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(The FASEB Journal. 2007;21:197-206.)
© 2007 FASEB

Prohibitin protects against oxidative stress in intestinal epithelial cells

Arianne L. Theiss*, Richard D. Idell*, Shanthi Srinivasan*, Jan-Michael Klapproth*, Dean P. Jones{dagger}, Didier Merlin* and Shanthi V. Sitaraman*

* Division of Digestive Diseases,

{dagger} Division of Pulmonary Medicine, Department of Medicine, Emory University, Atlanta, Georgia, USA

1Correspondence: Division of Digestive Diseases, 615 Michael St., Whitehead Biomedical Research Bldg. 265, Atlanta, GA 30322, USA. E-mail: atheiss{at}emory.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prohibitin (PHB) is an evolutionarily conserved and ubiquitously expressed protein whose expression or function in intestinal diseases is not known. In this study, we examined the expression and role of PHB in oxidative stress associated with inflammatory bowel disease. Our results show that PHB primarily localizes to the mitochondria in intestinal epithelial cells. Its expression is down-regulated during active human Crohn’s disease, experimental colitis in vivo, and oxidative stress in vitro. PHB overexpression increases the expression of glutathione-S-transferase {pi} and protects from oxidant-induced depletion of glutathione. Finally, PHB overexpression decreases accumulation of reactive oxygen metabolites, as well as increased permeability induced by oxidative stress in intestinal epithelial cells. Together, these results suggest that PHB constitutes a previously unrecognized cellular defense against oxidant injury. Thus, strategies to modulate PHB levels may constitute a novel therapeutic approach for intestinal inflammatory diseases, wherein oxidative stress plays a critical role in tissue injury and inflammation.—Theiss, A. L., Idell, R. D., Srinivasan, S., Klapproth, J.-M., Jones, D. P., Merlin, D., Sitaraman, S. V. Prohibitin protects against oxidative stress in intestinal epithelial cells.


Key Words: epithelial permeability • inflammatory bowel diseases • glutathione


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PROHIBITIN (PHB) IS A UBIQUITOUSLY expressed and highly conserved protein that was originally determined to play a predominant role in inhibiting cell-cycle progression and inhibit cellular senescence (1 2 3) . It’s best described function is as a chaperone protein involved in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes (4 , 5) . PHB is essential for normal mitochondrial development, and its deficiency in Caenorhabditits elegans is associated with inhibition of mitochondrial biogenesis and senescence (6) . PHB is also present in various cellular compartments, including the mitochondria, nucleus, and plasma membrane (7 8 9) . In the nucleus, it may serve as a modulator of transcriptional activity (9 , 10) . At the plasma membrane, it may function as a binding partner for yet uncharacterized ligands. The only study to assess PHB in intestinal epithelial cells showed that cell surface-associated PHB binds to Vi capsular polysaccharide of Salmonella typhi and inhibits the inflammatory response to S. typhi, suggesting that PHB may regulate inflammation in the intestine (11) . The expression and role of PHB in intestinal diseases are not known. Given that PHB is a regulator of mitochondrial function, we examined the potential role of PHB in modulating oxidative stress during inflammatory bowel disease (IBD).

The two main forms of IBD, Crohn’s disease, and ulcerative colitis, are characterized by recurring inflammation of the gastrointestinal tract presenting with abdominal pain, diarrhea, and mucosal damage. The etiology of IBD remains unclear, but it is thought to result from an inappropriate and exaggerated mucosal immune response driven by constituents of the normal mucosal microflora, that is in part genetically determined (12) . Increased production of free radicals and/or impaired antioxidant defense capabilities have been demonstrated in both animal models and human IBD, indicating a central contribution for reactive oxygen metabolites (ROM) released by epithelial cells and neutrophils in the onset, progression, and pathological consequences of disease (13 , 14) . Increased ROM levels have been shown to disrupt mucosal barrier function of intestinal epithelial cells characterized by increased permeability and impaired wound healing (15 , 16) . An imbalanced antioxidant response in the mucosa of IBD patients is thought to contribute to the pathogenesis and progression of the inflammatory process (17 18 19 20 21) , and antioxidant therapy improves colitis in multiple experimental models (22 23 24 25) . However, little is currently known regarding the intracellular proteins involved in combating oxidative stress in intestinal epithelial cells. In this study, we examined the expression and role of PHB in modulating oxidative stress in the intestine.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Primary antibodies: Rabbit polyclonal PHB antibody (Ab) was obtained from Lab Vision Corporation (Fremont, CA), rabbit polyclonal COX IV Ab, and mouse monoclonal cytokeratin 8 from Abcam (Cambridge, MA), mouse monoclonal antiapoptosis-inducing factor (AIF), mouse monoclonal cytokeratin 18 Ab and rabbit polyclonal antiactin antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit polyclonal anti-STAT3 Ab from Cell Signaling Technology (Danvers, MA), mouse monoclonal ß-tubulin Ab from Sigma-Aldrich Corp. (St. Louis, MO), and mouse monoclonal glutathione-S-transferase {pi} from DakoCytomation, Inc (Carpinteria, CA). Secondary antibodies: peroxidase-conjugated goat anti-mouse or goat anti-rabbit from Bio-Rad Laboratories (Hercules, CA), Alexa-Fluor 568 phalloidin and ToPRO-3 iodide (642/661) from Molecular Probes (Carlsbad, CA), and fluorescein FITC-conjugated AffiniPure goat anti-rabbit IgG and goat anti-mouse IgG from Jackson ImmunoResearch Laboratory (West Grove, PA). One-step RT-polymerase chain reaction (RT-PCR) kit was purchased from Qiagen (Valencia, CA).

Cell Culture
The Caco2-BBE human intestinal epithelial cell line was used as an in vitro model of polarized intestinal epithelium (26) . All cells were grown as confluent monolayers in Dulbecco’s modified Eagle medium (DMEM) supplemented with penicillin (40 mg/l), 10% fetal calf serum, and plasmocin (1.75 mg/l; InvivoGen, San Diego, CA). Cells were plated on permeable supports (pore size 0.4 µm; Transwell-Clear polyester membranes; Costar Life Sciences, Acton, MA). Experiments, including H2O2 treatment were performed as follows: serum-deprived cells were treated with 0.5 mM H2O2 on the apical side of the monolayers and incubated for 1 h. This dose was chosen since previous studies have shown that 0.5 mM H2O2 treatment resulted in maximal disruption of the mucosal barrier in Caco2 cells when testing doses 0.1 mM to 2.5 mM H2O2 (15) . Initial experiments showed no difference between 0.5 mM H2O2 and 1.0 mM H2O2 added apically or basolaterally, and therefore, subsequent experiments used 0.5 mM H2O2 added apically. For recovery experiments, cells were treated with 0.5 mM H2O2 for 1 h, H2O2 was subsequently removed, cells were rinsed and allowed to recover in media for various amounts of time, after which total protein was collected. All experiments were performed on Caco2-BBE cells between passages 58–72.

Human material
The diagnosis of IBD was based on clinical, endoscopic, and histological criteria. Clinical data for IBD patients were obtained by medical record review. Infectious colitis was ruled out by stool cultures. The collection of samples was approved by the Institutional Review Board of Emory University. Mucosal biopsy specimens were obtained during routine endoscopy that was performed after written informed consent was obtained. Biopsy samples were taken from involved areas of the colon in cluster of differentiation (CD) patients. Control biopsy samples were collected from volunteers undergoing colonoscopy for colorectal cancer screening who had no overt pathology including polyps. Biopsy specimens were snap frozen in Optimal Cutting Temperature immediately after endoscopic resection and stored at –80°C for confocal staining or homogenized in PBS containing 1% Triton X-100, 1% Nonidet P-40 (v/v), 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and 1 µl/ml protease inhibitor mixture III (Boehringer Mannheim, Indianapolis, IN) to obtain protein extracts for Western immunoblotting. To obtain mitochondrial fractions, colonic mucosal resection specimens were obtained from 4 patients who underwent colonic resection for colon cancer. Care was taken to obtain mucosa away from the tumor margin. Specimens were homogenized in mitochondrial isolation buffer (70 mM sucrose, 220 mM D-mannitol, 2 mM HEPES, pH 7.4), and centrifuged at 1000 g for 5 min. The supernatant was then centrifuged at 10,500 g for 15 min to obtain a soluble cytosolic fraction from the pellet containing mitochondria. The fractions were then subjected to Western blot analysis.

Mice
Two independent experimental models of colitis were used to assess PHB expression during intestinal inflammation: the interleukin (IL)-10–/– and dextran sodium sulfate (DSS) models. Oxidative stress has been shown to play a pathogenic role in the development of colitis in both these models (25 , 27 28 29) . IL-10–/– mice spontaneously develop chronic, T cell-mediated, transmural colitis, which shares many features with human Crohn’s disease. Indeed, on the basis of this model, there have been several clinical trials using IL-10 treatment for IBD in human patients (30) . However, because of the inconsistency in the development of colitis in IL-10–/– mice, Berg and colleagues have described rapid development of colitis in IL-10–/– mice treated with a nonselective NSAID (27 , 31) . Accordingly, mice were treated with piroxicam, 200 ppm in the diet for 4 wk to induce colitis. For the DSS model, wild-type (WT) mice given 3.0% DSS in their drinking water for 5 days exhibit acute mucosal inflammation characterized by epithelial ulceration and subsequent infiltration of neutrophils and other acute inflammatory cells that mimics several features of human IBD. Male WT and IL-10–/– mice (6–8 wk, 12–16 g) on the inbred C57BL/6 background were purchased from The Jackson ImmunoResearch Laboratory. All mice were group-housed in standard cages under a controlled temperature (25°C) and photoperiod (12:12 h light-dark cycle) and were allowed standard chow and tap water ad libitum. They were allowed to acclimate to these conditions for at least 7 days before inclusion in experiments. All procedures were in accordance with the Emory University Institutional Animal Care, Authorization no. 146-2002. DSS-colitis was induced in WT mice by the addition of 3.0% (w/v) DSS (MW 40 kDa; ICN Biochemicals, Aurora, OH) to the drinking water for 5 days. Control mice were given normal drinking water throughout the study period. Mean DSS/water consumption and body weight were assessed daily during the treatment period. Colitis was assessed by clinical score (body weight, presence of occult blood, and stool consistency), as well as histological score (crypt damage, ulceration, and inflammatory infiltration) using hematoxylin and eosin (H&E)-stained colon sections as described (32) . Myeloperoxidase activity was determined as described (32) .

Confocal microscopy
Ten-micrometer cryostat sections of biopsied human mucosa or Caco2-BBE cells grown to confluency on filters were fixed in buffered 4% paraformaldehyde for 20 min, blocked in 2% BSA, incubated with respective primary antibodies overnight at 4°C, washed with PBS, and subsequently incubated with fluoresceinated secondary antibodies for 1 h at room temperature. Cell monolayers and human sections were also counterstained with rhodamine/phalloidin to visualize actin. Samples were mounted in p-phenylenediamine/glycerol (1:1) and analyzed by confocal microscopy (Zeiss dual-laser confocal microscope), as described previously (33) .

Electron microscopy
Confluent Caco2-BBE cells were fixed with 4% paraformaldehyde in 0.1 M PBS for 20 min. For Immunogold labeling, cells were washed with PBS, incubated in 0.1% sodium borohydrade in PBS for 15 min to reduce residual aldehyde, washed with PBS, permeabilized with 0.05% Triton X-100 in PBS for 10 min, blocked with buffer containing 5% normal goat serum, 5% BSA, and 0.1% cold water fish-skin gelatin, incubated with primary Ab or isotype control Ab (rabbit IgG), diluted with PBS containing 0.2% acetylated BSA (Aurion; Wageningen, The Netherlands), washed with PBS and subsequently incubated for 12 h at 4°C in goat anti-rabbit ultrasmall gold conjugates (Aurion). Cells were washed with PBS and fixed with 2.5% gluteraldehyde in 0.1 M PBS. Aurion R-gent SE-electron microscopy kit was used for silver enhancement of ultrasmall gold particles following the manufacturer’s protocol. Cells were then fixed with 0.5% osmium tetroxide in 0.1 M PBS for 15 min, dehydrated, and embedded in epoxy resin for EM. Transmural sections were cut 70 nm thick and visualized on a transmission electron microscope (model H-7500; Hitachi, Pleasanton, CA).

SDS-PAGE and Western immunoblot analysis
Protein samples were separated by SDS-PAGE using Laemmli’s 2 x SDS sample buffer and 12% polyacrylamide gels followed by electrotransfer to nitrocellulose membranes. Membranes were incubated with primary antibodies at 4°C overnight and subsequently incubated with corresponding peroxidase-conjugated secondary antibodies. Membranes were washed, and immunoreactive proteins were detected using enhanced chemiluminescence (Denville Scientific, Metuchen, NJ) and exposed to high-performance chemiluminescence film (Denville). Blots were reprobed with antiactin or anti-ß-tubulin as a loading control. Films were analyzed by densitometry, and signal intensity was quantitated using a gel documentation system (Alpha Innotech, San Leandro, CA).

RNA isolation and RT-PCR
Total RNA was isolated from monolayers of Caco2-BBE cells using TRI reagent (Molecular Research Center, Cincinnati, OH). Total RNA was then used to amplify PHB, glutathione S-transferase (GST){pi}, and ß-tubulin using RT-PCR (Qiagen One Step RT-PCR kit). The primers used for RT-PCR were designed using human nucleotide sequences available in the GenBank database. PHB (sense: 5'-ggcctggccttagctgttgc-3', antisense: 5'-cgctctatgaggtcgtcgc-3'); GST{pi} (sense: 5'-ggtgaatgacggcgtgg-3', antisense: 5'-cgttgccattgatggggagg-3'); ß-tubulin (sense: 5'-ccagctggtggagaatacgg-3', antisense: 5'-cgaacatctgctgggtgagc-3').

cDNA cloning of intestinal PHB and overexpression of PHB in Caco2-BBE cells
A single PHB polymerase chain reaction (PCR) product corresponding to the entire coding region of PHB (818 bp) was generated from Caco2-BBE cells. The PCR product was ligated into a pcDNA4/HisMax vector (Invitrogen, Carlsbad, CA) using the Quick Ligation Kit (New England Biolabs, Ipswich, MA) and sequenced from the T7 priming site (Emory University sequencing facility, Atlanta, GA). The clone from Caco2-BBE cells had identical sequence as human PHB (34) . Caco2-BBE cells were transfected with PHB/pcDNA4 using lipofectamine 2000 (Invitrogen).

DCF assay
The nonionic, nonpolar 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen) crosses the cell membrane and is hydrolyzed by intracellular esterases to nonfluorescent dichlorofluorescin (DCFH). DCFH is converted to fluorescent 2',7'-DCF (DCF) in the presence of intracellular reactive oxygen metabolites. Caco2-BBE cells transfected with PHB/pcDNA4 or vector were loaded with 50 µM H2DCFDA for 10 min, washed, and exposed to 0.5 mM H2O2 for 1 h. DCF fluorescence is therefore a measure of intracellular reactive oxygen species (ROS) produced by the cell, as well as H2O2 added exogenously that has diffused across the cell membrane. Fluorescence was quantitated according to the manufacturer’s protocol.

GSH assay
Caco2-BBE cells transfected with PHB/pcDNA4 or vector were allowed to grow to confluency on filters (72 h posttransfection). Cells were serum-deprived overnight and treated apically with 0.5 mM H2O2 for 1 h. Following treatment, cells were lysed in buffer containing 5% perchloric acid (PCA), 0.2 M boric acid, and 10 µM {gamma}-Glu-Glu and centrifuged at 16,000 g for 5 min. Supernatants were analyzed for GSH by HPLC and pellets were dissolved in 1 M sodium hydroxide and assayed to determine total protein concentration by Bradford assay. Final values of GSH are given as nanomoles per milligram protein.

Measurement of transepithelial electrical resistance (TEER) and macromolecular permeability
Caco2-BBE cells transfected with PHB/pcDNA4 or vector were allowed to grow to confluency on filters (72 h posttransfection). TEER was measured with an epithelial voltohmmeter (Millicell-ers, Millipore, Billerica, MA). For permeability assays, cells were treated apically with ± 0.5 mM H2O2 for 30 min or 60 min in the presence of 10 mg/ml FITC-dextran (MW 4 kDa; Sigma-Aldrich Corp.). The apical and basolateral reservoirs were sampled at the indicated time points and FITC-dextran concentration quantified via spectrofluorimetry ({lambda}ex=492 nm, {lambda}em=510 nm). Values are shown as nanograms per milliliter per minute FITC-dextran present in the basolateral reservoir.

Statistical analysis
Values are expressed as means ± SE. Statistical analysis was performed using unpaired Student’s t test. A P value < 0.05 was considered statistically significant in all analyses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PHB is primarily located in the epithelium of human colon and in the mitochondrial fraction of human mucosal biopsies
Sections of human colonic mucosa obtained as biopsies during routine colonoscopies were used to assess the localization of PHB in normal colon. PHB is primarily located in epithelial cells with less expression found in the underlying lamina propria (Fig. 1 A). To determine the subcellular localization of PHB in normal human colonic mucosa, mitochondrial and cytosolic fractions were isolated from mucosal biopsies and subjected to Western immunoblotting for PHB. PHB is predominantly expressed in the mitochondria of cells present in the normal human mucosa, containing both epithelial cells and lamina propria cells (Fig. 1B ).


Figure 1
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  		Figure 1. PHB is primarily located in the epithelium of human colon and in the mitochondrial fraction of human mucosal biopsies. A) Confocal imaging of PHB and actin localization in normal human colon from mucosal biopsies. PHB is primarily expressed in epithelial cells. Magnification, x40. B) Mitochondrial (M) and soluble cytosolic (C) extracts were isolated from normal colonic mucosal biopsies. The majority of PHB is expressed in the mitochondrial fraction as shown by Western immunoblot analysis. Blots were subsequently probed with anti-COX IV Ab as a mitochondrial marker. Results are representative of 4 patients.

PHB is predominantly expressed in the mitochondrial fraction of the model intestinal epithelial cell line, Caco2-BBE
To determine the subcellular localization of PHB in intestinal epithelial cells, polarized monolayers of the model intestinal epithelial cell line Caco2-BBE were exposed to anti-PHB Ab and visualized by confocal microscopy. The most intense staining for PHB colocalizes with apoptosis-inducing factor (AIF), a mitochondrial marker protein, in the perinuclear region of the cell (Fig. 2 A). Immunoblotting for PHB in mitochondrial, cytosolic, and nuclear fractions from Caco2-BBE cells shows that PHB expression is exclusively in the mitochondria with no expression in the nuclear extracts (Fig. 2B ). To further confirm the localization of PHB, we performed EM of the Caco2-BBE monolayer. As seen in Fig. 2C and consistent with the confocal imaging and biochemical data, Immunogold labeling for PHB localizes to mitochondria.


Figure 2
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  		Figure 2. PHB is preferentially located in the mitochondria of Caco2-BBE intestinal epithelial cells. A) Confocal imaging of PHB and AIF (mitochondrial marker) in polarized Caco2-BBE cells. PHB and AIF primarily colocalize in mitochondria (PHB-AIF merge) while little PHB is localized in the nucleus (PHB-nuclei merge). Cells incubated with normal rabbit serum and an antiactin Ab to visualize cells were used as a negative control. Magnification, x40. B) PHB expression was determined in cytosolic (C) vs. nuclear (N) fractions from Caco2-BBE cells (40 µg per lane). Mitochondria (M) was further isolated from soluble cytosolic (C) fractions and probed with anti-PHB, anti-AIF as a mitochondrial marker, or anti-STAT3 as a control to ensure protein loading. C) EM showing that the localization of PHB by Immunogold labeling is predominantly in mitochondria (arrows). M, mitochondria; N, nucleus. Scale bar = 300 nm.

PHB protein expression is decreased in colonic mucosal biopsies from Crohn’s disease patients and during experimental colitis in vivo
Given the robust expression of PHB in normal human intestinal epithelia, we next assessed the expression levels of PHB during intestinal inflammation. Total protein was isolated from biopsies obtained from patients undergoing screening colonoscopies (8 independent patients) and patients with active Crohn’s disease (10 independent patients) and analyzed for PHB protein expression. As shown in Fig. 3 A, PHB expression is reduced in the colonic mucosa of Crohn’s disease patients with active inflammation compared to normal mucosa. Cytokeratin 18 was included as a marker for epithelial cells to ensure that the decrease in PHB in total colonic lysates is not due a lack of epithelial cells during colitis. Cytokeratin 18 levels are unchanged indicating that decreased PHB likely reflects its expression during active CD.


Figure 3
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  		Figure 3. PHB protein expression is decreased in colonic mucosal biopsies from Crohn’s disease patients and during experimental colitis in vivo. A) Representative western immunoblots for PHB and actin (loading control) in total protein extracts isolated from normal colon or Crohn’s disease inflamed colon. Cytokeratin 18 is included as a marker for epithelial cells to ensure that the decrease in PHB in total colonic lysates is not due a lack of epithelial cells during colitis. Each patient biopsy is plotted as an individual dot in the histogram. n ≥ 8 per condition; *P < 0.05 vs. control. B) Western blot analysis showing PHB and ß-tubulin (loading control) protein abundance in total protein isolated from colon of control mice, mice given 3% DSS for 5 days, and IL-10 –/– mice. Histograms show mean ± SEM relative to control animals analyzed on the same blot. **P < 0.01 vs. control; n = 4 for control and DSS; *P < 0.05 vs. control; n = 3 for IL-10 –/–.

To determine whether PHB levels are also decreased in experimental colitis, we next analyzed PHB protein expression in two distinct mouse models of intestinal inflammation: the IL-10–/– and dextran sodium sulfate (DSS) models. IL-10–/– mice spontaneously develop chronic, T cell mediated, transmural colitis that shares many features with human Crohn’s disease (31) . WT mice given 3.0% DSS in their drinking water for 5 d exhibit acute mucosal inflammation characterized by epithelial ulceration and subsequent infiltration of neutrophils and other acute inflammatory cells that mimics several features of human IBD. Whole colonic protein extracts were isolated from control mice, mice administered DSS, and IL-10–/– mice. PHB protein expression is reduced in both DSS and IL-10–/– mouse models of intestinal inflammation compared to control mice (Fig. 3B ), similar to human Crohn’s disease. Although cytokeratin 18 levels are variable across animal models of colitis, it is clear that the decrease in PHB in total colonic lysates is not due a lack of epithelial cells during inflammation as indicated by cytokeratin 18 expression.

PHB protein abundance is decreased by oxidative stress in vitro and recovers after subsequent removal of oxidative stress
Since PHB is expressed in the mitochondria, we tested whether levels of PHB in intestinal epithelial cells were altered by oxidative stress in vitro. Confluent Caco2-BBE monolayers were treated apically with 0.5 mM H2O2, which was previously shown to be an in vitro model of oxidative stress and to disrupt mucosal barrier function (15) . Cells treated with H2O2 showed a decrease in PHB protein expression compared to control cells (Fig. 4 A), similar to results obtained in patients with active Crohn’s disease and in animal models of colitis. On the subsequent removal of H2O2, PHB expression returns to baseline after 12 h of recovery (Fig. 4B ). These data demonstrate that PHB expression is decreased in intestinal epithelial cells during oxidative stress and that PHB levels recover after removal of oxidative stress.


Figure 4
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  		Figure 4. PHB protein expression is decreased during oxidative stress in vitro and recovers after subsequent removal of oxidative stress. A) Confluent monolayers of Caco2-BBE cells grown on filters were treated apically with 0.5 mM H2O2 for 1 h as an in vitro model of oxidative stress. Total protein extracts were probed for anti-PHB Ab and antiactin to confirm equal loading. Histograms show mean ± SEM relative to control cells. *P = 0.05 vs. control; n = 8. B) Confluent monolayers of intestinal epithelial cells were treated with 0.5 mM H2O2 on the apical side. Following treatment for 1 h, H2O2 was removed, cells were rinsed and allowed to recover for various amounts of time. Representative western immunoblots showing PHB and ß-tubulin protein abundance in total cell lysates. Histograms show mean ± SEM relative to control cells. *P < 0.05 vs. control; ** P < 0.005 vs. control; n = 4 per treatment.

PHB overexpression protects against H2O2-induced GSH decrease in Caco2-BBE cells
GSH is the major endogenous antioxidant scavenger that protects cells from oxidative stress through its ability to bind to and reduce hydrogen peroxide and ROS, thereby preventing macromolecular damage. To determine the effect of PHB expression on GSH levels, Caco2-BBE cells overexpressing PHB were treated apically with 0.5 mM H2O2 and GSH levels were compared to cells transfected with empty vector. Under conditions in which the amount of GSH reacting with oxidative compounds exceeds the capacity of the amount produced by the cell, depletion of GSH can result in cell damage. Such depletion of GSH occurs in cells transfected with vector when treated with H2O2 (Fig. 5 ). Interestingly, cells overexpressing PHB are protected from H2O2-induced decrease in GSH. In fact, PHB overexpressing cells show significantly higher levels of GSH compared to no treatment control cells (Fig. 5) . These results indicate that under conditions of oxidative damage, PHB overexpression increases the levels of the antioxidant GSH.


Figure 5
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  		Figure 5. PHB overexpression protects against H2O2-induced GSH decrease in Caco2-BBE cells. Cells were transfected with PHB or vector (V) for 72 h followed by treatment with 0.5 mM H2O2 apically for 1 h. GSH in total protein was measured by HPLC. H2O2 treatment causes a reduction in GSH levels in vector transfected cells. PHB overexpression significantly increases GSH levels during H2O2-induced oxidative stress. *P < 0.05 vs. V no treatment; n = 6 per treatment.

PHB overexpression induces expression of GST{pi}, a member of the GST family of antioxidant enzymes
Since GSTs catalyze the binding of electrophiles to GSH (35) , we determined whether PHB overexpression could increase expression of GST{pi}, the most abundant GST isoform expressed in the colon and associated with oxidative stress in the intestine (36 37 38) . Caco2-BBE cells overexpressing PHB were compared to cells transfected with vector for expression of GST{pi}. PHB overexpression induced expression of GST{pi} at the mRNA and protein levels (Fig. 6 A and B). Together, these results suggest that PHB modulates the GSH/glutathione S-transferase antioxidant system in intestinal epithelial cells.


Figure 6
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  		Figure 6. PHB overexpression induces expression of GST{pi} in Caco2-BBE cells. A) RT-PCR amplification of GST{pi}. Total RNA from Caco2-BBE cells transfected with vector (V) or PHB was subjected to reverse transcription followed by PCR amplification using GST{pi} or ß-tubulin specific primers. RT-PCR for PHB confirms PHB overexpression compared to vector transfected cells. Gel is representative of three independent experiments. n = 3 per treatment. B) Representative Western immunoblots showing GST{pi} or ß-tubulin (loading control) expression in total protein lysates from Caco2-BBE cells overexpressing PHB or vector. Blots were subsequently probed for PHB to ensure overexpression in PHB transfected cells. Histograms show mean ± SEM of GST{pi} protein expression relative to V control cells. **P < 0.005 vs. V; n = 3 per treatment.

PHB overexpression inhibits accumulation of ROM and protects against oxidant-induced permeability changes in intestinal epithelial cells
To determine whether PHB overexpression alters neutralization of exogenous H2O2 that has diffused across the cell membrane into the cell, cells were loaded with the oxidation-sensitive dye H2DCFDA, treated with H2O2 for 1 h and assayed for DCF fluorescence. Compared to untreated cells, H2O2 induced a 3.3-fold increase in DCF fluorescence in vector transfected cells (Fig. 7 A). In comparison, cells overexpressing PHB and treated with H2O2 exhibited significantly less DCF fluorescence (2.0-fold increase vs. untreated cells) suggesting decreased ROM accumulation by PHB.


Figure 7
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  		Figure 7. PHB overexpression inhibits accumulation of ROM and protects against oxidant-induced permeability changes in intestinal epithelial cells. A) DCF fluorescence was measured in Caco2-BBE cells transfected with PHB or vector (V) for 72 h and subsequently treated with 0.5 mM H2O2 apically for 1 h. *P < 0.05 vs. V no treatment; #P < 0.05 vs. V + H2O2; n ≥ 5 per treatment. B) Monolayers of Caco2-BBE cells overexpressing PHB or vector (V) were treated apically with 0.5 mM H2O2 in the presence of FITC-dextran. Histograms show mean ± SEM of nanograms per milliliter per minute FITC-dextran in the basolateral reservoir. **P = 0.005 vs. PHB + H2O2; n ≥ 11 per treatment.

Given that increased permeability contributes to oxidant-induced disruption of the mucosal barrier (15) and that overexpression of PHB is associated with the induction of GSH and GST{pi} as well as decreased ROM, we determined whether PHB overexpression could protect against H2O2-induced increased intestinal epithelial cell permeability. As expected, H2O2 treatment increased permeability in cells overexpressing vector compared to no treatment control cells (Fig. 7B ). Interestingly, cells overexpressing PHB were protected from H2O2-induced increased permeability and showed similar rates of FITC translocation as untreated cells (Fig. 7B ). In terms of transepithelial electrical resistance (TEER), PHB overexpression protected against the H2O2-induced decrease in TEER, reflecting our permeability data (vector: 213.3±15.0, vector+H2O2: 187.7±5.8, PHB: 204.0±5.8, PHB+H2O2: 203.7±5.1 Ohms/cm2. Mean±SE.; n=3 per condition). Together, these data show that PHB protects against oxidant-induced permeability changes in intestinal epithelial cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PHB was first cloned in 1991 and has since been described to have antiproliferative properties and act as a potential tumor suppressor (39 40 41 42 43) . Expression of PHB is altered in gastric, cervical, and breast cancer (42 , 44 45 46) and loss of PHB expression along with other tumor suppressors on chromosome 17q is associated with colorectal cancer (47) . PHB has also been shown to regulate the ras/raf-mediated signaling pathway in cancer cells (8) and in breast cancer cells PHB translocates to mitochondria in response to chemotherapy and leads to apoptosis (10) . Little is known, however, regarding the role of PHB during inflammation in any organ system.

PHB has been reported to be localized typically in the mitochondria or nucleus depending on cell type and situation (9 , 48 , 49) . A single study has shown its localization to lipid rafts of plasma membrane in Caco2 cells (11) . Using confocal imaging, cell fractionation, and EM, we demonstrate that PHB localizes to the mitochondrial fraction in Caco2-BBE cells. Our findings are consistent with several studies in yeast and mammalian cells showing localization of PHB to the mitochondria but differ from the study showing PHB in membrane lipid rafts. In this study, Sharma et al. (11) used a sucrose density gradient to demonstrate the localization of PHB in lipid rafts in Caco2 cells. However, the authors could not demonstrate caveolin or ganglioside GM-1 staining to confirm the light fractions and formal biochemical or cell fractionation studies were not done to further study localization of PHB. In addition, there was significant PHB expression in the bottom of the sucrose gradient suggesting predominant expression in the cytosolic fraction. Although a small amount of PHB may be present in plasma membrane lipid rafts, we demonstrate that the majority of PHB is present in the mitochondria of Caco2 cells.

Mitochondrial PHB forms a high MW complex with B cell receptor-associated protein 37 (Bap37) and stabilizes subunits of mitochondrial respiratory enzymes in yeast (5) . It is well established that expression of PHB is associated with cellular senescence (11 , 50 , 51) . This association prompted Mishra et al. (3) to hypothesize that decreased PHB levels may be associated with the accumulation of damage from oxygen radicals. This is consistent with data showing that PHB expression is decreased in ex situ lung tissue exposed to hyperoxia (52) . However, a direct association between PHB and oxidative stress has never been shown. Here, we show that PHB overexpression increases neutralization of exogenous intracellular H2O2. Furthermore, we show that PHB expression is reduced in intestinal epithelial cells by treatment with H2O2, an in vitro model of oxidative stress. These data have a direct implication on the understanding of the pathogenesis of human inflammatory bowel disease wherein oxidative stress has been shown to be increased in inflamed mucosa and oxidant-induced damage is thought to play a significant role in inflammation and tissue damage that characterize this disease (13 , 14) . Furthermore, the antioxidant response in the mucosa of IBD patients is imbalanced and overwhelmed by increased oxidants, (17 18 19 20 21 , 53) and antioxidant therapy improves colitis in multiple experimental models (22 23 24 25) . Similar to human IBD, DSS and IL-10–/– models of colitis are associated with increased oxidative stress (17 , 25 , 28 , 29) . Our data suggest that PHB may play a direct role in oxidative stress in IBD.

In our study, we tested whether PHB alters the levels of GSH, the major antioxidant molecule. As expected, hydrogen peroxide decreased the levels of GSH in intestinal epithelial cells. However, cells overexpressing PHB showed no oxidant-induced depletion in GSH and instead showed increased GSH levels. Since GSH is such a potent antioxidant, even small changes correlate with biological effects, suggesting that the small change in GSH levels in our study is biologically significant. This suggests that increased expression of PHB can combat oxidant-induced decrease of GSH, a potent antioxidant that reduces hydrogen peroxide levels. Furthermore, PHB overexpression induces expression of GST{pi}, an enzyme that catalyzes the conjugation of electrophiles to GSH. GST{pi} is the most abundant isoform expressed in the small intestine and colon and has been associated with oxidative stress (36 , 54) . Indeed GST{pi} polymorphisms are associated with age of onset and severity of IBD leading to colectomy (55) . Since induction of the GSH/glutathione S-transferase antioxidant system is a conserved response of cells to oxidative stress (35) our data indicates a role of PHB in regulating antioxidant expression and response to oxidants.

The mechanism by which PHB increases GSH and GST{pi} levels is unknown. Given that PHB can localize to the nucleus and modulate transcription (9 , 10) , PHB may increase transcription of antioxidant proteins during conditions of oxidative stress by binding to the antioxidant response element that is present in the promoter region of many antioxidant proteins. Both GST{pi} and glutamyl cysteine synthase (enzyme involved in GSH synthesis) promoters have antioxidant response elements in their promoters. Alternatively, PHB may bind to antioxidant proteins to stabilize them during oxidative stress as it does with some mitochondrial proteins (37) .

Increased oxidative stress has been shown to disrupt mucosal barrier function of intestinal epithelial cells characterized by increased permeability (15 , 16) . Since PHB induces expression of the antioxidants GSH and GST{pi} and reduces H2O2-induced ROM accumulation, we assessed whether PHB overexpression could protect against oxidant-induced increased permeability in intestinal epithelial cells. Indeed, cells overexpressing PHB showed no increase in permeability by treatment with hydrogen peroxide compared to cells overexpressing vector. The mechanism by which H2O2 induces barrier dysfunction is thought to include altered expression, phosphorylation, and/or redistribution of junctional proteins such as occludin and ZO-1 (56 57 58) . We are currently exploring the mechanism(s) by which PHB reverses oxidant-induced barrier dysfunction.

Together, our results demonstrate that increased PHB expression in intestinal epithelial cells can combat the damaging effects of oxidative stress on mucosal barrier function. Reduced levels of PHB during intestinal inflammation may be one underlying factor that leads to oxidant-induced loss of mucosal integrity and function. Since overexpression of PHB can protect against the damaging effects of oxidative stress, restoring or increasing PHB expression in IBD patients is a potential therapeutic strategy to prevent mucosal barrier disruption by increased oxidants.


   ACKNOWLEDGMENTS
 
We thank Dr. Hong Yi for technical assistance with EM. This work was supported by National Institutes of Health, Diabetes, Digestive and Kidney Diseases under a center grant (R24-DK-064399) ROI-DKO 6411 (S. Sitaraman).

Received for publication July 6, 2006. Accepted for publication August 22, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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