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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Substance P-Stimulated Interleukin-8 Expression in Human Colonic Epithelial Cells Involves Protein Kinase C Activation

Gastrointestinal Neuropeptide Center, Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts (H.-W.K., D.Z., Y.Z., S.S., C.P.); and INCELL Corporation, San Antonio, Texas (M.P.M.)

Received April 13, 2005; accepted May 23, 2005.

	Abstract

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Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of nuclear factor B (NF-B)-driven proinflammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-B activation and interleukin-8 (IL-8) production are not clear. Here, we examined participation of protein kinase C (PKC) in SP-induced IL-8 production in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells). SP (10^-7 M) induced an early (1 min) phosphorylation of the PKC isoforms PKC, PKC, and PKC, followed by I-B kinase, IB, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 µM) blocked SP-induced IB and p65 phosphorylation and IL-8 production. The PKC inhibitor rottlerin at a low concentration (1 µM), but not pseudosubstrate PKC and PKC inhibitors (10 µM), significantly reduced IL-8 secretion. PKC silencing by RNA interference reduced PKC protein expression and SP-induced PKC phosphorylation that was associated with diminished IL-8 promoter and NF-B luciferase activities in response to SP. Moreover, overexpression of wild-type PKC increased SP-induced IL-8 promoter- and NF-B-driven luciferase activities that were rottlerin-sensitive. We conclude that PKC plays an important role in SP-induced proinflammatory signaling in human colonocytes.

Ligand binding to NK-1R stimulates phosphoinositide hydrolysis (Rollandy et al., 1989), mobilizes Ca²⁺ (Pradier et al., 1993), and activates mitogen-activated protein kinases (MAPKs) (Luo et al., 1996). SP stimulation also activates MAPKs and causes cell proliferation in human astrocytoma and colonic epithelial cells via pathways involving transactivation of epidermal growth factor receptor (EGFR) (Castagliuolo et al., 2000; Koon et al., 2004). Yang et al. (2002) reported that in human tracheal smooth muscle cells, SP stimulates MAPKs associated with cell proliferation through the protein kinase C (PKC) pathway. In spiral ganglion neurons, PKCs may also interact with Ca²⁺ mobilization to induce MAPK activation in response to SP (Lallemend et al., 2003). The PKC superfamily is classified into three subfamilies based on their domain structure and their ability to respond to Ca²⁺ and DAG (Newton and Johnson, 1998). Although the conventional PKC isoforms , I, II, and are regulated by Ca²⁺ and DAG, the novel PKC isoforms , , , and respond to DAG, but do not involve Ca²⁺. The third group of PKC isoforms includes the atypical PKC isoforms , /, and µ, which are regulated by Ca²⁺ or DAG. It was previously shown that, among these PKCs, SP could transiently induce tyrosine phosphorylation of PKC in rat parotid acinar cells (Soltoff and Toker, 1995) and Ser729 phosphorylation of PKC in U373 MG human astrocytoma cells and primary rat astrocytes (Lieb et al., 2003). However, the physiological significance of activation of these PKCs is not known yet.

In addition to the MAPK pathway and protein kinase C activation, SP also activates the NF-B pathway, which triggers release of IL-8 production in both U373 MG cells (Lieb et al., 1997) and human colonic epithelial cells transfected with NK-1R (Zhao et al., 2002). Although different isoforms of PKCs have been associated with the NF-B pathway in response to various extracellular stimuli, whether PKCs are involved in SP's proinflammatory signaling is not known. In this study, we sought to investigate whether SP activates PKCs in nontransformed human colonic epithelial NCM-460 cells overexpressing NK-1R and examine their involvement in SP-stimulated NF-B activation and IL-8 gene expression. We used colonocytes overexpressing NK-1R because previous studies demonstrated increased NK-1R mucosal expression in intestinal inflammation in both animal models (Castagliuolo et al., 1998; Pothoulakis et al., 1998; Stucchi et al., 2003; Weinstock et al., 2003) and humans with inflammatory bowel disease (Goode et al., 2000; Renzi et al., 2000). Our results present novel evidence that SP rapidly and persistently induces threonine phosphorylation of PKC, and this activation is critical for SP-induced NF-B activation and IL-8 gene expression.

	Materials and Methods

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Cell Cultures. Nontransformed human colonic epithelial NCM460 cells overexpressing NK-1R (NCM460-NK-1R) were made as previously described by us (Zhao et al., 2002). NCM460-NK-1R cells were used in studies investigating both the proinflammatory (Zhao et al., 2002) and proliferative (Koon et al., 2004) responses to SP. Cells were cultured in M3D medium (INCELL Corporation, San Antonio, TX) containing 10% fetal calf serum (Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin (Invitrogen) solution.

Pharmacological Experiments. NCM460-NK-1R cells were seeded in 12-well plates (2 x 10⁶ cells/well) overnight in M3D medium containing 10% fetal calf serum and 1% penicillin/streptomycin (Invitrogen). Cells were pretreated with rottlerin (0.1–1 µM mallotoxin), caffeic acid phenethyl ester (CAPE; 2–10 µg/ml), specific PKC pseudosubstrate peptide inhibitor (10 µM Myr-LHQRRGAIKQAKVHHVKC-NH₂), and PKC pseudosubstrate peptide inhibitors (10 µM EAVSLKPT) for 30 min or with phorbol-12-myristate-13-acetate (PMA; 10 µM) overnight (Calbiochem, San Diego, CA). Cells were then stimulated with SP or 0.1% trifluorocetic acid (vehicle control) for various time points in Western blot experiments and 4 h in ELISA and luciferase experiments. Trifluorocetic acid had no effect on IL-8 secretion or PKC-NF-B signaling (data not shown).

IL-8 Measurements. IL-8 protein levels in conditioned media were determined by double-ligand ELISA using goat anti-(human IL-8) (R&D Systems, Minneapolis, MN) as previously described (Zhao et al., 2002). Results were expressed as mean ± S.E.M. (nanograms per milliliter).

Western Blot Analyses. SP-treated cells were lysed in 1x lysis buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 0.01% bromophenol blue, and 1% 2-mercaptoethanol). Equal amounts of cell extracts were fractionated by 10% SDS-PAGE, and proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA) at 400 milliamps for 2 h at 4°C. Membranes were blocked in 5% nonfat milk in Tris-buffered saline/Tween 20 (50 mM Tris, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20) and then incubated with antibodies against phospho-PKC (Ser729) (Santa Cruz Biochemicals, Santa Cruz, CA), -actin (Sigma-Aldrich, St. Louis, MO), phospho-PKC (Thr505), phospho-PKC (Thr538), phospho-IB kinase (IKK/ Ser180/Ser181), phospho-p65 (Ser536), and phospho-IB (Ser32/36) (Cell Signaling Technology Inc., Beverly, MA). Horseradish peroxidase-labeled antibodies were detected by enhanced chemiluminescence (Pierce Chemical, Rockford, IL). The image of the signal was exposed to X-ray film (Fujifilm, Tokyo, Japan).

PKC and p65 Knockdown by siRNA Transfection. Cells were seeded in 12-well plates (1 x 10⁵ cells/well) overnight and transiently transfected with equal amounts of PKC siRNA, p65 siRNA, or control siRNA (Santa Cruz Biochemicals) using X-tremeGENE Transfection Reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Specific down-regulation of endogenous PKC/p65 protein expression was confirmed by Western blot analyses using a PKC/p65-specific antibody and the -actin antibody.

Luciferase Assay. Cells were seeded in 12-well plates (2 x 10⁵ cells/well) overnight and transiently cotransfected using Lipofectamine 2000 transfection reagent (Invitrogen) with siRNA of the PKC, p65, or control siRNA (Santa Cruz Biochemicals). In the same experiments, cells were also cotransfected with an IL-8 promoter luciferase construct, an NF-B luciferase construct (a gift from Dr. Tom Mitchell and Dr. Bill Sugden, University of Wisconsin, Madison, WI), or with a control luciferase construct (pRL-TK; Promega, Madison, WI). For cotransfection experiments with a wild-type PKC-overexpressing plasmid (American Type Culture Collection, Manassas, VA) or its negative control, pCMV- galactosidase plasmid (backbone of PKC-overexpressing construct) (BD Biosciences Clontech, Palo Alto, CA), the Effectene transfection reagent (QIAGEN, Valencia, CA) was used. Transfected cells were serum-starved for 24 h, followed by exposure to SP for 4 h. Firefly and Renilla luciferase activities in cell extracts were measured using a dual-luciferase reporter assay system (Promega). The relative luciferase activity was then calculated by normalizing IL-8 promoter/NF-B driven firefly luciferase activity to control Renilla luciferase activity. Results are expressed as mean ± S.E.M. (n = 6).

Statistical Analyses. ELISA and luciferase assay results were analyzed using the Prism professional statistics software program (GraphPad Software Inc., San Diego, CA). Analyses of variance were used for intergroup comparisons.

	Results

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SP Induces Phosphorylation of PKC, PKC, and PKC. Since SP can transiently stimulate PKC tyrosine (Soltoff and Toker, 1995) and PKC serine phosphorylation (Lieb et al., 2003), we first examined whether SP activates these as well as other PKC members in human colonic epithelial cells by Western blot analyses using phospho-specific PKC antibodies. To do this, NCM460-NK-1R cells were stimulated with SP (10^-7 M) for 1 to 60 min, and equal amounts of cell protein were used to determine phosphorylation of various PKCs. We found that SP potently induced PKC threonine phosphorylation by 1 min that remained activated through 60 min (Fig. 1A). PKC phosphorylation was dose-dependent, with detectable SP induction at 10^-9 M (Fig. 1B). SP also induced transient PKC phosphorylation within 1 to 5 min as well as PKC phosphorylation at 1 to 2 min and returned to basal levels after 2 min, whereas the -actin signal indicates equal protein loading (Fig. 1A). However, SP did not induce phosphorylation of other PKCs including PKC, PKC, PKC, PKC, and PKD/PKCµ (data not shown).

View larger version (63K): [in this window] [in a new window]   Fig. 1. SP induces phosphorylation of PKC, PKC, and PKC in human nontransformed colonocytes expressing NK-1R. A, serum-starved NCM460-NK-1R cells were exposed to SP (10-7 M) for the indicated time points. Cells were lysed, and equal amounts of protein were fractionated on 10% SDS-PAGE gels to determine the levels of phospho-PKC, phospho-PKC, phospho-PKC, and -actin. B, serum-starved NCM460-NK-1R cells were exposed to the indicated concentrations of SP for 2 min. Cells were lysed, and equal amounts of protein were fractionated on 10% SDS-PAGE to determine the levels of phospho-PKC and -actin. Results are representative of three independent experiments.

SP Stimulates Phosphorylation of IKKs, IB, and p65 Subunit. To find out whether rapid activation of PKC, PKC, and PKC is kinetically followed by activation of signaling molecules involved in the NF-B pathway, the same time course samples were also subjected to Western blot analyses using antibodies against phosphorylated IKKs, IB, and NF-B p65. The results indicate that SP (10^-7 M) induced subsequent phosphorylation of IKKs, IB, and p65 at 5 to 10 min after SP stimulation (Fig. 2). These results suggest that activation of the NF-B pathway is downstream of PKC, PKC, and PKC phosphorylation in response to SP.

View larger version (47K): [in this window] [in a new window]   Fig. 2. SP stimulates IKK, IB, and p65 subunit phosphorylation. Serum-starved NCM460-NK-1R cells were exposed to SP (10-7 M) for the indicated time points. Cells were lysed, and equal amounts of protein were fractionated on 10% SDS-PAGE to determine the levels of phospho-IKK, phospho-IB, phospho-p65, and -actin. Results are representative of three independent experiments.

PKC Is Involved in SP-Induced NF-B-Mediated IL-8 Gene Expression.

View larger version (18K): [in this window] [in a new window]   Fig. 3. SP stimulates NF-B-mediated IL-8 gene expression. A, NCM460-NK-1R cells were incubated in serum-free M3D media for 24 h and then pretreated with CAPE (2–10 µg/ml) for 30 min, followed by SP (10-7 M) stimulation for 4 h. B, NCM460-NK-1R cells were transiently transfected with siRNAs of NF-B or negative control together with IL-8 promoter or NF-B firefly reporter plasmids. All cells were also transfected with control pRL-TK plasmids for Renilla luciferase signal. The transfected cells were serum-starved overnight and treated with SP (10-7 M) for 4 h. Cell extracts were prepared to measure IL-8 promoter (BI) or NF-B (BII) luciferase activity. Data are expressed as mean percentage relative firefly luciferase activity (normalized to control group without SP stimulation, which was normalized as 100%) ± S.E.M. and are representative of six independent samples. **, P < 0.01; ***, P < 0.001. C, p65 siRNA or control siRNA-transfected NCM460-NK-1R cells were stimulated with SP (10-7 M) for 15 min and then lysed. Equal amounts of protein were fractionated on 10% SDS-PAGE to determine the levels of phospho-p65, p65, and -actin. Results are representative of three independent experiments. D, serum-starved NCM460-NK-1R cells were pretreated with PMA (10 µM). After 24 h, the medium was either left unchanged or replaced with fresh serum-free M3D media for 1 h, followed by SP (10-7 M) stimulation for 4 h. Conditioned media were collected and IL-8 levels were measured by ELISA. Data are expressed as means ± S.E.M. (n = 6). **, P < 0.01; ***, P < 0.001.

To determine whether SP-stimulated NF-B-dependent IL-8 expression involves activation of PKCs, we first applied prolonged treatment with the PKC activator PMA that depletes protein expression of various PKCs, including PKC. Overnight PMA treatment led to high amounts of IL-8 in the media, even without SP stimulation (Fig. 3D), whereas addition of SP did not produce any further increase in IL-8 levels. When the PMA conditioned medium was replaced by fresh medium at 1 h before SP stimulation to remove residual IL-8, IL-8 levels returned to basal levels. Moreover, after the depletion of PKC, SP failed to significantly increase IL-8 levels, indicating that PKC depletion completely inhibited SP-induced IL-8 secretion (Fig. 3B).

Next, we determined whether PKC or PKC are involved in SP-stimulated NF-B-dependent IL-8 synthesis by pretreating NCM460-NK-1R cells with PKC and PKC pseudosubstrate peptide inhibitors (10 µM) for 30 min, followed by SP stimulation for 4 h. Our results showed that the PKC and PKC pseudosubstrate peptide inhibitors had no significant effect in SP-induced IL-8 production (Fig. 4A). To validate the inhibitory effects of these pseudosubstrate inhibitors, Western blot analyses were used to measure SP-induced PKC and PKC protein phosphorylation, compared with their nonphosphorylated proteins and -actin. As shown in Fig. 4B, both these inhibitors significantly reduced SP-induced phosphorylation of PKC and PKC, without affecting their protein expression levels. These results indicate that PKC and PKC are not involved in SP-induced IL-8 synthesis.

View larger version (37K): [in this window] [in a new window]   Fig. 4. PKC, but not other PKCs, mediates SP-induced IL-8 secretion in human nontransformed colonocytes expressing NK-1R. A, serum-starved NCM460-NK-1R cells were pretreated with specific PKC and PKC pseudosubstrate peptide inhibitors (10 µM) for 30 min, followed by SP (10-7 M) stimulation for 4 h. Conditioned media were collected for IL-8 ELISA measurements. Data are expressed as means ± S.E.M. and are representative of six independent samples. B, serum-starved NCM460-NK-1R cells were pretreated with specific PKC and PKC pseudosubstrate peptide inhibitors (10 µM) for 30 min, followed by SP (10-7 M) stimulation for 1 to 5 min. Cells were lysed, and equal amounts of protein were fractionated on 10% SDS-PAGE to determine the levels of phosphorylated and nonphosphorylated PKC and PKC proteins and -actin. Results are representative of three independent experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 5. PKC is required for SP-induced activation of signaling molecules of the NF-B pathway. A, serum-starved NCM460-NK-1R cells were pretreated with rottlerin (0.1–1 µM) or vehicle (DMSO) for 30 min, followed by SP (10-7 M) exposure for 4 h. Conditioned media were collected, and IL-8 levels were measured by ELISA. Data are expressed as means ± S.E.M. (n = 6). ** and ***, P < 0.01 and P < 0.001, respectively. B, serum-starved NCM460-NK-1R cells were pretreated with rottlerin (0.1–1 µM) or vehicle (DMSO) for 30 min, followed by SP (10-7 M) exposure for the indicated time points. Cells were lysed, and equal amounts of protein were fractionated on 10% SDS-PAGE to determine the levels of phospho-PKC (2 min), phospho-IKK (15 min), phospho-p65 (15 min), and -actin. Results are representative of three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 6. PKC siRNA significantly inhibited SP-induced IL-8 promoter and NF-B luciferase activities. NCM460-NK-1R cells were transiently transfected with a PKC siRNAs or its control siRNA together with an IL-8 promoter or an NF-B firefly reporter plasmid. All cells were also transfected with control pRL-TK plasmids for Renilla luciferase signal. The transfected cells were serum-starved overnight and treated with SP (10-7 M) for 4 h. Cell extracts were prepared to measure IL-8 promoter (A) or NF-B (B) luciferase activity as described under Materials and Methods. Data are expressed as mean ± S.E.M. (n = 6). ***, P < 0.001. C, PKC siRNA or control siRNA-transfected NCM460-NK-1R cells were lysed, and equal amounts of protein were fractionated on 10% SDS-PAGE to determine the levels of phospho-PKC, PKC, and -actin. Results are representative of three independent experiments.

Overexpression of Wild-Type PKC Potentiates SP-Induced IL-8 Promoter and NF-B Luciferase Activities. The role of PKC in SP-induced NF-B activation was further characterized by the transfection of a PKC wild-type plasmid that overexpresses PKC. Without transfection of wild-type PKC, SP significantly increased IL-8 promoter activity by 4.9-fold (P < 0.001) (Fig. 7A) and NF-B luciferase activity by 1.8-fold (P < 0.01) (Fig. 7B). However, in colonocytes transfected with PKC, SP-induced IL-8 promoter and NF-B luciferase activities were increased by 4.5- and 3-fold, respectively (P < 0.001) (Fig. 7, A and B). The baseline activity of PKC wild-type transfected NCM460-NK-1R cells without SP stimulation had increased IL-8 promoter and NF-B luciferase activities (by 11-fold, P < 0.001; 4-fold, P < 0.01, respectively). Consistent with our above results, rottlerin (1 µM) significantly blocked SP-induced IL-8 promoter (P < 0.001) and NF-B luciferase (P < 0.01) activity with or without overexpression of wild-type PKC (Fig. 7, A and B). Taken together, our findings clearly demonstrate that PKC activation is an important step for SP-induced NF-B activation and subsequent IL-8 gene expression.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Overexpression of wild-type PKC potentiates SP-induced IL-8 promoter and NF-B luciferase activities. NCM460-NK-1R cells were transiently transfected with a pCMV--galactosidase control or a wild-type PKC-overexpressing plasmid together with an IL-8 promoter or an NF-B reporter construct plus an internal control pRL-TK plasmid. Transfected cells were serum-starved overnight and then pretreated with either rottlerin (1 µM) or vehicle (DMSO) for 30 min followed by SP (10-7 M) exposure for 4 h. Cell extracts were prepared to measure IL-8 promoter (A) or NF-B (B) luciferase activity. Data are expressed as means ± S.E.M. (n = 6). ** and ***, P < 0.01 and P < 0.001, respectively.

	Discussion

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We found that in colonocytes, SP causes PKC-threonine phosphorylation, a critical event involved in PKC activation. Results from several different experimental approaches support the importance of PKC in SP-mediated, NF-B-driven IL-8 release. Thus, SP-mediated IL-8 production is selectively inhibited by low doses (<1 µM) of rottlerin, a specific pharmacological antagonist of PKC (Wang et al., 2003), as well as by down-regulation of PKC expression, including PKC. The selective roles of PKC in this SP response were further confirmed by inhibition of endogenous PKC expression by a specific PKC siRNA and by transfection of a wild-type PKC-expressing plasmid that enhances SP-induced NF-B activation as well as IL-8 transcription. Our results also indicate that SP can rapidly but transiently activate PKC and PKC but has no effect on PKC, PKC, PKC, PKC, and PKD/PKCµ phosphorylation. However, our results with specific pseudosubstrate peptide inhibitors do not support a functional role for PKC and PKC in SP-induced IL-8 secretion. Although SP had been shown to induce Ca²⁺ mobilization in U373MG cells (Renzi et al., 2000), SP-induced IL-8 synthesis was not affected by pretreatment of NCM-460-NK-1R cells with the intracellular Ca²⁺ calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (40 µM), the Ca²⁺ channel blocker 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl (100 µM), or the Ca²⁺-dependent PKC inhibitor Go6976 (40 µM) (data not shown). Taken together, our results clearly demonstrate that among several PKC isoforms, Ca²⁺-independent PKC plays a critical role in SP-mediated, NF-B-dependent IL-8 gene expression. Consistent with our observations, the importance of PKC in regulating the NF-B pathway has also been reported in several other systems. For example, lysophosphatidic acid-induced NF-B and IL-8 expression is dependent on PKC activation in human bronchial epithelial cells (Cummings et al., 2004). PKC is also involved in TNF-induced NF-B-dependent gene expression in airway epithelial cells as inhibition of PKC either by rottlerin or by a dominant negative PKC mutant-attenuated TNF-induced IL-8 promoter transcriptional activity (Page et al., 2003).

Our previous results indicated an important role for Rho GTPases in SP-induced NF-B activation and IL-8 secretion in human NCM460-NK-1R colonocytes (Zhao et al., 2002). Recent reports also suggested that Rho A is upstream of PKC, including PKC in colonic epithelial cells (Massoumi et al., 2002), and intestinal smooth muscle cells (Harnett et al., 2005) Therefore, it is likely that SP-induced PKC activation and IL-8 secretion are also mediated via the Rho family of GTPases in NCM460 colonocytes. SP also induces MAPK via EGFR transactivation in human NCM460 and U373MG (Koon et al., 2004), but pharmacological inhibition of EGFR by AG1478 (2 µM) and ERK1/2 by PD98059 (25 µM) failed to affect SP-induced IL-8 secretion (data not shown), suggesting that SP-induced IL-8 secretion does not involve the EGFR/MAPK pathway.

Although NF-B plays a pivotal role in intestinal inflammation (Jobin and Sartor, 2000), the role of PKCs, especially PKC, has not been systematically evaluated. PKC was found to be one of important mediators during 2,4,6-trinitrobenzenesulfonic acid-induced colitis in rats as the PKC inhibitors staurosporine and GF-109203X reduced 2,4,6-trinitrobenzenesulfonic acid-induced changes in mucosal PKC activity and the degree of mucosal damage (Brown et al., 1999). The selective PKC inhibitor rottlerin was found to inhibit proinflammatory cytokine production such as TNF and IL-1 in human monocytes (Kontny et al., 2000). Also, PKC was activated in lung epithelial cells with asbestos-induced inflammation (Lounsbury et al., 2002). Our results provide strong evidence that the proinflammatory effect of SP in intestinal inflammation could be mediated via PKC. Thus, inhibition of PKC may represent a potential therapeutic approach for treatment of diseases associated with intestinal inflammation.

	Footnotes

 
This work was supported by National Institutes of Health Grant DK 47343 (to C.P.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.088013.

ABBREVIATIONS: SP, substance P; NK-1R, neurokinin-1 receptor; NF, nuclear factor; IL-8, interleukin-8; TNF, tumor necrosis factor ; MAPK, mitogen-activated protein kinase; EGFR, epidermal growth factor receptor; PKC, protein kinase C; DAG, diacylglycerol; CAPE, caffeic acid phenethyl ester; PMA, phorbol-12-myristate-13-acetate; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; IKK, IB kinase complex; siRNA, small interfering RNA; Go6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; PD98059, 2'-amino-3'-methoxyflavone; DMSO, dimethyl sulfoxide.

Address correspondence to: Dr. Charalabos Pothoulakis, Beth Israel Deaconess Medical Center, Division of Gastroenterology, Dana 501, 330 Brookline Avenue, Boston, MA 02215. E-mail: cpothoul{at}bidmc.harvard.edu

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