Noriyuki Kaji

Protease-activated receptor-2 induces proinflammatory cytokine and chemokine gene expression in canine keratinocytes.

Although the molecular basis of the allergenicity remains to be fully elucidated, the ability of allergens to elicit allergic responses is at least partly attributed to their proteolytic activity. Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is activated by site-specific proteolysis by serine proteases and is known to mediate inflammatory processes in various tissues. In this study, we investigated the effects of trypsin, a major serine protease, and a human PAR-2 agonist peptide (SLIGKV-NH2) on proinflammatory cytokine and chemokine gene expression in the canine keratinocyte cell line CPEK. The expression of PAR-2 mRNA and protein in CPEK cells was detected by RT-PCR and Western blotting, respectively. The localization of PAR-2 in CPEK was examined by immunofluorescence. The mRNA expression levels of proinflammatory cytokines and chemokines were quantified by real-time RT-PCR. The free intracellular Ca(2+) concentration was measured using the Ca(2+)-sensitive fluorescent dye. CPEK cells constitutively expressed PAR-2 mRNA and protein. Stimulation of CPEK cells with trypsin induced significant upregulation of the mRNA expression levels of tumor necrosis factor alpha (TNF-α, P<0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF, P<0.01), thymus and activation regulated chemokine (TARC/CCL17, P<0.01), and interleukin 8 (IL-8/CXCL8, P<0.01). Similarly, the PAR-2 agonist peptide increased the mRNA expression levels of TNF-α (P<0.05), GM-CSF (P<0.05), TARC/CCL17 (P<0.05), and IL-8/CXCL8 (P<0.05) in CPEK cells. Both trypsin and the PAR-2 agonist peptide increased the intracellular Ca(2+) concentration and PAR-2 internalization. These results suggest that PAR-2 activation can augment inflammatory cytokine and chemokine expression in canine keratinocytes, and it may initiate allergic inflammation through the proteolytic activity of allergens in canine atopic dermatitis.

Epidermal growth factor is a critical regulator of the cytokine IL-33 in intestinal epithelial cells.

IL‐33 is a novel cytokine that is believed to be involved in inflammation and carcinogenesis. However, its source, its production and its secretion process remain unclear. Recently, we have reported that IL‐33 is up‐regulated in dextran sulfate sodium (DSS) colitis in mice.

Nitric oxide-induced oxidative stress impairs pacemaker function of murine interstitial cells of Cajal during inflammation

The pacemaker function of interstitial cells of Cajal (ICC) is impaired during intestinal inflammation. The aim of this study is to clarify the pathophysiological mechanisms of ICC dysfunction during inflammatory condition by using intestinal cell clusters. Cell clusters were prepared from smooth muscle layer of murine jejunum and treated with interferon-gamma and lipopolysaccharide (IFN-γ+LPS) for 24h to induce inflammation. Pacemaker function of ICC was monitored by measuring cytosolic Ca(2+) oscillation in the presence of nifedipine. Treatment with IFN-γ+LPS impaired the pacemaker activity of ICC with increasing mRNA level of interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 in cell clusters; however, treatment with these cytokines individually had little effect on pacemaker activity of ICC. Treatment with IFN-γ+LPS also induced the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and resident macrophages, but not in ICC. Pretreatment with NOS inhibitor, L-NAME or iNOS inhibitor, 1400W ameliorated IFN-γ+LPS-induced pacemaker dysfunction of ICC. Pretreatment with guanylate cyclase inhibitor, ODQ did not, but antioxidant, apocynin, to suppress NO-induced oxidative stress, significantly suppressed the impairment of ICC function induced by IFN-γ+LPS. Treatment with IFN-γ+LPS also decreased c-Kit-positive ICC, which was prevented by pretreatment with L-NAME. However, apoptotic ICC were not detected in IFN-γ+LPS-treated clusters, suggesting IFN-γ+LPS stimulation just changed the phenotype of ICC but not induced cell death. Moreover, ultrastructure of ICC was not disturbed by IFN-γ+LPS. In conclusion, ICC dysfunction during inflammation is induced by NO-induced oxidative stress rather than NO/cGMP signaling. NO-induced oxidative stress might be the main factor to induce phenotypic changes of ICC.

EGF is a critical regulator of the interleukin‐33 in intestinal epithelial cells

IL‐33 is a novel cytokine that is believed to be involved in inflammation and carcinogenesis. However, its source, its production and its secretion process remain unclear. Recently, we have reported that IL‐33 is up‐regulated in dextran sulfate sodium (DSS) colitis in mice.

Time-dependent changes in inhibitory action of lipopolysaccharide on intestinal motility in rat

Endotoxin causes gastrointestinal motility disorder. Aim of this study is to clarify inhibitory mechanisms of lipopolysaccharide (LPS) on smooth muscle contraction in rat ileum. Ileal tissues were isolated from control rat or from LPS-induced peritonitis model rat. Treatment with LPS inhibited carbachol (CCh)-mediated contraction in a time-dependent manner. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes were also upregulated, but iNOS expression was preceded by a rising of COX-2. All subtypes of prostaglandin E2 (PGE2) receptors (EP1-EP4) were expressed in ileum, and PGE2 and selective EP2 or EP4 agonist inhibited CCh-mediated contraction. Selective iNOS inhibitor did not reverse LPS-induced inhibition of contraction by CCh at 1 and 2 hr, but reduced the inhibitory action at 4 hr after the LPS treatment. COX-2 inhibitor reversed the inhibitory action by LPS in all exposure time. Finally, in ileal tissues isolated from peritonitis model rat, iNOS expression was upregulated only at 4 hr after LPS administration, resulting in enhanced inhibitory action of LPS against CCh-induced contraction. In conclusion, LPS induces COX-2 to produce PGE2, which initially activates EP2 and/or EP4 on smooth muscle cells to inhibit the contractility in early phase of LPS exposure. Moreover, in late phase of LPS treatment, iNOS is expressed to produce NO, which in turn inhibited the contraction by CCh. The inhibitory cascade is similar in the ileum isolated from peritonitis model rat, indicating time-dependent changes of inhibitory action by LPS on intestinal motility in peritonitis.

Disruption of the pacemaker activity of interstitial cells of Cajal via nitric oxide contributes to postoperative ileus

Interstitial cells of Cajal (ICC) serve as intestinal pacemakers. Postoperative ileus (POI) is a gastrointestinal motility disorder that occurs following abdominal surgery, which is caused by inflammation‐induced dysfunction of smooth muscles and enteric neurons. However, the participation of ICC in POI is not well understood. In this study, we investigated the functional changes of ICC in a mouse model of POI.

The essential role of phospho-T38 CPI-17 in the maintenance of physiological blood pressure using genetically modified mice

PKC‐potentiated phosphorylation‐dependent inhibitory protein of protein phosphatase 1 (CPI‐17), an endogenous myosin phosphatase inhibitory protein, is considered a key molecule for Ca2+ sensitization of the contractile apparatus. Here, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR‐associated protein 9 to generate CPI‐17‐deficient [knockout (KO)] and threonine 38 (T38)‐phosphoresistant mice [threonine mutant into alanine (TA)], and then effects of CPI‐17 on vascular contractility in vitro and mean blood pressure (MBP) in vivo were investigated. In isolated thoracic aorta, phorbol 12,13‐dibutyrate induced a sustained contraction of wild‐type (WT) mice, whereas no contraction showed from TA or KO mice. A high concentration of KCl solution‐induced contraction was not different between transgenic and WT mice. In contrast, phenylephrine (PE)‐induced contractions in both mutant strains were significantly smaller than those of WT mice in association with a low level of myosin phosphorylation, suggesting that at least part of PE‐induced contraction is regulated by phosphorylation of CPI‐17 at T38. Finally, the physiologic role of CPI‐17 in the regulation of blood pressure was investigated using radio telemetry. MBP was decreased significantly in both transgenic mice, even with a compensatory increase in heart rate. In summary, we generated KO and constitutively phospho‐resistant mouse models of CPI‐17 for the first time. p‐CPI‐17 at T38, possibly by PKC, could be important to maintain vascular contractility and blood pressure in vivo.— Yang, Q., Fujii, W., Kaji, N., Kakuta, S., Kada, K., Kuwahara, M., Tsubone, H., Ozaki, H., Hori, M. The essential role of phospho‐T38 CPI‐17 in the maintenance of physiological blood pressure using genetically modified mice. FASEB J. 32, 2095–2109 (2018). www.fasebj.org

The anti-inflammatory pathway regulated via nicotinic acetylcholine receptors in rat intestinal mesothelial cells

Regulation of inflammation in intestinal mesothelial cells in the abdominal cavity is important for the pathogeny of clinical conditions, such as postoperative ileus, peritonitis and encapsulating peritoneal sclerosis. Here we have examined the inflammatory effect of lipopolysaccharide (LPS) and the anti-inflammatory effect of nicotinic acetylcholine receptor stimulation in rat intestinal mesothelial cells. LPS upregulated mRNA expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS). The α7, α9 and α10 subunits of nicotinic acetylcholine receptor were detected in intestinal mesothelial cells. Nicotine (10 nM) significantly inhibited LPS-induced mRNA expression of IL-1β and iNOS, but not TNF-α and MCP-1. In addition, the α7 nicotinic acetylcholine receptor selective agonist, PNU-282987 (10 nM), significantly inhibited LPS-induced mRNA expression of IL-1β but not TNF-α, iNOS and MCP-1. Finally, we found that enteric nerves adhered to intestinal mesothelial cells located under the ileal serosa. In conclusion, intestinal mesothelial cells react to LPS to induce the production of nitric oxide from iNOS. The anti-inflammatory action of intestinal mesothelial cells expressing α7nAChR may be mediated via their connectivity with enteric nerves.

Anti-emetic drug maropitant induces intestinal motility disorder but not anti-inflammatory action in mice

Maropitant is a neurokinin 1 receptor (NK1R) antagonist that is clinically used as a new anti-emetic drug for dogs. Substance P (SP) and its receptor NK1R are considered to modulate gastrointestinal peristalsis. In addition, SP works as an inflammatory mediator in gastrointestinal diseases. Aim of this study is to clarify the effects of maropitant on intestinal motility and inflammation in mice. Ex vivo examination of luminal pressure-induced intestinal motility of whole intestine revealed that maropitant (0.1–10 µM) increased frequency of contraction, decreased amplitude of contraction and totally inhibited motility index in a concentration-dependent manner. We measured intestinal transit in vivo by measuring transportation of orally administered luminal content labeled with phenol red. Our results demonstrated that maropitant (10 mg/kg, SC) delayed intestinal transit. Geometric center value was significantly decreased in maropitant-treated mice. Anti-inflammatory effects of maropitant against leukocytes infiltration into the intestinal smooth muscle layer in post-operative ileus (POI) model mice were measured by immunohistochemistry. In POI model mice, a great number of CD68-positive macrophages or MPO-stained neutrophils infiltrated into the inflamed muscle region of the intestine. However, in the maropitant treated mice, the infiltration of leukocytes was not inhibited. The results indicated that maropitant has ability to induce disorder of intestinal motility in mice, but has no anti-inflammatory action in the mouse of a POI model. In conclusion, in mice, maropitant induces disorder of intestinal motility in vivo.

Serotonin 3 receptor signaling regulates 5-fluorouracil–mediated apoptosis indirectly via TNF-α production by enhancing serotonin release from enterochromaffin cells

Antagonists of the 5‐hydroxytryptamine (serotonin) 3 receptor (5‐HT3R) have anti‐inflammatory and anti‐apoptotic activities, but the detailed, underlying mechanisms are not well understood. We focused on anti‐apoptotic activities via 5‐HT3R signaling to clarify the underlying mechanisms. Mice were administered 5‐fluorouracil (5‐FU), which induced apoptosis in intestinal epithelial cells. Coadministration with 5‐HT3R antagonists or agonists tended to decrease or increase the number of apoptotic cells, respectively. In serotonin 3A receptor (5‐HT3AR) null (HTR3A−/−) mice, the number of apoptotic cells induced by 5‐FU was decreased compared with that in wild‐type (WT) mice. Bone marrow (BM) transplantation was performed to determine if BM‐derived immune cells regulated 5‐FU‐induced apoptosis, but they were found to be unrelated to this process. Data from 5‐HT3AR/enhanced green fluorescent protein reporter mice revealed that 50% of enterochromaffin (EC) cells expressed 5‐HT3AR, but the number of apoptotic cells induced by 5‐FU in the intestinal crypt organoids of HTR3A−/− mice was not altered compared with WT mice. In contrast, plasma 5‐HT concentrations in WT mice but not in HTR3A−/− mice administered 5‐FU were increased significantly. In conclusion, 5‐HT3R signaling may enhance 5‐HT release, possibly from EC cells intravascularly, or paracrine, resulting in increases in plasma 5‐HT concentration, which in turn, enhances apoptotic activities induced by 5‐FU—Mikawa, S., Kondo, M., Kaji, N., Mihara, T., Yoshitake, R., Nakagawa, T., Takamoto, M., Nishimura, R., Shimada, S., Ozaki, H., Hori, M. Serotonin 3 receptor signaling regulates 5‐fluorouracil‐mediated apoptosis indirectly via TNF‐α production by enhancing serotonin release from enterochromaffin cells. FASEB J. 33, 1669–1680 (2019). www.fasebj.org