Articles in PresS. Am J Physiol Gastrointest Liver Physiol (July 3, 2003).10.1152/ajpgi.00412.2002
PEROXYNITRITE INHIBITS ENTEROCYTE PROLIFERATION AND MODULATES Src KINASE ACTIVITY IN VITRO
Douglas A. Potoka MD , Jeffrey S. Upperman MD , Xiao-Ru Zhang MD , Joshua R. Kaplan , Seth J. Corey MD†, Anatoly Grishin Ph.D., Ruben Zamora Ph.D. , Henri R. Ford MD
Department of Surgery, Children's Hospital of Pittsburgh, and the University of Pittsburgh School of Medicine, Pittsburgh, PA †
Department of Hematology and Oncology, Children's Hospital of Pittsburgh, Pittsburgh, PA
Acknowledgement: This work was supported by grants #RO1-AI-14032 and AI-49473 from the National Institutes of Health, Bethesda, MD
Running Head: Enterocyte Proliferation and Src Kinase Activity
Correspondence to:
Henri R. Ford, MD Children's Hospital of Pittsburgh 3705 Fifth Avenue Pittsburgh, PA 15213 Tel: (412) 692-7291 Fax: (412) 692-5008 E-mail:
[email protected]
Copyright (c) 2003 by the American Physiological Society.
1 ABSTRACT: Overproduction of nitric oxide (NO) or its toxic metabolite, peroxynitrite (ONOO-), following endotoxemia promotes gut barrier failure, in part, by inducing enterocyte apoptosis. We hypothesized that ONOO- may also inhibit enterocyte proliferation by disrupting the Src tyrosine kinase signaling pathway, thereby blunting repair of the damaged mucosa. We examined the effect of ONOO- on enterocyte proliferation and Src kinase activity. Sprague-Dawley rats were challenged with LPS or saline, while IEC-6 cells were treated with ONOO- or decomposed ONOO- in vitro. Enterocyte proliferation in vivo and in vitro was measured by BrdU or 3Hthymidine incorporation. Src kinase activity in cell lysates was determined at various times. LPS challenge in vivo, and ONOO- treatment in vitro inhibited enterocyte proliferation. ONOOtreatment blunted the activity of Src and its downstream target, FAK in a time- dependent manner. ONOO- blocked mitogen (FBS, EGF)-induced enterocyte proliferation and Src phosphorylation while increasing Src nitration. Thus, ONOO- may promote gut barrier failure not only by inducing enterocyte apoptosis, but also by disrupting signaling pathways involved in enterocyte proliferation.
Key Words: Cell Signaling, Src Kinase, Focal Adhesion Kinase
2
INTRODUCTION: Sustained overproduction of nitric oxide (NO) in the gastrointestinal tract has been demonstrated in a variety of inflammatory conditions in vivo (4, 9, 14, 26, 27, 29, 39, 40). Evidence suggests that such excess NO production may promote cellular injury and disrupt the intestinal epithelial barrier. Previously, we have shown that the inducible isoform of NO synthase (iNOS) is upregulated in the intestinal epithelium of newborn infants with necrotizing enterocolitis (NEC) (14). The iNOS protein co-localizes with enterocyte apoptosis at the villus tips and with immunoreactivity to 3-nitrotyrosine, a molecular marker of reactive nitrogen intermediates such as peroxynitrite (ONOO-), in the intestinal epithelium and lamina propria. Furthermore, iNOS upregulation, nitrotyrosine immunoreactivity and enterocyte apoptosis have also been demonstrated in conditions such as endotoxemia, inflammatory bowel disease (IBD), Helicobacter pylori gastritis, as well as in animal models of colitis and ileitis (4, 9, 12, 26, 27, 29, 39-41). These findings suggest that ONOO-, a toxic metabolite formed by the reaction of NO with superoxide, may be a key mediator of mucosal injury and gut barrier failure in vivo. One mechanism by which ONOO- may promote mucosal injury is by inducing enterocyte apoptosis. Indeed, intestinal epithelial cell apoptosis has been shown to occur in a variety of conditions associated with gastrointestinal inflammation including endotoxemia, NEC, IBD, and H. pylori gastritis (4, 14, 26, 27, 40). Administration of iNOS inhibitors or NO scavengers decreased enterocyte apoptosis and immunoreactivity to 3-nitrotorosine and ameliorated gut barrier failure after endotoxemia (12, 41). Moreover, we and others have demonstrated that ONOO- can induce enterocyte apoptosis in vitro (33, 37, 44). However, a central feature of the intestinal epithelial barrier is its ability to repair itself following mucosal injury. The integrity of
3 the intestinal mucosa depends upon the continued proliferation, migration, and differentiation of the crypt enterocytes (48). Interference with these processes may promote or exacerbate mucosal injury. For example, decreased proliferation of crypt enterocytes has been shown to be part of the characteristic morphological changes seen in the intestine following burn injury (46). We propose that in conditions associated with overproduction of NO or ONOO-, gut barrier failure may result from an imbalance between accelerated epithelial injury and blunted tissue repair mechanisms. The effect of ONOO- on the proliferation side of this equation has not been previously explored. The regulation of intestinal epithelial proliferation and differentiation is not completely understood (31). The Src family of non-receptor tyrosine kinases may play a key role in enterocyte proliferation. For example, higher levels of Src protein are detected in proliferating crypt enterocytes than in villus enterocytes, and Src activity is elevated in colon cancer cells and in areas of epithelial dysplasia in ulcerative colitis (6-8, 42). Activation of Src kinase results in phosphorylation of downstream targets such as Shc, phosphatidylinositol-3-kinase (PI3-K), and focal adhesion kinase (FAK), which then diverge along distinct signaling pathways that lead to cellular proliferation, migration or differentiation (10, 17, 38). Regulation of Src function is dependent upon Src phosphorylation at key tyrosine residues. Thus, the Src signaling pathway may be a potential site for modulation by ONOO-, since ONOO- is known to nitrate tyrosine residues (3). We hypothesized that ONOO- may inhibit enterocyte proliferation by disrupting Src kinase-dependent signaling pathways. In this study, we examined the effect of LPS on enterocyte proliferation in a rat model of endotoxemia in vivo, and the effect of ONOO- on enterocyte proliferation and Src kinase activity in vitro, using the IEC-6 rat intestinal epithelial cell line.
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Materials and Methods: Reagents ONOO- and decomposed ONOO- were obtained from Alexis Biochemicals (San Diego, CA). Recombinant human EGF was obtained from R&D Systems (Minneapolis, MN). 5-Bromo-2’Deoxyuridine (BrdU) and the superoxide generator, pyrogallol, were obtained from Sigma (St. Louis, MO). The NO donor, S-nitroso-N-acetyl penicillamine (SNAP), was synthesized from NaNO2 and N-acetyl-D, L-penicillamine (Sigma Chemical Co., St. Louis, MO) as previously described (15). Polyclonal Src antibody (sc-18), rabbit polyclonal FAK antibody, and monoclonal phosphotyrosine antibody (sc-7020) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal phospho-FAK (Tyr576/577) antibody was obtained from Cell Signaling Technology (Beverly, MA). Polyclonal nitrotyrosine antibody (06-284) was obtained from Upstate Biotechnology (Lake Placid, NY). The pMv-Src vector carrying the chicken v-Src gene under the control of the CMV promoter, was a generous gift of Dr. Ed Prochownik, Children’s Hospital of Pittsburgh (CHP, Pittsburgh). The pcDNA3.1 control vector was purchased from Invitrogen (Carlsbad, CA).
Measurement of Enterocyte Proliferation in vivo Male Sprague-Dawley rats (Harlan Sprague Dawley Inc., Indianapolis, Indiana) weighing between 250 to 300 grams were acclimatized for a minimum of 1 week prior to experimentation. The experimental protocol was approved by the Animal Research and Care Committee of the Children’s Hospital of Pittsburgh. The animals were challenged with 10 mg/kg of LPS (Escherichia coli 0127:B8, Sigma) intraperitoneally (i.p.) or with equal volume of saline (12). After 23 hr, the rats received 50 mg/kg BrdU i.p., and then were killed one hour later. Segments of terminal ileum and colon were harvested, fixed in formalin, immunostained with anti-BrdU antibody (Amersham, Arlington Heights, IL) and then counterstained with hematoxylin.
5 Enterocyte proliferation was determined by counting the number of BrdU positive cells per crypt (15 crypts) using light microscropy (Olympus BH-2 microscope).
Cell Culture The rat small intestinal epithelial cell line, IEC-6, was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were grown in tissue culture medium consisting of Dulbeco’s modified Eagle’s medium with 4.5 gm/L glucose (Bio-Whittaker, Walkersville, MD) supplemented with 5% fetal bovine serum (Bio-Whittaker), 0.02 mM glutamine (GIBCO; Grand Island, NY), 0.1 U/ml insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO) at 37 °C and 10% CO2. Cells from passages 3 through 26 were used for experiments.
ONOO- Treatment ONOO- stock solution was stored at –80 °C. The concentration of ONOO- was monitored spectrophotometrically before each experiment by measuring absorbance at 302 nm (
302 nm
= 1670 M-1cm-1). Solutions of 10 mM ONOO- in 0.3 N NaOH were used for experiments.
For all experiments, decomposed ONOO- was used for the negative control, and made in similar concentrations in 0.3 N NaOH as for ONOO-. IEC-6 cells were washed twice with Phosphatebuffered Saline (PBS; GIBCO) prior to ONOO- treatment. ONOO- or decomposed ONOO- was then added to the side of the well, and then mixed into the solution by swirling for 20 seconds.
In vitro Proliferation Assay Approximately 75% confluent IEC-6 monolayers grown in 96 well-plates were cultured in
6 serum-free medium for 48 hr. After washing the monolayers with PBS, the cells were then treated with 50 µM ONOO- or with 50 µM decomposed ONOO- for 5 or 10 min. After treatment, the culture media was replaced with regular media containing serum and 3H-thymidine (1 µCi/well). Following incubation at 37oC and 5% CO2 for 6 hr, cells were harvested and 3H-thymidine incorporation was measured by liquid scintillation spectrometry.
To determine the effect of ONOO- on FBS- or EGF-induced proliferation, 75% confluent IEC-6 monolayers in 2-chamber slides were grown in serum-free tissue culture medium at 37 °C and 10% CO2 for 48 hr. The monolayers were washed twice with PBS, and then treated with 50 µM ONOO- or with 50 µM decomposed ONOO- for 10 min. The monolayers were then washed with 1 ml of PBS, and then treated with 1 ml of regular tissue culture medium, 1 ml of serum-free tissue culture medium, or 1 ml of serum-free medium plus 200 ng/ml human recombinant EGF or 5% FBS for 4 hr at 37 °C and 10% CO2. Following this incubation, 10 µM BrdU was added to each well for one hour at 37 °C and 10% CO2. After incubation, the upper chambers were removed from the slides, and BrdU incorporation was analyzed immunohistochemically using a commercially prepared BrdU staining kit (Zymed; San Francisco, CA). Slides were examined using a Olympus BH-2 microscope. The degree of proliferation was defined as the proportion of BrdU positive cells per 5-10 high-power fields.
Src Kinase Activity Assay IEC-6 monolayers grown to approximately 75 % confluence were washed with PBS twice and then treated with buffer or with 50 µM ONOO- or decomposed ONOO- for 1, 5, 15, and 30 min. After each treatment time, cells were isolated from the plate by scraping, washed in PBS and
7 resuspended in 200 µl of lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 1% NP-40, 10% glycerol, 10 ng/ml aprotinin, 10 ng/ml leupeptin, 2 mM sodium orthovanadate, and 1 mM PMSF) for 30-45 min at 4 °C. The supernatant was collected after centrifugation at 9,500 x g for 20 min. Protein concentration was determined using the bicinchoninic acid (BCA) assay. For each sample (200 µg protein in 200 µl of lysis buffer), 1 µl of Src polyclonal antibody was added for overnight immunoprecipitation of Src at 4 °C. To each sample, 15 µl of Protein A sepharose beads were then added for a 1 hr. incubation, with constant turning at 4 °C. The beads were then pelleted by microcentrifugation for 20 seconds. The beads were then washed twice with PBS, 1% NP-40 (1 ml per wash) at 4 °C, followed by two washes with TNE buffer (50 mM Tris, 100 mM NaCl, and 1 mM EDTA) at room temperature. Following washes, 20 µl of kinase assay buffer (HEPES, pH 7.4, MgCl2, MnCl2, and 32P- ATP) was added to each sample for a 30 min incubation at room temperature. Following this incubation, 30 µl of Laemmli sample buffer was added to each sample. Each sample was then boiled for 5 min, and samples were loaded on a 10% polyacrilamide gel and run overnight. Phosphorylated proteins were detected by autoradiography. This assay detects the phosphorylation of immunoprecipitated Src, and thus determines the degree of Src activation. For studies examining the effect of ONOO- on EGF or FBS induction of Src activity, IEC-6 cells were grown to approximately 75% confluence, as described above. The cells were first serum-starved in serum-free medium at 37 °C and 10% CO2 for 48 hr. The cells were washed twice with PBS, and then treated with 50 µM ONOO- or with 50 µM decomposed ONOO- for 10 min. After treatment, the monolayers were washed with 1 ml of PBS, and then replaced with 5 ml of serum-free medium or with 5 ml of serum-free medium plus 200 ng/ml
8 human recombinant EGF or 5% FBS for 15 min at 37 °C and 10% CO2. Src kinase assays were then performed as described above.
Transfection experiments IEC-6 cells grown on 100 mm Petri dishes to approximately 75% confluence were transfected with 10 µg plasmid DNA using the Lipofectamine reagent (Invitrogen) as directed by the manufacturer. IEC-6 cells were transiently transfected with pMv-Src or pcDNA3.1 vector control. After 24 hr, cells were collected and seeded into 96-well plates at a density of 10,000 cells per well. After cells attached, they were starved in serum-free medium for 48 h. 3Hthymidine (1 µCi/well) was then added together with ONOO- (50 µM), or equivalent amount of decomposed ONOO-, or Src family inhibitor PP1 (10 µM), or solvent alone. Following incubation at 37oC and 5% CO2 for 6 hr, cells were harvested and 3H-thymidine incorporation was measured by liquid scintillation spectrometry.
Apoptosis Assay Apoptosis was determined using flow cytometry with annexin V-FITC and propidium iodide (PI) staining as previously described (33, 47).
Western Blot Analysis Proteins from cell monolayers were electropheresed on 8 to 12% SDS-PAGE gels in a Bio-Rad minigel apparatus and blotted onto nitrocellulose membranes as previously described (33). After the membranes were blocked for 1 hr with 5% milk in PBS with 0.1% Tween 20, blots were probed for 1 hr at room temperature with primary antibody (1:500 dilution). As
9 secondary antibody, horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse IgG was used and detection was performed by enhanced chemiluminescence (Pierce, Rockford IL).
Immunoprecipitation Treated cells in 100 mm petri dishes or 6-well plates were washed twice with ice-cold PBS and lysed in a total of 200 µl of lysis buffer per sample. After incubation at 4 °C with turning for 30-45 min, the supernatant was collected after centrifugation at 9,500 x g for 20 min. Aliquots of protein (150 to 200 µg) were prepared in 200 µl lysis buffer. Primary antibody (5 µg) was added to each sample for a one hour incubation with constant turning at 4 °C, followed by addition of 25 µl of either protein A sepharose or protein A/G sepharose (based on primary antibody used) to each sample for an overnight incubation with constant turning at 4 °C. The beads were then pelleted by centrifugation at 350 x g for 5 min at 4 °C. The beads were washed four times with lysis buffer at 4°C. After the final wash, the beads were resuspended in 30 µl of Laemmli sample buffer, boiled for 3 min, and electrophoresed on 8 to 12% SDS-PAGE gels as described above. Western blot analysis was completed as described (33).
Statistical Analysis All data are expressed as means ± standard errors of the means (SEM). For multiple comparisons, analysis of variance (ANOVA) was used to determine significant differences between groups. Significant differences between individual groups were determined using Fisher’s exact probability test. P-values less than or equal to 0.05 were considered significant.
RESULTS:
10 Effect of LPS challenge on enterocyte proliferation in vivo We have previously demonstrated that administration of LPS to Sprague-Dawley rats resulted in intestinal iNOS upregulation, enterocyte apoptosis, and gut barrier dysfunction (12, 41). Administration of an iNOS inhibitor or a NO scavenger ameliorated the derangement in epithelial barrier function and reduced enterocyte apoptosis and immunoreactivity to 3nitrotyrosine. To determine whether endotoxemia also impaired enterocyte proliferation, we measured BrdU incorporation in the intestinal epithelium of rats challenged with 10 mg/kg LPS or with an equal volume of saline. The number of actively proliferating (BrdU positive) cells per crypt (15 crypts analyzed) in the terminal ileum was 0.14 ± 0.38 in LPS-treated rats compared to 4.45 ± 1.92 in saline-treated rats (p250 µM) inhibited activity of Src kinase family members (25). Thus, the effect of ONOO- on Src activity may be concentration-dependent and cell type-dependent. In contrast to these previous reports, we also correlated our Src kinase activity results with corresponding changes in cellular proliferation. Although there are estimates of the rate of ONOO- production by isolated alveolar macrophages in vitro, the complex biochemistry and short half-life of ONOO- make it difficult to measure rates of intestinal mucosal ONOO- production during inflammation (18). In addition,
20 because of the short half-life and resultant limited diffusion of ONOO-, the local concentration of ONOO- to which any specific enterocyte within the intestinal mucosa will be exposed may vary widely. Thus, the choice of ONOO- concentrations to use for experiments is largely empiric. We have used a concentration of ONOO- (50 µM) which is significantly lower than that used by other investigators in different cell types because the lower concentrations of ONOO- are more likely to be physiologically relevant. In addition, we developed a treatment regimen that did not induce apoptosis in our IEC-6 cells, and therefore did not confound the effects of ONOO- on proliferation and cell signaling pathways. In summary, we have shown that ONOO- inhibits proliferation and blocks the proliferative response to FBS, EGF and v-Src transfection in an enterocyte cell line. These events are associated with decreased Src and FAK phosphorylation. Thus, in addition to causing mucosal injury by inducing enterocyte apoptosis, ONOO- can contribute to mucosal inflammation and gut barrier dysfunction by inhibiting enterocyte proliferation via the Src kinase signaling pathway.
21
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26 FIGURE LEGENDS Figure 1: Effect of ONOO- on IEC-6 cell proliferation. IEC-6 cells grown to 75% confluence were cultured in serum-free medium for 48 hr. The monolayers were then treated with 50 µM ONOO- or with 50 µM decomposed ONOO- for 5 min. After treatment, the culture media was replaced with regular media containing serum and 3H-thymidine and incorporation was measured as described in Methods. An experiment is shown (mean of 5 wells) that is representative of 3 independent experiments with similar results. Treatment with 50 µM ONOOsignificantly inhibited IEC-6 cell proliferation (* p=0.001 vs. buffer and decomposed ONOO-).
Figure 2: Effect of ONOO- on IEC-6 cell apoptosis. IEC-6 cells were treated with 62.5, 125, 250 or 500 µM SNAP for 12 hr followed by the addition of 1 mM pyrogallol or buffer control for 24 hr. Apoptosis was determined via flow cytometry with annexin V-FITC and propidium iodide staining (n=5 experiments). There was a significant increase in IEC-6 apoptosis in cells treated with a combination of SNAP plus pyrogallol (* p
