1Disease Pathogenesis Program, Children's Memorial Institute for Education and Research, Chicago; and 2Department of Pathology, Children's Memorial Hospital, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60614
Submitted 5 March 2003 ; accepted in final form 19 June 2003
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ABSTRACT |
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intestinal epithelial cell; reactive oxygen metabolites; premRNA splicing machinery; SR proteins; signal transduction
The mammalian nucleus is a well-organized multi-functional organelle (reviewed in Ref. 24). It contains numerous morphologically defined subcompartments such as nucleoli, Cajal (coiled) bodies, promyelocytic leukemia bodies, and speckles (17, 26, 27, 43). Among them, nuclear speckles accumulate pre-mRNA splicing factors and their associated molecules (30). A growing body of evidence suggests that nuclear speckles are sites of pre-mRNA splicing component storage and/or assembly (21, 28, 31, 65). They are highly dynamic structures that respond specifically to activation of nearby genes (22, 28, 65). In living cells, components in speckles are in continuous flux (3, 14, 28, 31). Under circumstantial conditions such as heat shock and inhibition of transcriptional and/or splicing activity, the nuclear speckles undergo reorganization (6, 34, 44). However, it has not been explored whether physiological or pathophysiological stimuli induce morphological changes on nuclear speckles in intestinal epithelial cells.
Serine- and arginine-rich (SR) proteins are a family of protein factors that play important roles in both constitutive and regulated splicing (reviewed in Refs. 15, 18, 25). They are characterized by the presence of one or more RNA recognition motifs (RRM) and a COOH-terminal domain rich in arginine and serine residues (RS domain). The RRM functions to recognize and bind substrates for SR proteins, whereas the RS domain anchors proteins essential for assembling splicing machinery (63). The RS domain is extensively phosphorylated by tightly controlled mechanisms in cells (10, 20, 28, 40, 64). Immunofluorescence localization studies have demonstrated that SR proteins are primarily localized in nuclear speckles (1, 16, 44). They are dynamic components. When the RS domain is modulated by phosphorylation, SR proteins are redistributed within the nucleus or accumulate in the cytoplasm (11, 23). In addition, both hyper- and hypophosphorylation of SR proteins appears to influence protein-protein interactions and to be linked to their physiological functions in vivo (35, 38, 62).
Recently, proteins involved in the pre-mRNA splicing, including several heterogeneous nuclear ribonucleoproteins (hnRNP) and SR proteins, have been found to be phosphorylated in response to extracellular stimulations (12, 36, 46, 60). These changes have been proposed to regulate splice site selection in pre-mRNA alternative splicing, which is recognized as the cause or the consequence of numerous human diseases such as genetic disorders, tumors, and inflammatory injuries (reviewed in Ref. 45). In fact, many inflammatory mediators, such as tumor necrosis factor (TNF), interleukin-1 (IL-1
), and LPS, cause changes in alternative splicing patterns of numerous genes (45), which are part of inflammatory responses. However, a large gap exists in our understanding of how the pre-mRNA splicing process is targeted by inflammatory stimuli.
In this study, we aimed to investigate whether stimulation of intestinal epithelial cells with monochloramine affects nuclear speckles and their components. Moreover, we explored the mechanism by which NH2Cl modulates the architecture of nuclear speckles and phosphorylation of SR proteins in intestinal epithelial cells. We showed that NH2Cl induces aggregation of nuclear speckles and selectively upregulates phosphorylation of SRp30 in colonic epithelial cells (both Caco-2 and HT-29 lines) in a dose-dependent manner. This effect is mediated via a distinctive signal pathway involved in protein kinase C (PKC) in intestinal epithelial cells. These findings have determined the link between physiologically relevant stimuli and premRNA splicing machinery in intestinal epithelial cells.
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MATERIALS AND METHODS |
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Cell culture. Caco-2 and HT-29 cells (human intestinal epithelial cell lines derived from colonic adenocarcinoma) were used in this study. They were purchased from American Type Culture Collection (ATCC, Rockville, MD). Caco-2 cells (passages 2-20 after receipt from ATCC) were grown in minimum essential medium (Eagle) supplemented with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum at 37°C in a CO2-humidified incubator. HT-29 cells (passages 20-35 after receipt from ATCC) were maintained in Dulbecco's modified Eagle's minimum essential medium containing 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum.
Preparation of MAb 104. MAb 104 hybridoma cells produce mouse IgM monoclonal antibody against phosphoepitope of human SR proteins (40, 64). The cell line was purchased from ATCC. The hybridoma cells were grown in Iscovere's modified Dulbecco's medium supplemented with 20% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. The culture medium was renewed every 3 days. To produce the antibody, hybridoma cells were cultured with Hybridoma-SFM (GIBCO) for 5 days and centrifuged at 800 g afterward. The culture supernatant was collected and stored at -70°C until use.
Preparation of NH2Cl. Briefly, NH2Cl was synthesized fresh before each experiment by drop-wise addition of 200 µl of NaOCl to ice-cold 10 ml of solution containing NH4Cl (20 mM) and Na2HPO4 (5 mM). Thereafter, the mixture was measured photometrically by the optical density of 242 nm, and the concentration of NH2Cl in the mixture was calculated by using a method described by Thomas et al. (57).
Measurement of cell injury. Dual staining living cells with Hoechst 33258 and propidium iodide (PI) was used to measure cell injury. Briefly, cells were subjected to various treatments. Afterward, living cells were carefully washed with fresh culture medium and consequently incubated for 10 min in the presence of Hoechst 33258 (5 µg/ml) and PI (5 µg/ml) respectively. Dual fluorescence-stained culture were visualized with an inverted fluorescence microscope (model DM IRB, Leica, Germany) equipped with various florescent filters. Images were acquired with a digital camera (model C4742-95, Hamamatsu) using OpenLab software (Improvision, Lexington, MA).
Cytotoxicity measurement. A standard method setup in the lab was used (51). Briefly, cells were grown in 96-well plates (2 x 104 cells/well). After various pretreatments, the medium was replaced by fresh medium containing monochloramine and incubated for an additional 4 h. The medium supernatant was then transferred to an Eppendorf centrifuge tube and centrifuged for 5 min at 700 g. Then, the cytotoxic effect of monochloramine was determined by measuring lactate dehydrogenase (LDH) activity in supernatant, an index of cell injury, using a detection kit supplied by Roche Molecular Biochemicals. The assay was performed in an optically clear 96-well flat-bottomed microtiter plate, following the protocol provided by the manufacturer. At the end of the assay, the optical density of each well was determined by using a UVMAX kinetic microplate reader at a test wavelength of 490 nm and a reference wavelength of 650 nm. Each plate contained appropriate blank control wells containing medium and assay reagent but no cells. With each assay, one group of cells was lysed with 1% Triton X-100 and LDH activity in the medium was used as the total cellular LDH activity.
Indirect immunofluorescence and microscopy. Cells grown on coverslips were subjected to various treatments. Afterward, cells were washed three times with ice-cold phosphate-buffered saline (PBS) and then fixed in precold methanol (-20°C) for 10 min, followed by PBST (PBS containing 0.05% Triton X-100) for 10 min at room temperature. Cells were then blocked for 30 min in PBS containing 1% horse serum. The pretreated slides were incubated with properly diluted primary antibodies for 30 min at room temperature. After being washed with PBS three times, slides were incubated with diluted secondary antibodies (1:500) conjugated with Cy3 or FITC for 30 min, washed with PBS, and finally mounted with FluorSave (Calbiochem).
Primary antibodies used were MAb SC35 (1:1,000 dilution), mouse IgG monoclonal antibody localizing speckled structure (16, 44), MAb H5 (1:100 dilution), and mouse IgM monoclonal antibody that recognizes the active form of the RNA polymerase II (i.e., RNA polymerase II phosphorylated at serine 2 of the COOH-terminal domain of the largest pol II subunit) (9, 37).
Secondary antibodies used were Cy3-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-mouse IgM.
The stained cells were examined using an upright fluorescence microscope (model MD R, Leica, Germany) equipped with various fluorescent filters. Images were acquired with a digital camera (model C4742-95, Hamamatsu), and Open-Lab software was used to analyze image such as quantitation of fluorescence on MAb H5-labeled cells. Quantitative analysis was performed by inspection of n = 100 cells per sample. Image was assembled using Adobe Photoshop 7.0.
Western blotting. Cells were lysed in a buffer containing 2 mM Tris-Cl (pH 7.6), 30 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 1 mM Na3VO4, and 1% Notidet-40. After centrifugation of the cell lysate at 10,000 g for 10 min at 4°C, the supernatant was mixed with an equal volume of 2x Laemmli's buffer and boiled for 5 min. Thirty micrograms of protein were resolved on 4-20% SDS-PAGE gel along with molecular weight standards. The protein was then transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) as described before (52). The membranes containing sample proteins were used for immunodetection of phosphorylated SR proteins. Briefly, blots pre-incubated with PBS containing 5% skim milk were reacted with primary antibody (1:2 diluted culture supernatant of MAb 104 hybridoma) for 2 h at room temperature. After incubation, the blot was washed four times with PBS containing 0.05% Tween 20 (PBS-T) and then incubated with PBS-T containing 1:1,000 diluted HRP-conjugated goat anti-mouse IgM for 1 h at room temperature. After additional washing with PBS-T, immune complexes on the blot were visualized using the ECL system. Blots were stripped and reprobed with MAb against E-Cadherin following a standard procedure (51).
PKC assay. PKC assay system supplied by Promega was used and the protocol provided by the manufacturer was followed. Briefly, cells were lysed with a buffer containing 50 mM Tris-Cl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1% (vol/vol) Notidet-40, 30 mM sodium pyrophosphate, 50 mM NaF, and complete protease inhibitor cocktail (1 tablet/10 ml), and homogenized. Afterwards, cell lysate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was processed for chromatography with a minicolumn containing Whatman DE 52 according to a procedure suggested by Promega. PKC activity in eluted protein extracts was determined by measuring the incorporation of 32P from [-32P]ATP into a synthetic peptide substrate according to manufacturer's recommendations. Total PKC specific activity (picomoles per minute) was normalized to total protein.
Statistics. Data were expressed as means ± SE. Analysis of variance, and one-way analysis of variance (ANOVA) followed by Fisher's protected least significant difference post hoc test were used to assess the significant of differences. P < 0.05 was considered significant.
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RESULTS |
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Previous studies have shown that MAb SC35 identifies a speckled pattern in the nucleus of mammalian cells, which corresponds to the nuclear subcompartment associated with regulatory proteins for premRNA splicing (44). Thus intestinal epithelial cells including HT-29 and Caco-2 lines were stained with MAb SC35 using indirect immunofluorescent histochemical staining to determine the link between NH2Cl stimulation and the pre-mRNA splicing machinery. As shown in Fig. 2, nontreated HT-29 (Fig. 2A) or Caco-2 (Fig. 2D) cells stained with MAb SC35 showed a bright fluorescence of nuclear speckles. The speckled pattern occupied a portion of the nucleoplasm excluding the nucleoli and was identical to one found in Hela cells by us (Zhu et al., unpublished data) and others (16, 44). In contrast, fluorescence was not observed in cells when the primary antibody was eliminated from the staining solution (data not shown). NH2Cl at concentrations that did not cause human colonic epithelial cell injury induced aggregation of nuclear speckles in both HT-29 (Fig. 2B) and Caco-2 (Fig. 2E) within 2 h. In control groups, cells treated with culture medium containing vehicles such as NaOCl (0.2 mM for HT-29 cells and 0.5 mM for Caco-2 cells) did not show changes on structure of nuclear speckles (Fig. 2, C and F). In addition, the culture medium containing 1.5 mM NH4Cl and 0.4 mM Na2HPO4 did not emulate the changes induced by NH2Cl (data not shown). The data suggest that cell-permeable oxidants such as NH2Cl influence the dynamic structure of nuclear speckles in intestinal epithelial cells.
Inhibition of RNA polymerase II (pol II) transcription is known to result in disruption of nuclear speckle compartment (6, 28, 44). To examine whether treatment with NH2Cl affects transcriptional status in intestinal epithelial cells, HT-29 and Caco-2 cells were stained with MAb H5 (an antibody recognizing transcriptionally active RNA polymerase II, Refs. 9, 37) using indirect immunofluorescent histochemical staining. In either untreated or NH2Cl-treated HT-29 and Caco-2 cells, MAb H5 stained the entire portion of the nucleus (data not shown). This observation indicates that pol II remains in a transcriptionally active phosphorylation state during stimulation by NH2Cl. A quantitative analysis of the fluorescence of cells stained with MAb H5 indicated that simulation with NH2Cl did not affect the intensity (data not shown). The result suggests that treatment of intestinal epithelial cells with NH2Cl at concentrations within physiological range does not result in the reduction of transcriptional activity of pol II in the cells.
NH2Cl selectively induces phosphorylation of SRp30 in intestinal epithelial cells. Several SR proteins were found to be hyperphosphorylated in mammalian nuclei (40, 64). The phosphorylation status of SR proteins is associated with organization of nuclear speckles and their functions (see review in Refs. 15, 18, 25). Previous studies have shown that MAb 104 recognizes only phosphorylated SR proteins including SRp75, SRp55, SRp40, and SRp30 of the human species (40, 63). The antibody has been widely used to study the phosphorylation state of SR proteins by numerous investigators (7, 36, 41, 59). To determine the effect of NH2Cl on the phosphorylation of SR proteins, HT-29 cells were incubated with various concentrations of NH2Cl for 2 h, and cell lysates were subjected to SDS-PAGE and Western blotting with MAb104. As shown in Fig. 3A, several SR proteins including SRp75, SRp55, and SRp40 were persistently phosphorylated in unstimulated HT-29 cells. Stimulation of HT-29 cells with NH2Cl produced a dose-dependent increase in phosphorylation of SRp30 within 2 h, whereas the phosphorylation status of other SR proteins including SRp75, SRp55, and SRp40 were not changed after treatment with NH2Cl, suggesting that SRp30 is targeted by NH2Cl treatment. Therefore, we focused on SRp30 in later experiments. Using Caco-2 cells, it was also demonstrated that treatment with NH2Cl resulted in an increase of phosphorylation on SRp30 in a dose-dependent manner (Fig. 3B). Moreover, the effect of NH2Cl on phosphorylation of SRp30 in HT-29 or Caco-2 cells was in a time-dependent fashion (Fig. 4, A and B). Control experiments performed with cell culture medium containing either NaOCl or the NH4Cl/Na2HPO4 did not evoke phosphorylated SRp30 (data not shown). Taken together, the data indicate that NH2Cl specifically induces phosphorylation of SRp30 in intestinal epithelial cells.
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NH2Cl activates PKC, and the selective PKC inhibitor attenuates NH2Cl-induced phosphorylation of SRp30 and aggregation of nuclear speckles in intestinal epithelial cells. To elucidate the signal pathway mediating the effect of NH2Cl on nuclear speckles and SRp30 phosphorylation, HT-29 and Caco-2 cells were subjected to a pretreatment of bisindolylmaleimide I (FX, 5 µM, a cell-permeable selective PKC inhibitor; Ref. 58) or H89 (10 µM, a cell-permeable selective PKA inhibitor, Ref. 8) for 30 min, respectively, followed by NH2Cl stimulation. After treatment, cells were processed for indirect immunofluorescent staining to determine changes in nuclear speckles or Western blotting with MAb104 to detect the phosphorylation status of SRp30. As shown in Fig. 5A, FX specifically blocked the effect of NH2Cl on reorganization of nuclear speckle in both HT-29 and Caco-2 cells. In contrast, treatment with H89 did not affect the effect of NH2Cl on nuclear speckles (Fig. 5A). Moreover, pretreatment with FX attenuated the NH2Cl-induced phosphorylation of SRp30 in HT-29 cells (Fig. 5B, lane 4), whereas H89 did not block the effect of NH2Cl on the phosphorylation of SRp30 (Fig. 5B, lane 3) in HT-29 cells. In addition, the vehicle (i.e., DMSO) did not influence the NH2Cl effect (Fig. 5B, lane 5). Similar results were obtained using Caco-2 cells (data not shown).
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To confirm that stimulation of intestinal epithelial cells with NH2Cl results in PKC activation, we carried out a PKC assay experiment using HT-29 cells. As shown in Fig. 5C, treatment with NH2Cl (0.2 mM) for 60 min caused a significant induction of PKC activity in HT-29 cells. Together, the available data indicate that a signal pathway involved in PKC mediates the NH2Cl-induced reorganization of nuclear speckle and SRp30 phosphorylation.
PMA mimics NH2Cl effect on nuclear speckle aggregation and SRp30 phosphorylation in intestinal epithelial cells. To further determine whether activation of PKC links to reorganization of nuclear speckles, HT-29 and Caco-2 cells were treated with PMA (100 nM, a classic PKC activator) for 2 h, fixed with methanol, and processed for indirect immunofluorescent staining with MAb SC35. As shown in Fig. 6A, PMA-induced nuclear speckle reorganization in both HT-29 and Caco-2 cells. The pattern was identical to one induced by NH2Cl treatment. Furthermore, SRp30 was examined by Western blotting to determine its changes in response to PMA treatment. Using MAb104, it was demonstrated that PMA induced phosphorylation of SRp30 in HT-29 (lane 3 in Fig. 6B) or Caco-2 cells (lane 3 in Fig. 6C), whereas SRp30 was barely detected in cell lysates isolated from vehicle-treated cells. These observations showed that PMA mimics the effect of NH2Cl on the aggregation of nuclear speckle and phosphorylation of SRp30 in intestinal epithelial cells.
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DISCUSSION |
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The speckled pattern is a unique structure associated with gene expression within mammalian nuclei. They accumulate various proteins essential for the pre-mRNA splicing process (30). The components presented in speckles are in continuous influx (21, 28, 31, 65). Although each speckle maintains its position in the nucleus, Spector et al. have demonstrated that nuclear speckles undergo reorganization during mitosis (44). Moreover, inhibition of the transcriptional activity by either heat shock or transcriptional inhibitors results in speckles becoming larger and more rounded (6, 28, 44). Taken together, previous investigations indicate that dynamic properties of nuclear speckles are associated with the physiological state of cells. In the present study, we examined the effect of cell-permeable oxidant on the speckled structure. As seen in Fig. 2, stimulation of intestinal epithelial cells with monochloramine at pathophysiologically relevant concentrations causes reorganization of nuclear speckles. Such an effect does not result from cytotoxicity of NH2Cl (Fig. 1). In addition, the cell-permeable oxidant used in the present study does not modulate the RNA polymerase II activity in intestinal epithelial cells. These data imply that the morphological change of nuclear speckles in response to cell-permeable oxidant stimulation is due to a specific mechanism without linking to global transcriptional activity and cell injury.
MAb104 recognizes a phosphorylated epitope residing within the RS domain of SR proteins ranging in apparent size from 20 to 75 kDa (63). Among SR proteins, SRp30 identified by MAb104 includes SC35 and SF2/ASF (40). They are essential factors for both constitutive and alternative splicing (15, 18, 25). Previous investigations have shown that the SRp30 is differentially phosphorylated in a tissue-specific manner (40). Caceres et al. demonstrated that variations in the intracellular levels of SF2/ASF, a member of SRp30, govern alternative splicing in cells (4). Furthermore, several investigators have found that phosphorylation status of SRp30 influences both constitutive and alterative splicing (15, 18, 25). In the present study, we showed that phosphorylation of SRp30 rather than other SR proteins in HT-29 or Caco-2 cells was increased after treatment with NH2Cl. This observation suggests that NH2Cl selectively enhances phosphorylation of SRp30 in intestinal epithelial cells. In addition, increased phosphorylation on SRp30 correlated with NH2Cl-induced morphological changes on nuclear speckles. Because the level of phosphorylation controls subnuclear distribution of SR proteins and the reorganization of speckle patterns (2, 11, 23, 28, 29), we hypothesized that NH2Cl-induced phosphorylation of SRp30 might be associated with NH2Cl-triggered aggregation of nuclear speckles in intestinal epithelial cells. In addition, induction of phosphorylation of SRp30 may contribute to oxidant-modulated gene expression in intestinal epithelial cells.
The mechanism whereby NH2Cl affects nuclear speckles and SRp30 in intestinal epithelial cells is unknown. Previous studies showed that phosphorylation of SR proteins results in either their diffusion in the nucleoplasm (2, 28, 29) or the relocation of SR proteins to speckles (23), which subsequently causes reorganization of nuclear speckles. Thus trafficking of SR proteins with speckles is controlled by mechanisms involved in the phosphorylation process and influences speckled structures. Recently, protein kinases, namely SRPKs and Clk/Sty, have been discovered (10, 20, 61). They directly phosphorylate RS domains of SR proteins and are able to cause the redistribution of splicing factors within the nuclear speckles (10, 11, 20, 61). Currently, little is known about how SR protein kinases are regulated in cells. It has been hypothesized that the activity of these kinases is modulated through mechanisms involving regulation of their subcellular localization (20, 33, 42). Recently, Mylonis and Giannakouros have found that protein kinase CK2 directly phosphorylates SRPK1 and enhances its activity (32a), suggesting that SR protein kinases are regulated at multiple levels in vivo. In the present study, we found that NH2Cl modulates nuclear speckles and SRp30 via PKC but not PKA activation (Fig. 5). Activation of PKC in intestinal epithelial cells using PMA mimics the effect of NH2Cl on nuclear speckles and phosphorylation of SRp30 (Fig. 6). Our findings suggest that extracellular stimulations might regulate pre-mRNA splicing machinery in intestinal epithelial nucleus via distinct signaling mechanisms.
Recent evidence indicates that stress stimuli induce alteration of subcellular distribution of hnRNPs and modulation of alternative splicing via MKK-p38 signal cascade (60), whereas mitogens such as insulin target SRp40 through PI3-kinase dependent pathways and, consequently, result in alternative splicing of PKCII (36). In addition, FAS activation induces dephosphorylation of SR proteins through a signaling pathway involving in ceramide and protein phosphatase 1 (7). The present study reveals that PKC activation plays an important role in cell-permeable oxidants targeting nuclear events associated with splice site selection. Taken together, the available data suggest that extracellular stimuli regulate pre-mRNA processing and/or its associated nuclear subcompartments through signaling cascades in a stimulus-dependent fashion. Interestingly, many inflammatory stimuli can regulate pre-mRNA processing (45). Further studies that determine signal cascades linked to pre-mRNA splicing will offer the opportunity for filling the gaps between extracellular stimulation and nuclear events involved in inflammation.
In summary, we demonstrated for the first time that the dynamic distribution of nuclear speckles in intestinal epithelial cells responds to stimulation with cell-permeable oxidants such as NH2Cl. We further demonstrated that NH2Cl selectively induces phosphorylated SRp30 in intestinal epithelial cells. The effect of NH2Cl on nuclear speckles and SRp30 is mediated via a distinctive pathway involving PKC activation. Previous studies have shown that oxidants modulate redox-sensitive signal pathways in various cells (13). They are essential participants in cell signaling and regulation, in addition to inducing cell injury via oxidizing various intracellular molecules (53). Stimulation of intestinal epithelial cells with a cell-permeable oxidant such as monochloramine causes changes of physiological status of mucosa in vivo and in vitro (5). The demonstration of the link between cell-permeable oxidant and pre-mRNA splicing machinery in intestinal epithelial cells by the present study could provide insights into the mechanisms involved in coordinating oxidant-triggered pathophysiological responses in intestinal mucosa. Furthermore, we propose that the effect of cell-permeable oxidants on nuclear speckles and SRp30 might be associated with NH2Cl-potentiated inflammatory responses in the GI tract.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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