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Osman J, Savari S, Chandrashekar NK, Bellamkonda K, Douglas D, Sjölander A. Cysteinyl leukotriene receptor 1 facilitates tumorigenesis in a mouse model of colitis-associated colon cancer. Oncotarget 2018; 8:34773-34786. [PMID: 28410235 PMCID: PMC5471010 DOI: 10.18632/oncotarget.16718] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2016] [Accepted: 03/20/2017] [Indexed: 12/19/2022] Open
Abstract
Cysteinyl leukotriene receptor 1 (CysLT1R) has been shown to be up-regulated in the adenocarcinomas of colorectal cancer patients, which is associated with a poor prognosis. In a spontaneous model of colon cancer, CysLT1R disruption was associated with a reduced tumor burden in double-mutant female mice (ApcMin/+/Cysltr1-/-) compared to ApcMin/+ littermates. In the current study, we utilized a genetic approach to investigate the effect of CysLT1R in the induced azoxymethane/dextran sulfate sodium (AOM/DSS) model of colitis-associated colon cancer. We found that AOM/DSS female mice with a global disruption of the Cysltr1 gene (Cysltr1-/-) had a higher relative body weight, a more normal weight/length colon ratio and smaller-sized colonic polyps compared to AOM/DSS wild-type counterparts. The Cysltr1-/- colonic polyps exhibited low-grade dysplasia, while wild-type polyps had an adenoma-like phenotype. The Cysltr1-/- colonic polyps exhibited significant decreases in nuclear β-catenin and COX-2 protein expression, while the normal crypts surrounding the polyps exhibited increased Mucin 2 expression. Furthermore, Cysltr1-/- mice exhibited an overall reduction in inflammation, with a significant decrease in proinflammatory cytokines, polyp 5-LOX expression and infiltration of CD45 leukocytes and F4/80 macrophages. In conclusion, the present genetic approach in an AOM/DSS model further supports an important role for CysLT1R in colon tumorigenesis.
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Affiliation(s)
- Janina Osman
- Division of Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Sayeh Savari
- Division of Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Naveen Kumar Chandrashekar
- Division of Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Kishan Bellamkonda
- Division of Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Desiree Douglas
- Division of Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Anita Sjölander
- Division of Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
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Arthur S, Sundaram U. Protein kinase C-mediated phosphorylation of RKIP regulates inhibition of Na-alanine cotransport by leukotriene D(4) in intestinal epithelial cells. Am J Physiol Cell Physiol 2014; 307:C1010-6. [PMID: 25231108 DOI: 10.1152/ajpcell.00284.2014] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Leukotriene D4 (LTD4) is an important immune inflammatory mediator that is known to be elevated in the mucosa of chronically inflamed intestine and alter nutrient absorption. LTD4 inhibits Na-alanine cotransport in intestinal epithelial cells by decreasing the affinity of the cotransporter ASCT1. LTD4 is known to increase intracellular Ca(++) and cAMP concentrations. However, the intracellular signaling mechanism of LTD4-mediated ASCT1 inhibition is unknown. In the present study, pretreatment with calcium chelator BAPTA-AM or inhibition of Ca(++)-dependent protein kinase C (PKC), specifically PKCα, resulted in the reversal of LTD4-mediated inhibition of ASCT1, revealing the involvement of the Ca(++)-activated PKC pathway. PKCα is known to phosphorylate Raf kinase inhibitor protein (RKIP), thus activating its downstream signaling pathway. Immunoblotting with anti-RKIP-Ser(153) antibody showed an increase in phosphorylation levels of RKIP in LTD4-treated cells. Downregulation of endogenous RKIP showed no decrease in ASCT1 activity by LTD4, thus confirming its involvement in ASCT1 regulation. Phosphorylation of RKIP by PKC is known to activate different signaling pathways, and in this study it was found to activate cAMP-activated protein kinase A (PKA) pathway. Although protein abundance of ASCT1 was not altered in any of the experimental conditions, there was an increase in the levels of phosphothreonine in ASCT1 protein, thus showing that phosphorylation changes were responsible for the altered affinity of ASCT1 by LTD4. In conclusion, LTD4 inhibits ASCT1 through PKC-mediated phosphorylation of RKIP, leading to the subsequent activation of PKA pathway, possibly through β2-andrenergic receptor activation.
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Affiliation(s)
- Subha Arthur
- Department of Clinical and Translational Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia
| | - Uma Sundaram
- Department of Clinical and Translational Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, West Virginia
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Savari S, Vinnakota K, Zhang Y, Sjölander A. Cysteinyl leukotrienes and their receptors: Bridging inflammation and colorectal cancer. World J Gastroenterol 2014; 20:968-977. [PMID: 24574769 PMCID: PMC3921548 DOI: 10.3748/wjg.v20.i4.968] [Citation(s) in RCA: 76] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/06/2013] [Revised: 11/16/2013] [Accepted: 12/06/2013] [Indexed: 02/06/2023] Open
Abstract
Long-standing inflammation has emerged as a hallmark of neoplastic transformation of epithelial cells and may be a limiting factor of successful conventional tumor therapies. A complex milieu composed of distinct stromal and immune cells, soluble factors and inflammatory mediators plays a crucial role in supporting and promoting various types of cancers. An augmented inflammatory response can predispose a patient to colorectal cancer (CRC). Common risk factors associated with CRC development include diet and lifestyle, altered intestinal microbiota and commensals, and chronic inflammatory bowel diseases. Cysteinyl leukotrienes are potent inflammatory metabolites synthesized from arachidonic acid and have a broad range of functions involved in the etiology of various pathologies. This review discusses the important role of cysteinyl leukotriene signaling in linking inflammation and CRC.
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Savari S, Liu M, Zhang Y, Sime W, Sjölander A. CysLT(1)R antagonists inhibit tumor growth in a xenograft model of colon cancer. PLoS One 2013; 8:e73466. [PMID: 24039952 PMCID: PMC3764114 DOI: 10.1371/journal.pone.0073466] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2012] [Accepted: 07/22/2013] [Indexed: 12/17/2022] Open
Abstract
The expression of the inflammatory G-protein coupled receptor CysLT1R has been shown to be upregulated in colon cancer patients and associated with poor prognosis. The present study investigated the correlation between CysLT1R and colon cancer development in vivo using CysLT1R antagonists (ZM198,615 or Montelukast) and the nude mouse xenograft model. Two drug administration regimens were established. The first regimen was established to investigate the importance of CysLT1R in tumor initiation. Nude mice were inoculated with 50 µM CysLT1R antagonist-pretreated HCT-116 colon cancer cells and received continued treatment (5 mg/kg/day, intraperitoneally). The second regimen aimed to address the role of CysLT1R in tumor progression. Nude mice were inoculated with non-pretreated HCT-116 cells and did not receive CysLT1R antagonist treatment until recordable tumor appearance. Both regimens resulted in significantly reduced tumor size, attributed to changes in proliferation and apoptosis as determined by reduced Ki-67 levels and increased levels of p21WAF/Cip1 (P<0.01), cleaved caspase 3, and the caspase-cleaved product of cytokeratin 18. Decreased levels of VEGF (P<0.01) and reduced vessel size (P<0.05) were also observed, the latter only in the ZM198,615-pretreatment group. Furthermore, we performed a series of in vitro studies using the colon cancer cell line HCT-116 and CysLT1R antagonists. In addition to significant reductions in cell proliferation, adhesion and colony formation, we observed induction of cell cycle arrest and apoptosis in a dose-dependent manner. The ability of Montelukast to inhibit growth of human colon cancer xenograft was further validated by using two additional colon cancer cell lines, SW-480 and HT-29. Our results demonstrate that CysLT1R antagonists inhibit growth of colon cancer xenografts primarily by reducing proliferation and inducing apoptosis of the tumor cells.
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Affiliation(s)
- Sayeh Savari
- Division of Cell and Experimental Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden
| | - Minghui Liu
- Division of Cell and Experimental Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden
| | - Yuan Zhang
- Division of Cell and Experimental Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden
| | - Wondossen Sime
- Division of Cell and Experimental Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden
| | - Anita Sjölander
- Division of Cell and Experimental Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden
- * E-mail:
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Jupp J, Hillier K, Elliott DH, Fine DR, Bateman AC, Johnson PA, Cazaly AM, Penrose JF, Sampson AP. Colonic expression of leukotriene-pathway enzymes in inflammatory bowel diseases. Inflamm Bowel Dis 2007; 13:537-46. [PMID: 17230539 DOI: 10.1002/ibd.20094] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND Leukotrienes derived from the 5-lipoxygenase pathway are proinflammatory lipid mediators that possibly play a role in inflammatory bowel diseases. The expression of 5-lipoxygenase pathway proteins has not previously been examined in colonic mucosa in inflammatory bowel disease. RESULTS Quantitative immunohistochemical analyses showed that, compared to those of the control subjects (n = 9), colonic biopsies from patients with active inflammatory bowel disease (n = 17) had 3- to 7-fold higher mean counts of cells expressing 5-lipoxygenase (P = 0.03), 5-lipoxygenase-activating protein (P = 0.005), and the leukotriene A(4) hydrolase (P = 0.004), which make up the biosynthetic pathway of the potent neutrophil chemotaxin leukotriene B(4). Immunoexpression of the leukotriene C(4) synthase was unaltered (P > 0.2). The increased representation of leukotriene B(4)-pathway enzymes was associated with higher counts of neutrophils (P = 0.0001), macrophages (P = 0.03), eosinophils (P = 0.0004), CD8(+) T cells (P < 0.001), activated T cells (P < 0.05), and B cells (P < 0.05) but not of mast cells (P > 0.9). These eicosanoid and cellular changes were most marked in the subgroup of patients with ulcerative colitis (n = 9), and were absent in patients with quiescent disease (n = 6). The anomalies in the 5-lipoxygenase pathway were accompanied as expected by more cells immunostaining for cytokine-inducible COX-2 (P = 0.004, n = 17), but this study also revealed a greater number of cells expressing COX-1 in the samples from the patients in the ulcerative colitis subgroup (P = 0.03, n = 9). CONCLUSIONS The 5-lipoxygenase data provide a cellular basis for increased tissue synthesis of the leukotriene B(4), as reflected in the colonic mucosa and rectal dialysates of patients with active inflammatory bowel disease, which contributes to neutrophil influx and colonic injury. The COX-1/COX-2 data highlight the ambiguous functional role of prostanoid pathways in inflammatory bowel diseases.
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Affiliation(s)
- James Jupp
- Division of Infection, Inflammation & Repair, University of Southampton School of Medicine, Southampton General Hospital, Southampton, United Kingdom
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Ohd JF, Wikström K, Sjölander A. Do leukotrienes increase cell viability in human intestinal epithelial cells? ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2003; 507:193-8. [PMID: 12664585 DOI: 10.1007/978-1-4615-0193-0_30] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/01/2023]
Abstract
In this preliminary report we present data showing that leukotrienes increase the baseline cell viability in human intestinal epithelial cells and that LTB4 partially reverses the morphological hallmarks of non-necrotic cell death induced by the COX-2 specific inhibitor NS-398. The proposed signaling mechanisms regulating these events are summarized in fig. 3. Please view the work on LT signal transduction in these cells by Thodeti et al. in this volume.
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Affiliation(s)
- John F Ohd
- Department of Laboratory Medicine, Division of Experimental Pathology, Lund University, Malmö General Hospital, Entrance 78, Malmö, SE-205 02 Sweden
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Ohd JF, Nielsen CK, Campbell J, Landberg G, Löfberg H, Sjölander A. Expression of the leukotriene D4 receptor CysLT1, COX-2, and other cell survival factors in colorectal adenocarcinomas. Gastroenterology 2003; 124:57-70. [PMID: 12512030 DOI: 10.1053/gast.2003.50011] [Citation(s) in RCA: 135] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
Abstract
BACKGROUND & AIMS The effects of leukotriene (LT) D(4) on intestinal epithelial cells govern events that are involved in cell survival and colon cancer, notably increased expression of cyclooxygenase (COX)-2 and enhanced production of prostaglandin E(2). We investigated possible correlations between distribution of the recently described LTD(4) receptor CysLT(1)R and factors previously shown to be up-regulated by LTD(4) as well as clinicopathologic traits. METHODS Immunohistochemistry and in situ hybridization were performed on tissue arrays, which were made using colorectal cancer samples from 84 patients. RESULTS CysLT(1)R was significantly correlated to COX-2, 5-lipoxygenase, and Bcl-x(L). Male subjects more often exhibited high levels of this receptor relative to female subjects, and Dukes' B patients with elevated CysLT(1)R expression showed markedly poorer survival than those with low-level expression. Furthermore, this was paralleled by an increased viability of CysLT(1)R-overexpressing cells in a colon cancer cell line. CONCLUSIONS Our results further implicate the involvement of LTs in colorectal carcinoma. Based on our present and earlier findings, we propose that LT/CysLT(1)R signaling facilitates survival of colon cancer cells, which may affect disease outcome. Like COX-2, LTs are accessible targets for pharmacologic treatment.
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Affiliation(s)
- John F Ohd
- Division of Experimental Pathology, Department of Laboratory Medicine, Malmö University Hospital, Lund University, SE-205 02 Malmö, Sweden
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Manning BP, Sharkey KA, Mawe GM. Effects of PGE2 in guinea pig colonic myenteric ganglia. Am J Physiol Gastrointest Liver Physiol 2002; 283:G1388-97. [PMID: 12388206 DOI: 10.1152/ajpgi.00141.2002] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
PGE(2) is a proinflammatory mediator that can influence many cell types. This study was conducted to determine whether PGE(2) alters the electrical activity of distal colonic myenteric neurons, because colitis is typically associated with altered motility and changes in neural signaling may be involved. The electrical properties of intact myenteric neurons were evaluated with intracellular microelectrodes. Acute application of PGE(2) elicited a prolonged depolarization in both AH and S neurons with little effect on input resistance or electrical excitability. PGE(2) effects were suppressed by tetrodotoxin (TTX) or neurokinin (NK) receptor antagonists, indicating that PGE(2) acts directly and indirectly to depolarize colonic neurons. PGE(2)-evoked depolarization was concentration dependent (approximately 3 microM EC(50)) and was attenuated by the E prostanoid (EP)1/2 receptor antagonist, AH-6809. When preparations were maintained for 48 h in the presence of the stable PGE(2) analog PGE(2)-ethanolamide (10 microM), neurons exhibited a significant membrane depolarization and enhanced excitability. These results suggest that PGE(2) can play a role in altered motility in colitis by evoking changes in the electrical properties of myenteric neurons.
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Affiliation(s)
- Brian P Manning
- Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, Vermont 05405, USA
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Massoumi R, Larsson C, Sjölander A. Leukotriene D4 induces stress-fibre formation in intestinal epithelial cells via activation of RhoA and PKCδ. J Cell Sci 2002; 115:3509-15. [PMID: 12154081 DOI: 10.1242/jcs.115.17.3509] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
The intestinal epithelial barrier, which is regulated by the actin cytoskeleton, exhibits permeability changes during inflammation. Here we show that activation of the CysLT1 receptor by the inflammatory mediator leukotriene D4 (LTD4) causes a rapid increase in stress-fibre formation in intestinal epithelial cells. This effect was mimicked by cytotoxic necrotising factor-1 (CNF-1)-induced activation of RhoA,overexpression of constitutively active RhoA (L63-RhoA) and phorbol-ester-induced activation of protein kinase C (PKC). In accordance,inhibition of RhoA, by C3 exoenzyme or by dominant-negative RhoA (N19-RhoA),as well as GF109203X-induced inhibition of PKC, suppressed the LTD4-induced stress-fibre formation. Introduction of the dominant-negative regulatory domain of PKCδ, but not the corresponding structures from PKCα, βII or ϵ, blocked the LTD4-induced stress-fibre formation. Evaluating the relationship between PKCδ and RhoA in LTD4-induced stress-fibre formation,we found that C3 exoenzyme inhibited the rapid LTD4-elicited translocation of PKCδ to the plasma membrane. Furthermore, CNF-1-induced stress-fibre formation was blocked by GF109203X and by overexpression of the regulatory domain of PKC-δ, whereas PKC-induced stress-fibre production was not affected by N19-RhoA. We conclude that PKC-δ is located downstream of RhoA and that active RhoA and PKCδ are both necessary for LTD4-induced stress-fibre formation.
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Affiliation(s)
- Ramin Massoumi
- Experimental Pathology, Department of Laboratory Medicine, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden
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Holma R, Salmenperä P, Riutta A, Virtanen I, Korpela R, Vapaatalo H. Acute effects of the cys-leukotriene-1 receptor antagonist, montelukast, on experimental colitis in rats. Eur J Pharmacol 2001; 429:309-18. [PMID: 11698051 DOI: 10.1016/s0014-2999(01)01330-9] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Cysteinyl leukotrienes play a part in inflammatory reactions such as inflammatory bowel diseases. The aim of the present study was to evaluate the acute effects of a cys-leukotriene-1 receptor antagonist, montelukast, on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats. Montelukast (5, 10 or 20 mg kg(-1) day(-1)), a 5-lipoxygenase inhibitor, zileuton (50 or 100 mg kg(-1) day(-1), a positive control), or the vehicle was administered intracolonically to the rats twice daily throughout the study, starting 12 h before the induction of colitis with TNBS. The severity of colitis (macroscopic and histological assessment, as well as myeloperoxidase activity), the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, and eicosanoid production in colonic tissue incubation were assessed 24 and 72 h after colitis induction. Montelukast increased prostaglandin E(2) production at 24 h and tended to reduce the cyclooxygenase-2 protein expression at 72 h, but did not influence the severity of colitis. Zileuton failed to decrease the inflammatory reaction in spite of reduced leukotriene B(4) production at 72 h. The results suggest that drugs that block cysteinyl leukotriene receptors have limited potential to ameliorate acute TNBS-induced colitis, but that they exert some beneficial effects which make them capable of modulating the course of colitis.
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Affiliation(s)
- R Holma
- Institute of Biomedicine, Pharmacology, Biomedicum Helsinki, P.O. Box 63, FIN-00014 University of Helsinki, Helsinki, Finland
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Ohd JF, Wikström K, Sjölander A. Leukotrienes induce cell-survival signaling in intestinal epithelial cells. Gastroenterology 2000; 119:1007-18. [PMID: 11040187 DOI: 10.1053/gast.2000.18141] [Citation(s) in RCA: 80] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
Abstract
BACKGROUND & AIMS Inflammatory bowel conditions, particularly ulcerative colitis, are associated with an increased incidence of neoplastic transformation. High levels of proinflammatory leukotrienes (LTs) and up-regulated expression of cyclooxygenase (COX)-2 are characteristic of inflammation. Moreover, COX-2 has been implicated in cell survival and early colon carcinogenesis. Other aspects of interest for intestinal cell viability are the levels of beta-catenin and the antiapoptotic protein Bcl-2. We investigated the possibility that LTs participate in the regulation of these survival factors. METHODS We used the human intestinal epithelial cell line Int 407 and the rat intestinal epithelial cell line IEC-6. Immunoblotting was applied to ascertain protein expression and distribution, and enzyme immunoassay methodology was used to measure prostaglandin E(2) (PGE(2)) production. Apoptotic ability was assessed by trypan blue exclusion, Hoechst staining, DNA fragmentation, and a caspase-3 activity assay. RESULTS LTD(4) and LTB(4), but not LTC(4), caused a time- and dose-dependent increase in expression and/or membrane accumulation of COX-2, beta-catenin, and Bcl-2, as well as PGE(2) production. Apoptosis assays showed that the effects of LTs on these transformation-associated proteins correlated well with the ability of these LTs to reduce programmed cell death. CONCLUSIONS The results suggest that inflammatory conditions are associated with the expression and distribution of proteins that are characteristic of transformed cells; such conditions may involve a signaling mechanism comprising an altered rate of apoptosis.
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Affiliation(s)
- J F Ohd
- Division of Experimental Pathology, Department of Laboratory Medicine, Lund University, University Hospital Malmö, Malmö, Sweden
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Massoumi R, Sjölander A. The inflammatory mediator leukotriene D4 triggers a rapid reorganisation of the actin cytoskeleton in human intestinal epithelial cells. Eur J Cell Biol 1998; 76:185-91. [PMID: 9716265 DOI: 10.1016/s0171-9335(98)80033-2] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Epithelial cells play an important role in maintaining the intestinal mucosa barrier, a barrier that is impaired in several inflammatory conditions. The mechanisms behind this impairment are not known, but it can be presumed that structural alterations of the epithelial cells are involved. In support of this notion, we here show the inflammatory mediator leukotriene D4 (LTD4) triggered first a rapid (10 s) increase and immediately thereafter (30 s) a sustained decrease in the cellular filamentous actin (F-actin) level in intestinal epithelial cells. The initial LTD4-induced increase in F-actin content was effectively blocked by preincubating the cells with either pertussis toxin or the tyrosine kinase inhibitor genistein. A possible involvement of the tyrosine kinase-dependent phosphatidylinositol-3-kinase (PI-3-kinase) in the polymerisation of actin was supported by the observations that LTD4 induced a translocation to a membrane fraction of PI-3-kinase and by the findings that wortmannin, a PI-3-kinase inhibitor, totally abolished both this translocation of PI-3-kinase as well as the initial LTD4-induced polymerisation of actin. In addition, pertussis toxin and genistein, both known to interfere with the LTD4-induced calcium signal, completely or partially reversed the actin-depolymerising effect of LTD4. The calcium ionophore ionomycin (30s) induced actin depolymerisation to the same extent as LTD4 (30 s) did, but lacked the initial effect on actin polymerisation. In cells loaded with the calcium chelator MAPT, LTD4 induced a normal actin polymerisation response but the subsequent depolymerisation was completely inhibited. Similar results were obtained when the cells were preincubated with the protein kinase A inhibitor Rp-cAMPS, which has been shown to impair the LTD4-induced calcium signal in these epithelial cells. The present results show that the inflammatory mediator LTD4 triggers a reorganisation of the actin network in intestinal epithelial cells that is likely to contribute to the impairment of the intestinal barrier function.
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Affiliation(s)
- R Massoumi
- Department of Laboratory Medicine, Lund University, Malmö/Sweden
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Ohd JF, Adolfsson JL, Sjölander A. Leukotriene D4-induced signalling events in human epithelial cells: G alpha i3 activation and translocation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 1998; 433:99-102. [PMID: 9561113 DOI: 10.1007/978-1-4899-1810-9_20] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Our model of LTD4-induced signal transduction in epithelial cells is summarised in Figure 2. Extending what is already known about LTD4 signalling in epithelial cells, we identified the Gi3-protein as the crucial PTX sensitive G-protein and found that it is translocated to what might be a cytoskeletal fraction. This finding suggests a subtle response to LTD4, mediated via the bifurcation at the alpha/beta gamma junction. Although little is known about the role of epithelial cells in inflammation, it has been shown that such cells produce the potent chemoattractant LTB4 and the proinflammatory 5-HETE in response to intracellular accumulation of Ca2+ 24. The target protein(s) and the effect(s) of the translocation of the activated G alpha i3-proteins, as well as the possible role of the beta/gamma-subunits of Gi3, remain to be elucidated.
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Affiliation(s)
- J F Ohd
- Department of Laboratory Medicine, Lund University, Wallenberg Laboratory, UMAS, Malmö, Sweden
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