribose) polymerase [PARP]-specific antibodies and DNA Elisa +/- Hsp72 siRNA.
  
  
  RESULTS
  L-glutamine specifically augmented Hsp72 transcript abundance and HSF-1 DNA binding during heat shock. No glutamine-dependent differences in HSF-1 phosphorylation, trimerization, nuclear localization during heat shock, or HSF-1 minimal promoter activity were observed. Nevertheless, the presence of L-glutamine was an important determinant of wild-type Hsp72 promoter transcriptional activation. Reduced Hsp72 was associated with increased camptothecin-induced caspase-3 and PARP cleavage in glutamine-deficient cells. siRNA treated cells were less resistant to camptothecin.
  
  
  CONCLUSIONS
  Taken together, the data suggest that glutamine does not affect the classical pathway of HSF-1 activation and that glutamine-dependent upstream trans -factor binding elsewhere in the Hsp72 promoter or coactivator recruitment may determine Hsp72 abundance. L-glutamine potentiation of Hsp72 is associated with increased epithelial resistance to apoptotic injury.
authors: Gastrointestinal Diseases Research Unit, Department of Medicine, Queen's University, Kingston, Ontario, Canada.
authors: Mark J
authors: MJ
authors: Ropeleski
authors: Jacob
authors: J
authors: Riehm
authors: Kathy A
authors: KA
authors: Baer
authors: Mark W
authors: MW
authors: Musch
authors: Eugene B
authors: EB
authors: Chang
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D005973:Glutamine / Q000494:pharmacology*; D050884:HSP72 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000235:genetics*; D018869:Heat-Shock Response / Q000187:drug effects; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D008156:Luciferases / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000502:physiology; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D050884:HSP72 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors; D005973:Glutamine; D008156:Luciferases; D002166:Camptothecin