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RECEPTORS AND SIGNAL TRANSDUCTION
pathway activation
1Department of Surgery and 2Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, Texas
Submitted 27 September 2005 ; accepted in final form 10 January 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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inhibitor, and by small interfering RNA (siRNA) directed against PKC-, suggesting the regulation of PKD activity by upstream PKC-. Activation of PKD was also blocked by a Rho kinase (ROK)-specific inhibitor, Y-27632, as well as by C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H2O2-induced PKC- phosphorylation was inhibited by C3 treatment, further suggesting that PKC- is downstream of Rho/ROK. Interestingly, H2O2-induced intestinal cell apoptosis was enhanced by PKD siRNA. Together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells and that this activation is regulated by upstream PKC- and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications for intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants).
Rho kinase; protein kinase C-
Protein kinase D (PKD), initially referred to as PKC-µ (14), is a serine-threonine kinase with a distinctive structure and substrate specificity different from the PKC family members (34). PKD has been implicated in many important intracellular signal transduction pathways via PKC-dependent mechanisms. Importantly, PKD is activated by oxidative stress and induces survival pathways in fibroblast cells (33, 36, 37). However, its potential role in the regulation of oxidative stress-induced intestinal epithelial cell injury is not known. PKC-, a novel PKC isoform, is activated by a variety of apoptotic stimuli in different cellular systems. Although the majority of studies indicate that PKC- is involved in the induction of cell apoptosis, there are other reports indicating a role of PKC- in cell survival and in antiapoptotic responses (6).
The Rho family small GTPases, including well-known Rho proteins (RhoA and RhoB), Rac1, and Cdc42, are members of the Ras superfamily of GTPases. The Rho family regulates many biological processes, including cytoskeletal regulation, membrane trafficking, cell adhesion, cell polarization, and transcriptional activity (1, 7). The Rho family has been implicated in cell growth and apoptosis in response to diverse stimuli (2, 18). The Rho kinase (ROK), including isoforms 
and 
, is an effector of Rho proteins. It has been shown that membrane blebbing during apoptosis is mediated through caspase-mediated activation of ROK- (9). However, the exact role of these cellular signaling kinases in oxidative stress-induced intestinal epithelial cell injury is not known.
In the present study, we show activation of PKD by H2O2 treatment in intestinal epithelial cells. This activation of PKD is regulated by PKC- and Rho/ROK pathways, which also play a critical antiapoptotic role during oxidative stress-mediated intestinal epithelial cell injury. Using small interfering RNA (siRNA) targeting PKD, we also demonstrate the protective role of PKD during H2O2-induced intestinal epithelial cell apoptosis. Together, our findings suggest an important protective role for PKD in intestinal cells in response to oxidative damage. These results have potential clinical implications for the management of diseases (such as NEC) that are related to ROS-mediated injury.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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siRNA were synthesized by Custom SMARTPool siRNA Design Service of Dharmacon (Lafayette, CO). The nonspecific control siRNA was also obtained from Dharmacon. Recombinant glutathione-S-transferase (GST)-C3 and GST control proteins were purified from Escherichia coli with constructs provided by Dr. Keith Burridge (University of North Carolina, Chapel Hill, NC). GF-109203X (GFX), Ro 31-8220, rottlerin, and Y-27632 were from Biomol Research Laboratories (Plymouth Meeting, PA). Syntide-2 and Gö-6983 were from Calbiochem (La Jolla, CA). 2',7'-Dichlorofluorescin diacetate (DCFH-DA) was from Sigma (St. Louis, MO). PKD, PKC-, poly(ADP-ribose)polymerase (PARP), and caspase-3 polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-PKD (Ser744/748) antibody was from Cell Signaling Technology (Beverly, MA). Anti-phospho-PKC- (Tyr311) antibody was from Stressgen Biotechnologies (San Diego, CA). Secondary antibodies were from Pierce (Rockford, IL). Alexa Fluor 488 antibody for fluorescent staining was from Molecular Probes (Eugene, OR). The enhanced chemiluminescence (ECL) system for Western immunoblot analysis was from Amersham (Arlington Heights, IL). The concentrated protein assay dye reagent was from Bio-Rad (Hercules, CA). Tissue culture media and reagents were from GIBCO-BRL (Grand Island, NY). All other reagents were of molecular biology grade and were purchased from Sigma. Cell culture and transfection. The RIE-1 cell line (a generous gift from Dr. Kenneth D. Brown, Cambridge Research Station, Babraham, Cambridge, UK) is a diploid, nontransformed, cryptlike cell line derived from rat small intestine (5). The IEC-6 cell line (purchased from American Type Culture Collection; Manassas, VA) was derived from normal rat intestinal crypt cells and was developed and characterized by Quaroni et al. (26). For all experiments, RIE-1 cells were used from passages 18 to 29, and IEC-6 cells were used from passages 23 to 31. Both cell lines were found to be free of Mycoplasma contamination using the polymerase chain reaction method. Cells were maintained in DMEM supplemented with 5% FBS in 5% CO2 at 37°C. For experimental purposes, cells were plated in 100-mm dishes and grown to near confluence. Cells were treated with the indicated concentrations of H2O2 at 37°C. For inhibitor studies, cells were pretreated with inhibitors for 30 min and then treated with H2O2 in combination with inhibitors for another 30 min. siRNA or GST-C3 protein was transfected by electroporation (400 V, 500 µF for siRNA; 450 V, 25 µF for GST-C3 protein) with GenePulser XCell (Bio-Rad).
Immunoprecipitation, in vitro kinase assays, and Western blotting.
Immunoprecipitation and in vitro kinase assays were performed as described previously (21). In brief, proteins (50 µg) were incubated with PKD antibodies (1:50) on a shaker for 2 h at 4°C, followed by another 2-h incubation with 30 µl of protein A Sepharose beads at 4°C. The immunocomplexes were suspended in 20 µl of kinase buffer, and a kinase reaction, with or without 2.5 µg of syntide-2 as a substrate, was started by adding 5 µCi of [
-32P]ATP and incubated for 10 min at 30°C. Reactions were stopped by the addition of 2x Tris-glycine sample buffer. Samples were denatured by boiling for 5 min and separated by NuPAGE 4–12% Bis-Tris gels. Gels were incubated in Gel-Dry drying solution (Invitrogen) for 5 min and dried at 60°C for 60 min, followed by exposure to X-ray film. For Western blotting, equal amounts of protein were resolved on NuPAGE Bis-Tris gels and electrophoretically transferred onto polyvinylidene difluoride membranes; the membranes were incubated with primary antibodies overnight at 4°C, followed by secondary antibodies conjugated with horseradish peroxidase. Membranes were developed using the ECL detection system.
Immunofluorescent staining and fluorescence microscopy. Cells were grown in chamber slides. Three days after seeding, cells were treated either with or without H2O2 (500 µM) for 15 min. After treatment, cells were fixed with 4% paraformaldehyde for 20 min at 37°C. After three washes with PBS, the cells were permeabilized with 0.3% Triton X-100 for 15 min at 37°C and blocked with 1% BSA-PBS for 20 min. The cells were incubated with rabbit polyclonal anti-PKD antibody diluted 1:100 with 1% BSA-PBS for 1 h at room temperature or overnight at 4°C. Cells were washed three times with PBS and incubated with Alexa Fluor 488-conjugated anti-rabbit secondary antibody diluted 1:500 in 1% BSA-PBS. The fluorescence of PKD immunoreactivity was observed under a fluorescence microscope.
Cell death assay and measurement of ROS. RIE-1 cells (4 x 104 cells/well) were seeded in 24-well plates for 24 h. Cells were transfected with siRNA and then grown for an additional 72 h. Cells were treated with various doses of H2O2 in growth medium for 3 h or with 500 µM H2O2 over a time course. Both adherent and floating cells were collected and washed three times with cold PBS. Quantitation of apoptosis was performed with a Cell Death Detection ELISAplus kit (Roche Applied Science; Indianapolis, IN), following the manufacturer's instructions. ROS were measured with a modification of methods described by Wang et al. (39). In brief, 6.4 x 103 cells were plated in 96-well plates. After 24 h, cells were incubated with the fluorescent probe DCFH-DA at 50 µM for 30 min. DCFH-DA was removed, and cells were washed twice with PBS and incubated with 500 µM H2O2 in DMEM containing 5% FBS for 3 h. DCFH-DA fluorescence was determined at an excitation of 485 nm and an emission of 520 nm using a fluorescence microplate reader (FLUOstar Optima; BMG Labtech, Durham, NC).
Statistical analysis. All experiments were repeated at least three times, and data are reported as means ± SE. Data were analyzed using the Kruskal-Wallis test because of heterogeneous variability in each group. All tests were assessed at the 0.05 level of significance. All statistical computations were conducted with the SAS system (release 8.2; SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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First, RIE-1 cells were treated with or without various concentrations of H2O2 (10–500 µM) for 15 min. PKD phosphorylation was noted with 250 µM H2O2, with increased phosphorylation at 500 µM (Fig. 1A, top). Next, RIE-1 cells were treated with 500 µM H2O2 over a time course. PKD activation occurred rapidly at 5 min, with a significant increase at 10 min after treatment with H2O2 (Fig. 1A, bottom). Similar results in IEC-6 cells confirmed that PKD activation in response to H2O2 was not limited to RIE-1 cells (Fig. 1B), suggesting that the activation of PKD by H2O2 occurs in intestinal epithelial cells, consistent with previous studies in other cell types (33, 36, 37).
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H2O2 stimulates apoptosis in RIE-1 cells. H2O2 is a known intracellular stress agent that induces apoptosis (19). We next examined whether H2O2 increases apoptosis in RIE-1 cells. Cells were treated with various dosages of H2O2 for 3 h. Then, using the dose that produces significant cell apoptosis (i.e., 500 µM), we also performed a time course study. As shown in Fig. 2A, treatment of RIE-1 cells with 500 µM H2O2 did not induce cell death until 3 h after treatment. Next, RIE-1 cells were treated with different doses of H2O2 for 3 h (Fig. 2B). The results demonstrate that H2O2 induces intestinal epithelial cell apoptosis in a dose-dependent manner.
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PKD siRNA enhances H2O2-induced cell death.
PKD activation in HeLa cells was shown to protect against oxidative stress-induced cell death (33), but whether PKD activation also protects intestinal epithelial cells from oxidative stress-induced cell apoptosis is unknown. To further determine whether PKD plays a role in the protection of RIE-1 cells from H2O2-induced apoptosis we transfected PKD siRNA into RIE-1 cells, and 3 days later cells were treated with 500 µM H2O2 for 3 h. Western blotting demonstrated that the inhibition of PKD expression by siRNA significantly decreased endogenous PKD levels and concurrently PKD phosphorylation compared with control siRNA (Fig. 3A). The cell death assay demonstrated that H2O2 treatment enhanced intestinal epithelial cell apoptosis by 
1.5-fold compared with cells without H2O2 treatment in control siRNA-transfected cells (Fig. 3B). More important, H2O2 treatment induced cell death up to fivefold compared with PKD siRNA-transfected cells in the absence of H2O2, strongly suggesting that the suppression of endogenous PKD enhances H2O2-induced intestinal epithelial cell apoptosis. Additionally, this increase in H2O2-induced apoptosis in PKD siRNA-transfected RIE-1 cells was confirmed by increased activation of caspase-3 and PARP cleavage (Fig. 3C).
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-selective inhibitor rottlerin (13) as well as the ROK inhibitor Y-27632 (10). H2O2-induced PKD activation in RIE-1 cells was significantly attenuated in the presence of all of these inhibitors (Fig. 4A, top). The membrane was stripped and reprobed with total PKD antibody to monitor loading equality (Fig. 4A, middle). Similar results were obtained in IEC-6 cells (Fig. 4B, top and middle). Quantitative analysis confirmed that H2O2 treatment increases PKD phosphorylation by 40.3% and 42.9% in RIE-1 and IEC-6 cells, respectively; these increases were attenuated to baseline level by all of the PKC inhibitors and the ROK inhibitor (Fig. 4, A and B, bottom). These data further demonstrate that the activation of PKD is PKC or Rho/ROK pathway dependent in intestinal epithelial cells.
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PKC- has been implicated in both pro- and antiapoptotic mechanisms (15). Because of the significant inhibition of PKD activation by rottlerin, a PKC--selective inhibitor, we wanted to confirm the involvement of PKC- in oxidative stress-mediated PKD activation in intestinal epithelial cells. Rottlerin effectively inhibits PKC- kinase activity at a concentration of 5–10 µM (13). To examine the sensitivity of PKD phosphorylation to rottlerin, RIE-1 cells were pretreated with various doses of rottlerin for 30 min and then a combination of H2O2 and rottlerin for another 30 min. Western blot analysis was performed with anti-phospho-PKD (Ser744/748) antibody. H2O2-induced PKD phosphorylation was decreased with the 2.5 µM dosage of rottlerin and markedly decreased with 5 µM (Fig. 5A). This result was confirmed in IEC-6 cells (Fig. 5B). To further demonstrate a specific role of PKC- in the regulation of PKD activity in H2O2-induced cell apoptosis, siRNA targeting PKC- was transfected into RIE-1 cells for 3 days. The expression of PKC- protein was significantly decreased by PKC- siRNA compared with control siRNA (Fig. 5C). Furthermore, PKD activation was significantly inhibited by PKC- siRNA in either the presence or the absence of H2O2 compared with the control siRNA (Fig. 5C). To further support the specific nature of PKC- regulation of PKD activation, we next performed an in vitro PKD kinase assay with syntide-2, a specific PKD substrate. Consistent with the PKD protein expression level, PKC- siRNA also significantly decreased PKD kinase activity (Fig. 5C).
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in H2O2-induced intestinal cell apoptosis, a cell death assay was performed in RIE-1 cells transfected with either PKC- or control siRNA (Fig. 5D). In cells transfected with control siRNA, H2O2 treatment enhanced cell death approximately twofold compared with cells in the absence of H2O2. In contrast, H2O2 treatment induced cell death up to fourfold compared with cells in the absence of H2O2 and transfected with PKC- siRNA (Fig. 5D). We also determined caspase-3 and PARP cleavage in RIE-1 cells transfected with PKC- siRNA and found that PKC- siRNA resulted in increased active procaspase-3 as well as PARP cleavage compared with control siRNA (Fig. 5E). Together, these results strongly suggest the regulation of PKC- on PKD activity in H2O2-induced cell apoptosis in intestinal epithelial cells and, moreover, demonstrate a specific role of PKC- in the regulation of PKD activation during H2O2-induced intestinal epithelial cell apoptosis. Rho/ROK pathway regulation of PKD activity during H2O2-induced apoptosis. ROK has been implicated in cell membrane blebbing, which often occurs when cells become committed to apoptosis (27). However, the importance of ROK in apoptosis remains to be elucidated (17). Because of the inhibition of PKD phosphorylation by the ROK inhibitor Y-27632 (Fig. 4), we next examined whether the Rho/ROK pathway is indeed involved in H2O2-induced PKD activation. For this purpose, both RIE-1 and IEC-6 cells were treated with various concentrations of Y-27632 to examine the sensitivity of PKD activity, and Western blots were performed with anti-phospho-PKD (Ser744/748) antibody to detect PKD activation. PKD phosphorylation was inhibited slightly at 5 µM but significantly at 15 and 30 µM Y-27632, suggesting relatively specific regulation of PKD activation by ROK (Fig. 6A). Similar results were obtained in IEC-6 cells (Fig. 6B). Next, RIE-1 cells were treated with 500 µM H2O2, alone or in combination with Y-27632, and apoptosis was measured (Fig. 6C). Treatment with Y-27632 significantly enhanced H2O2-induced cell death, which is consistent with the protective role of PKD in H2O2-induced apoptosis.
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was shown to be a regulator of PKD phosphorylation downstream of the Rho/ROK pathway in the human pancreatic carcinoid cell line BON (22). On the basis of the findings presented here that PKC- plays an antiapoptotic role in H2O2-induced cell death and regulation of PKD phosphorylation, we further determined the sequential regulation of PKD phosphorylation by PKC- and Rho/ROK pathway. The membrane was reprobed with anti-phospho-PKC- (Tyr311) antibody to detect PKC- tyrosine phosphorylation, which is often noted in apoptosis (Fig. 6D, middle). As expected, PKC- tyrosine phosphorylation at 311 was also decreased by C3 treatment. This result demonstrated that PKD phosphorylation was regulated by PKC- downstream of the Rho/ROK pathway, consistent with previous results by Li et al. (22). Together, these results provide evidence that the signaling pathway of H2O2-induced PKD activation requires the Rho/ROK pathway and PKC- is downstream of this pathway in intestinal epithelial cells. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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- and Rho/ROK-dependent mechanisms to function as an important antiapoptotic pathway during oxidative stress-mediated intestinal epithelial cell injury. ROS include a group of molecules such as H2O2, superoxide anion (O2–), singlet oxygen (O2), and hydroxyl radical (·OH). Cells possess endogenous antioxidant enzyme systems to protect against the deleterious effects of free oxygen radicals. Therefore, cells undergo oxidative stress when levels of ROS exceed the counterregulatory antioxidant capacity. The exposure of cells to oxidative stress requires cellular responses to avoid lethal damage through induction of necrosis or apoptosis (3). Furthermore, oxidative stress can concurrently stimulate protective cellular functions as a compensatory response to stressful stimuli. The PKD enzymes have recently been implicated in diverse cellular functions, including Golgi organization and plasma membrane-directed transport, metastasis, immune responses, apoptosis, and cell proliferation (28). Activation of PKD by oxidative stress has been recently reported in a variety of cell types (30–33, 36, 37, 40). Using HeLa cells as a model, Storz and Toker (33) recently showed that oxidative stress induces tyrosine phosphorylation both in the PH domain and in the activation loop serine residues leading to PKD activation (30–33). PKD phosphorylation at Ser744/748 was induced in oxidative stress mediated by menadione, a superoxide generator, in hepatocytes as a protective cell survival response (40). However, oxidative stress-induced activation of PKD has never been reported in intestinal epithelial cells.
In the present study, we have demonstrated that activation of PKD in H2O2-induced oxidative stress in RIE-1 and IEC-6 cells and that the activation of PKD protects against H2O2-induced cell apoptosis. Consistent with previous reports in Swiss 3T3 fibroblasts (36, 37) and HeLa cells (30–33), we also noted phosphorylation of PKD at the activation loop Ser744/748 induced by H2O2 treatment in intestinal epithelial cells. More important, we found that suppression of PKD by siRNA rendered RIE-1 cells more sensitive to oxidative stress-induced cell apoptosis. Therefore, our study demonstrates an important role of PKD in protection against oxidative stress-induced intestinal epithelial cell injury.
The function of PKC-, a member of the novel PKC family, depends on the cell type and specific stimulus. PKC- has been considered a redox-sensitive kinase and is known to play a key role in promoting apoptotic cell death in various cell types (6). However, more recent studies have demonstrated a role of PKC- either in cell survival or in antiapoptosis (16, 20, 24, 38). In human neutrophils, PKC- is a critical factor of TNF-related antiapoptotic signaling. Inhibition of PKC- resulted in the inhibition of TNF-mediated inhibition of apoptosis (16). Using a colon cancer cell line, COLO 205, Lewis et al. (20) demonstrated that PKC- inhibition is sufficient for caspase-3-independent apoptosis. Similarly, PKC- plays a critical role in the antiapoptotic effect of basic FGF in granulosa cells (24). Storz and Toker (33) proposed a model in which two distinct signaling pathways converge at the level of PKD; the Src-Abl pathway, which controls tyrosine phosphorylation and activation loop phosphorylation of PKD in response to oxidative stress, is mediated by the PKC pathway, specifically, the PKC- isoform (30–32). On the other hand, tyrosine-phosphorylated PKC- was required for H2O2-induced phosphorylation of vascular endothelial growth factor receptor-3, which is activated in response to H2O2 and promotes endothelial cell survival (38). In our present study, we utilized the PKC--selective inhibitor rottlerin as well as PKC- siRNA to examine the role of PKC- in the regulation of oxidative stress-induced PKD activity in intestinal epithelial cells. We found that PKC- siRNA promoted H2O2-induced cell death, suggesting an antiapoptotic role of PKC- in intestinal epithelial cells. We also showed that PKD phosphorylation at the activation loop Ser744/748 was significantly inhibited by rottlerin and PKC- siRNA, demonstrating that PKC- is an upstream kinase of PKD, consistent with the findings in HeLa cells.
The Rho family and its effector ROK have been implicated in the regulation of apoptosis (2, 8). The human RhoA, CDC42, and Rac-1 proteins and their guanine nucleotide exchange factor Dbl efficiently induce the transcriptional activity of NF-
B (25), which is clearly capable of controlling the expression of several antiapoptotic factors to enhance cell survival (35). This indirect evidence suggests that Rho family proteins play a role in the cellular survival pathway. In this study, we showed that exposure of RIE-1 or IEC-6 cells to Y-27632, a selective ROK inhibitor, induces the inhibition of H2O2-induced PKD phosphorylation. To our knowledge, this is the first report describing that H2O2-induced PKD activation is regulated by the Rho/ROK pathway. Furthermore, treatment of RIE-1 cells with Y-27632 enhanced H2O2-induced cell death, suggesting a role of the Rho/ROK pathway in the protective effect of PKD against H2O2-induced cell apoptosis. Moreover, PKD phosphorylation was completely blocked by C3 toxin, a specific Rho protein inhibitor, and the PKC- phosphorylation at Tyr311 was also decreased by C3 treatment, suggesting the regulation of PKD activity by PKC- downstream of Rho/ROK pathway.
In summary, the results presented in this report demonstrate a critical role of PKD activation in oxidative stress-induced intestinal epithelial cell injury. The results also demonstrate that PKD activity is regulated by PKC- downstream of the Rho/ROK pathway. Here we propose a model for a PKD-mediated cell survival pathway in oxidative stress-induced intestinal epithelial cell injury. Oxidative stress is strongly implicated in various diseases, including NEC; the potent activation of PKD may provide new mechanistic insights into both physiological and pathological processes in intestinal epithelial cells.
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ACKNOWLEDGMENTS |
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This paper was presented in part at the annual meeting of the American Gastroenterological Association (May 18–24, 2004, New Orleans, LA) and was previously published in abstract form (Gastroenterology 126: A147, 2004).
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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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REFERENCES |
|---|
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Aznar S and Lacal JC. Rho signals to cell growth and apoptosis. Cancer Lett 165: 1–10, 2001.[CrossRef][ISI][Medline]
3. Bergamini CM, Gambetti S, Dondi A, and Cervellati C. Oxygen, reactive oxygen species and tissue damage. Curr Pharm Des 10: 1611–1626, 2004.[CrossRef][ISI][Medline]
4. Bernotti S, Seidman E, Sinnett D, Brunet S, Dionne S, Delvin E, and Levy E. Inflammatory reaction without endogenous antioxidant response in Caco-2 cells exposed to iron/ascorbate-mediated lipid peroxidation. Am J Physiol Gastrointest Liver Physiol 285: G898–G906, 2003.
5. Blay J and Brown KD. Characterization of an epithelioid cell line derived from rat small intestine: demonstration of cytokeratin filaments. Cell Biol Int Rep 8: 551–560, 1984.[CrossRef][ISI][Medline]
6. Brodie C and Blumberg PM. Regulation of cell apoptosis by protein kinase C. Apoptosis 8: 19–27, 2003.[CrossRef][ISI][Medline]
7. Burridge K and Wennerberg K. Rho and Rac take center stage. Cell 116: 167–179, 2004.[CrossRef][ISI][Medline]
8. Coleman ML and Olson MF. Rho GTPase signalling pathways in the morphological changes associated with apoptosis. Cell Death Differ 9: 493–504, 2002.[CrossRef][ISI][Medline]
9. Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A, and Olson MF. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol 3: 339–345, 2001.[CrossRef][ISI][Medline]
10. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000.[CrossRef][ISI][Medline]
11. Dypbukt JM, Ankarcrona M, Burkitt M, Sjoholm A, Strom K, Orrenius S, and Nicotera P. Different prooxidant levels stimulate growth, trigger apoptosis, or produce necrosis of insulin-secreting RINm5F cells. The role of intracellular polyamines. J Biol Chem 269: 30553–30560, 1994.
12. Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, and Johannes FJ. Inhibition of protein kinase Cµ by various inhibitors. Differentiation from protein kinase C isoenzymes. FEBS Lett 392: 77–80, 1996.[CrossRef][ISI][Medline]
13. Gschwendt M, Muller HJ, Kielbassa K, Zang R, Kittstein W, Rincke G, and Marks F. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun 199: 93–98, 1994.[CrossRef][ISI][Medline]
14. Johannes FJ, Prestle J, Eis S, Oberhagemann P, and Pfizenmaier K. PKCµ is a novel, atypical member of the protein kinase C family. J Biol Chem 269: 6140–6148, 1994.
15. Kanthasamy AG, Kitazawa M, Kanthasamy A, and Anantharam V. Role of proteolytic activation of protein kinase C in oxidative stress-induced apoptosis. Antioxid Redox Signal 5: 609–620, 2003.[CrossRef][ISI][Medline]
16. Kilpatrick LE, Sun S, and Korchak HM. Selective regulation by -PKC and PI 3-kinase in the assembly of the antiapoptotic TNFR-1 signaling complex in neutrophils. Am J Physiol Cell Physiol 287: C633–C642, 2004.
17. Kobayashi K, Takahashi M, Matsushita N, Miyazaki J, Koike M, Yaginuma H, Osumi N, Kaibuchi K, and Kobayashi K. Survival of developing motor neurons mediated by Rho GTPase signaling pathway through Rho-kinase. J Neurosci 24: 3480–3488, 2004.
18. Lacal JC. Regulation of proliferation and apoptosis by Ras and Rho GTPases through specific phospholipid-dependent signaling. FEBS Lett 410: 73–77, 1997.[CrossRef][ISI][Medline]
19. Lee WC, Choi CH, Cha SH, Oh HL, and Kim YK. Role of ERK in hydrogen peroxide-induced cell death of human glioma cells. Neurochem Res 30: 263–270, 2005.[CrossRef][ISI][Medline]
20. Lewis AE, Susarla R, Wong BC, Langman MJ, and Eggo MC. Protein kinase C is not activated by caspase-3 and its inhibition is sufficient to induce apoptosis in the colon cancer line, COLO 205. Cell Signal 17: 253–262, 2005.[CrossRef][ISI][Medline]
21. Li J, Hellmich MR, Greeley GH Jr, Townsend CM Jr, and Evers BM. Phorbol ester-mediated neurotensin secretion is dependent on the PKC- and - isoforms. Am J Physiol Gastrointest Liver Physiol 283: G1197–G1206, 2002.
22. Li J, O'Connor KL, Hellmich MR, Greeley GH Jr, Townsend CM Jr, and Evers BM. The role of protein kinase D in neurotensin secretion mediated by protein kinase C-/- and Rho/Rho kinase. J Biol Chem 279: 28466–28474, 2004.
23. Oliver FJ, de la Rubia G, Rolli V, Ruiz-Ruiz MC, de Murcia G, and Murcia JM. Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. J Biol Chem 273: 33533–33539, 1998.
24. Peluso JJ, Pappalardo A, and Fernandez G. Basic fibroblast growth factor maintains calcium homeostasis and granulosa cell viability by stimulating calcium efflux via a PKC -dependent pathway. Endocrinology 142: 4203–4211, 2001.
25. Perona R, Montaner S, Saniger L, Sanchez-Perez I, Bravo R, and Lacal JC. Activation of the nuclear factor-B by Rho, CDC42, and Rac-1 proteins. Genes Dev 11: 463–475, 1997.
26. Quaroni A, Wands J, Trelstad RL, and Isselbacher KJ. Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell Biol 80: 248–265, 1979.
27. Riento K and Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 4: 446–456, 2003.[CrossRef][ISI][Medline]
28. Rykx A, De Kimpe L, Mikhalap S, Vantus T, Seufferlein T, Vandenheede JR, and Van Lint J. Protein kinase D: a family affair. FEBS Lett 546: 81–86, 2003.[CrossRef][ISI][Medline]
29. Shirai Y and Saito N. Activation mechanisms of protein kinase C: maturation, catalytic activation, and targeting. J Biochem (Tokyo) 132: 663–668, 2002.
30. Storz P, Doppler H, Johannes FJ, and Toker A. Tyrosine phosphorylation of protein kinase D in the pleckstrin homology domain leads to activation. J Biol Chem 278: 17969–17976, 2003.
31. Storz P, Doppler H, and Toker A. Activation loop phosphorylation controls protein kinase D-dependent activation of nuclear factor B. Mol Pharmacol 66: 870–879, 2004.
32. Storz P, Doppler H, and Toker A. Protein kinase C selectively regulates protein kinase D-dependent activation of NF-B in oxidative stress signaling. Mol Cell Biol 24: 2614–2626, 2004.
33. Storz P and Toker A. Protein kinase D mediates a stress-induced NF-B activation and survival pathway. EMBO J 22: 109–120, 2003.[CrossRef][ISI][Medline]
34. Valverde AM, Sinnett-Smith J, Van Lint J, and Rozengurt E. Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain. Proc Natl Acad Sci USA 91: 8572–8576, 1994.
35. Van der Woude CJ, Kleibeuker JH, Jansen PL, and Moshage H. Chronic inflammation, apoptosis and (pre-)malignant lesions in the gastro-intestinal tract. Apoptosis 9: 123–130, 2004.[CrossRef][ISI][Medline]
36. Waldron RT, Rey O, Zhukova E, and Rozengurt E. Oxidative stress induces protein kinase C-mediated activation loop phosphorylation and nuclear redistribution of protein kinase D. J Biol Chem 279: 27482–27493, 2004.
37. Waldron RT and Rozengurt E. Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C. J Biol Chem 275: 17114–17121, 2000.
38. Wang JF, Zhang X, and Groopman JE. Activation of vascular endothelial growth factor receptor-3 and its downstream signaling promote cell survival under oxidative stress. J Biol Chem 279: 27088–27097, 2004.
39. Wang X, Simpkins JW, Dykens JA, and Cammarata PR. Oxidative damage to human lens epithelial cells in culture: estrogen protection of mitochondrial potential, ATP, and cell viability. Invest Ophthalmol Vis Sci 44: 2067–2075, 2003.
40. Wang Y, Schattenberg JM, Rigoli RM, Storz P, and Czaja MJ. Hepatocyte resistance to oxidative stress is dependent on protein kinase C-mediated down-regulation of c-Jun/AP-1. J Biol Chem 279: 31089–31097, 2004.
41. Yamamoto K, Kushima R, Kisaki O, Fujiyama Y, and Okabe H. Combined effect of hydrogen peroxide induced oxidative stress and IL-1 on IL-8 production in Caco-2 cells (a human colon carcinoma cell line) and normal intestinal epithelial cells. Inflammation 27: 123–128, 2003.[CrossRef][ISI][Medline]
42. Yuan J, Slice LW, and Rozengurt E. Activation of protein kinase D by signaling through Rho and the subunit of the heterotrimeric G protein G13. J Biol Chem 276: 38619–38627, 2001.
43. Zimmermann KC, Bonzon C, and Green DR. The machinery of programmed cell death. Pharmacol Ther 92: 57–70, 2001.[CrossRef][ISI][Medline]
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P < 0.05 vs. control siRNA and H2O2. C: RIE-1 cells were transfected with PKD or control siRNA for 72 h and then treated with H2O2 (500 µM) for 3 h. Both attached and floating cells were collected, lysed, and examined using Western blot analysis for the expression of caspase-3 or PARP cleavage products; 
