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Am J Physiol Gastrointest Liver Physiol 292: G1429-G1438, 2007. First published February 15, 2007; doi:10.1152/ajpgi.00315.2006
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NEUROREGULATION AND MOTILITY

Intestinal inflammation downregulates smooth muscle CPI-17 through induction of TNF-{alpha} and causes motility disorders

Takashi Ohama,1 Masatoshi Hori,1 Eiichi Momotani,2 Yoichiro Iwakura,3 Fengling Guo,4 Hiroko Kishi,4 Sei Kobayashi,4 and Hiroshi Ozaki1

1Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo; 2Paratuberculosis and Inflammatory Bowel Disease Research Team, National Institute of Animal Health, Tsukuba; 3Center for Experimental Medicine, Institute of Medical Science, The University of Tokyo, Tokyo; and 4Department of Molecular Physiology and Medical Bioregulation, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan

Submitted 18 July 2006 ; accepted in final form 26 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Motility disorders are frequently observed in intestinal inflammation. We previously reported that in vitro treatment of intestinal smooth muscle tissue with IL-1beta decreases the expression of CPI-17, an endogenous inhibitory protein of smooth muscle serine/threonine protein phosphatase, thereby inhibiting contraction. The present study was performed to examine the pathophysiological importance of CPI-17 expression in the motility disorders by using an in vivo model of intestinal inflammation and to define the regulatory mechanism of CPI-17 expression by proinflammatory cytokines. After the induction of acute ileitis with 2,4,6,-trinitrobenzensulfonic acid, CPI-17 expression declined in a time-dependent manner. This decrease in CPI-17 expression was parallel with the reduction of cholinergic agonist-induced contraction of smooth muscle strips and sensitivity of permeabilized smooth muscle fibers to Ca2+. Among the various proinflammatory cytokines tested, TNF-{alpha} and IL-1beta were observed to directly inhibit CPI-17 expression and contraction in cultured rat intestinal tissue. Moreover, both TNF-{alpha} and IL-1beta inhibited CPI-17 expression and contraction of smooth muscle tissue isolated from wild-type and IL-1{alpha}/beta double-knockout mice. However, IL-1beta treatment failed to inhibit CPI-17 expression and contraction in TNF-{alpha} knockout mice. In beta-escin-permeabilized ileal tissues, pretreatment with anti-phosphorylated CPI-17 antibody inhibited the carbachol-induced Ca2+ sensitization in the presence of GTP. These findings suggest that CPI-17 was downregulated during intestinal inflammation and that TNF-{alpha} plays a central role in this process. Downregulation of CPI-17 may play a role in motility impairments in inflammation.

smooth muscle contraction; tumor necrosis factor-{alpha}

CPI-17 WAS FIRST ISOLATED from the pig aorta as an endogenous inhibitory protein of Ser/Thr protein phosphatase (8, 9). Activation of CPI-17 is regulated by phosphorylation of Thr38, and several kinases, including protein kinase C, Rho kinases, zipper-interacting protein kinase, protein kinase N, and integrin-linked kinase, are capable of doing this (11, 21, 25, 28, 32). Activated CPI-17 directly binds to the catalytic subunit of myosin phosphatase (PP1c{delta}), thereby inhibiting myosin phosphatase activity and increasing the relative amount of phosphorylated myosin light chain protein and augmenting smooth muscle contraction (7). Thus inhibition of myosin phosphatase plays a pivotal role in agonist-induced smooth muscle contraction. Various receptor agonists, such as cholinergic agents, histamine, endothelin, and angiotensin II, activate CPI-17 in tandem with induction of myosin phosphorylation and smooth muscle contraction at a given intracellular Ca2+ concentration (21, 22, 32). Although the mechanism by which CPI-17 induces smooth muscle contraction is well established, little is understood about the pathophysiological significance of this.

Motility disorders in the inflamed gastrointestinal tract are clinically important because they can lead to systemic disease. Both increased and decreased smooth muscle contractility were observed in intestinal inflammation. In a nematode infectious model, such as Trichinella spiralis-infected gut inflammation, smooth muscle contractility was increased (4, 41). On the other hand, in an intestinal inflammation induced by 2,4,6,-trinitrobenzensulfonic acid (TNBS), surgical manipulation, experimental obstruction, hemorrhagic shock, and peritonitis, smooth muscle contractility was decreased (12, 16, 23, 26, 30, 42). One of the most well-investigated mechanisms of inhibition of motility is the change in enteric neural circuitry (29). As for the myogenic mechanism for decreased smooth muscle contractility of inflamed intestinal tissues, there are some reports that suggest the changing activity of channels and receptors (1, 15, 18, 20, 27, 37). However, the role of endogenous signaling molecules for smooth muscle contraction in dysmotility of intestine has not been examined in the setting of inflammation.

Mucosal immune responses to intestinal inflammation, such as inflammatory bowel disease (IBD), have been well examined (5). Although little is known about immune responses in the region of intestinal smooth muscle, some reports suggest that there is a unique immune system in the intestinal smooth muscle region independent of mucosal region (6, 33–35, 38). Our group (40) recently demonstrated upregulation of proinflammatory cytokines, such as IL-1beta and IL-6, in the inflamed ileal muscle of a dilated region of intestine in a rat model of Hirschsprung's disease. Based on this finding, proinflammatory cytokines might directly affect the myogenic contractile apparatus, thus impairing the function of intestinal smooth muscle cells. Previously, our group (32) clarified that long-term treatment of ileal smooth muscle tissues with IL-1beta inhibits contraction through downregulation of CPI-17. This result suggests the possibility that CPI-17 strongly contributes to gastrointestinal motility disorders during inflammation. However, the pathophysiological significance of CPI-17 remains unclear.

In the present study, we sought to determine the role of smooth muscle CPI-17 in the motility disorders associated with intestinal inflammation. Furthermore, we examined the effects of various cytokines on this process using cytokine knockout (KO) mice. We observed that a decrease in CPI-17 expression within smooth muscle contributes to decreased motility in an animal model of intestinal inflammation and that TNF-{alpha} plays a crucial role in the CPI-17 downregulation.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. C57BL/6J IL-1{alpha}/beta double-KO mice (14) and C57BL/6J TNF-{alpha} KO mice (17) were bred and maintained in our laboratory. C57BL/6J mice (Charles River Japan) were used as control mice. Male Wistar rats (200–250 g) were purchased from Charles River Japan. Animal care and treatment were conducted in accordance with the institutional guidelines of the University of Tokyo. Experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Tokyo.

Organ culture procedure. Organ culture was performed as previously described (32). Briefly, male Wister rats and male C57BL/6J mice were euthanized by a sharp blow to the neck, followed by exsanguination. A segment of the ileum was detached from the mesentery and placed in sterile Hanks' balanced salt solution. Strips were teased along the natural line of cleavage from the longitudinal smooth muscle. The strips, which included both the circular and longitudinal smooth muscle layer, were then transferred to culture dishes with medium 199 supplemented with 1% antibiotic and antimicotic and L-glutamine. The culture dishes were incubated at 37°C in an atmosphere of 95% air and 5% CO2. The incubation medium was replaced every day.

Induction of acute intestinal inflammation. Male Wistar rats and male C57BL/6J mice were anesthetized, and the abdomen was opened by midline laparotomy. The lower segment of the ileum was gently exteriorized, and a 2- to 3-cm tract was isolated using clips, after which ileitis was induced by injecting 100 mg/kg TNBS within a 50% ethanol saline solution into the lumen. Saline was used as the vehicle.

Measurement of muscle tension. Measurement of longitudinal and circular muscle tension was performed as previously described (32). Briefly, muscle tension was isometrically recorded with a force displacement transducer. Each of the muscle strips was cut into 3 x 5-mm pieces and attached to a holder under a resting tension of 10 mN. After equilibration for 15 min in a bath, each strip was repeatedly exposed to the high-K+ (72.7 mM) solution until stable responses were observed. Contractile force was expressed in terms of millinewtons per square millimeter of cross-sectional area.

{alpha}-Toxin-permeabilized muscle. Permeabilized muscle was prepared by treating the intestinal smooth muscle strips with Staphylococcus aureus {alpha}-toxin (180 µg/ml, 25°C, 30 min; Sigma) as previously described (32). The relaxing solution contained Mg2+-dimethanesulfonate (MgMs2; 7.1 mM), KMs (108.0 mM), ATP (5.9 mM), creatine phosphate (2.0 mM), PIPES (20.0 mM), EGTA (2.0 mM), creatine phosphokinase (10 U/ml), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 µM), and E-64 (1 µg/ml) (pH 6.8). Free Ca2+ concentrations were adjusted by adding appropriate amounts of CaMs2. The apparent binding constant of EGTA for Ca2+ was considered to be 1 µM. Longitudinal contractile force was measured by an isometric transducer under a resting tension of 1 mN at room temperature (22–24°C).

beta-Escin-permeabilized muscle. The permeabilization with beta-escin of small strips (400–600 x 400–600 µm) of rat ileal smooth muscle was performed as previously described (24). Isometric tension of longitudinal muscle was measured using a force transducer in a well on a bubble plate. The solution was changed by sliding the bubble plate to an adjacent well. When steady responses to high K+ were observed, the strips were incubated for 10 min in normal relaxing solution (in mM: 74.1 KMs, 2 MgMs2, 4.5 MgATP, 1 EGTA, 10 creatine phosphate, and 30 PIPES). The strips were incubated with 20 µM beta-escin for 20–30 min in normal relaxing solution. Calmodulin (1 µM) was added to the activating solution. Ca2+ sensitization was elicited by 1 µM carbachol plus 100 µM GTP at pCa 6.3. Either normal IgG (1:200) or anti-Thr38-phosphorylated CPI-17 antibody (1:200) was pretreated for 20 min.

Western blot analysis. Western blot analysis was performed as previously described (32). Ileal muscle strips devoid of mucosa were homogenized in homogenizing buffer to extract protein. The homogenizing buffer used to extract CPI-17 contained 500 mM KCl, 0.1 mM EGTA, and 50 mM Tris·HCl at pH 6.8. Homogenization solution contained 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Total protein (20 µg) was mounted in each lane to detect CPI-17. PBS containing 5% skim milk was used as a blocking buffer and treated for 30 min at room temperature. Anti-CPI-17 antibody [CPI-17 (N-20), 1:250 dilution; Santa Cruz Biotechnology, Santa Cruz, CA] was treated as the first antibody in the blocking buffer overnight at 4°C and biotinylated anti-goat IgG (H+L; 1:1,000 dilution; Vector Laboratories, Burlingame, CA) as the secondary antibody for 1 h at room temperature. Afterwards horseradish peroxidase-streptavidin (1:1,000 dilution; Zymed Laboratories) was treated for 1 h at room temperature. CPI-17 was detected using an ECL Plus Western blotting detection system (Amersham Biosciences, Amersham, UK). Bands of CPI-17 were visualized using an LAS-1000 (FUJIFILM, Tokyo, Japan).

ELISA. Ileal smooth muscle tissue without the mucosal layer was homogenized as described under Western blot analysis using 50 mM Tris (pH 7.3) containing 5 mM EDTA as homogenization buffer with various protease inhibitors. The supernatants were collected and used for ELISA. To perform the protein assays, we used rat TNF-{alpha} Quantikine ELISA, rat IL-1beta Quantikine ELISA, and rat IL-6 Quantikine ELISA (R&D Systems). Each sample was assayed in duplicate. The standard was diluted in homogenization buffer with a protease inhibitor cocktail. Apart from this, the assay was performed as recommended by the manufacturer.

RT-PCR. Total RNA was extracted from the smooth muscle strips by using the acid guanidinium isothiocyanate-phenol-chloroform method, and the concentration of RNA was adjusted to 1 µg/µl with RNase-free distilled water. Semiquantitative RT-PCR was performed, during which first-strand cDNA was synthesized using a random 9-mer primer and avian myeloblastosis virus (AMV) reverse transcriptase XL at 30°C for 10 min, followed by 55°C for 45 min, 99°C for 5 min, and 4°C for 5 min. PCR amplification was performed using the hot starting method with Taq Gold (Perkin-Elmer, Branchburg, NJ). The following oligonucleotide sequences were used as primers to amplify the target genes: 5'-acggcatggatctcaaagac-3' (TNF-{alpha} sense) and 5'-aacacccattcccttcacag-3' (TNF-{alpha} antisense), 5'-ctacagttctgccattgacc-3' (IL-1{alpha} sense) and 5'-ttgagcgctcacgaacagtt-3' (IL-1{alpha} antisense), 5'-agctctccacctcaatggac-3' (IL-1beta sense) and 5'-tggggaaggcattagaaaca-3' (IL-1beta antisense), and 5-'tgttcctacccccaatgtgt-3' (GAPDH sense) and 5'-ccctgttgctgtagccgtat-3' (GAPDH antisense). Amplification was performed by initial denaturation at 95°C for 10 min, followed by 30 amplification cycles including 40 s at 94°C, 60 s at 56°C, and 40 s at 72°C. The PCR products were then separated by electrophoresis on 2% agarose gel containing 0.1% ethidium bromide. The possibility of DNA contamination was excluded by PCR using total RNA and excluding the reverse transcription step. The resultant fluorescent bands were visualized using an ultraviolet transilluminator with FAS-III (Toyobo, Tokyo, Japan), after which band density was measured using NIH Image software.

Statistical analyses. Results are expressed as means ± SE. Comparisons between the control and test groups were performed using one-way ANOVA, followed by Dunnett's multiple-comparison test. For all evaluations, P values of <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TNBS-induced intestinal inflammation inhibits CPI-17 expression and motility. Alterations in CPI-17 protein expression in smooth muscle tissue isolated from rats with TNBS-induced acute ileitis were analyzed by Western blot analysis. As shown in Fig. 1, CPI-17 expression within the smooth muscle layer gradually declined in TNBS-treated rats, reaching a minimum 3 days after the treatment. In accordance with the reduced inflammation, CPI-17 expression made a recovery. Therefore, we examined the effects of inflammation on smooth muscle contraction at 3 days after treatment with TNBS. In the rat model of TNBS-induced ileitis, the amplitude of both longitudinal and circular smooth muscle contractions induced by carbachol was decreased (Fig. 2). Relative contraction of longitudinal smooth muscle induced by 1 µM carbachol-induced contraction that was normalized with the force induced by high-K+ (72.7 mM) solution was inhibited by treatment with TNBS (vehicle: 134.4 ± 4.8%, TNBS: 110.0 ± 4.2%, n = 4, P<0.01), suggesting that carbachol-induced contraction was more significantly inhibited than high K+-induced contraction. At day 7, 1 µM carbachol-induced contractions of the TNBS-induced ileitis tissues were recovered to the level of vehicle-treated tissues (vehicle: 32.4 ± 2.2 mN/mm2, TNBS: 31.0 ± 2.1 mN/mm2, n = 4).


Figure 1
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  		Fig. 1. 2,4,6,-Trinitrobenzensulfonic acid (TNBS)-induced inflammation causes downregulation of CPI-17 protein within ileal smooth muscle. Male Wistar rats were treated with vehicle or TNBS. Time-dependent changes in CPI-17 protein expression during intestinal inflammation were measured by Western blot analysis. Results are expressed as means ± SE of 4–6 experiments. **P < 0.01, significantly different from vehicle.

 

Figure 2
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  		Fig. 2. TNBS-induced inflammation decreases the force of muscarinic agonist-induced ileal smooth muscle contraction. Male Wistar rats were treated with vehicle or TNBS. Carbachol-induced contractions of longitudinal (A) and circular smooth muscle (B) were measured 3 days after TNBS treatment. Results are expressed as means ± SE of 4–6 experiments. **P < 0.01, significantly different from vehicle.

 
Receptor agonist-induced contraction is greatly dependent on sensitization of the contractile elements to cytosolic Ca2+. guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S), a nonhydrolyzable GTP analog, is known to induce Ca2+ sensitization that is mediated by RhoA (10, 13). Because our group previously clarified the involvement of CPI-17 in RhoA-mediated pathway (32), we next examined the effect of TNBS-induced inflammation on Ca2+ sensitization in {alpha}-toxin-permeabilized ileal smooth muscle. As shown in Fig. 3, in the permeabilized muscle prepared from tissue treated with vehicle, GTP{gamma}S sensitized the contractions under the constant cytosolic Ca2+ concentration (1 µM). The Ca2+ sensitization elicited by GTP{gamma}S at 1 µM Ca2+ was significantly inhibited by TNBS treatment.


Figure 3
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  		Fig. 3. TNBS-induced inflammation reduces guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S)-induced Ca2+ sensitization of {alpha}-toxin-permeabilized ileal smooth muscle. Male Wistar rats were treated with vehicle or TNBS. GTP{gamma}S was applied for 5 min before induction of contraction with 1 µM Ca2+. A: typical trace demonstrating Ca2+ sensitization, selected from 14 trials. B: quantitative data demonstrating Ca2+ sensitization elicited by GTP{gamma}S at a given cytosolic Ca2+ concentration (1 µM). 100% force represents the contraction induced by 1 µM Ca2+ alone. Results are expressed as means ± SE of 7 experiments. **P < 0.01, significantly different from vehicle.

 
TNF-{alpha} and IL-1beta downregulate CPI-17 and reduce the force of contraction. In previous work, our group (32) chose IL-1beta as an inducer of inflammatory reaction in smooth muscle tissue. Because cytokines are part of a complex network, we explored the effects of other inflammatory cytokines on smooth muscle function in this study. First, we measured the production of proinflammatory cytokines within the smooth muscle layer of the rat intestine following induction of inflammation. As shown in Fig. 4, TNF-{alpha}, IL-1beta, and IL-6 were observed to increase within the smooth muscle layer of the rat ileum 1–3 days after treatment with TNBS.


Figure 4
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  		Fig. 4. TNBS-induced inflammation increases the expression of proinflammatory cytokines within ileal smooth muscle. Male Wistar rats were treated with vehicle or TNBS. Cytokine expression within the smooth muscle layer [TNF-{alpha} (A), IL-1beta (B), and IL-6 (C)] was measured by ELISA 1 to 3 days after TNBS treatment. Results are expressed as means ± SE of 4–16 experiments. *P < 0.05 and **P < 0.01, significantly different from control.

 
Next, we used an organ culture system to identify which cytokines directly affect smooth muscle function. When isolated smooth muscle from Wistar rats was treated with TNF-{alpha} (20 ng/ml) and IL-1beta (10 ng/ml) for 5 days, CPI-17 expression significantly declined, whereas IL-6 (10 ng/ml) and IL-10 (10 ng/ml) did not inhibit CPI-17 expression. Carbachol-induced contraction was consistently inhibited by TNF-{alpha} and IL-1beta but not by IL-6 and IL-10 (Fig. 5). Because our group (32) had previously clarified that IL-1beta decreased CPI-17 expression and carbachol-induced contraction, we next examined whether IL-1beta acts directly or indirectly through the production of TNF-{alpha} in organ culture system. When rat ileal smooth muscle tissue was treated with IL-1beta (10 ng/ml) for 1 day, TNF-{alpha} protein in medium increased 17-fold (control: 1.4 ± 1.3 ng/ml, IL-1beta: 24.6 ± 4.0 ng/ml, n = 4) and then fell by day 3 (control: 1.7 ± 0.7 ng/ml, IL-1beta: 8.0 ± 2.3 ng/ml, n = 4). These results suggest that both TNF-{alpha} and IL-1beta are implicated in the CPI-17 downregulation after gut inflammation.


Figure 5
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  		Fig. 5. TNF-{alpha} and IL-1beta directly decrease CPI-17 expression and smooth muscle contraction in an in vitro organ culture system. The effects of TNF-{alpha} (20 ng/ml), IL-1beta (10 ng/ml), IL-6 (10 ng/ml), and IL-10 (10 ng/ml) were analyzed. Ileal smooth muscle tissue isolated from Wistar rats was cultured for 5 days with or without cytokines. A: protein expression of CPI-17, which was measured using Western blot analysis. CPI-17 band densities were normalized to the control (Cont.). B: absolute force of 1 µM carbachol-induced contraction. Results are expressed as means ± SE of 5 experiments. N.S., not significantly different. **P < 0.01, significantly different from vehicle.

 
IL-1beta decreases CPI-17 expression and smooth muscle contraction via TNF-{alpha}. In view of the above findings, we questioned whether TNF-{alpha} and IL-1beta act on smooth muscle cells in parallel and/or via a pathway involving sequential activation. To clarify this point, we next investigated the effect of TNF-{alpha} and IL-1beta on CPI-17 expression and smooth muscle contraction using TNF-{alpha} KO mice and IL-1{alpha}/beta double-KO mice. Ileal smooth muscle tissue isolated from these mice was treated with TNF-{alpha} (20 ng/ml) and IL-1beta (10 ng/ml) for 3 days in organ culture. As shown in Fig. 6A, TNF-{alpha} reduced CPI-17 expression in all ileal smooth muscle preparations. On the other hand, although IL-1beta significantly inhibited CPI-17 expression in control C57BL/6J mice and IL-1{alpha}/beta double-KO mice, it failed to do so in TNF-{alpha} KO mice. Along with the decline in CPI-17 expression, the extent of carbachol-induced contraction was significantly inhibited by treatment with either TNF-{alpha} or IL-1beta in ileal smooth muscle tissue isolated from control C57BL/6J mice. TNF-{alpha} inhibited the carbachol-induced contraction in both TNF-{alpha}- and IL-1{alpha}/beta-deficient mice. Although IL-1beta reduced the carbachol-induced contraction in IL-1{alpha}/beta douuble-KO mice, it did not change in ileal smooth muscle tissue isolated from TNF-{alpha} KO mice (Fig. 6, B and C).


Figure 6
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  		Fig. 6. IL-1beta decreases CPI-17 expression and smooth muscle contraction through the action of TNF-{alpha}. The effect of TNF-{alpha} (20 ng/ml) and IL-1beta (10 ng/ml) on CPI-17 expression (A) and force of 1 µM carbachol-induced contraction (B) was measured in wild-type C57BL/6J mice, TNF-{alpha} knockout (KO) mice, and IL-1{alpha}/beta double-KO mice. Ileal smooth muscle tissue was isolated from these mice and cultured for 3 days with or without cytokines. Open, shaded, and solid bars represent tissue cultured without cytokines, with TNF-{alpha}, and with IL-1{alpha}/beta, respectively. CPI-17 band densities were normalized to the control. Results are expressed as means ± SE of 4–10 experiments. *P < 0.05 and **P < 0.01, significantly different from control.

 
TNF-{alpha} is essential for downregulation of CPI-17 in an in vivo model of ileitis. We further explored the importance of TNF-{alpha} in downregulation of CPI-17 in vivo. Ileal inflammation was induced by injection of TNBS in control C57BL/6J mice, TNF-{alpha} KO mice, and IL-1{alpha}/beta double-KO mice. First, we confirmed that intestinal inflammation upregulates TNF-{alpha}, IL-1{alpha}, and IL-1beta mRNA within the smooth muscle layer of the ileum (Fig. 7). Semiquantitative RT-PCR showed an increase in TNF-{alpha} mRNA following treatment with TNBS in control C57BL/6J mice and IL-1{alpha}/beta double-KO mice (note that IL-1{alpha} and IL-1beta were undetectable in IL-1{alpha}/beta double-KO mice). IL-1{alpha} and IL-1beta mRNA were also increased in control C57BL/6J mice and TNF-{alpha} KO mice (note that TNF-{alpha} was undetectable in TNF-{alpha} KO mice). TNBS treatment reduced CPI-17 expression in control C57BL/6J mice and in IL-1 {alpha}/beta double-KO mice, whereas the expression remained unchanged following induction of ileal inflammation in TNF-{alpha} KO mice (Fig. 8A). Moreover, the carbachol-induced contraction was significantly inhibited in IL-1{alpha}/beta double-KO mice following TNBS treatment but did not change in TNF-{alpha} KO mice (Fig. 8, B and C).


Figure 7
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  		Fig. 7. TNBS-induced inflammation increases TNF-{alpha} mRNA expression in IL-1{alpha}/beta KO mice. Intestinal inflammation was induced by TNBS in wild-type C57BL/6J mice, TNF-{alpha} KO mice, and IL-1{alpha}/beta double-KO mice. Each mouse was treated with vehicle or TNBS for 2 days. The effect of TNBS-induced inflammation on mRNA expression [TNF-{alpha} (B), IL-1{alpha} (C), and IL-1beta(D)] within the smooth muscle region was measured using semiquantitative RT-PCR analysis. A: images of typical bands for cytokines. Band densities were normalized to that of GAPDH. Results are expressed as means ± SE of 10 experiments. U.D., undetectable. *P < 0.05 and **P < 0.01, significantly different from control.

 

Figure 8
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  		Fig. 8. TNF-{alpha} is essential for downregulation of CPI-17 in an in vivo model of ileitis. Intestinal inflammation was induced by TNBS in wild-type C57BL/6J mice, TNF-{alpha} KO mice, and IL-1{alpha}/beta double-KO mice. A: each mouse was treated with vehicle or TNBS. CPI-17 expression was measured by Western blot, and band densities were normalized to the level of protein found within freshly isolated control tissue. Carbachol-induced contraction was measured at 3 days after TNBS treatment in IL-1{alpha}/beta KO (B) and TNF-{alpha} KO mice (C). Results are expressed as means ± SE of 4–8 experiments. **P < 0.01, significantly different from control.

 
CPI-17 plays a key role in carbachol-induced contraction of ileal smooth muscle. To clarify the physiological importance of CPI-17 in carbachol-induced contraction of ileal smooth muscle, we utilized beta-escin-permeabilized tissues. As shown in Fig. 9, when the permeabilized tissues were treated with normal IgG as negative control, carbachol (1 µM) with GTP (100 µM) elicited Ca2+ sensitization that nearly doubled. However, after incubation with anti-Thr38-phosphorylated CPI-17 IgG, the carbachol-induced Ca2+ sensitization was almost completely inhibited.


Figure 9
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  		Fig. 9. CPI-17 plays a key role in contraction of ileal smooth muscle. Ileal smooth muscle tissues of Wistar rats were permeabilized with beta-escin. Normal IgG or antibody against Thr38-phosphorylated CPI-17 was applied for 20 min before and during induction of contraction with 1 µM carbachol and 100 µM GTP at a given cytosolic Ca2+ concentration (pCa 6.3). A: a representative trace demonstrating Ca2+ sensitization. Filled circles indicate mixing of buffer. B: quantitative data demonstrating Ca2+ sensitization elicited by 1 µM carbachol and 100 µM GTP at pCa 6.3 at a given cytosolic Ca2+ concentration (pCa 6.3). 100% force represents the contraction induced by pCa 6.3 alone. Results are expressed as means ± SE of 3–4 experiments. **P < 0.01, significantly different from normal IgG.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we attempted to clarify the pathophysiological role of smooth muscle CPI-17, an inhibitory protein of smooth muscle myosin phosphatase, in the motility disorders as a result of intestinal inflammation. Our main findings were 1) reduced CPI-17 expression upon exposure to proinflammatory cytokines in in vitro and in vivo models of intestinal inflammation and 2) marked suppression of CPI-17 downregulation, as well as the associated decrease in smooth muscle contractility, in the complete absence of TNF-{alpha}.

It is generally recognized that receptor stimulation activates the Ca2+ sensitivity of the smooth muscle via phosphorylation of MYPT1 and CPI-17, the endogenous inhibitory protein for Ser/Thr protein phosphatase, and induces smooth muscle contraction (39). Our group (32) previously reported that in intestinal smooth muscle, Ca2+ sensitization due to muscarinic stimulation is mainly mediated by CPI-17 rather than MYPT1. Moreover, our group found that long-term treatment of smooth muscle with IL-1beta attenuates ileal smooth muscle contraction by decreasing CPI-17 activity (32). Thus a correlation was clearly demonstrated between alterations in CPI-17 expression and smooth muscle contractile function in inflamed intestinal smooth muscle in this study. We observed downregulation of CPI-17 expression in TNBS-induced acute ileitis model animals at 3 days. CPI-17 expression was recovered at 7 days after TNBS induction. In keeping with these results, the force of carbachol-induced contractions was markedly reduced at 3 day and recovered at 7 day in inflamed smooth muscle. Together with these findings, the fact that carbachol-induced contraction was more greatly inhibited than high K+-induced contraction supports the notion that CPI-17 downregulation is important in the smooth muscle dysmotility, because the carbachol-induced contraction is more strongly dependent on the Ca2+ sensitization mechanism. In intestinal smooth muscle, our group (32) also previously reported that the Ca2+ sensitization is mediated by CPI-17 activation through G protein-coupled RhoA pathway after receptor stimulation. As described in RESULTS, GTP{gamma}S, a nonhydrolyzable GTP analog, activates RhoA and induces Ca2+ sensitization (10, 13). Therefore, an inhibition of GTP{gamma}S-induced Ca2+ sensitization in TNBS-treated rat {alpha}-toxin-permeabilized tissue, as observed in the present study, supports the involvement of CPI-17 in decreased motility associated with intestinal inflammation. Moreover, in the present study we confirmed the importance of CPI-17 in ileal smooth muscle contraction by using anti-Thr38-phosphorylated CPI-17 antibody to block CPI-17 activity in beta-escin-permeabilized tissues. These results suggest that CPI-17 downregulation is involved in ileal inflammation-induced dysmotility.

The gastrointestinal immune system tends to involve primarily the mucosal layer, and a number of reports have demonstrated the production of various cytokines and/or chemokines from the mucosal region (5). However, several recent reports (6, 33–35, 38, 40) support the view that there is a unique immune system in the intestinal muscle region. In the present study, we focused on the inflamed muscle layer to monitor the changes in expression of proinflammatory cytokines (TNF-{alpha}, IL-1{alpha}, IL-1beta, and IL-6). Our results indicate that each of these cytokines within the smooth muscle layer is transiently upregulated during TNBS-induced acute inflammation. These results suggest that smooth muscle cells may be directly exposed to these cytokines during intestinal inflammation in vivo. The fact that CPI-17 expression made a recovery in accordance with reduced cytokine production suggests that these cytokines may be a key regulator of CPI-17 expression. Therefore, we further examined which cytokines are likely responsible for downregulation of CPI-17 and dysfunction of smooth muscle by using an organ culture system and found that TNF-{alpha} and IL-1beta, but not IL-6 and IL-10, directly affect smooth muscle tissue and decrease CPI-17 expression.

Since cytokines form a complex network, we utilized TNF-{alpha} KO and IL-1{alpha}/beta double-KO mice to examine the effects of various cytokines on CPI-17 expression. The results obtained from organ culture studies indicate that IL-1beta regulates CPI-17 expression and smooth muscle contraction through the action of TNF-{alpha}, since the direct inhibitory effects of IL-1beta on CPI-17 expression and smooth muscle contraction were absent in smooth muscle tissues from TNF-{alpha} KO mice. In an in vivo ileitis model, TNBS treatment reduced CPI-17 expression in control C57BL/6J mice and in IL-1{alpha}/beta double-KO mice, whereas expression remained unchanged following induction of ileal inflammation in TNF-{alpha} KO mice. In an in vitro organ culture study, we demonstrated that IL-1beta generated TNF-{alpha}, which resulted in the suppression of CPI-17 expression and smooth muscle contraction. In an in vivo TNBS-induced inflammation model, it is possible that not only IL-1beta but also other cytokines may contribute to the induction of TNF-{alpha}. Consistently, we observed upregulation of TNF-{alpha} mRNA in smooth muscle tissues of TNBS-treated IL-1{alpha}/beta double-KO mice. Based on these findings, TNF-{alpha}, but not IL-1beta, is thought to directly affect intestinal smooth muscle tissues and plays a key role in the decreased smooth muscle contractility-associated CPI-17 downregulation. We further confirmed that IL-1beta upregulated TNF-{alpha} production in smooth muscle tissue culture system at 1 day after the treatment. However, CPI-17 downregulation and decreased motility are observed from 3 days as previously described (32). This time lag between TNF-{alpha} upregulation and CPI-17 downregulation was also observed in an in vivo ileitis model in this study. It is possible that CPI-17 downregulation surfaces a few days after TNF-{alpha} exposure because of its half-life.

In this study, we demonstrated the decrease in smooth muscle contraction in an in vivo model of TNBS-induced intestinal inflammation. Consistently, Moreels et al. (30) demonstrated that longitudinal contractile force was attenuated by TNBS treatment at 1.5 days, which then returned to control levels by 7 days. On the other hand, Collins and colleagues (4, 41) reported an increase of longitudinal smooth muscle contraction in a Trichinella spiralis-infected gut inflammation model. They suggested that the hypercontractility is mediated by an increase in PGE2 following the induction of Th2 cytokines, such as IL-4 and IL-13 (2, 3). Because previous reports from our laboratory and others suggested that the TNBS-induced gut inflammation is mediated mainly by Th1 cytokines such as IL-1beta, TNF-{alpha}, and IL-12 (19, 31), the different contractile responses may be explained by the different cytokine profile.

In intestinal motility disorders caused by inflammation, there could be various factors, from histological to functional causes, to disturb intestinal motility. Although myogenic mechanism for decreased motility of inflamed intestinal tissue has not been well examined, there are some reports suggesting the mechanism at a level of smooth muscle function, such as decreased activity of voltage-dependent L-type Ca2+ channel (1, 18, 20, 27, 36), increased activity of ATP-sensitive K+ channel (15), and inhibition of coupling of M3 receptor to its G protein (37). Our finding suggests that CPI-17 downregulation, in cooperate with these receptor/channel abnormalities, causes decreased smooth muscle contraction in intestinal inflammation.

In conclusion, the present study demonstrates that CPI-17, an inhibitory protein of Ser/Thr protein phosphatase, is downregulated during intestinal inflammation and that TNF-{alpha} plays a central role in the inflammatory response mediating CPI-17 downregulation. This CPI-17 downregulation may play a role in decreased motility in intestinal inflammation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by a Grant-in-aid for scientific research from the Japanese Ministry of Education, Program for the Promotion of Basic Research Activities for Innovative Biosciences and the Yakult Bioscience Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Ozaki, Dept. of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The Univ. of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan (e-mail: aozaki{at}mail.ecc.u-tokyo.ac.jp)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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