Volume 129, Issue 1, Pages 170-184 (July 2005) Anti-apoptotic Effects of L-Glutamine—Mediated Transcriptional Modulation of the Heat Shock Protein 72 During Heat Shock  Mark J. Ropeleski, Jacob Riehm, Kathy A. Baer, Mark W. Musch, Eugene B. Chang  Gastroenterology  Volume 129, Issue 1, Pages 170-184 (July 2005) DOI: 10.1053/j.gastro.2005.05.017 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 L-glutamine specifically potentiates the induction of Hsp72 protein during heat shock. (A) Effect of various L-glutamine (L-gln) concentrations on immunodetectable Hsp72 expression. IEC-18 cells were exposed to various L-gln concentrations for 6 hours, heat shocked at 42°C for 23 minutes, followed by a 2-hour recovery. Compared with 0 mmol/L treated cells (lane 2), treatment with 2 mmol/L and 4 mmol/L L-gln (lanes 3 and 4) led to significant increases in Hsp72 expression. Samples from cells of a different passage are also depicted here (lanes 5–8). Nonheat-shocked negative controls are shown in lanes 1 and 5. (B) D-glutamine has no effect on heat-inducible Hsp72 accumulation. IEC-18 cells were exposed to varying concentrations of L-gln as in panel (A) or equimolar amounts of the D-isomer of gln. Nonheat-shock control C (lane 1); 5% fetal bovine serum control S (lane 2); cells treated with 0 mmol/L, 2 mmol/L, and 4 mmol/L L-gln (lanes 3–5) or 0, 2, or 4 mmol/L D-gln (lanes 6–8). No potentiation was observed in D-gln treated cells. (C) L-arginine and glycine fail to recapitulate the L-glutamine effect. HS, heat shock. Figure is representative of 3 separate experiments. Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 L-glutamine potentiates the induction of Hsp72 mRNA during heat shock. (A) L-glutamine affects heat-induced Hsp72 transcript induction. IEC-18 cells were exposed to various L-gln concentrations or 5% serum for 6 hours. After heat shock at 42°C for 23 minutes (lanes 1–4), cells recovered for 2 hours at 37°C prior to RNA isolation. Nonheat-shock controls are shown in lanes 5–8. Compared with 0 mmol/L L-gln-treated cells (lane 2), treatment with 2 and 4 mmol/L L-gln (lanes 3 and 4) led to increased 2.8- and 2.3-kb Hsp72 transcript abundance. Treatment with 5% serum (lane 1) led to an intermediate accumulation of Hsp72 transcript compared with the L-gln effect. No significant inducible transcript accumulation was seen in similarly treated nonheat-shocked cells (lanes 5–8). (B) L-glutamine has no effect on c-fos gene induction by 5% serum in IEC-18 cells. After a 6-hour incubation with and without L-gln, cells were exposed to 5% FBS for the indicated times. No effect was seen on the immediate early c-fos gene response. Membranes were stripped and probed for GAPDH. Figure is representative of 3 separate experiments. Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 L-glutamine potentiation of Hsp72 abundance. (A) L-glutamine potentiates the induction of Hsp72 protein during heat shock. The presence of 4 mmol/L L-gln led to a significant increase in heat-induced Hsp72 protein expression, which represented a 3.9-fold difference (P < .05 by Tukey-Kramer multiple comparisons test) (*P < .05) (n = 4). (B) L-glutamine potentiates the induction of Hsp72 mRNA during heat shock. Significant changes in Hsp72 mRNA abundance were observed in 2 mmol/L L-gln-treated cells and 4 mmol/L L-gln-treated cells postheat shock, which represented 3.04-fold (P < .05 by Bonferroni multiple comparisons test) and 6.6-fold (P < .01 by Bonferroni multiple comparisons test) differences, respectively, compared with 0 mmol/L L-gln-treated heat shocked cells. Serum did not have a significant potentiating effect during heat shock. (*P < .05; **P < .01) (n = 3). Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 L-glutamine potentiation of Hsp72 promoter-reporter activity. IEC-18 cells were transfected with the distal 1500 bp of the human Hsp72 promoter. The presence of 4 mmol/L L-glutamine (L4/72/hs) during heat shock led to significantly greater heat-inducible reporter activity, with a 2.67-fold increase (***P < .001 by Bonferroni multiple comparisons test) in Hsp72 promoter-driven heat-inducible luciferase activity compared with 0 mmol/L L-gln heat-shocked cells (L0/72/hs) (n = 4). The presence of 4 mmol/L D-gln (D4/72/hs) had no effect on Hsp72 promoter activity. Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 L-glutamine does not affect HSF-1 trimerization during heat shock. (A) Immunodetection of HSF-1 trimers by nonreducing native 5%–20% polyacrylamide gel electrophoresis. Regardless of the presence of gln, equal amounts of heat-inducible HSF-1 trimers could be demonstrated following heat shock (lanes 6–8). In the absence of heat shock, no detectable HSF-1 trimerization was detected (lanes 4 and 5). Native molecular weight markers (lanes 1–3: F, apo-ferritin [900-kilodalton dimer and 450-kilodalton monomer]; U, urease [575-kilodalton hexamer]; BSA, bovine serum albumin [66 kilodaltons]). (B) Coomasie blue staining of blot shown in A. Figure is representative of 4 separate experiments. Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Retarded complexes in electromobility shift assays represent HSF-1/HSE protein DNA complexes during heat shock. HS, heat shock control; 100Xcold, cold competition with unlabeled competitor; anti-HSF-1, preincubation with rabbit polyclonal anti-HSF-1; rabbit IgG, preincubation with rabbit preimmune serum; ns, nonspecific band. Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 L-glutamine potentiates HSF-1 DNA binding detected by EMSA in heat-shocked IEC-18 cells. (A) L-glutamine affects protein/DNA complex abundance in electromobility shift assays. After preincubation with various concentrations of L-gln, cells were heat shocked, followed by immediate total cell extract isolation. S, 5% fetal bovine serum; 0, 0 mmol/L L-gln; 2, 2 mmol/L L-gln; 4, 4 mmol/L L-gln; P, free probe without extract; HS, heat shock. Representative of 4 experiments. (B) Cumulative effects of L-glutamine effect on HSF-1 DNA binding. The presence of 4 mmol/L L-gln for 6 hours resulted in a 3.52-fold (P < .01 by Bonferroni multiple comparisons test) increase in HSF-1 DNA binding compared with 0 mmol/L L-gln heat-shocked cells, whereas 2 mmol/L L-gln led to a 2.46-fold increase (P < .05 by Bonferroni multiple comparisons test) in DNA binding during heat shock compared with 0 mmol/L treated cells. The incubation with 5% serum for 6 hours did not yield statistically significant results. (*P < .05; **P < .01) (n = 3). Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Degree and duration of binding of HSF-1 to HSE is L-gln specific. (A) Prolongation of detectable HSF-1 binding in L-gln-treated cells. After exposure to 4 mmol/L L-gln for 6 hours, cells were heat shocked and extracts prepared immediately (STAT) or at 5–10-minute intervals thereafter upon return to 37°C. Compared with 0 mmol/L treated cells, the presence of 4 mmol/L L-gln led to a greater degree of DNA binding as well as the prolongation of the duration of detectable HSF-1 binding up to 30 minutes after return to 37°C compared with untreated heat shocked cells. (B) D-gln fails to significantly potentiate HSF-1 DNA binding during heat shock. S, 5% fetal bovine serum; 0, 0 mmol/L D-gln; 2, 2 mmol/L D-gln; 4, 4 mmol/L D-gln. Figure representative of 3 separate experiments. Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 L-glutamine does not modulate the degree of heat shock-induced transcriptional activation of HSF-1. (A) No of effect of L-gln on inducible phosphorylation of HSF-1. IEC-18 cells were labeled with 32Pi for the duration of the 6-hour exposure to either 4 mmol/L L-gln, 4 mmol/L D-gln, or 0 mmol/L L-gln. Compared with nonheat shock controls, a distinctive heat-inducible band corresponding to immunoprecipitated activated HSF-1 could be detected, which was unaffected by L- or D-glutamine. Figure representative of 4 separate experiments. (B) No modulation effect of L- or D-gln on heat-induced HSF-1 transcriptional activity. No glutamine-dependent differences in HSF-1 minimal promoter activity were detected in transiently transfected IEC-18 cells. L0/HSE/hs, heat shock without glutamine; L4/HSE/hs, heat shock with 4 mmol/L L-gln; D4/HSE/hs, heat shock with 4 mmol/L D-gln. Compiled from 4 separate series of triplicate transfections. Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 10 Altered nuclear translocation of activated HSF-1 does not account for glutamine-dependent effects on Hsp72 gene induction. (A) Immunodetectable HSF-1 is not altered by heat shock (HS), by 5% serum (S), or by alterations in extracellular L-gln. (B) Nuclear localization of activated HSF-1 during heat shock is not affected by extracellular glutamine concentrations. In the absence of heat shock, equal amounts of HSF-1 are detected in cytoplasmic fractions, regardless of glutamine concentration (lanes 1 and 2). During heat shock, there is a disappearance of HSF-1 from the cytoplasmic compartment (lanes 3 and 4) paralleled by the appearance of HSF-1 in the nuclear fraction (lanes 7 and 8). HSF-1 is barely detectable in the cytoplasmic compartment postheat shock (lanes 5 and 6). Figure representative of 3 experiments. Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 11 L-glutamine potentiation of Hsp72 induction by L-glutamine is associated with decreased caspase 3 activation. Two hours postheat shock, cells were exposed to 20 μmol/L camptothecin. The glutamine effect on Hsp72 was associated with the delayed appearance and decreased abundance of (A) cleaved caspase-3 (17–19 kilodaltons) and (B) large and small cleaved fragments of PARP. Figure representative of 3 identical experiments. Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 12 L-gln increases heat induction of Hsp72 in human intestinal epithelial H4 cells and protects H4 cells from camptothecin-induced apoptosis. (A) Cells were transfected with human Hsp72 siRNA (lanes 5–8) or transfection reagent alone (lanes 1–4) for 36 hours prior to exposure to different L-gln concentrations (0 or 4 mmol/L L-gln in serum-free DMEM for 6 hours). When appropriate, cells were heat shocked (42°C, 23 minutes) and then returned to cell culture incubator to recover for 10 hours. Hsp72 siRNA was effective in attenuating basal and inducible Hsp72 compared with transfection reagent-only controls. Hsc73 expression was used to control for protein loading and demonstrates specificity of the siRNA effect (n = 3). (B) Under conditions described in A, 2 hours postheat shock, all cells were exposed to camptothecin 20 μmol/L for 8 hours. Western blotting for Hsp72 revealed that camptothecin treatment did not affect the pattern of Hsp72 induction shown in A and that siRNA was effective in attenuating Hsp72 expression. (C) DNA fragmentation was measured in lysates of cells for all 8 conditions shown in B, using the Cell Death Detection ELISA assay. Values shown are the differences in absorbance (at 450–490 nm) between cells treated with and without camptothecin and are means ± SE for 4 separate experiments. Images shown are representative of those of 4 separate experiments. (*P < .05; ***P < .01) (n = 4). Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 13 L-glutamine potentiates the effect of SEB toxin on H4 Hsp72 expression. H4 cells were pretreated with 0 or 4 mmol/L L-gln prior to exposure to SEB toxin 1 μg/mL for 6 hours. L-gln pretreatment resulted in a 40% increase in Hsp72 expression compared with 0 mmol/L-treated cells. (*P < .05 by Bonferroni multiple comparisons test). Gastroenterology 2005 129, 170-184DOI: (10.1053/j.gastro.2005.05.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions