Volume 124, Issue 5, Pages 1358-1368 (May 2003) Interleukin-11-induced heat shock protein 25 confers intestinal epithelial-specific cytoprotection from oxidant stress  Mark J Ropeleski, Jun Tang, Margaret M Walsh-Reitz, Mark W Musch, Eugene B Chang  Gastroenterology  Volume 124, Issue 5, Pages 1358-1368 (May 2003) DOI: 10.1016/S0016-5085(03)00282-8

Figure 1 IL-11 induces rapid phosphorylation and activation of STAT3 at Tyr705 in IEC-18, YAMC, NIH3T3, and MDCK-HR cells. Serum-deprived cells grown in 0.01% FBS for 12 hours were stimulated with IL-11 (100 ng/mL) for 10–120 minutes. Cells were washed and total cell lysates were prepared as previously outlined. Identical membranes were incubated separately with antibodies specific for (Tyr705) phospho-STAT3 and total STAT3. Rapid phosphorylation of STAT3 occurred within 10 minutes of IL-11 stimulation, indicating that all cells expressed functional IL-11Rα chain (C, 0 minutes unstimulated control; C120′, 120 minutes unstimulated control). The figure is representative of 3 separate experiments with cells from different passages. Gastroenterology 2003 124, 1358-1368DOI: (10.1016/S0016-5085(03)00282-8)

Figure 4 IL-11 induces intestinal epithelial-specific induction of immunodetectable hsp25 expression. (A ) IEC-18 and YAMC intestinal epithelial cells were exposed to IL-11 50 ng/mL for 24 hours while 3T3 cells were used as nonepithelial controls and MDCK cells were used as nonintestinal epithelial controls. HS IEC-18 cells were used as positive controls. Analysis by SDS PAGE and Western blotting revealed that only intestinal epithelial cells (IEC-18 and YAMC) responded to IL-11 by increasing hsp25 expression. No effect was seen in 3T3, MDCK cells, or RLE-6TN cells. +, IL-11 50ng/mL for 24 hours; −, unstimulated controls. (B) RLE-6TN pulmonary epithelial cells did not express hsp25 in response to IL-11 despite IL-11R functionality demonstrated by STAT3 phosphorylation. Gastroenterology 2003 124, 1358-1368DOI: (10.1016/S0016-5085(03)00282-8)

Figure 2 IL-11 induces immunodetectable hsp25 expression in a time-dependent manner. (A ) IEC-18 cells were exposed to IL-11 50ng/mL for 6–72 hours and 10μg of total cell lysates were analyzed by SDS-PAGE. A significant increase in hsp25 expression was observed by 6 hours. Values represent results from 3 independent experiments using cells from different passages. Heat-shocked (HS) IEC-18 cells (IEC-18 cells heat shocked at 42°C for 23 minutes followed by a 2-hour recovery) were used as positive controls and the constitutively expressed Hsc73, which does not respond to IL-11, was used for normalization of densitometric analysis. No effect of IL-11 on hsp72 expression was observed. (B) A more elaborate time course reveals that no induction of hsp25 by IL-11 is seen prior to 4 hours, unlike HS controls where significant induction appears within 2 hours (see Figure 2A representative of n =3). (C ) A significant increase in hsp25 expression was seen by 6 hours, and was maximal by 24 hours which was used as 100% reference for densitometric analysis. Data are expressed as the mean ± SEM where ∗P < 0.05 and ∗∗∗P < 0.001 with respect to unstimulated controls. Gastroenterology 2003 124, 1358-1368DOI: (10.1016/S0016-5085(03)00282-8)

Figure 3 IL-11 induces immunodetectable hsp25 in a dose-dependent manner. IEC-18 cells were exposed to various doses of IL-11 in ng/mL for 24 hours, total cell protein lysates were analyzed by SDS-PAGE and Western blotting. A dose-dependent increase in hsp25 expression was observed while no changes in inducible hsp72 expression were observed. Results were normalized to the constitutively expressed hsc73, which is not affected by IL-11 treatment. HS, IEC-18 cells heat shocked at 42°C for 23 minutes followed by a 2-hour recovery. The image shown here is representative of 3 separate experiments. Gastroenterology 2003 124, 1358-1368DOI: (10.1016/S0016-5085(03)00282-8)

Figure 5 Anti-sense to hsp25 eliminates basal, IL-11, and heat-induced hsp25 expression. IEC-18 clones transfected with the pCEP4 expression vector containing the full-length rat cDNA for hsp25 sub-cloned in the reverse direction were selected under hygromycin B for stable anti-sense expression. Clone A4 depicted here was used in further studies to determine hsp25 specific protective effects of IL-11 during oxidant stress. Empty pCEP4 vector controls are also depicted here, where no effect of transfection was observed on hsp25 expression. +, IL-11 50 ng/mL for 24 hours; −, no stimulation; HS, 42°C for 23 minutes followed by a 2-hour recovery. Gastroenterology 2003 124, 1358-1368DOI: (10.1016/S0016-5085(03)00282-8)

Figure 6 IL-11 protects intestinal epithelial viability from oxidant stress in 51Cr release assays. Empty vector IEC-18 transfectants and IEC-18 cells expressing stable anti-sense to hsp25 were labeled with 51Cr following a 24-hour exposure to IL-11 50 ng/mL as outlined previously. Cytotoxicity was assayed as a function of percent of 51Cr released after a 1-hour challenge with 0.3–0.6mmol/L of the oxidant monochloramine. Unstimulated empty vector IEC-18 transfectants were used as controls. Data are expressed as the mean ± SEM. ∗∗∗P <0.001. ns, no statistical significance. Gastroenterology 2003 124, 1358-1368DOI: (10.1016/S0016-5085(03)00282-8)

Figure 7 (A ) IL-11 fails to protect NIH-3T3 cells from oxidant stress in 51Cr release assays. The effects of IL-11 on 3T3 cell resistance to oxidative stress were examined in 51Cr release assays as described in the Materials and Methods section. Unlike assays with IEC-18 cells, 100ng/mL of IL-11 for 24 hours failed to confer any cytoprotection. Because of the relative resistance to oxidant injury compared with IEC-18 cells, the effects of IL-11 were also examined at 1 and 3 mmol/L doses of monochloramine. (B) IL-11 fails to protect IEC-18 cells at time points before the induction of hsp25. The ability of IL-11 100ng/mL to confer cytoprotection to IEC-18 cells in 51Cr release assays was examined after 2 hours of incubation, at time points that antedate the induction of hsp25. Compared with incubation with IL-11 for 24 hours, no significant reduction in 51Cr release was detected. Data are shown as mean and SEM of 3 experiments. ∗∗∗P <0.001 with respect to IEC-18 cells treated with IL-11 for 2 hours. Gastroenterology 2003 124, 1358-1368DOI: (10.1016/S0016-5085(03)00282-8)

Figure 8 (A ) IL-11 stimulation leads to the accumulation of hsp25 in Triton ×-100 insoluble fractions. IEC-18 cells were exposed to IL-11 and cell pellets were resuspended in 0.5% Triton ×-100 lysis buffer followed by centrifugation. Insoluble pellets were resuspended in SDS solubilization buffer and 20 μg of each sample were analyzed by SDS PAGE. Hsp25 detection was carried out as described previously. Blot is representative of 4 separate experiments. (B) Heat shock leads to a rapid shift of hsp25 to Triton ×-100 insoluble fractions. IEC-18 cells were heat shocked at 43°C for 30 minutes followed by a recovery period at 37°C. Triton ×-100 insoluble proteins (5μg/lane) were analyzed by SDS-PAGE and Western blotting. (C ) Heat shock induces hsp25 phosphorylation in IEC-18 cells. Cells were heat-shocked for 30 minutes at 43°C and immediately harvested for isoelectric focusing 2-dimensional gel electrophoresis. Samples were run in the first dimension in 4% polyacrylamide tube gels containing 5% ampholytes(pH 3–10) and 55% wt/vol high-grade urea. Samples were electrophoresed in the second dimension on 12.5% gels, transferred to polyvinylidene difluoride membranes, and immunoblotted to detect phosphorylated hsp25 isoforms. a, native form; b, monophosphorylated form; c, diphosphorylated form (n = 2). (D) IL-11 treatment of IEC-18 cells does not lead to hsp25 phosphorylation. IEC-18 cells were treated for 4 and 24 hours before harvesting. Two-dimensional isoelectric focusing gel electrophoresis was carried out as described in Figure 8C. No alteration in hsp25 phosphorylation was detected. a, native form, b, monophosphorylated form; c, diphosphorylated form. Blot is representative of 2 experiments. Gastroenterology 2003 124, 1358-1368DOI: (10.1016/S0016-5085(03)00282-8)