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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Departments of Anatomy and Physiology (N.N.R., L.C.F., S.G.) and Clinical Sciences (K.S., D.N., K.W., J.D.L.), College of Veterinary Medicine, Kansas State University, Manhattan, Kansas
Received June 26, 2007; accepted February 14, 2008.
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Abstract |
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; Tomisato et al., 2004). Despite exhaustive investigation, the mechanisms responsible for NSAID-associated GI damage are not completely understood. Evidence gathered to date suggests that NSAIDs may promote ulcer formation not only by inhibiting mucosal cyclooxygenase (COX) and decreasing cytoprotective prostaglandins (PGs) but also by adversely influencing intestinal microflora, neutrophil recruitment, surface hydrophobicity, and epithelial restitution (Lichtenberger, 2001; Little et al., 2007). Although the inhibition of COX isoforms has received much attention and investigation as the basis of GI toxicity, it is acknowledged widely that COX inhibition cannot account for the complete spectrum of NSAID-induced toxicity seen clinically (Lichtenberger, 2001; Tomisato et al., 2004; Little et al., 2007).
The GI epithelium has several functions, the most important of which are uptake of nutrients and separation of the internal milieu from the external environment. Surface epithelial cells are continuously exposed to noxious agents and abrasive ingesta that may cause mucosal injury. In general, superficial mucosal defects are repaired rapidly by migration of epithelial cells from proliferative zones into the wound. This process, mucosal restitution, represents a primary repair modality in the GI tract and allows resealing of the epithelial barrier within minutes or hours via reformation of tight junctions between cells (Dignass, 2001). Restitution is a well coordinated event that is dependent on cell migration but independent of cell proliferation and differentiation (Dignass, 2001).
The migration of intestinal epithelial cells is modulated by a wide range of cytoplasmic effectors associated with a number of signaling pathways (Dignass, 2001; Pai et al., 2001). Cellular components involved in the regulation of resting membrane potential, intracellular calcium dynamics, focal adhesion components, and cytoskeletal integrity have been identified as common sites of action for interventions that impair intestinal restitution and inhibit ulcer healing (McCormack and Johnson, 2001; Pai et al., 2001; Rao et al., 2002). It is unfortunate that there is a lack of knowledge about the global effects of NSAIDs on the components of these complex signal transduction networks. Our work presented here and elsewhere extends knowledge about the mechanisms underlying the adverse GI effects of NSAIDs. In a recent study, we have demonstrated that inhibition of cell migration by NSAIDs is related to drug effects on potassium channel expression and trafficking (Freeman et al., 2007). The current study was conducted to characterize the effects of NSAIDs on gene expression and to identify novel mediators of NSAID toxicity.
To address these goals, IEC-6 cells were exposed to a series of NSAIDs with variable potential for GI toxicity and known effects on cell migration. Total RNA was isolated, and transcriptional responses were evaluated using high-density cDNA array analysis. Our results suggested that NSAIDs with adverse effects on epithelial restitution affect the expression of genes associated with signaling pathways that influence the migration of intestinal epithelial cells. Specific targets of NSAID activity included the calpains, cysteine proteases involved in numerous cellular processes such as cell migration and invasion. The work presented herein demonstrates the power of microarray analysis to provide new insights into the molecular basis of NSAID toxicity.
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Materials and Methods |
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Treatment Protocols. We assessed the effects of NSAIDs with variable ulcerogenic potential on gene expression. Experimental treatments included: vehicle control (0.1% DMSO), indomethacin (10 or 100 µM), NS-398 (10 or 100 µM), or SC-560 (1 µM). The conditions used in this investigation are based on previous work documenting predictable effects on cell migration at noncytotoxic concentrations of drug.
Indomethacin is a nonselective COX inhibitor that has been shown to induce ulcers in drug-treated animals (Tavares, 2000; Kato et al., 2001; Peskar et al., 2001). We have shown previously that chronic (72 h) exposure to 100 µM indomethacin decreases cell migration in wounded intestinal epithelial monolayers (Freeman et al., 2007), and others have demonstrated that 250 to 500 µM indomethacin inhibits re-epithelialization of wounded gastric epithelial cell monolayers (Pai et al., 2001). The highest concentration of indomethacin used in this study (100 µM) is consistent with plasma and tissue drug concentrations achieved after administration of therapeutic doses to rats and humans (Frey and El-Sayed, 1977; Suzuki et al., 1997; Kokoska et al., 1998). Moreover, this concentration of indomethacin has been shown to inhibit cellular PGE2 synthesis in vitro without affecting cell viability or inducing apoptosis in monolayer cultures of epithelial cells (Kokoska et al., 1998; Tavares, 2000; Pai et al., 2001; Tomisato et al., 2004).
NS-398 is a relatively selective inhibitor of COX-2 that is used experimentally (Tavares, 2000; Kato et al., 2001). Administration of NS-398 alone does not induce gastric lesions in drug-treated animals (Tomisato et al., 2004); however, this drug has been shown to delay ulcer healing (Brzozowski et al., 2001; Peskar et al., 2001). We have shown previously that chronic exposure (72 h) to 100 µM NS-398 decreases cell migration in wounded intestinal epithelial monolayers (Freeman et al., 2007), and others have shown comparable inhibition of re-epithelialization of wounded gastric epithelial cell monolayers (Pai et al., 2001). The highest concentration of NS-398 used in the current investigation (100 µM) has been shown to inhibit synthesis of PGE2 in vitro without affecting viability or inducing apoptosis in cultured epithelial cells (Tavares, 2000; Pai et al., 2001; Tomisato et al., 2004).
SC-560 (1 µM) inhibits COX-1 but not COX-2 activity in vitro (Smith et al., 1998). SC-560 does not induce ulcers in animals treated with dosages sufficient to inhibit mucosal PGE2 production (Peskar et al., 2001; Tanaka et al., 2001). We have shown previously that chronic exposure (72 h) to 1 µM SC-560 has no inhibitory effects on cell migration in wounded intestinal epithelial monolayers (Freeman et al., 2007).
Cell Culture. The IEC-6 cell line, developed by Quaroni et al. (1979), was purchased from the American Type Culture Collection. IEC-6 culture conditions were similar to those described previously (Freeman et al., 2007). The basic culture medium consisted of DMEM supplemented with heat-inactivated fetal bovine serum (5%), insulin (10 µg/ml), and gentamicin (50 µg/ml). Cells were maintained in 75-cm2 tissue culture flasks at 37°C in a humidified atmosphere of 5% CO2 in air. Cell passages 16 to 20 were used for all experiments to minimize the effects of passage. Cells were seeded at a density of approximately 6.25 x 104 cells/cm2 on 35-, 60-, or 100-mm plates thinly coated with Matrigel (BD Biosciences) and were grown to a confluent monolayer prior to the introduction of media containing experimental treatments. Cells were treated for 72 h with NSAIDs or the vehicle control prior either to harvesting RNA for microarray and qRT-PCR analysis or to lysing cells for immunoblot analysis.
Additional experiments were performed using IEC-Cdx2 cells (Suh and Traber, 1996). In this stably transfected cell line, the forced expression of the Cdx2 gene in IEC-6 cells induces a differentiated phenotype. The LacSwitch expression vector system (Stratagene, La Jolla, CA) is used to direct conditional expression of the Cdx2 gene, with isopropyl β-D-thiogalactoside serving as the inducer for gene expression. IEC-Cdx2 cell stocks were maintained in the same basic culture medium used for IEC-6 and grown in media supplemented with 4 mM isopropyl β-D-thiogalactoside for 16 days to induce the conditional expression of Cdx2 before experiments.
Affymetrix Gene Chip Analysis. RNA was isolated from treated IEC-6 cells using a commercially available kit (RNeasy Micro Kit; QIAGEN, Hilden, Germany). The quality and quantity of RNA were assessed by microfluidic electrophoresis (Bioanalyzer; Agilent Technologies, Palo Alto, CA) and a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE), respectively. Total RNA (10 µg) was processed and hybridized to separate high-density nucleotide gene chips for microarray analysis according to the manufacturer's standards. The gene chip used, Rat RAE 230-2.0 (Affymetrix, Santa Clara, CA), provides comprehensive coverage of the rat genome by interrogating over 31,042 transcripts and variants including more than 28,000 well documented rat genes. Sample processing for RNA amplification, cDNA synthesis, labeling, and hybridization were carried out using the Small Sample Labeling Protocol version II developed by the Kansas University Medical Center-Microarray Facility (http://www2.kumc.edu/siddrc/microarray/Protocols.html). Probe arrays were scanned at a wavelength of 570 nm with a gene array scanner (Agilent, Microarray Core-Kansas University Medical Center, KS). All array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches on the chip surface. The methodology followed complies with the MIAME (Minimum Information about a Microarray Experiment) guidelines, which are provided at http://www.mged.org/Workgroups/MIAME/miame.html. The control and experimental conditions (DMSO vehicle control, indomethacin, NS-398, and SC-560) were repeated four times to ensure a robust analysis.
Signal intensities were quantified by pixel intensity, and expression signals were analyzed using the Affymetrix Data Acquisition Software, Gene Chip Operating Software 1.4 (Affymetrix). Statistical algorithms [detection, change call, signal log ratio (SLR)] were then used to identify differential gene expression in control and experimental samples. Data were imported into the Ingenuity Pathway Analysis (IPA) program (Ingenuity Systems Inc., Redwood City, CA; http://www.ingenuity.com/products/pathways_analysis.html).
Probe sets were assigned as either present (P), absent (A), or marginal (M) based on detection p values (P, p < 0.05; M, p = 0.05–0.065; A, p > 0.065). Additional analysis was conducted if and only if three of four samples in either the control or experimental groups were called present. Pair-wise comparisons between individual experimental and control arrays were made to generate an SLR value for each transcript. Student's t test was done between the SLR values to determine the significance of these genes (using a p value of 0.05). -Fold change was calculated from the median of the SLRs; the cut-off for -fold change was 1.5 for up- or down-regulated genes. This analysis by SLR cancels out the differences in individual intensities at the probe level.
IPA was used to analyze the data in the context of molecular mechanisms and to identify the signaling networks altered by NSAID treatment in an unbiased, gene-by-gene fashion. IPA uses a curated database and analysis system designed to determine how proteins work together to effect cellular changes. Using a global approach, IPA was used first to analyze common and distinct properties of all altered genes in relationship to one another. Identified genes were next mapped to the functional networks available in the IPA database and ranked by score. The score is the probability that a collection of genes equal to or greater than the number in a network could be achieved by chance alone. Initial results were mined further to detect alterations in the expression of mRNA transcripts encoding proteins related to the maintenance of cell migration.
Confirmation of Differential Gene Expression. Quantitative RT-PCR was used to confirm changes in mRNA expression levels detected by microarray analysis for selected genes. These included calpain 8, calpain 2, calpain 1, and calpastatin; 18S served as the internal standard. Primers were designed based on the reported rat sequences with the assistance of the Primer3 software (Whitehead Institute for Biomedical Research, Cambridge, MA). The primers used in these experiments (Invitrogen and Integrated DNA Technologies, Inc., Coralville, IA) are listed in Table 1.
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The SuperScript III Platinum One-Step Quantitative RT-PCR system (Invitrogen), in combination with sequence specific primers and a dual-labeled fluorogenic probe (Integrated DNA Technologies), was used for the analysis of calpastatin message. The 18S primers were obtained from a commercially available kit (TaqMan, ribosomal RNA; Applied Biosystems, Foster City, CA), The real-time cycling protocol consisted of first-strand cDNA synthesis for 15 min at 50°C, initial activation for 2 min at 95°C, and then 40 cycles of denaturation for 15 s at 95°C, followed by annealing/extension for 30 s at 60°C. Single products of appropriate size were demonstrated by gel electrophoresis in the presence of ethidium bromide. All RT-PCR reactions were duplicated for each experimental condition from three to four separate RNA isolations.
Before quantitative analysis, 10-fold dilutions of RNA were used as template for each primer pair to determine their amplification efficiency. The resulting Ct values were then fit by linear regression to a logarithmic scale. The slope was then used to determine primer efficiency using eq. 1:
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Differences in the mRNA levels between experimental conditions were determined using the 
Ct values obtained from our qRT-PCR experiments in conjunction with the calculated efficiencies of each primer set. Using eq. 2:
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CtGOI is the Ct for the gene of interest, and Ct18S is the Ct for the 18S internal controls, we compared the relative expression levels of calpains 8, 2, and 1 and calpastatin for the following treatment groups: DMSO vehicle control, indomethacin, NS-398, and SC-560. 18S served as the internal standard. Western Blot Analysis. Whole cell lysates were collected using a commercially available isolation buffer (radioimmunoprecipitation assay; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and protein content was determined by Micro BCA Protein Assay (Pierce, Rockford IL). Cell lysates were separated by SDS-polyacrylamide gel electrophoresis, transferred to membranes, and probed sequentially with appropriate primary and secondary antibodies. Primary antibodies were obtained commercially (Triple Point Biologic, Forest Grove, OR) and used at the following dilutions: calpain 8, 1:750; calpain 2, 1:500; calpain 1, 1:500; and calpastatin, 1:750. Primary antibodies were diluted into the blocking buffer [5% nonfat milk in Tris-buffered saline with Tween 20 (0.1%)]; Tris-buffered saline was made from a commercially available 10x stock (Bio-Rad, Hercules, CA; no. 170-6435). Membranes were incubated in primary antibody overnight at 4°C. Stabilized goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Pierce) was diluted 1:1000 into the blocking buffer, and membranes were exposed to secondary antibody for 1 h at room temperature. Immunocomplexes were visualized using an enhanced chemiluminescence detection system (SuperSignal West Femto Maximum Sensitivity Substrate; Pierce).
Equal loading of protein was confirmed by stripping and reprobing membranes with antibody directed against actin (Sigma-Aldrich). Our previous work validated the use of actin as a loading control in experiments that included NSAID-treated IEC-6 monolayers (Freeman et al., 2007). Densitometry was performed using imaging software (AlphaEaseFC; Alpha Innotech, San Leandro, CA). The expression of calpains 8, 2, and 1 and calpastatin was normalized to actin for comparisons between treatment groups. Summary data shown is based on Western blots performed using cell lysates from at least three independent experiments using all treatment groups.
Cell Migration Assay. The effect of calpain inhibition on IEC-6 cell migration was assessed using an established wounding assay (Freeman et al., 2007). In brief, cells were seeded on Matrigel basement membrane until a confluent monolayer was established. A razor blade was used to sharply remove approximately one-third of the monolayer and create a border line (scratch line) after 24 (calpain inhibitors) or 72 (NSAIDs) h of exposure to media containing either vehicle or a treatment. Micrographs (Nikon ACT-1; Nikon, Tokyo, Japan) were captured after creation of this defect and 4 h later. Cell migration was measured in these captured images using analysis software to document restitution (Nova Prime; Bioquant, Nashville, TN). A standardized rectangular region of interest (31,200 µm2) was created and positioned at the scratch line; the percentage of this region occupied by migrating cells was measured over time. Two to three fields per replicate were examined and measured on three different monolayers per treatment.
The treatments used included the NSAIDs described previously and the calpain inhibitors N-acetyl-Leu-Leu-Nle-CHO (ALLN) and N-acetyl-Leu-Leu-methioninal (ALLM), alone and in combination at the concentrations indicated in the figure legends. The extent of migration postwounding for various treatments was normalized to the appropriate drug-free control for presentation to facilitate comparisons. The vehicle control for the calpain inhibitors was 0.1% ethanol in experiments where these drugs were used alone and 0.01% ethanol in experiments where the calpain inhibitors were used in combination with NSAIDs.
Statistical Analysis. Microarray experiments were performed in quadruplicate, and statistical analyses were performed as described in the previous sections. The data resulting from qRT-PCR (-fold change), immunoblot analysis, and migration assays are expressed as mean ± S.E. Significant differences between treatment groups were identified by one-way analysis of variance, and multiple comparisons were made using the least significant difference procedure (Statistix; Analytical Software, Tallahassee, FL). Differences were considered to be significant when p < 0.05. The numbers of replicates per treatment group and independent experiments associated with specific studies are provided in the figures or the accompanying legends. Data in graphs are presented as mean ± S.E.M.
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Ingenuity Pathway Analysis. Genes identified as significantly influenced by NSAIDs were mapped to the functional networks available in the IPA database and ranked by score. The top network affected by indomethacin had a score of 17 and contained 11 focus genes that exhibited ±2.0-fold change (Supplemental Fig. S1). The top three functions of this network were cellular movement, cell morphology, and cell-to-cell signaling and interaction. The top network influenced by NS-398 had a score of 18 and contained 12 focus genes that exhibited ±2.0-fold change (Supplemental Fig. S2). The top three functions of this network were lipid metabolism; DNA replication, recombination, and repair; and developmental disorder. The top network affected by SC-560 had a score of 3 and contained one focus gene that exhibited ±2.0-fold change. The top three functions of this network were cell-to-cell signaling and interaction, tissue development, and cell morphology. The genes present in the top networks affected by indomethacin, NS-398, and SC-560 and the associated -fold changes are listed in Table 2.
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There were a few genes common to the top networks influenced by the two NSAIDs known to inhibit intestinal cell migration. For example, microarray analysis showed 
–2.5-fold down-regulation of transgelin and pleiotrophin and +2.5-fold up-regulation of fatty acid binding protein 4 by both indomethacin and NS-398. It was also notable that the NSAIDs with inhibitory effects on cell migration altered the expression of genes involved in calpain-dependent signaling. NS-398 caused –3.2-fold change in the expression of calpain 8 and less dramatic down-regulation of other calpains in the top network, calpain 2 and the calpain small subunit 1. Calpain 1 was not part of the NSAID-affected networks identified by IPA; however, the calpain 1 gene was present and apparently down-regulated –1.3-fold by NS-398. Indomethacin was associated with a +1.4-fold change in the expression of calpastatin, the endogenous inhibitor of the calpain proteases.
Additional experiments were conducted to define further the effects of NS-398, indomethacin, and SC-560 on the expression of calpain 8, calpain 2, calpain 1, and calpastatin. The decision to pursue this line of inquiry was influenced by our functional data. Inhibition of calpains in migrating cells has been shown to produce distortions to the migrating front with retention of cellular protrusions at the rear of migrating cells (Lokuta et al., 2003; Franco et al., 2004). It is interesting to note that NSAID treatment induced this suite of morphological changes in migrating IEC-6 cells (Fig. 1).
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The data obtained from qRT-PCR assay of calpain 1 mRNA expression exhibited more variability than those obtained for either calpain 8 or calpain 2. As a result, two series of experiments using different primer sets were performed (Table 1). Neither demonstrated a significant effect on NSAID treatment on calpain 1 expression; the results obtained using primer set 2 are featured in Fig. 2C. None of the NSAIDs tested affected significantly the expression of calpastatin mRNA (p > 0.05; data not shown).
Western Blots. To further characterize the influence of NSAIDs on the expression of the calpain family of proteins, we examined the relative expression of calpain 8, calpain 2, calpain 1, and calpastatin proteins in IEC-6 cells exposed to indomethacin, NS-398, and SC-560. NS-398 and indomethacin inhibited the protein expression of calpains 8 and 2 in IEC-6 cells when used at concentrations of either 100 (Fig. 3) or 10 (Supplemental Fig. S3) µM. Indomethacin inhibited expression of calpain 1 when used at 100 but not 10 µM, whereas NS-398 inhibited expression of calpain 1 when used at either concentration. There was no change in the protein expression of calpastatin after treatment with any NSAID at the highest concentration tested (Supplemental Fig. S4).
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The Effect of Calpain Inhibition on Cell Migration. Concentration-response relationships were established for NS-398, indomethacin, and two calpain inhibitors, ALLM (also known as calpain inhibitor II) and ALLN (also known as calpain inhibitor I). Concentration-dependent inhibition of cell migration was observed in IEC-6 monolayers exposed to either ALLN or ALLM (Fig. 4). Moreover, migrating cells in the monolayers exposed to these calpain inhibitors appeared to have retained cell membrane protrusions at the rear of the migrating front, suggestive of inhibition of adhesion complex turnover (Fig. 4). As expected from our previous work (Freeman et al., 2007), 72 h of exposure to either indomethacin or NS-398 also inhibited IEC-6 cell migration in a concentration-dependent manner (Fig. 5).
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Comparison of IEC-6 with IEC-cdx2 Cells. IEC-6-Cdx2 cells exhibit the ultrastructural characteristics of differentiated villus enterocytes and an increased rate of migration in response to wounding compared with the parent IEC-6 cell line (Rao et al., 2002). We have shown previously that NSAIDs affect cell migration similarly in IEC-6 and IEC-Cdx2 cells (Freeman et al., 2007). We were interested in ascertaining whether NSAIDs also influence the expression of calpain proteases in these more differentiated intestinal epithelial cells. To this end, immunoblot analysis was used to assess the expression of calpain protein in IEC-Cdx2 monolayers exposed to NSAIDs for 72 h. As shown in Fig. 6, indomethacin and NS-398, but not SC-560, decreased the expression of calpains 8, 2, and 1 by IEC-Cdx2 cells.
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). Efforts to develop less toxic NSAIDs led to the introduction of COX-2 inhibitors with decreased GI complications; however, epidemiological data show that the GI benefits of these drugs may be offset by the increased cardiovascular toxicity associated with selective inhibition of COX-2 (Motsko et al., 2006). The development of safe, effective NSAIDs will remain elusive until the multi-factorial basis for the drugs' GI toxicity is better understood. To this end, we used a multistep functional genomic strategy to identify novel signaling pathways that contribute to NSAID inhibition of GI epithelial cell migration.
We used a series of techniques to identify changes in mRNA and protein expression induced in IEC-6 monolayers by three NSAIDs with variable pharmacodynamic profiles and established effects on epithelial migration (Freeman et al., 2007). First, high-density microarray analysis was used to screen for novel effects of NSAIDs on gene expression. In particular, we sought to identify changes in gene expression associated with exposure to indomethacin and NS-398, two NSAIDs that inhibit epithelial migration in vitro and contribute to ulcer persistence in vivo, but unassociated with exposure to SC-560, an NSAID without adverse effects on GI epithelial migration. IPA facilitated the interpretation of the microarray findings by enabling data analyses in context of biologically relevant signaling pathways. Microarray analysis was followed by qRT-PCR and immunoblotting to confirm changes in the mRNA and protein expression of selected genes. Finally, pharmacological antagonism of candidate genes provided additional support for their functional significance.
The IEC-6 cell line was used for a number of reasons. First, these nontransformed cells have been used extensively to study intestinal epithelial cell migration, and there is abundant knowledge about the signal transduction pathways involved in their migration (McCormack and Johnson, 2001; Guo et al., 2002; Rao et al., 2002; Freeman et al., 2007). In addition, the IEC-6 cell line has been used previously by laboratories investigating wound healing through microarray analysis, and, as a result, genomic alterations have been linked to protein expression and/or to phenotypic change (Hafner et al., 2005; Liu et al., 2005). Finally, we have documented previously the effects of NSAIDs on IEC-6 cell migration under experimental conditions identical to those used here (Freeman et al., 2007).
The high-density microarray and IPA revealed three genes common to the top networks influenced by indomethacin and NS-398: pleiotrophin (–2.5-fold down-regulation), transgelin (–2.5-fold down-regulation), and fatty acid binding protein 4 (+2.5-fold up-regulation). These genes have not been identified in previous analyses of wound healing-associated genes in IEC-6 cells (Hafner et al., 2005; Liu et al., 2005). However, there are some functional data implicating these proteins in cell migration, invasion, and wound healing (Gunnersen et al., 2000; Ohlsson et al., 2005; Martin et al., 2006). Although pleiotrophin, transgelin, and fatty acid-binding protein 4 represent potential drug targets, we did not investigate their roles in NSAID inhibition of intestinal epithelial migration.
We were more intrigued by the changes induced by NSAIDs in the expression of genes encoding the calpain proteases and their endogenous inhibitor calpastatin. The calpain family of cysteine proteases regulates many cellular processes, including migration (Franco and Huttenlocher, 2005). Calpains are involved in cell migration specifically through directing the disassembly/reassembly of cytoskeletal elements and cell-cell adhesions, thereby permitting cells to flatten, spread, and detach rear adhesions (Franco and Huttenlocher, 2005). Microarray analysis suggested that NS-398 induced a 3-fold down-regulation of calpain 8 and less dramatic down-regulation of other calpains and the calpain small subunit 1. Indomethacin had no apparent effect on the expression of calpain mRNAs but did up-regulate the expression of calpastatin by +1.4-fold.
We attempted to validate drug-induced changes in the expression of calpains 8, 2, and 1 as well as calpastatin using qRT-PCR and immunoblotting to assess expression of mRNA and protein. We focused on those signaling molecules, either because of their expression in the gastrointestinal tract or because of their association with signal transduction in migrating cells (Franco and Huttenlocher, 2005; Hata et al., 2006). The results of qRT-PCR validated our microarray findings that NS-398 induced down-regulation of calpain 8 and calpain 2 mRNA. However, we were unable to demonstrate either down-regulation of calpain 1 mRNA by NS-398 or up-regulation of calpastatin mRNA by indomethacin using this technique.
Many factors may contribute to inconsistencies between the results obtained by microarray versus qPCR. In a systematic study of these factors, lower correlations between microarray and qPCR data were found for genes exhibiting low levels of change (<1.4-fold) than for those showing more significant up- or down-regulation (Morey et al., 2006). This may have contributed to our inability to correlate qRT-PCR data with microarray data for calpain 1 and calpastatin, given that the microarray analysis indicated only –1.3-fold down-regulation of calpain 1 by NS-398 and +1.4-fold up-regulation of calpastatin by indomethacin. The contribution of other factors cannot be ruled out, although care was taken to minimize problems associated with experimental variables such as RNA quality and primer efficiency.
Western blotting was used subsequently to assess the effects of NSAIDs on the protein expression of calpain and calpastatin because determination of mRNA levels by either microarray or qPCR analysis may not accurately predict protein expression. Large-scale studies that have examined the correlation between mRNA and proteins across gene products and cell line have shown significant discrepancies that do not result from measurement errors, but rather reflect the biology of gene expression (Tew et al., 1996; Tian et al., 2004). Protein expression is regulated not only at transcription, but also post-transcription, at translation and post-translation.
We observed NSAID-induced changes in protein expression that were both congruent and incongruent with the measurements of mRNA. Most significantly, we found that indomethacin and NS-398, the NSAIDs that inhibit epithelial cell migration, decreased the expression of calpain 8, calpain 2, and calpain 1 proteins in IEC-6 cells. In contrast, SC-560, the NSAID without adverse effects on migration, had no effect on the expression of calpain protein by IEC-6 cells. Similar results were obtained using the more differentiated intestinal epithelial cell line IEC-Cdx2. Functional data were also consistent with decreased expression of calpain protein in cells treated with either NS-398 or indomethacin because both drugs also induced morphologic changes suggestive of retained adhesions at the rear of migrating IEC-6 cells. This phenomenon was documented previously in fibroblasts and neutrophils after inhibition of calpain expression or activity (Lokuta et al., 2003; Franco et al., 2004).
We demonstrated that pharmacological inhibition of calpains retarded IEC-6 migration in a fashion similar to that seen after exposure to indomethacin and NS-398. The calpain inhibitors ALLM and ALLN decreased IEC-6 cell migration in a concentration-dependent manner. Moreover, the morphological changes associated with calpain inhibition were similar to those seen with NS-398 and indomethacin. When IEC-6 cells were treated with ALLM or ALLN in combination with either NS-398 or indomethacin, the drug effects on cell migration were additive, as anticipated from the mechanisms of action. NS-398 and indomethacin affect calpain activity by decreasing the expression of calpain proteins. ALLM and ALLN antagonize calpain activity by binding to the active site on the protease domain of expressed protein. The combined effects of NSAIDs and calpain inhibitors reported in this investigation are consistent with but do not prove the hypothesis that calpain inhibition may contribute to NSAID antagonism of GI epithelial migration.
To our knowledge, this is first report demonstrating decreased calpain expression in intestinal epithelial cells treated with NS-398 and indomethacin. However, at least three previous studies have described decreases in calpain expression, release, or activity after treatment with NSAIDs. In studies using chondrocytic cells, NS-398 suppressed TNF-
-stimulated expression and release of calpain, whereas indomethacin suppressed TNF--stimulated release of calpains (Fushimi et al., 2004). In that investigation, the mechanism whereby the NSAIDs stimulated calpain release was presumed to depend on COX inhibition because PGE2 and EP2 agonists were shown to accelerate calpain release. The mechanisms underlying NSAID suppression of TNF--stimulated calpain expression were not addressed experimentally (Fushimi et al., 2004). In another, unrelated investigation, indomethacin was shown to reduce the activity of spinal cord calpains in vitro (Banik et al., 2000). In that work, the concentrations of indomethacin required to suppress calpain activity were in the millimolar range and thus higher than the concentrations typically needed to inhibit COX. It is interesting to note that indomethacin showed similar concentration-dependent inhibition of purified calpain activity (Banik et al., 2000). Recent work has demonstrated that NSAID inhibition of COX-2 cleavage through COX-independent mechanisms probably involve cysteine proteases (Mancini et al., 2007). Taken together, the results from these three investigations suggest that the mechanisms that underlie NSAID effects on calpains are complex.
Our findings are consistent with the hypothesis that NSAID effects on calpains can contribute to the drugs' pharmacodynamic and toxicodynamic profiles. However, the experiments we have performed to date are insufficient to implicate a cellular mechanism of action for NSAID inhibition of calpain expression in intestinal epithelial cells. It is clear that this will be an important future focus for our laboratory.
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Acknowledgements |
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Footnotes |
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N.N.R. and K.S. contributed equally to this work.
Parts of this work were presented at the following meeting: Lillich JD, Silver K, Raveendran NN, and Freeman LC (2007) Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit IEC-6 cell migration and calpain protein expression. 2007 Experiment Biology Meeting; 2007 Apr 20–May 2; Washington, DC. Federation of American Societies for Experimental Biology, Bethesda, MD.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; GI, gastrointestinal; COX, cyclooxygenase; PG, prostaglandin; IEC, intestinal epithelial cell; SC-560, 5-(4-chlorphenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; NS-398, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide; DMSO, dimethyl sulfoxide; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; SLR, signal log ratio; IPA, Ingenuity Pathway Analysis; Ct, threshold cycle; ALLN, N-acetyl-Leu-Leu-Nle-CHO; ALLM, N-acetyl-Leu-Leu-methioninal; Indo, indomethacin.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. James D. Lillich, Department of Clinical Sciences, VCS-Q-203 Moiser Hall, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506. E-mail: lillich{at}vet.ksu.edu
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80 kDa. Summary data (left panels) depict calpain expression normalized to the actin loading control using densitometry for calpain 8 (A; n = 4), calpain 2 (B; n = 3), and calpain 1 (C; n = 4). *, significant differences from control at p < 0.05.
