AJP - GI AJP: Cell Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 291: G178-G188, 2006. First published February 9, 2006; doi:10.1152/ajpgi.00304.2005
0193-1857/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/G178    most recent
00304.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parhar, K.
Right arrow Articles by Ropeleski, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parhar, K.
Right arrow Articles by Ropeleski, M. J.

TRANSLATIONAL PHYSIOLOGY

Short-chain fatty acid mediated phosphorylation of heat shock protein 25: effects on camptothecin-induced apoptosis

Kuljit Parhar,1 Kathy A. Baer,1 Kristy Parker,1 and Mark J. Ropeleski1,2

1Department of Medicine, Gastrointestinal Diseases Research Unit and 2Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada

Submitted 5 July 2005 ; accepted in final form 31 January 2006

ABSTRACT

Although short-chain fatty acid (SCFA)-induced heat shock protein 25 (Hsp25) is associated with increased cellular resistance to injury, withdrawal of lumenal butyrate in vivo is associated with intestinal epithelial injury and apoptosis. Recognizing that SCFA-dependent posttranslational modification of Hsp25 may involve altered Hsp25 phosphorylation, we hypothesized that butyrate regulates Hsp25 phosphorylation and secondarily affects cellular responses to apoptosis-inducing agents. Intestinal epithelial crypt IEC-18 cells were treated with butyrate, propionate, or the histone deacetylase inhibitor trichostatin A for 6–24 h. Immunolocalization of Hsp25 was examined by confocal laser microscopy. Hsp25 phosphorylation was characterized using two-dimensional isoelectric focusing gel electrophoresis. Hsp25 accumulation in cytoskeletal- and mitochondrial-enriched fractions was examined by immunoblotting. The activation of p38 MAP kinase was determined using phospho-specific antibodies and MAPKAPK 2 kinase assays. The effects of SCFA on apoptosis were studied by ELISA detection of cleaved DNA and using antibodies recognizing cleaved caspase-3. Five-millimolar butyrate induced no significant injury to IEC-18 cells. Hsp25 did not accumulate in Triton X-100-insoluble cytoskeletal fractions with butyrate treatment but did localize to mitochondria in a p38 MAP kinase-dependent manner. Hsp25 phosphorylation was induced by butyrate, propionate, and trichostatin A. Butyrate-mediated changes in Hsp25 phosphorylation coincide with the activation of the p38 MAP kinase and MAPKAPK 2. Butyrate, propionate, and low-dose trichostatin A confer significant protection from camptothecin-induced apoptosis, which was not reversed by the p38 inhibitor SB203580. We conclude that butyrate-mediated phosphorylation of Hsp25 is associated with significant resistance to apoptosis, which appears to be independent of p38-mediated targeting of Hsp25 to mitochondria.

MAP kinase; isoelectric focusing; kinase assay; caspase; histone deacetylase; DNA cleavage

SHORT-CHAIN FATTY ACIDS (SCFA) are short carbon chain fatty acid by-products of the bacterial fermentation and metabolism of undigested dietary fiber (9). Through the association of increased dietary fiber intake with a reduced colorectal cancer risk, a large body of literature has emerged describing specific effects of SCFA such as butyrate on colon cancer behavior in vitro and in vivo (4, 44, 59). In this setting, butyrate's effects include the induction of both cyclin-dependent kinase inhibitors and markers of differentiation with ensuing cell-cycle arrest and the upregulation of death receptor signaling pathways (7, 12, 21, 24). Emerging data also ascribe an important role to butyrate and its derivatives as adjuncts to conventional chemotherapy (8, 40, 52). Yet, butyrate also exerts important physiological effects in vivo in the normal physiological state that remain incompletely understood. Not only are butyrate and other SCFA important sources of energy for the colonocyte (54, 55), they also convey important trophic signals to the mucosa of the colon and distal small bowel (15, 16). In the absence of lumenal sources of SCFA, inflammation and ulceration of the diverted colon have been described. These changes have been reversed successfully with topical SCFA treatments (14, 19, 29). Furthermore, the absence of lumenal sources of butyrate leads to worsened mucosal damage in the dextran sulfate sodium (DSS) model of colitis (45, 46, 61). Putative effects of butyrate in this setting include butyrate-mediated antagonism of proinflammatory signaling as well as upregulation of anti-apoptotic survival signals at the level of the colonic and distal ileal epithelium in vivo. Proposed epithelial anti-apoptotic effects of butyrate in the nontransformed setting in vivo are supported by studies in which the absence of butyrate is associated with upregulated epithelial expression of Bax and significant epithelial apoptosis (23, 35, 36, 37, 42).

It has previously been reported that the bowel flora and dietary fiber precursors required for the synthesis of SCFA are important determinants of the basal expression of heat shock protein (Hsp25) in intestinal epithelial cells of the colon and distal small intestine in vivo in the rat (3, 30, 53). In vitro, SCFA have been shown to induce Hsp25 in a dose- and time-dependent manner that is transcriptionally regulated (58) and that protects rat intestinal epithelial crypt IEC-18 cells from injury during monochloramine oxidant challenge (53). Hsp25 has also been shown to exert other effects on cellular function that can increase cellular resistance to injury. These include chaperone-mediated protein stabilization (10, 56), the regulation of intracellular reactive oxygen species and glutathione (2, 49, 50), the antagonism of apoptosis (34), and the stabilization of the actin cytoskeleton (22, 27, 28). It is known that the phosphorylation state of Hsp25 is a primary determinant of its physiological function (31–33); yet, the posttranslational regulation of Hsp25 expression in butyrate-treated intestinal epithelial cells remains unexplored. We hypothesized that SCFA regulate Hsp25 phosphorylation, which might account for butyrate's effect on intestinal epithelial apoptosis in the normal distal ileum and colon. Using the nontransformed diploid IEC-18 crypt cell line, we exploited its minimal basal expression of Hsp25 and present novel findings which demonstrate that butyrate not only induces Hsp25 but also regulates its phosphorylation through both p38 MAP kinase-dependent and p38 MAP kinase-independent pathways. We demonstrate anti-apoptotic protective effects of butyrate, propionate, and trichostatin A in camptothecin-treated cells which do not appear to depend on p38 MAP kinase-mediated phosphorylation of Hsp25 nor the targeting of Hsp25 to the mitochondria.

MATERIALS AND METHODS

Reagents. All chemicals were of molecular biology grade and obtained from Fisher Scientific (Hanover Park, IL) unless otherwise stated. All cell culture reagents were obtained from Gibco (Grand Island, NY) unless otherwise stated. Fetal bovine serum was obtained from Sigma Aldrich (St. Louis, MO). pH 3–10 immobilized pH gradient (IPG) strips were obtained from Bio-Rad (Hercules, CA). The p38 MAP kinase (SB203580) and MEK1 (PD98059) inhibitors, the SCFA butyrate and propionate, the histone deacetylase inhibitor trichostatin A, DTT, iodoacetamide, anisomycin, cycloheximide and the topoisomerase 1 inhibitor camptothecin were all obtained from Sigma Aldrich. Human recombinant TNF-{alpha} was purchased from PeproTech (Rocky Hill, NJ).

Cell culture. The rat IEC-18 intestinal epithelial crypt cell line (CR-1589; ATCC, Manassas, VA) was grown as previously described (57). The cells were passed every 3–4 days with 0.05% trypsin and 0.53 mM EDTA. Experiments were performed 48 h postpassage when cells were approaching confluence. Cells were treated with butyrate (5–20 mM), propionate (5 mM), or the histone deacetylase inhibitor trichostatin A (0.1 or 0.03 µg/ml). For inhibitor studies, cells were pretreated with the p38 MAP kinase inhibitor SB203580 (10 µM in DMSO) or the MEK1 inhibitor PD98059 (50 µM in DMSO) for 2 h before treatment with SCFA.

Cell viability experiments. IEC-18 cells were plated and allowed to grow for 48 h as described before being exposed to butyrate at 5–20 mM for 24–72 h. At the indicated time-points, cells were examined by phase-contrast microscopy and photographed. For cell viability assays, floating cells and adherent cells were collected from control plates at 0 and 24 h and from plates treated with 5 mM butyrate for 24 h. Viability was determined by Trypan blue dye exclusion.

Confocal indirect immunofluorescence. Cells were grown on 18 x 18-mm glass coverslips in 6-well cell culture plates. Cells were treated with 5 mM butyrate for 24 h. Prior to fixation, cells were washed in K-PIPES buffer (pH 6.5; 80 mM K-PIPES, 5 mM EDTA, and 2 mM MgCl2). Using a pH shift method to preserve the three-dimensional structure of the cells, we carried out fixation in 3.75% formaldehyde K-PIPES buffer for 5 min at 37°C followed by 3.75% formaldehyde in NaBO4 buffer (pH 11.0, 100 mM NaBO4) for 10 min at room temperature. Following washes in PBS, cells were permeabilized in PBS-0.1% Triton X-100 for 5 min at room temperature. Coverslips were blocked in 1.5% goat serum in PBS for 30 min at room temperature and incubated overnight at 4°C with rabbit polyclonal antibody to Hsp25 (Stressgen Biotechnology, Victoria, BC, Canada) diluted 1:100 with PBS-1.5% (vol/vol) goat serum. Following washes in PBS, coverslips were incubated for 1 h at room temperature with a Cy3-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch, West Grove, PA) diluted 1:5,000 in PBS. After washing, coverslips were mounted on glass slides using 1,4-diazabicyclo[2.2.2]octane (DABCO, Sigma Aldrich) and sealed. Localization of Hsp25 was analyzed using an Olympus Fluoview 200 laser-scanning confocal microscope equipped with a 633-nm HeNe laser at x40 magnification. Images were compiled from a Z-series consisting of 8 µm total thickness.

Immunoblotting. After various treatments, cells were rinsed twice with ice-cold PBS, scraped on ice, pelleted, snap-frozen, and stored at –70°C until use. Cell pellets were resuspended in lysis buffer containing 10 mM Tris (pH 7.3), 5 mM MgCl2, 1x complete protease inhibitor cocktail (Roche Biochemicals, Indianapolis, IN), 50 U/ml DNase I (GE Healthcare, Baie d'Urfe, Quebec, Canada), and 5 µl/ml RNase cocktail (Ambion, Austin, TX). Aliquots were taken for protein determination using the BCA method (BCA protein assay reagent kit, Sigma Aldrich). Lysates were added to 0.5 vol/vol of 3x Laemmli stop solution and heated to 95°C for 10 min. SDS-PAGE and protein transfer was carried out as previously described (57). Phospho-specific antibodies to p38 MAP kinase (Thr180/Tyr182) (Cell Signaling Technologies, Beverly, MA) were used according to the manufacturer's instructions. Blots were stripped using 200 mM glycine-0.05% Tween-20 (pH 2.5) for 1 h at 60°C and reblotted with antibody to detect total p38 MAP kinase. Anti-beta-actin (Sigma Aldrich) was used at 1:5,000, and anti-cytochrome c (Cell Signaling Technologies) was used at 1:1,000. Anti-MAPKAPK 2 and anti Hsp25 were purchased from Stressgen and used at a dilution of 1:1,000 and 1:5,000, respectively. All primary antibodies were diluted in TBS-0.05% Tween-20 (TBS-T) with 5% wt/vol low-fat milk, and membranes were incubated overnight at 4°C on a shaker platform. After washing in TBS-T, blots were incubated with donkey anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary antibodies (Jackson Immunoresearch) at 1:10,000 in TBS-T. Blots were developed using the SuperSignal West Pico chemiluminescent system (Pierce Biotechnology, Rockford, IL) and detected using Kodak Biomax light film.

Isolation of mitochondrial fractions. Mitochondrial fractions were prepared using the Pierce mitochondrial isolation kit (Pierce Biotechnology) according to the manufacturer's instructions. Mitochondrial pellets were resuspended in 2% 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS) in TBS-T and diluted in 0.5 vol/vol of 3x Laemmli buffer and heated at 95°C 5 min prior to SDS-PAGE. Immunoblotting for cytochrome c and Hsp25 was then carried out as described above.

MAPKAPK 2 kinase assays. Cells were pretreated with inhibitors where indicated for 2 h before being stimulated with butyrate for 24 h. Cells were harvested in solubilization buffer (20 mM HEPES, pH 7.2, 100 mM NaCl, 5 mM EGTA, 2 mM EDTA, 20 mM NaF, 25 mM beta-glycerophosphate, and 1% Nonidet P-40; with freshly added 1x complete protease inhibitor cocktail, 1 mM sodium orthovanadate, 1 mM DTT, and 1 µg/ml microcystin), sonicated twice for 10 s, and centrifuged at 10,000 g for 15 min. Protein concentrations were determined using the Bradford assay (Bio-Rad). One milligram of protein lysate was precleared by incubating with 5 µl of nonspecific rabbit IgG on a rotator at 4°C for 1 h. Fifty microliters of Pansorbin cells (Calbiochem, La Jolla, CA) were then added and incubated on a rotator at 4°C for 1 h followed by a 2,000 g spin for 1 min. Supernatants were incubated overnight with 7 µg of anti-MAPKAPK 2 antibody on a rotator at 4°C. Immune complexes were collected using 50 µl of a 50% protein-G agarose slurry (Upstate Biotechnologies, Lake Placid, NY) for 1 h. The immune complexes were washed twice with solubilization buffer and twice with assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM beta-glycerophosphate, 20 mM MgCl2, 5 mM EGTA, 2 mM EDTA, 1 mM DTT, and 1 mM sodium orthovanadate). Kinase assays were carried out in assay dilution buffer in a final volume of 40 µl that included the immune complexes, 1 µg of recombinant heat shock protein 27 (Hsp27) (Stressgen) as the substrate, 5 µM PKI (a PKA inhibitor, 6–22 amide) purchased from Calbiochem, 20 µM RO318220 methanesulfonate salt (an inhibitor of G protein-coupled receptor kinase, PKC, MAPKAP1beta, p70 S6 kinase) purchased from Sigma Aldrich, and 0.5 µg of ATP (250 µM {gamma}-[32P]ATP, 1 µCi; GE Healthcare) for 20 min at 30°C. Samples were then boiled in 5x sample buffer and resolved by SDS-PAGE. Gels were subsequently stained using Coomassie blue, dried, and exposed to film overnight at –70°C.

Two-dimensional isoelectric focusing gel electrophoresis. Pilot studies were carried out according to the method of O'Farrell using individually poured isoelectric focusing tube gels as previously described (57). Subsequently, experiments were carried out using IPG strips. Cell lysates were obtained as described above and were diluted 1:1 in denaturing two-dimensional (2-D) electrophoresis rehydration buffer (8 M urea, 2% CHAPS, 0.1% bromophenol blue) and frozen at –70°C until use. Protein solutions containing 50 µg of protein in 0.5% Bio-Lyte (pH 3–10, Bio-Rad) and 100 mM DTT were applied to 7-cm Bio-Rad pH 3–10 IPG strips by passive rehydration. Proteins were focused for 10,000 V-h using a Bio-Rad Protean isoelectric cell. After focusing, the strips were incubated in equilibration buffer (0.375 M Tris pH 8.8, 6 M urea, 2% SDS, 20% glycerol) containing first 20 mg/ml DTT for 15 min and then 25 mg/ml iodoacetamide for 15 min. Strips were loaded onto 12.5% acrylamide gels, electrophoresed, and later transferred using 1x Towbin buffer (25 mM Tris, pH 8.8, 192 mM glycine) with 20% methanol to Immobilon-P membranes (Millipore, Bedford, MA). Membranes were then blocked at room temperature in TBS-T with 5% wt/vol nonfat milk for 1 h and then incubated with Hsp25 antibody (Stressgen Biotechnology) at 1:5,000 in TBS-T with 5% nonfat milk overnight at 4°C on a shaker platform. After washing in TBS-T, blots were incubated with donkey anti-rabbit IgG HRP-linked secondary antibodies (Jackson Immunoresearch) at 1:10,000 in TBS-T. Blots were developed using the Pierce SuperSignal West Pico substrate and detected using a Perkin-Elmer ProXpress chemiluminescent detection system or Kodak Biomax light film.

Apoptosis assays. Proapoptotic stress was induced in IEC-18 cells by the topoisomerase 1 inhibitor camptothecin at 20 µM and TNF-{alpha} (20 ng/ml)/cycloheximide (25 µg/ml) treatment (5). Apoptosis was detected by immunoblotting as described above using antibodies specific to procaspase-3 and cleaved caspase-3 (Aspartate 276) (Cell Signaling Technologies). Lysates from cytochrome c-stimulated Jurkat T cells were used as positive controls. In parallel, cells were lysed, and changes in cleaved genomic DNA were determined by ELISA using the cell death detection kit (Roche Biochemicals) according to the manufacturer's instructions.

Data analysis. Where indicated, one-way analysis of variance (ANOVA) was used to test the statistical significance of differences between groups using InStat software (GraphPad, San Diego, CA). Results are means ± SE of 3–4 experiments.

RESULTS

IEC-18 cells tolerate 5 mM butyrate in vitro without evidence of apoptosis. In previous studies, 5 mM butyrate conferred resistance to oxidant injury in the IEC-18 cell (53). Yet, butyrate is also known to demonstrate proapoptotic properties in certain cell types, many of which are transformed in nature. These variable effects may reflect differences between in vitro and in vivo models and may be determined by the cellular compartment of origin along the vertical axis of the crypt-villus unit in vivo and the transformed nature of certain cell types in vitro. Thus we examined the effect of various concentrations of butyrate over time in the IEC-18 cell so as to confirm that our time-point of interest and concentration (24 h) was not associated with signs of cell death. As shown in Fig. 1, 5 mM butyrate was well-tolerated at 24 h through to 72 h. Although the 10 mM butyrate concentration was well-tolerated at 24 h, by 48 h increased numbers of floating cells were observed. By 72 h, clear evidence of cell death was present in 10 mM treated cells. This effect was seen at 48 h in the 20 mM treated cells where there was clear evidence of increased numbers of floating cells. At 72 h, striking cell death occurred, which limited cell and protein recovery. This latter effect was also associated with a persistent pH change, suggesting that low pH was likely contributing to the butyrate toxicity and was reflective of an exceeded buffering capacity of the bicarbonate-buffered culture media. Other HEPES-containing buffer formulations were not examined here.


Figure 1
View larger version (65K):
[in this window]
[in a new window]
  		Fig. 1. Phase-contrast microscopy of butyrate-treated intestinal epithelial crypt IEC-18 cells indicates dose-dependent toxicity over time. Confluent cells were exposed to 5, 10, or 20 mM butyrate for 24 to 72 h. Phase-contrast images were then obtained to delineate degrees of toxicity in vitro. Five millimolar butyrate was well-tolerated compared with 10 and 20 mM concentrations. Magnification x40. Figure is representative of three separate experiments.

 
Subsequently, we confirmed the lack of toxicity of 5 mM butyrate using Trypan blue exclusion and cytometry. First, floating cells were collected, and cell counts revealed no increase in the number of floating cells with butyrate treatment (data not shown). Next, we compared cell viability between controls and butyrate-treated adherent cells. As shown in Fig. 2A, treatment with 5 mM butyrate for 24 h resulted in no significant decrease in cell viability compared with controls. Recognizing reports that butyrate has proapoptotic effects in various transformed cell lines in vitro, we proceeded to determine the effects of 5 mM butyrate on caspase-3 cleavage and cytochrome c release. As shown in Fig. 2B, treatment with 5 mM butyrate over 24–72 h failed to increase detectable cleaved caspase-3. Cytochrome c-treated Jurkat T cells were used as a positive control. In support of this data, when mitochondria were separated and cytoplasmic fractions were examined for cytochrome c release, no 5 mM butyrate-inducible increase in cytoplasmic cytochrome c was detected (Fig. 2C). Subsequently, we examined the conditions depicted in Fig. 1 for corroborating evidence of apoptosis. As shown in Fig. 2D, prolonged treatment with 10 and 20 mM butyrate resulted in the appearance of cleaved caspase-3. No significant increase in caspase-3 cleavage was detected in 5 mM treated cells compared with controls in three separate experiments. In parallel, we confirmed the induction of Hsp25 by butyrate treatment. Five millimolar butyrate resulted in a significant increase in Hsp25 expression at 24 h that did not increase over 72 h. Higher butyrate concentrations resulted in increased Hsp25 expression at the expense of greater toxicity to the epithelia, suggesting that the protective effects of maximal induction of Hsp25 may be overcome under conditions of severe cellular stress. Whether preconditioning with lower concentrations of butyrate might result in an Hsp25-dependent survival effect against more toxic concentrations similar to the heat stress/heat shock paradigm is unclear at this time, and further studies are needed to address this. In the absence of clear-cut toxicity to the IEC-18 cell, we used 5 mM butyrate induction of Hsp25 in IEC-18 cells to model the physiological effects of butyrate in vivo and to examine more closely the posttranslational regulation of Hsp25 by butyrate.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. Butyrate effects on cell viability and induction of apoptosis in IEC-18 cells. Cells were exposed to 5–20 mM butyrate for the indicated periods. A: %viability of adherent cells was assessed using Trypan blue exclusion. The induction of apoptosis was examined by immunoblot using antibodies specific to cleaved caspase-3 (B) and cytochrome c (C), as well as antibodies that recognize both pro and cleaved forms of caspase-3, heat shock protein 25 (Hsp25), and beta-actin (D). For determination of cytochrome c release during butyrate treatment, cytosolic and mitochondrial fractions were compared. In B, Cyt c + and – indicates cytochrome c-treated Jurkat T cell control lysates. Figure is representative of three separate experiments.

 
Butyrate targets Hsp25 to mitochondrial cellular fractions. We first examined the effect of butyrate on the cellular localization of Hsp25 using confocal microscopy. As shown in Fig. 3, treatment with butyrate led to a diffuse increase of Hsp25 expression in the cell. Yet, as indicated by the arrows, the stippled appearance of Hsp25 in the perinuclear and surrounding area suggested that mitochondria may be an intracellular address for Hsp25 in butyrate-treated IEC-18 cells. Negative-control experiments were carried out in the absence of primary antibody and revealed no nonspecific background signal (data not shown). Recognizing that Hsp25 demonstrates enrichment in the cytoskeletal compartment during cellular stress to regulate key elements such as actin (20) and microtubules (6, 25), we examined Hsp25 expression in Triton X-100-insoluble fractions in butyrate-treated cells over time. As shown in Fig. 4A, treatment with butyrate did not result in any significant accumulation in cytoskeletal fractions over 24 h. This contrasts with the control experiment shown in Fig. 4B, where rapid accumulation of Hsp25 in Triton X-100-insoluble fractions occurs during heat stress at 43°C. In light of the confocal microscopy findings and previous reports of the role of Hsp25 in the antagonism of intrinsic apoptotic pathway activation (43, 47, 51), we examined the effects of butyrate on Hsp25 accumulation in mitochondrial-enriched fractions. As shown in Fig. 5, A and B, butyrate treatment resulted in the accumulation of Hsp25 in mitochondrial fractions by 12 h. This accumulation at 24 h persisted through 72 h. Cytochrome c detection in mitochondrial samples was used as a loading control.


Figure 3
View larger version (40K):
[in this window]
[in a new window]
  		Fig. 3. Confocal immunofluorescent localization of Hsp25 in butyrate-treated IEC-18 cells. Cells were grown on coverslips and exposed to butyrate for 24 h. After processing as described in MATERIALS AND METHODS, Hsp25 expression indicated by Cy3 excitation demonstrates that compared with control (A), treatment with butyrate leads to the diffuse accumulation of butyrate within the cell (B). Focal areas of stippled Hsp25 expression (arrows) suggest that a proportion of Hsp25 may be targeted to organelles such as mitochondria. Magnification x40; n = 3; Z = 4 µm (A) 5 µm (B).

 

Figure 4
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 4. Treatment with butyrate does not lead to Hsp25 accumulation in cytoskeletal cell fractions. To determine whether butyrate treatment targets Hsp25 to the cytoskeletal enriched cellular fractions, cells were lysed in Triton X-100 lysis buffer as outlined in MATERIALS AND METHODS. Triton X-100-insoluble fractions were analyzed by SDS-PAGE and immunoblotting for Hsp25 expression. Butyrate treatment for 24 h (A) resulted in no appreciable accumulation of Hsp25 in insoluble fractions, whereas heat shock for 45 min at 43°C (B) resulted in the rapid accumulation of Hsp25 in Triton X-100-insoluble fractions. Coomassie blue stain in A indicates equal protein loading. Figure is representative of three separate experiments.

 

Figure 5
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 5. Short-chain fatty acids (SCFAs) target Hsp25 protein to the mitochondrial compartment. Treatment with 5 mM butyrate led to the time-dependent enrichment of Hsp25 in mitochondrial fractions (A). Mitochondrial cytochrome c blotting and Coomassie blue staining were used to demonstrate equal protein loading. The targeting of Hsp25 to mitochondria was sustained and increased over 72 h (B). Figure is representative of three separate experiments.

 
Butyrate activates p38 MAP kinase signaling in IEC-18 cells. We proceeded to determine whether treatment with butyrate results in the activation of p38 MAP kinase in the IEC-18 cell. As shown in Fig. 6A, minimal to no basal phosphorylation of p38 MAP kinase was observed. Butyrate treatment led to the phosphorylation of p38 which appeared at ~6 h and lasted through 12 h with an eventual return to baseline. Blots were then stripped and reblotted with antibody recognizing total p38 MAP kinase expression. Data from two separate passages are shown in Fig. 6A. Since MAPKAPK 2 is a well-described downstream target of p38 as well as a physiological regulator of Hsp25 phosphorylation, we next examined the effect of butyrate treatment on MAPKAPK 2 kinase activity using recombinant Hsp27 (rHsp27) as a substrate. MAPKAPK 2 was immunoprecipitated and combined with rHsp27 in kinase assay buffer in the presence of {gamma}-[32P]ATP with inhibitors to block nonspecific protein kinase activity. As shown in Fig. 6B, the small amount of detectable basal activity present in IEC-18 cells was markedly increased by 5 mM butyrate at 24 h. p38 MAP kinase dependence of butyrate-mediated MAPKAPK 2 activation was demonstrated using the p38 MAP kinase inhibitor SB203580 (10 µM). Negative-control experiments were performed using preimmune rabbit serum to carry out the immunoprecipitation. In parallel experiments, 20-µg aliquots of precleared lysate were separated by SDS-PAGE, transferred to membranes, and blotted with MAPKAPK 2 antibody. As shown in Fig. 6C, treatment with butyrate or SB203580 had no effect on the abundance of MAPKAPK 2 isoforms which are detectable at 65 kDa and 45–50 kDa, respectively. Concurrently we examined the effects of the related SCFA, propionate, and the histone deacetylase inhibitor trichostatin A on Hsp25 targeting to the mitochondria. As shown in Fig. 6D, control cells shown in lane 3 show a small amount of basal cytoplasmic expression of Hsp25 that was absent from the mitochondrial compartment. Treatment with 5 mM propionate or 0.1 µg/ml of trichostatin A resulted not only in an increase in Hsp25 in the cytoplasmic fraction but also in a greater proportional increase in mitochondrial accumulation at 24 h. Lane 4 is the 5 mM butyrate control. Recognizing that the p38 MAP kinase signaling pathway is a canonical mechanism of posttranslational regulation of Hsp25, we examined how pretreatment with the p38 inhibitor SB203580 (10 µM) affects butyrate-induced accumulation of Hsp25 in mitochondrial fractions. As shown in lane 5, SB203580 treatment alone for 24 h did not modulate the low basal Hsp25 expression in the cytoplasmic compartment and did not affect mitochondrial fractions. Furthermore, SB203580 treatment did not result in obvious cellular toxicity at 24 h (data not shown). Pretreatment with SB203580 for 2 h prior to butyrate treatment significantly attenuated the accumulation in mitochondrial fractions (lane 6) while having no significant effect on butyrate-induced cytoplasmic accumulation of Hsp25. Treatment with DMSO alone (lane 7) without butyrate had no effect on basal Hsp25 expression.


Figure 6
View larger version (32K):
[in this window]
[in a new window]
  		Fig. 6. Butyrate treatment leads to the phosphorylation of p38 MAP kinase and activation of MAPKAPK 2. A: time-dependent activation of p38 MAP kinase by 5 mM butyrate was assessed by Western blot using phospho-specific antibodies to Thr180/Tyr182 p38 MAP kinase. Blots were stripped and reprobed for total p38 expression. Data indicate findings in two different passages. Cells treated with anisomycin were used as a positive control. B: signaling downstream of p38 was assessed by MAPKAPK 2 kinase assay using recombinant Hsp27 as a substrate (Neg = lysis and immunoprecipitation using a preimmune rabbit serum control; SB = 10 µM SB203580 2 h prior to butyrate treatment). In parallel, butyrate had no effect on total MAPKAPK 2 expression by Western blot (C). Other SCFAs as well as the histone deacetylase inhibitor trichostatin A were examined (D) where their effects on Hsp25 expression were compared between cytoplasmic and mitochondrial fractions. Lane 1 (Pr) = 5 mM propionate x 24 h; lane 2 (TrA) = 0.1 µg/ml trichostatin A x 24 h; lane 3 (C) = unstimulated control; lane 4 = 5 mM butyrate (B) x 24 h; lane 5 (SB) = 10 µM SB203580 x 24 h; lane 6 (B/SB) = 5 mM butyrate + 10 µM SB203580 x 24 h; lane 7 (DMSO) = DMSO alone x 24 h. Figure is representative of three separate experiments.

 
SCFA induce and phosphorylate Hsp25 in IEC-18 cells. Given the demonstration of butyrate-dependent activation of p38 MAP kinase and MAPKAPK 2, 2-D isoelectric focusing gel electrophoresis was used to examine butyrate mediated changes in Hsp25 phosphorylation. Initial studies were carried out using the method of O'Farrell using individually poured denaturing gels containing ampholytes as previously described (57). As shown in Fig. 7A, treatment with butyrate led to the appearance of an acidic shift of Hsp25 expression with the appearance of monophosphorylated and diphosphorylated forms of Hsp25. We then went on to carry out the remainder of our studies using pH 3–10 IPG strips. As shown in Fig. 7B, the butyrate effect was recapitulated. A heat shock control also demonstrated the appropriate shift as previously demonstrated (57). We did observe greater basal expression of Hsp25 and the monophosphorylated form in control cells shown in Fig. 7A. This discrepancy likely reflects differences in film exposures times to chemiluminescent substrates, but more so that pilot studies shown in Fig. 7A were carried out using tube gels that could accommodate between 100 and 125 µg of protein lysate. Fifty micrograms of cell protein lysate was loaded per IPG strip.


Figure 7
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 7. Butyrate treatment leads to the accumulation of phosphorylated Hsp25 in IEC-18 cells. A: using the method of O'Farrell, lysates were run on prepoured tube gels across a pH 3–10 gradient as described in MATERIALS AND METHODS. Separation of proteins according to molecular weight followed by immunoblotting for Hsp25 expression led to the detection of Hsp25 expression in the 1) unphosphorylated, 2) monophosphorylated, and 3) diphosphorylated forms. B: responses were confirmed using the immobilized pH gradient strip approach. HS = heat shock. Figure is representative of three experiments using each technique.

 
We proceeded to determine whether the changes in Hsp25 phosphorylation induced by butyrate were p38 MAP kinase dependent. As shown in Fig. 8A, compared with butyrate treatment for 24 h, the pretreatment with 10 µM SB203580 resulted in the disappearance of the butyrate-inducible diphosphorylated form of Hsp25 (lane 3) with only the monophosphorylated form remaining (lane 2), suggesting that other butyrate-activated signaling pathways may also phosphorylate Hsp25. Recognizing that ERK-1/2 signaling has been proposed as an activator of Hsp25 phosphorylation in the heat shock condition (26) and that butyrate treatment leads to ERK-1/2 phosphorylation in IEC-18 cells (M. Ropeleski, unpublished observation), we determined the effect of the MEK 1 inhibitor PD98059 on butyrate-induced Hsp25 phosphorylation but found no effect (data not shown). In an attempt to determine whether the regulation of Hsp25 phosphorylation is a property characteristic of SCFA in general vs. their intrinsic capacity to inhibit histone deacetylases, we examined the effect of 5 mM propionate and 0.1 µg/ml of the histone deacetylase inhibitor trichostatin A on Hsp25 phosphorylation. As shown in Fig. 8B, both propionate and trichostatin A exert a pattern of Hsp25 phosphorylation comparable to the effect of butyrate.


Figure 8
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 8. SCFAs and histone deacetylase inhibitors induce phosphorylation of Hsp25. Two-dimensional (2-D) isoelectric focusing gel electrophoresis followed by separation of proteins according to molecular weight and immunoblotting detected Hsp25 expression (A) in the 1) unphosphorylated, 2) monophosphorylated, and 3) diphosphorylated forms as a result of butyrate treatment. B: the butyrate effect on Hsp25 phosphorylation is shared by the SCFA propionate (5 mM) and the histone deacetylase inhibitor trichostatin A (0.1 µg/ml). Figure is representative of three separate experiments.

 
Butyrate treatment antagonizes activation of intrinsic apoptotic pathway signaling. In an attempt to delineate the physiological implications of butyrate-mediated Hsp25 induction and phosphorylation, we considered previous reports on Hsp25 antagonism of apoptosis in other experimental systems (43, 47, 48, 51). We examined the effects of butyrate pretreatment for 24 h prior to induction of apoptosis. We first examined extrinsic death receptor-mediated apoptosis using TNF-{alpha} (20 ng/ml) and cycloheximide (25 µg/ml) as previously described (5). Butyrate pretreatment afforded no protective effect and actually led to worsened cell death as seen by phase-contrast microscopy (data not shown). We suspect that this may reflect an inhibitory effect of butyrate on NF{kappa}B activation. We then turned our attention from the extrinsic pathway to the intrinsic pathway and examined butyrate effects on camptothecin-induced apoptosis. After 8 h of exposure to camptothecin, butyrate-treated IEC-18 cells appeared to tolerate the insult with fewer floating cells compared with IEC-18 cells exposed to camptothecin only. Similar effects were seen with 5 mM propionate and 0.03 µg/ml of trichostatin A. As shown in Fig. 9, the pretreatment with butyrate resulted in a marked attenuation of caspase-3 cleavage at 8, 12, and 24 h postcamptothecin treatment.


Figure 9
View larger version (24K):
[in this window]
[in a new window]
  		Fig. 9. Pretreatment with 5 mM butyrate attenuates caspase-3 cleavage induced by camptothecin. Treatment of IEC-18 cells with 20 µM camptothecin results in the appearance of cleaved caspase-3 (17–19 kDa) by 4–8 h. All cells were pretreated with butyrate for 24 h prior to exposure to camptothecin for the indicated times (4–24 h). Cyt c = cytochrome c-treated Jurkat T cell lysate positive control. Figure is representative of three separate experiments.

 
We next proceeded to compare the effects of 5 mM propionate and 0.03 µg/ml trichostatin A pretreatment on camptothecin-induced caspase-3 cleavage. As shown in Fig. 10A, pretreatment with 5 mM propionate led to a significant attenuation of caspase-3 cleavage over 24 h. Similarly, in cells pretreated with trichostatin A at a dose of 0.03 µg/ml, a reduced dose to limit cellular toxicity, decreased caspase-3 cleavage is seen at 8 and 24 h. The effect of butyrate pretreatment on camptothecin-induced caspase-3 cleavage was supported further when apoptosis was determined by chromogenic ELISA detection of cleaved genomic oligonucleosomal DNA. As shown in Fig. 11A, butyrate fails to induce significant DNA cleavage in IEC-18 cells. Pretreatment with 5 mM butyrate for 24 h prior to camptothecin resulted in a 47.6% reduction in the abundance of cleaved DNA compared with the camptothecin alone group (mean absorbance camptothecin 8 h, 4.43 ± 1.143 vs. butyrate 24 h/camptothecin 8 h, 2.32 ± 0.57; P < 0.05 by Student-Newman-Keuls; n = 3). In a separate experiment, we determined the effect of propionate treatment on camptothecin-mediated apoptosis. As shown in Fig. 11B, propionate offered an even greater protection from apoptosis, resulting in a 63.2% decrease (mean absorbance camptothecin 8 h, 4.43 ± 1.143 vs. propionate 24 h/camptothecin 8 h, 1.62 ± 0.43; P < 0.01 by Student-Newman-Keuls; n = 3). In an attempt to characterize whether p38-mediated phosphorylation of Hsp25 was an important mediator of this physiological effect, we pretreated IEC-18 cells with 10 µM SB203580 for 2 h prior to the butyrate treatment. Treatment with SB203580 did not counteract the protective effect of butyrate, suggesting that an increase in diphosphorylated Hsp25 does not mediate the protective effects of butyrate in camptothecin-treated cells (data not shown). As shown in Fig. 11C when butyrate, propionate, and trichostatin A were compared in parallel, we found that the greatest protection (a 78.3% decrease) against camptothecin-induced DNA cleavage was conferred by propionate (mean absorbance camptothecin 8 h, 13.72 ± 2.77 vs. propionate 24 h/camptothecin 8 h, 2.98 ± 0.2; P < 0.01 by Student-Newman-Keuls; n = 3) compared with 0.03 µg/ml trichostatin A (–71%) (mean absorbance camptothecin 8 h, 13.72 ± 2.77 vs. 0.03 µg/ml trichostatin A/camptothecin 8 h, 4 ± 1.15; P < 0.01 by Student-Newman-Keuls; n = 3), which was slightly less effective. Butyrate was least protective with a 47% decrease in camptothecin-induced DNA cleavage (mean absorbance camptothecin 8 h, 13.72 ± 2.77 vs. butyrate 24 h/camptothecin 8 h, 7.33 ± 0.90; P < 0.05 by Student-Newman-Keuls; n = 3). Interestingly, our data demonstrate that IEC-18 cells are quite sensitive to the dose of trichostatin A, as 0.1 µg/ml induced a significant degree of DNA cleavage, whereas the dose of 0.03 µg/ml did not [mean absorbance 0.1 µg/ml trichostatin A, 7.96 ± 2.18 vs. control (P < 0.05 by Student-Newman-Keuls; n = 3) compared with 0.03 µg/ml trichostatin A 1.63 ± 0.6 vs. control (not significant by Student-Newman-Keuls)]. The effects of 0.1 µg/ml trichostatin A and camptothecin on DNA cleavage did not synergize when the two agents were combined.


Figure 10
View larger version (30K):
[in this window]
[in a new window]
  		Fig. 10. Pretreatment with 5 mM propionate or 0.03 µg/ml trichostatin A (TSA) attenuates caspase-3 cleavage induced by camptothecin. Pretreatment with 5 mM propionate (A) or 0.03 µg/ml trichostatin A (B) leads to a decrease in caspase-3 cleavage that persists through 24 h of camptothecin treatment. All cells were pretreated for 24 h prior to exposure to camptothecin for the indicated times (4–24 h) with subsequent cell lysis and immunoblotting for total and cleaved caspase-3 as described in MATERIALS AND METHODS. After stripping, membranes were reblotted for beta-actin expression as a loading control. Figure is representative of three separate experiments.

 

Figure 11
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 11. Pretreatment with SCFAs or histone deacetylase inhibitors attenuates DNA cleavage induced by camptothecin. A and B: cells were pretreated with 5 mM butyrate (A) or propionate (B) for 24 h prior to exposure to 20 µM camptothecin for 8 h. DNA cleavage was determined using the cell death ELISA per the manufacturer's instructions. A small amount of basal cell death occurs in control cells, and this was assigned the relative value of 1. *P < 0.05 and **P < 0.01 compared with camptothecin treatment alone. C: butyrate, propionate, and two doses of trichostatin A were compared in parallel. Propionate appears most effective (**P < 0.05 vs. camptothecin alone) with less protection from 0.03 µg/ml trichostatin A (**P < 0.01 vs. camptothecin alone), whereas butyrate is the least protective (*P < 0.05 vs. camptothecin alone). Trichostatin A at 0.1 µg/ml induces significant toxicity compared with control cells (#P < 0.05 vs. control), whereas 0.03 µg/ml exerts no significant toxicity (ns). The greatest toxicity is induced by camptothecin alone (###P < 0.001 vs. untreated control). Figure is representative of at least three separate experiments performed in triplicate.

 
DISCUSSION

A substantial body of literature supports a proapoptotic function of butyrate and dietary precursors of SCFA in various models of colon cancer both in vivo and in transformed epithelial cell lines cultured in vitro. Yet, the physiological functions of butyrate in vivo remain controversial in that, although SCFA may exert anticancer effects, the withdrawal of luminal colonic nutrient sources of SCFA also results in apoptosis of the normal epithelial lining and inflammation. Animal studies confirm increased epithelial apoptosis in vivo in fiber-deficient guinea pigs (23, 35, 39). In inflammation, butyrate also confers protective effects to the epithelium in the DSS model of colonic inflammation with resultant decreases in characteristic early epithelial damage and apoptotic fallout of crypts (61). The disparity in findings between transformed and nontransformed cellular responses to butyrate forms the basis of the "butyrate paradox" reviewed elsewhere (39).

Previous studies have demonstrated that dietary fiber sources of SCFA are important determinants of basal expression of Hsp25 in the colonocyte in vivo (53). In the IEC-18 cell in vitro, SCFA increase Hsp25 expression that confers Hsp25-dependent resistance to oxidant injury (53). The IEC-18 cell, which is derived from normal diploid nontransformed epithelial crypts in the rat, has been a useful vehicle to study the regulation of Hsp25 by butyrate, given that IEC-18 cells are not a cancer-derived cell line and express minimal basal Hsp25 compared with other transformed cell lines. As well, butyrate is characteristically proapoptotic in transformed epithelial cell lines, which makes the IEC-18 cell a rational model to study both SCFA-mediated regulation of Hsp25 and its physiological effects. The physiological role of the epithelial-specific expression of Hsp25 in the colonocyte and distal ileal enterocyte in vivo remains unclear. To date, intestinal and colonic epithelial cell-specific transgenic Hsp25 knockout mice, as well as nonphosphorylatable Hsp25 transgenic mutants, are unavailable for study. Given the putative anti-apoptotic effects of butyrate in vivo in the normal physiological setting, as well as the reported anti-apoptotic effects of Hsp25 overexpression, it is tempting to speculate that butyrate conveys part of its anti-apoptotic effect through the regulation of Hsp25. A body of literature identifies that the regulation of Hsp25 involves both transcriptional and posttranslational mechanisms. Butyrate-induced transcription of the Hsp25 gene has been noted in the IEC-18 cell (58) and in other cell types (18, 60), yet little is known about how butyrate regulates Hsp25 at the posttranslational level. We thus hypothesized that posttranslational regulation of Hsp25 by butyrate may determine the physiological effects of butyrate in models of epithelial apoptosis.

We have shown in Figs. 1 and 2 that IEC-18 cells tolerate treatment with 5 mM butyrate for up to 72 h without significant evidence of toxicity. Furthermore, we demonstrate that treatment with 5 mM butyrate for 24–72 h fails to induce significant caspase-3 cleavage or a significant increase in cleaved genomic DNA (Figs. 2 and 11). In our attempt to address the physiological implications of the butyrate effect on Hsp25, we uncovered that, unlike other stimuli such as heat stress, butyrate does not target Hsp25 to the cytoskeleton (Fig. 4) but rather to the mitochondrial compartment (Figs. 3 and 5) in a p38 MAP kinase-dependent manner (Fig. 6). This targeting effect was mimicked by propionate and trichostatin A and inhibited by the p38 MAP kinase inhibitor SB203580. We have demonstrated that butyrate and propionate, as well as the histone deacetylase inhibitor trichostatin A, induce Hsp25 phosphorylation. The appearance of phospho-Hsp25 coincides with butyrate-induced activation of p38 MAP kinase phosphorylation and activation of the downstream canonical pathway of MAPKAPK 2 activation as shown in the kinase assay in Fig. 6. In view of this mitochondrial targeting effect by butyrate, which appeared largely p38 MAP kinase dependent, we tested the hypothesis that p38-dependent phosphorylation of Hsp25 may serve as an important regulator of proapoptotic and anti-apoptotic balance in the intestinal epithelial cell. Since it was unclear whether Hsp25 phosphorylation and its accumulation in mitochondrial extracts was associated with the antagonism of the intrinsic or extrinsic apoptotic pathway, both were examined. As butyrate has been implicated as a protective factor in the DSS model of colitis, which is characterized by high levels of mucosal TNF-{alpha} (61), we examined extrinsic pathway activation by exploring the effects of butyrate pretreatment on TNF-{alpha}/cycloheximide-induced apoptosis. Butyrate pretreatment led to visibly more cell death, as observed by phase-contrast microscopy, compared with cells treated with TNF-{alpha}/cycloheximide alone. While this is intriguing and is the subject of ongoing studies, we speculate that this increase in cell death may reflect additional inhibitory effects of butyrate on NF{kappa}B-mediated survival signaling (1, 38, 62). We subsequently focused on the intrinsic apoptotic pathway using the topoisomerase 1 inhibitor camptothecin to trigger apoptosis. Pretreatment of IEC-18 cells with butyrate, propionate, and low-dose trichostatin A for 24 h led to a significant reduction in the amount of cleaved caspase-3 formation during camptothecin treatments for 4–24 h (Figs. 9 and 10). This effect was also associated with a significant decrease in camptothecin-mediated genomic DNA cleavage in butyrate-, propionate-, and low-dose trichostatin A-treated cells (Fig. 11). Although treatment with SB203580 resulted in a significant decrease in mitochondrial targeting of Hsp25 by butyrate, no effect of SB203580 pretreatment was observed on the protective effect of butyrate. This suggests that some of the protection afforded by butyrate in our model may be derived from the induction of Hsp25 in its unphosphorylated form that may be confined to the cytoplasmic compartment or from the butyrate-induced monophosphorylation that appears independent of p38 MAP kinase signaling. Although ERK pathway activation has been associated with Hsp25 phosphorylation (17, 26), the MEK1 inhibitor PD98059 had no effect on the butyrate-induced phosphorylation pattern observed on 2-D gels. Recent data have identified serine-82 of human Hsp27 as a target for PKD (13). Activation of the PKD pathway remains a physiologically plausible mechanism of p38-independent Hsp25 monophosphorylation induced by butyrate and is the subject of ongoing studies. Other putative pathways include the Akt/protein kinase B signaling pathway. Although our current data do not yet identify the extent that the abilities of butyrate, propionate, and trichostatin A to antagonize camptothecin-induced apoptosis depend on Hsp25 or phospho-Hsp25, the stage for a logical series of follow-up studies has been set. Furthermore, we must acknowledge that non-Hsp25-related protective effects of butyrate likely exist, as well and that butyrate-induced Hsp25-mediated resistance to apoptosis is likely not specific to camptothecin.

We have uncovered a novel pathway of Hsp25 regulation, whose physiological function remains incompletely understood. We speculate that the presence of high lumenal concentrations of butyrate and other SCFAs with a capacity to inhibit histone deacetylases in vivo contribute important anti-apoptotic cues that are integrated at the level of the epithelial cell in the colon and distal ileum, thus contributing to mucosal homeostasis. Having demonstrated the shared effects of butyrate, propionate, and trichostatin A on Hsp25 phosphorylation, we propose that alterations in the genomic histone code (41), characterized by altered patterns of histone acetylation as well as other posttranslational modifications, may serve as a signal resulting in the delayed (6–12 h) activation in p38 MAP kinase as shown in our studies. Alternatively, the activation of p38 and MAPKAPK 2 demonstrated here may reflect early changes in cell-cycle regulation that may or may not depend on Hsp25. Further studies are required to determine whether alterations in the histone code can lead to predictable patterns of activation of various signaling pathways in the intestinal epithelial cell.

GRANTS

K. Parker was the recipient of the 2004 Donna Lee Zamperion (nee Stahls) summer studentship from the Crohn's and Colitis Foundation of Canada. M. J. Ropeleski is funded through a Grant in Aid of Research from the Crohn's and Colitis Foundation of Canada and a Research Award from the Queen's University Department of Medicine.

FOOTNOTES


Address for reprint requests and other correspondence: M. J. Ropeleski, Queen's Gastrointestinal Diseases Research Unit, Hotel Dieu Hospital, 166 Brock St. Rm. C-477A, Kingston, Ontario, Canada K7L 5G2 (e-mail: ropelesm{at}hdh.kari.net)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

  1. Adam E, Quivy V, Bex F, Chariot A, Collette Y, Vanhulle C, Schoonbroodt S, Goffin V, Nguyen TL, Gloire G, Carrard G, Friguet B, De Launoit Y, Burny A, Bours V, Piette J, and Van Lint C. Potentiation of tumor necrosis factor-induced NF-{kappa}B activation by deacetylase inhibitors is associated with a delayed cytoplasmic reappearance of I{kappa}B{alpha}. Mol Cell Biol 23: 6200–6209, 2003.[Abstract/Free Full Text]
  2. Arrigo AP, Virot S, Chaufour S, Firdaus W, Kretz-Remy C, and Diaz-Latoud C. Hsp27 consolidates intracellular redox homeostasis by upholding glutathione in its reduced form and by decreasing iron intracellular levels. Antioxid Redox Signal 7: 414–422, 2005.[CrossRef][ISI][Medline]
  3. Arvans DL, Vavricka SR, Ren H, Musch MW, Kang L, Rocha FG, Lucioni A, Turner JR, Alverdy J, and Chang EB. Luminal bacterial flora determines physiological expression of intestinal epithelial cytoprotective heat shock proteins 25 and 72. Am J Physiol Gastrointest Liver Physiol 288: G696–G704, 2005.[Abstract/Free Full Text]
  4. Augenlicht LH, Mariadason JM, Wilson A, Arango D, Yang W, Heerdt BG, and Velcich A. Short chain fatty acids and colon cancer. J Nutr 132: 3804S-3808S, 2002.[Abstract/Free Full Text]
  5. Bhattacharya S, Ray RM, Viar MJ, and Johnson LR. Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-{alpha} in IEC-6 cells. Am J Physiol Gastrointest Liver Physiol 285: G980–G991, 2003.[Abstract/Free Full Text]
  6. Bluhm WF, Martin JL, Mestril R, and Dillmann WH. Specific heat shock proteins protect microtubules during simulated ischemia in cardiac myocytes. Am J Physiol Heart Circ Physiol 275: H2243–H2249, 1998.[Abstract/Free Full Text]
  7. Bonnotte B, Favre N, Reveneau S, Micheau O, Droin N, Garrido C, Fontana A, Chauffert B, Solary E, and Martin F. Cancer cell sensitization to fas-mediated apoptosis by sodium butyrate. Cell Death Differ 5: 480–487, 1998.[CrossRef][ISI][Medline]
  8. Chobanian NH, Greenberg VL, Gass JM, Desimone CP, Van Nagell JR, and Zimmer SG. Histone deacetylase inhibitors enhance paclitaxel-induced cell death in ovarian cancer cell lines independent of p53 status. Anticancer Res 24: 539–545, 2004.[Medline]
  9. Cook SI and Sellin JH. Review article: short chain fatty acids in health and disease. Aliment Pharmacol Ther 12: 499–507, 1998.[CrossRef][ISI][Medline]
  10. Cuesta R, Laroia G, and Schneider RJ. Chaperone hsp27 inhibits translation during heat shock by binding eIF4G and facilitating dissociation of cap-initiation complexes. Genes Dev 14: 1460–1470, 2000.[Abstract/Free Full Text]
  11. Dalle-Donne I, Rossi R, Milzani A, Simplicio P, and Colombo R. The actin cytoskeleton response to oxidants: from small heat shock protein phosphorylation to changes in the redox state of actin itself. Free Radic Biol Med 31: 1624–1632, 2001.[CrossRef][ISI][Medline]
  12. Domon-Dell C, Wang Q, Kim S, Kedinger M, Evers BM, and Freund JN. Stimulation of the intestinal Cdx2 homeobox gene by butyrate in colon cancer cells. Gut 50: 525–529, 2002.[Abstract/Free Full Text]
  13. Doppler H, Storz P, Li J, Comb MJ, and Toker A. A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D. J Biol Chem 280: 15013–15019, 2005.[Abstract/Free Full Text]
  14. Eggenberger JC and Farid A. Diversion colitis. Curr Treat Options Gastroenterol 4: 255–259, 2001.[Medline]
  15. Frankel WL, Zhang W, Singh A, Klurfeld DM, Don S, Sakata T, Modlin I, and Rombeau JL. Mediation of the trophic effects of short-chain fatty acids on the rat jejunum and colon. Gastroenterology 106: 375–380, 1994.[ISI][Medline]
  16. Friedel D and Levine GM. Effect of short-chain fatty acids on colonic function and structure. JPEN J Parenter Enteral Nutr 16: 1–4, 1992.[Abstract]
  17. Gaitanaki C, Konstantina S, Chrysa S, and Beis I. Oxidative stress stimulates multiple MAPK signalling pathways and phosphorylation of the small HSP27 in the perfused amphibian heart. J Exp Biol 206: 2759–2769, 2003.[Abstract/Free Full Text]
  18. Garcia-Bermejo L, Vilaboa NE, Perez C, de BE, Calle C, and Aller P. Modulation of HSP70 and HSP27 gene expression by the differentiation inducer sodium butyrate in U-937 human promonocytic leukemia cells. Leuk Res 19: 713–718, 1995.[CrossRef][ISI][Medline]
  19. Geraghty JM and Talbot IC. Diversion colitis: histological features in the colon and rectum after defunctioning colostomy. Gut 32: 1020–1023, 1991.[Abstract/Free Full Text]
  20. Geum D, Son GH, and Kim K. Phosphorylation-dependent cellular localization and thermoprotective role of heat shock protein 25 in hippocampal progenitor cells. J Biol Chem 277: 19913–19921, 2002.[Abstract/Free Full Text]
  21. Giardina C, Boulares H, and Inan MS. NSAIDs and butyrate sensitize a human colorectal cancer cell line to TNF-{alpha} and Fas ligation: the role of reactive oxygen species. Biochim Biophys Acta 1448: 425–438, 1999.[Medline]
  22. Guay J, Lambert H, Gingras-Breton G, Lavoie JN, Huot J, and Landry J. Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27. J Cell Sci 110: 357–368, 1997.[Abstract]
  23. Hass R, Busche R, Luciano L, Reale E, and Engelhardt WV. Lack of butyrate is associated with induction of Bax and subsequent apoptosis in the proximal colon of guinea pig. Gastroenterology 112: 875–881, 1997.[CrossRef][ISI][Medline]
  24. Heerdt BG, Houston MA, and Augenlicht LH. Short-chain fatty acid-initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to mitochondrial function. Cell Growth Differ 8: 523–532, 1997.[Abstract]
  25. Hino M, Kurogi K, Okubo MA, Murata-Hori M, and Hosoya H. Small heat shock protein 27 (HSP27) associates with tubulin/microtubules in HeLa cells. Biochem Biophys Res Commun 271: 164–169, 2000.[CrossRef][ISI][Medline]
  26. Hirano S, Rees RS, and Gilmont RR. MAP kinase pathways involving hsp27 regulate fibroblast-mediated wound contraction. J Surg Res 102: 77–84, 2002.[CrossRef][Medline]
  27. Huot J, Houle F, Marceau F, and Landry J. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 80: 383–392, 1997.[Abstract/Free Full Text]
  28. Huot J, Houle F, Spitz DR, and Landry J. HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res 56: 273–279, 1996.[Abstract/Free Full Text]
  29. Kiely EM, Ajayi NA, Wheeler RA, and Malone M. Diversion procto-colitis: response to treatment with short-chain fatty acids. J Pediatr Surg 36: 1514–1517, 2001.[Medline]
  30. Kojima K, Musch M, Ren H, Boone D, Hendrickson B, Ma A, and Chang E. Enteric flora and lymphocyte-derived cytokines determine expression of heat shock proteins in mouse colonic epithelial cells. Gastroenterology 124: 1395–1407, 2003.[CrossRef][ISI][Medline]
  31. Lambert H, Charette SJ, Bernier AF, Guimond A, and Landry J. HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus. J Biol Chem 274: 9378–9385, 1999.[Abstract/Free Full Text]
  32. Landry J and Huot J. Regulation of actin dynamics by stress-activated protein kinase 2 (SAPK2)-dependent phosphorylation of heat-shock protein of 27 kDa (Hsp27). Biochem Soc Symp 64: 79–89, 1999.[Medline]
  33. Lavoie JN, Lambert H, Hickey E, Weber LA, and Landry J. Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27. Mol Cell Biol 15: 505–516, 1995.[Abstract]
  34. Li Z, Zhao X, and Wei Y. Regulation of apoptotic signal transduction pathways by the heat shock proteins. Sci China C Life Sci 47: 107–114, 2004.[Medline]
  35. Luciano L, Busche R, Von EW, and Reale E. Apoptosis induction in vivo and fate of apoptotic material in the colon of the guinea pig. Ital J Anat Embryol 106: 347–352, 2001.[Medline]
  36. Luciano L, Groos S, Busche R, Von Engelhardt W, and Reale E. Massive apoptosis of colonocytes induced by butyrate deprivation overloads resident macrophages and promotes the recruitment of circulating monocytes. Cell Tissue Res 309: 393–407, 2002.[Medline]
  37. Luciano L, Hass R, Busche R, Von Engelhardt W, and Reale E. Withdrawal of butyrate from the colonic mucosa triggers "mass apoptosis" primarily in the G0/G1 phase of the cell cycle. Cell Tissue Res 286: 81–92, 1996.[CrossRef][ISI][Medline]
  38. Luhrs H, Kudlich T, Neumann M, Schauber J, Melcher R, Gostner A, Scheppach W, and Menzel TP. Butyrate-enhanced TNF{alpha}-induced apoptosis is associated with inhibition of NF-{kappa}B. Anticancer Res 22: 1561–1568, 2002.[Medline]
  39. Lupton JR. Microbial degradation products influence colon cancer risk: the butyrate controversy. J Nutr 134: 479–482, 2004.[Abstract/Free Full Text]
  40. Maggio SC, Rosato RR, Kramer LB, Dai Y, Rahmani M, Paik DS, Czarnik AC, Payne SG, Spiegel S, and Grant S. The histone deacetylase inhibitor MS-275 interacts synergistically with fludarabine to induce apoptosis in human leukemia cells. Cancer Res 64: 2590–2600, 2004.[Abstract/Free Full Text]
  41. Margueron R, Trojer P, and Reinberg D. The key to development: interpreting the histone code? Curr Opin Genet Dev 15: 163–176, 2005.[CrossRef][ISI][Medline]
  42. Mentschel J and Claus R. Increased butyrate formation in the pig colon by feeding raw potato starch leads to a reduction of colonocyte apoptosis and a shift to the stem cell compartment. Metabolism 52: 1400–1405, 2003.[CrossRef][ISI][Medline]
  43. Merendino AM, Paul C, Vignola AM, Costa MA, Melis M, Chiappara G, Izzo V, Bousquet J, and Arrigo AP. Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress-mediated apoptosis: possible implication in asthma. Cell Stress Chaperones 7: 269–280, 2002.[CrossRef][Medline]
  44. Miller SJ. Cellular and physiological effects of short-chain fatty acids. Mini Rev Med Chem 4: 839–845, 2004.[ISI][Medline]
  45. Moreau NM, Martin LJ, Toquet CS, Laboisse CL, Nguyen PG, Siliart BS, Dumon HJ, and Champ MM. Restoration of the integrity of rat caeco-colonic mucosa by resistant starch, but not by fructo-oligosaccharides, in dextran sulfate sodium-induced experimental colitis. Br J Nutr 90: 75–85, 2003.[CrossRef][ISI][Medline]
  46. Okamoto T, Sasaki M, Tsujikawa T, Fujiyama Y, Bamba T, and Kusunoki M. Preventive efficacy of butyrate enemas and oral administration of Clostridium butyricum M588 in dextran sodium sulfate-induced colitis in rats. J Gastroenterol 35: 341–346, 2000.[CrossRef][Medline]
  47. Park SH, Cho HN, Lee SJ, Kim TH, Lee Y, Park YM, Lee YJ, Cho CK, Yoo SY, and Lee YS. Hsp25-induced radioresistance is associated with reduction of death by apoptosis: involvement of Bcl2 and the cell cycle. Radiat Res 154: 421–428, 2000.[ISI][Medline]
  48. Paul C, Manero F, Gonin S, Kretz-Remy C, Virot S, and Arrigo AP. Hsp27 as a negative regulator of cytochrome C release. Mol Cell Biol 22: 816–834, 2002.[Abstract/Free Full Text]
  49. Preville X, Gaestel M, and Arrigo AP. Phosphorylation is not essential for protection of L929 cells by Hsp25 against H2O2-mediated disruption actin cytoskeleton, a protection which appears related to the redox change mediated by Hsp25. Cell Stress Chaperones 3: 177–187, 1998.[CrossRef][ISI][Medline]
  50. Preville X, Salvemini F, Giraud S, Chaufour S, Paul C, Stepien G, Ursini MV, and Arrigo AP. Mammalian small stress proteins protect against oxidative stress through their ability to increase glucose-6-phosphate dehydrogenase activity and by maintaining optimal cellular detoxifying machinery. Exp Cell Res 247: 61–78, 1999.[CrossRef][ISI][Medline]
  51. Rane MJ, Pan Y, Singh S, Powell DW, Wu R, Cummins T, Chen Q, McLeish KR, and Klein JB. Heat shock protein 27 controls apoptosis by regulating Akt activation. J Biol Chem 278: 27828–27835, 2003.[Abstract/Free Full Text]
  52. Reid T, Valone F, Lipera W, Irwin D, Paroly W, Natale R, Sreedharan S, Keer H, Lum B, Scappaticci F, and Bhatnagar A. Phase II trial of the histone deacetylase inhibitor pivaloyloxymethyl butyrate (Pivanex, AN-9) in advanced nonsmall cell lung cancer. Lung Cancer 45: 381–386, 2004.[CrossRef][ISI][Medline]
  53. Ren H, Musch M, Kojima K, Boone D, Ma A, and Chang E. Short-chain fatty acids induce intestinal epithelial heat shock protein 25 expression in rats and IEC 18 cells. Gastroenterology 121: 631–639, 2001.[CrossRef][ISI][Medline]
  54. Roediger WE. The colonic epithelium in ulcerative colitis: an energy-deficiency disease? Lancet 2: 712–715, 1980.[ISI][Medline]
  55. Roediger WE. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 83: 424–429, 1982.[ISI][Medline]
  56. Rogalla T, Ehrnsperger M, Preville X, Kotlyarov A, Lutsch G, Ducasse C, Paul C, Wieske M, Arrigo AP, Buchner J, and Gaestel M. Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation. J Biol Chem 274: 18947–18956, 1999.[Abstract/Free Full Text]
  57. Ropeleski MJ, Tang J, Walsh-Reitz MM, Musch MW, and Chang EB. Interleukin-11-induced heat shock protein 25 confers intestinal epithelial-specific cytoprotection from oxidant stress. Gastroenterology 124: 1358–1368, 2003.[CrossRef][ISI][Medline]
  58. Ropeleski MJ, Ren H, Musch MW, and Chang EB. Short-chain fatty acids induce intestinal epithelial heat shock protein 25 by transcriptional mechanisms. Gastroenterology 122: A39, 2002.
  59. Scheppach W, Bartram HP, and Richter F. Role of short-chain fatty acids in the prevention of colorectal cancer. Eur J Cancer 31A: 1077–1080, 1995.[CrossRef]
  60. Tan S, Seow TK, Liang RC, Koh S, Lee CP, Chung MC, and Hooi SC. Proteome analysis of butyrate-treated human colon cancer cells (HT-29). Int J Cancer 98: 523–531, 2002.[CrossRef][ISI][Medline]
  61. Venkatraman A, Ramakrishna BS, Shaji RV, Kumar NS, Pulimood A, and Patra S. Amelioration of dextran sulfate colitis by butyrate: role of heat shock protein 70 and NF{kappa}B. Am J Physiol Gastrointest Liver Physiol 285: G177–G184, 2003.[Abstract/Free Full Text]
  62. Yin L, Laevsky G, and Giardina C. Butyrate suppression of colonocyte NF-{kappa}B activation and cellular proteasome activity. J Biol Chem 276: 44641–44646, 2001.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/G178    most recent
00304.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map