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• W1977440623 abstract "We have shown that substance P (SP) and its neurokinin-1 receptor (NK-1R) regulate intestinal angiogenesis by increasing expression of protein CYR61 (the cysteine-rich angiogenic inducer 61, or CCN1) in colonic epithelial cells. However, the mechanism involved in SP-induced CCN1 expression has not been studied, and the outcome of increased CCN1 expression in the development of colitis is not fully understood. Because histone deacetylase (HDAC) modulates transcription of several genes involved in inflammation, we investigated participation of HDAC in SP-induced CCN1 expression in human colonic epithelial NCM460 cells overexpressing NK-1R (NCM460-NK-1R) and in primary colonocytes. SP increased HDAC activity with deacetylation and dephosphorylation of nucleosome protein histone H3 in NCM460-NK-1R and/or primary colonocytes. Histone deacetylation and dephosphorylation was observed in colonic mucosa from irritable bowel disease patients. Similarly, colonic mucosal tissues from mice exposed to dextran sulfate sodium showed histone H3 deacetylation and dephosphorylation and increased HDAC activity that was reversed by the NK-1R antagonist CJ-12255. SP-induced increased CCN1 expression in NCM460-NK-1R cells was abolished by pharmacological HDAC inhibition. HDAC overexpression activated basal and SP-induced CCN1 promoter activity. Intracolonic CCN1 overexpression significantly ameliorated dextran sulfate sodium-induced colitis, with reduction of proinflammatory cytokine expression in mice. Thus, SP-mediated CCN1 expression in the inflamed human and mouse colon involves increased HDAC activity. Our results strongly suggest that increased CCN1 expression may be involved in mucosal healing during colitis. We have shown that substance P (SP) and its neurokinin-1 receptor (NK-1R) regulate intestinal angiogenesis by increasing expression of protein CYR61 (the cysteine-rich angiogenic inducer 61, or CCN1) in colonic epithelial cells. However, the mechanism involved in SP-induced CCN1 expression has not been studied, and the outcome of increased CCN1 expression in the development of colitis is not fully understood. Because histone deacetylase (HDAC) modulates transcription of several genes involved in inflammation, we investigated participation of HDAC in SP-induced CCN1 expression in human colonic epithelial NCM460 cells overexpressing NK-1R (NCM460-NK-1R) and in primary colonocytes. SP increased HDAC activity with deacetylation and dephosphorylation of nucleosome protein histone H3 in NCM460-NK-1R and/or primary colonocytes. Histone deacetylation and dephosphorylation was observed in colonic mucosa from irritable bowel disease patients. Similarly, colonic mucosal tissues from mice exposed to dextran sulfate sodium showed histone H3 deacetylation and dephosphorylation and increased HDAC activity that was reversed by the NK-1R antagonist CJ-12255. SP-induced increased CCN1 expression in NCM460-NK-1R cells was abolished by pharmacological HDAC inhibition. HDAC overexpression activated basal and SP-induced CCN1 promoter activity. Intracolonic CCN1 overexpression significantly ameliorated dextran sulfate sodium-induced colitis, with reduction of proinflammatory cytokine expression in mice. Thus, SP-mediated CCN1 expression in the inflamed human and mouse colon involves increased HDAC activity. Our results strongly suggest that increased CCN1 expression may be involved in mucosal healing during colitis. Substance P (SP), an 11-amino-acid neuropeptide member of the tachykinin family isolated by Chang and Leeman,1Chang M.M. Leeman S.E. Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P.J Biol Chem. 1970; 245: 4784-4790Abstract Full Text PDF PubMed Google Scholar is localized in the central nervous system,2Mantyh P.W. Neurobiology of substance P and the NK1 receptor.J Clin Psychiatry. 2002; 63: 6-10PubMed Google Scholar enteric nerves,3Costa M. Furness J.B. Franco R. Llewellyn-Smith I. Murphy R. Beardsley A.M. Substance P in nerve tissue in the gut.Ciba Found Symp. 1982; : 129-144PubMed Google Scholar sensory neurons,4Maggi C.A. Capsaicin-sensitive nerves in the gastrointestinal tract.Arch Int Pharmacodyn Ther. 1990; 303: 157-166PubMed Google Scholar and immune cells.5Bost K.L. Quantification of macrophage-derived substance P receptor mRNA using competitive polymerase chain reaction.Adv Exp Med Biol. 1995; 373: 219-223Crossref PubMed Scopus (17) Google Scholar SP binding to its high-affinity neurokinin-1 receptor (NK-1R) mediates important intestinal functions, including mucosal permeability,6Pothoulakis C. Castagliuolo I. LaMont J.T. Jaffer A. O'Keane J.C. Snider R.M. Leeman S.E. CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin.Proc Natl Acad Sci USA. 1994; 91: 947-951Crossref PubMed Scopus (255) Google Scholar chloride secretion,7Riegler M. Castagliuolo I. So P.T. Lotz M. Wang C. Wlk M. Sogukoglu T. Cosentini E. Bischof G. Hamilton G. Teleky B. Wenzl E. Matthews J.B. Pothoulakis C. Effects of substance P on human colonic mucosa in vitro.Am J Physiol. 1999; 276: G1473-G1483PubMed Google Scholar motility,8Holzer P. Holzer-Petsche U. Tachykinins in the gut Part I. Expression, release and motor function.Pharmacol Ther. 1997; 73: 173-217Crossref PubMed Scopus (311) Google Scholar and inflammation.9Koon H.W. Pothoulakis C. Immunomodulatory properties of substance P: the gastrointestinal system as a model.Ann N Y Acad Sci. 2006; 1088: 23-40Crossref PubMed Scopus (110) Google Scholar Interactions of SP and NK-1R promote inflammation via activation of cyclooxygenase-2 and secretion of PGE2 via JAK-STAT activation and by stimulating proinflammatory genes regulated by nuclear factor kappa B (NF-κB).10Koon H.W. Zhao D. Zhan Y. Rhee S.H. Moyer M.P. Pothoulakis C. Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells.J Immunol. 2006; 176: 5050-5059PubMed Google Scholar, 11Koon H.W. Zhao D. Zhan Y. Simeonidis S. Moyer M.P. Pothoulakis C. Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves protein kinase Cdelta activation.J Pharmacol Exp Ther. 2005; 314: 1393-1400Crossref PubMed Scopus (59) Google Scholar, 12Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves Rho family small GTPases.Biochem J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar Moreover, SP enhances mucosal healing by stimulating colonic epithelial cell proliferation that involves activation of metalloproteinases and transactivation of epithelial growth factor receptor (EGFR) and exerts anti-apoptotic effects via Akt phosphorylation in vivo and in vitro.13Castagliuolo I. Morteau O. Keates A.C. Valenick L. Wang C.C. Zacks J. Lu B. Gerard N.P. Pothoulakis C. Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor.Br J Pharmacol. 2002; 136: 271-279Crossref PubMed Scopus (54) Google Scholar, 14Koon H.W. Zhao D. Na X. Moyer M.P. Pothoulakis C. Metalloproteinases and transforming growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation and proliferation in human colonocytes.J Biol Chem. 2004; 279: 45519-45527Crossref PubMed Scopus (101) Google Scholar, 15Koon H.W. Zhao D. Zhan Y. Moyer M.P. Pothoulakis C. Substance P mediates antiapoptotic responses in human colonocytes by Akt activation.Proc Natl Acad Sci USA. 2007; 104: 2013-2018Crossref PubMed Scopus (90) Google Scholar We have previously reported that SP exerts proangiogenic effects by stimulating expression of CCN1 (recently redesignated as CYR61) in colonic epithelial cells.16Koon H.W. Zhao D. Xu H. Bowe C. Moss A. Moyer M.P. Pothoulakis C. Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar The CCN family consists of 30- to 40-kDa cysteine-rich proteins17Brigstock D.R. The CCN family: a new stimulus package.J Endocrinol. 2003; 178: 169-175Crossref PubMed Scopus (445) Google Scholar that stimulate cell proliferation, adhesion, apoptosis, extracellular matrix formation, angiogenesis, and tumor growth.18Moussad E.E. Brigstock D.R. Connective tissue growth factor: what's in a name?.Mol Genet Metab. 2000; 71: 276-292Crossref PubMed Scopus (442) Google Scholar CCN1 plays an essential role in vasculogenesis during embryogenesis.19Brigstock D.R. Regulation of angiogenesis and endothelial cell function by connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61).Angiogenesis. 2002; 5: 153-165Crossref PubMed Scopus (271) Google Scholar, 20Mo F.E. Muntean A.G. Chen C.C. Stolz D.B. Watkins S.C. Lau L.F. CYR61 (CCN1) is essential for placental development and vascular integrity.Mol Cell Biol. 2002; 22: 8709-8720Crossref PubMed Scopus (342) Google Scholar Up-regulation of CCN1 is also evident in the colonic tissues of ulcerative colitis (UC) patients.16Koon H.W. Zhao D. Xu H. Bowe C. Moss A. Moyer M.P. Pothoulakis C. Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar However, the mechanism of CCN1 expression in colonic epithelial cells has never been investigated, and, except for angiogenesis, the outcome of its increased expression in the pathogenesis of colitis is not fully understood. Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from histone proteins.21Zhang Y. Fang H. Jiao J. Xu W. The structure and function of histone deacetylases: the target for anti-cancer therapy.Curr Med Chem. 2008; 15: 2840-2849Crossref PubMed Scopus (52) Google Scholar Their actions are opposite to those of histone acetyltransferase.21Zhang Y. Fang H. Jiao J. Xu W. The structure and function of histone deacetylases: the target for anti-cancer therapy.Curr Med Chem. 2008; 15: 2840-2849Crossref PubMed Scopus (52) Google Scholar Histones are found in nuclei of eukaryotic cells; they package DNA into nucleosomes and represent important components of chromatin.22Miremadi A. Oestergaard M.Z. Pharoah P.D. Caldas C. Cancer genetics of epigenetic genes.Hum Mol Genet. 2007; 16: R28-R49Crossref PubMed Scopus (208) Google Scholar Histone H3 is a core histone that assembles DNA into nucleosomes.23Campos E.I. Reinberg D. Histones: annotating chromatin.Annu Rev Genet. 2009; 43: 559-599Crossref PubMed Scopus (644) Google Scholar HDACs can regulate gene transcription through deacetylation of histone,24Perez-Cadahia B. Drobic B. Davie J.R. H3 phosphorylation: dual role in mitosis and interphase.Biochem Cell Biol. 2009; 87: 695-709Crossref PubMed Scopus (91) Google Scholar indicating that histone H3 modifications are related to modulation of gene expression. Of note, previous results indicate that inhibition of HDAC results in amelioration of experimental colitis in mice,25Glauben R. Batra A. Fedke I. Zeitz M. Lehr H.A. Leoni F. Mascagni P. Fantuzzi G. Dinarello C.A. Siegmund B. Histone hyperacetylation is associated with amelioration of experimental colitis in mice.J Immunol. 2006; 176: 5015-5022PubMed Google Scholar suggesting that HDAC may regulate expression of inflammation-related genes. Given that SP is involved in colonic inflammation, we hypothesized that HDAC-related pathways may play a role in the SP-mediated colonic inflammation. Here, we report increased HDAC activity as well as histone H3 deacetylation and dephosphorylation in SP-exposed colonic epithelial cells, inflamed colon tissues of mice with experimental colitis, and colonic mucosa of patients with UC. HDAC activity in colonocytes is involved in SP-mediated CCN1 expression, and its overexpression in mouse colon reduces tissue damage in experimental colitis, implicating a healing role for CCN1 in the development of colitis. NCM460 cells overexpressing NK-1R (NCM460-NK-1R), previously generated by us,10Koon H.W. Zhao D. Zhan Y. Rhee S.H. Moyer M.P. Pothoulakis C. Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells.J Immunol. 2006; 176: 5050-5059PubMed Google Scholar, 11Koon H.W. Zhao D. Zhan Y. Simeonidis S. Moyer M.P. Pothoulakis C. Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves protein kinase Cdelta activation.J Pharmacol Exp Ther. 2005; 314: 1393-1400Crossref PubMed Scopus (59) Google Scholar, 14Koon H.W. Zhao D. Na X. Moyer M.P. Pothoulakis C. Metalloproteinases and transforming growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation and proliferation in human colonocytes.J Biol Chem. 2004; 279: 45519-45527Crossref PubMed Scopus (101) Google Scholar, 15Koon H.W. Zhao D. Zhan Y. Moyer M.P. Pothoulakis C. Substance P mediates antiapoptotic responses in human colonocytes by Akt activation.Proc Natl Acad Sci USA. 2007; 104: 2013-2018Crossref PubMed Scopus (90) Google Scholar were cultured in M3D medium (INCELL, San Antonio, TX) containing 10% fetal calf serum and 1% penicillin/streptomycin (both from Invitrogen, Carlsbad, CA).14Koon H.W. Zhao D. Na X. Moyer M.P. Pothoulakis C. Metalloproteinases and transforming growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation and proliferation in human colonocytes.J Biol Chem. 2004; 279: 45519-45527Crossref PubMed Scopus (101) Google Scholar Cells were treated with 0.1% trifluoroacetic acid as vehicle solution, SP, or human CCN1 protein (PeproTech, Rocky Hill, NJ) as indicated.16Koon H.W. Zhao D. Xu H. Bowe C. Moss A. Moyer M.P. Pothoulakis C. Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Human primary colonic epithelial cells were isolated from colonic epithelial tissues of healthy subjects and from involved regions of UC patients after informed consent in accordance with procedures established by the Cedars-Sinai Institutional Review Board (IRB 3358). Epithelial cells were obtained as described previously.26Mayer L. Shlien R. Evidence for function of Ia molecules on gut epithelial cells in man.J Exp Med. 1987; 166: 1471-1483Crossref PubMed Scopus (409) Google Scholar The top 0%/30% layer interface contained >90% pure epithelial cells. The primary cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% penicillin/streptomycin (both from Invitrogen). NCM460-NK-1R cells and human primary colonic epithelial cells were seeded on 24 well plates (2 × 106 cells/plate), unless otherwise specified. Cell viability had been checked by adherence to culture plates and exclusion of Trypan Blue dye. Male 8- to 10-week-old C57BL/6 mice (n = 6/group) (Charles River Laboratories International, Wilmington, MA) were maintained at the animal facility under standard conditions. Animal studies were approved by the institutional animal care and use committees of Beth Israel Deaconess Medical Center and the University of California at Los Angeles. Mice received standard pelleted chow and tap water ad libitum, except that the colitis group received water containing dextran sulfate sodium (DSS) 5% (w/v), as described previously.10Koon H.W. Zhao D. Zhan Y. Rhee S.H. Moyer M.P. Pothoulakis C. Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells.J Immunol. 2006; 176: 5050-5059PubMed Google Scholar, 15Koon H.W. Zhao D. Zhan Y. Moyer M.P. Pothoulakis C. Substance P mediates antiapoptotic responses in human colonocytes by Akt activation.Proc Natl Acad Sci USA. 2007; 104: 2013-2018Crossref PubMed Scopus (90) Google Scholar Groups of mice were also injected intraperitoneally with 200 μL PBS containing the NK-1R antagonist CJ-12255 (2.5 mg/kg/twice per day) or with PBS. After 5 days, mice were sacrificed by carbon dioxide euthanasia. Colon tissues were dissected and homogenized in immunoprecipitation buffer (Santa Cruz Biotechnology, Santa Cruz, CA), and equal amounts of protein (40 μg/lane) were subjected to Western blotting. Some colon tissues were also fixed in formalin and paraffin-embedded for immunohistochemistry. Mice were injected with either 30% ethanol in 100 μL as vehicle solution or with 100 mg/kg trinitrobenzene sulfonic acid (TNBS) in 30% ethanol intracolonically. A group of mice received sodium butyrate (0.3 mmol/kg of body weight) intraperitoneally every day for days 0, 1, and 2. After the end of day 2, colonic tissues were obtained for immunohistochemistry. HDAC activities in cell lysates or tissue homogenates in radioimmunoprecipitation assay buffer (∼50 μg/mL protein) were measured by colorimetric HDAC activity assay (BML-AK501; Enzo Life Sciences, Plymouth Meeting, PA) according to the manufacturer's instructions. Immunoassays of colonic levels of mouse tumor necrosis factor α (TNFα) (DY410), IL-6 (DY406), and KC (redesignated as CXCL1) (DY453; R&D Systems, Minneapolis, MN) were performed according to the manufacturer's instructions. Serum-starved NCM460-NK-1R colonocytes were stimulated by vehicle 0.1% trifluoroacetic acid or SP (1 to 100 nmol/L) for 4 to 8 hours. Chromatin immunoprecipitation for determining the molecular association of histone H3 and CCN1 gene DNA was performed with a Pierce Agarose ChIP kit (no. 26156; Thermo Fisher Scientific, Rockford, IL). Cells were fixed and lysed by the reagents provided in the kit. Histone H3-CCN1 DNA complex was precipitated by anti-histone H3 polyclonal antibody (Pierce PA5-16183; Thermo Fisher Scientific). The precipitated protein was digested by proteinase K and the DNA was eluted. CCN1 DNA was detected by CCN1 primer (AJRR81V; Applied Biosystems, Foster City, CA) via real-time PCR. The CCN1 custom PCR primer set was automatically designed by Applied Biosystems based on the CCN1 genome sequence from 5′-CACAATGAGCCAGATTGCCC-3′ on chromosome 1, genome position from 86046664 to 86046803. CCN1 or an enhanced green fluorescent protein (eGFP)-overexpressing construct was transfected to colonic tissues in vivo, as described previously.16Koon H.W. Zhao D. Xu H. Bowe C. Moss A. Moyer M.P. Pothoulakis C. Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Briefly, human CCN1 cDNA (clone TC310465; OriGene, Rockville, MD)-overexpressing construct mixed with a polyethyleneimine-based transfection reagent was intracolonically injected to anesthetized C57BL/6 mice. Mice were returned to consciousness and provided with 5% DSS in their drinking water or water alone for 5 days ad libitum. A separate group of mice were injected with the same amount of eGFP-expressing plasmid. The CCN1 transfection procedure was repeated on day 3, to increase transfection efficiency. Mice were monitored daily for body weight, stool consistency, and stool blood, with disease activity index DAI determined as described previously.27Cooper H.S. Murthy S.N. Shah R.S. Sedergran D.J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar Histological severity of colitis was determined by two investigators (H.W.K. and T.C.H.) in a blinded manner. Histology score measures combined parameters of crypt damage (score, 0 to 4), polymorphonuclear neutrophil (PMN) infiltrate (score, 0 to 3), edema (score, 0 to 3), erosion or ulceration (score, 0 to 3), and epithelial regeneration (score, 0 to 3), as described previously.28Ungaro R. Fukata M. Hsu D. Hernandez Y. Breglio K. Chen A. Xu R. Sotolongo J. Espana C. Zaias J. Elson G. Mayer L. Kosco-Vilbois M. Abreu M.T. A novel Toll-like receptor 4 antagonist antibody ameliorates inflammation but impairs mucosal healing in murine colitis.Am J Physiol Gastrointest Liver Physiol. 2009; 296: G1167-G1179Crossref PubMed Scopus (146) Google Scholar Human irritable bowel disease (IBD) cDNA plates containing cDNA from 6 healthy subjects and 22 UC patients were obtained from OriGene (CCRT501). The PCR reactions were performed in an ABI Prism 7700 sequence detection system (Applied Biosystems, Carlsbad, CA) as described previously.16Koon H.W. Zhao D. Xu H. Bowe C. Moss A. Moyer M.P. Pothoulakis C. Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar The levels of mRNA were determined using the respective real-time primer set (HDAC1 Hs02621185_s1) obtained from Applied Biosystems according to the manufacturer's instructions. Levels were normalized to 18S mRNA using the respective primer set (Hs99999901_s1) and results were expressed as fold induction compared with their respective controls. All experiments were repeated three times. Cells were lysed in 1× lysis buffer (no. 7722; Cell Signaling Technology, Danvers, MA). Equal amounts of cell extracts were fractioned by 10% SDS-PAGE, and proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA) with a 0.2 micron pore size at 400 mA for 2 hours at 4°C. Membranes were blocked in 5% nonfat milk in TBST (50 mmol/L Tris pH 7.5, 0.15 mol/L NaCl, 0.05% Tween 20), and then were incubated with antibodies against phospho-histone H3 Ser 10 (no. 9701; Cell Signaling Technology), acetyl-histone H3 Lys 9 (no. 9649S; Cell Signaling Technology), histone H3 (no. 9715; Cell Signaling Technology), CCN1 (sc-13100; Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin (sc-47778; Santa Cruz Biotechnology). Horseradish peroxidase-labeled antibodies were detected by chemiluminescence (Fisher Scientific, Pittsburgh, PA), and signals were captured using an LAS4000 luminescent image analyzer (Fujifilm, Tokyo, Japan). In some experiments, Western blot bands were quantified by densitometry using Scion Image Analysis software version 1.1. The DNA fragments of the CCN1 promoter (1.1 kb) upstream of the translational start site were cloned by PCR from human colonocyte genomic DNA and, after confirmation of the sequence identity, were subcloned into a pGL3 vector (pGL3-CCN1). NCM460-NK-1R cells were seeded in 12-well plates (2 × 106 cells/plate) overnight and transiently transfected with the pGL3-CCN1 construct together with an internal control pRL-TK (Promega, Madison, WI), as described previously.16Koon H.W. Zhao D. Xu H. Bowe C. Moss A. Moyer M.P. Pothoulakis C. Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Transfected cells were serum-starved for 24 hours and then treated with SP for up to 24 hours. The relative promoter activity of CCN1 in equal amounts of cell extracts was measured using a dual luciferase reporter assay system (Promega). Empty pCMV6-XL5 vector, constructs overexpressing human HDAC1 (SC117054), human HDAC3 (SC112704), and human HDAC5 (SC124229) were purchased from OriGene. These constructs were transiently transfected to NCM460-NK-1R together with CCN1 promoter construct and pRL-TK control construct using Lipofectamine 2000 reagent (Invitrogen). Human colonic tissue frozen sections were purchased from OriGene. Colon tissue sections were incubated with blocking buffer, followed by incubation with a rabbit polyclonal anti-phospho-histone H3 (Ser 10) antibody (no. 9701; Cell Signaling Technology) or acetyl histone H3 (Lys 9) antibody (no. 9649S; Cell Signaling Technology) overnight at 4°C. After a washing, sections were incubated with donkey anti-rabbit IgG and slides were stained with an ABC kit for color development (sc-2018; Santa Cruz Biotechnology). Immunohistochemistry experiments were performed with assistance of the Histology Core Facility of the University of California Los Angeles. The severity of DSS colitis was estimated by measuring body weight loss, stool consistency, and presence of fecal blood, leading to a clinical colitis score (scale, 0 to 4), as described previously.13Castagliuolo I. Morteau O. Keates A.C. Valenick L. Wang C.C. Zacks J. Lu B. Gerard N.P. Pothoulakis C. Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor.Br J Pharmacol. 2002; 136: 271-279Crossref PubMed Scopus (54) Google Scholar, 15Koon H.W. Zhao D. Zhan Y. Moyer M.P. Pothoulakis C. Substance P mediates antiapoptotic responses in human colonocytes by Akt activation.Proc Natl Acad Sci USA. 2007; 104: 2013-2018Crossref PubMed Scopus (90) Google Scholar The severity of TNBS colitis was estimated by measuring macroscopic damage score (scale, 0 to 10), as described previously.13Castagliuolo I. Morteau O. Keates A.C. Valenick L. Wang C.C. Zacks J. Lu B. Gerard N.P. Pothoulakis C. Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor.Br J Pharmacol. 2002; 136: 271-279Crossref PubMed Scopus (54) Google Scholar Quantitative results are expressed as means ± SEM (as error bars within graphs). Results were analyzed using Prism professional statistics software program version 4 (GraphPad, La Jolla, CA). Student's t-tests were used for intergroup comparisons. P values of significant difference are reported within graphs. HDAC has been proposed as a key factor in the mediation of the inflammation.29Halili M.A. Andrews M.R. Sweet M.J. Fairlie D.P. Histone deacetylase inhibitors in inflammatory disease.Curr Top Med Chem. 2009; 9: 309-319Crossref PubMed Scopus (161) Google Scholar Inhibition of HDAC activity by pharmacological agents such as short chain fatty acid butyrate and red grape-derived resveratrol results in reduced inflammatory responses.30Zhang L. Jin S. Wang C. Jiang R. Wan J. Histone deacetylase inhibitors attenuate acute lung injury during cecal ligation and puncture-induced polymicrobial sepsis.World J Surg. 2010; 34: 1676-1683Crossref PubMed Scopus (62) Google Scholar, 31Rahman I. Marwick J. Kirkham P. Redox modulation of chromatin remodeling: impact on histone acetylation and deacetylation, NF-kappaB and pro-inflammatory gene expression.Biochem Pharmacol. 2004; 68: 1255-1267Crossref PubMed Scopus (417) Google Scholar Because SP modulates intestinal inflammation,9Koon H.W. Pothoulakis C. Immunomodulatory properties of substance P: the gastrointestinal system as a model.Ann N Y Acad Sci. 2006; 1088: 23-40Crossref PubMed Scopus (110) Google Scholar we examined whether this neuropeptide can modulate HDAC activity in human primary colonic epithelial cells. SP (100 nmol/L) stimulated HDAC activities only in the human primary colonic epithelial cells from involved colonic regions, but not in cells from normal or uninvolved regions, of UC and Crohn's disease (CD) patients (Figure 1A). This trend correlates with significantly higher expression level of SP receptor NK-1R in human primary colonic epithelial cells from involved colonic regions of UC and CD patients, compared with healthy control subjects (Figure 1B). High expression of NK-1R in human primary colonic epithelial cells from IBD patients is consistent with our previous finding of elevated NK-1R mRNA expression in the colonic tissues of IBD patients,16Koon H.W. Zhao D. Xu H. Bowe C. Moss A. Moyer M.P. Pothoulakis C. Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 32Goode T. O'Connell J. Anton P. Wong H. Reeve J. O'Sullivan G.C. Collins J.K. Shanahan F. Neurokinin-1 receptor expression in inflammatory bowel disease: molecular quantitation and localisation.Gut. 2000; 47: 387-396Crossref PubMed Scopus (119) Google Scholar making these primary cells suitable for studying SP-dependent pathways. The uninvolved colonic regions of UC and CD patients express low level of NK-1R (data not shown). Consistent with enhanced HDAC activity, we observed increased deacetylated and dephosphorylated histone H3 at the epithelial lining of the colonic biopsies obtained from UC and CD patients (Figure 1C). Cells below the epithelial lining of normal colon tissues remained acetylated and phosphorylated, indicating lower HDAC activity (Figure 1C). Moreover, the acetylation and phosphorylation state of cells in the lamina propria and submucosa were similar across all groups (Figure 1C). We next examined whether HDAC activity is dependent on the SP–NK-1R pathway, using a murine model of experimental DSS-induced colonic inflammation and an NK-1R specific antagonist (CJ-12255). As expected, DSS administration led to increase in colitis score, which was significantly reduced after CJ-12255 treatment (Figure 2B). Colonic levels of proinflammatory cytokines (TNFα, IL-6, and KC) in DSS-treated mice were significantly higher than those of water-treated mice, and they were significantly reduced by NK-1R antagonist CJ-12255 administration (Figure 2, D–F). Water-treated groups did not develop colitis, so their colitis scores are zero (Figure 2B) and the proinflammatory cytokine levels remain low (Figure 2, D–F). Similarly as in IBD patients, DSS-induced colitis in mice led to significantly higher colonic HDAC activity than was observed in the water-treated control group (Figure 2A). Administration of SP receptor antagonist (CJ-12255) significantly reduced colonic HDAC activity in the DSS-treated group (Figure 2A). CJ-12255 did not affect basal colonic HDAC activity among water-tr" @default.
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• W1977440623 date "2011-11-01" @default.
• W1977440623 modified "2023-10-06" @default.
• W1977440623 title "Substance P Induces CCN1 Expression via Histone Deacetylase Activity in Human Colonic Epithelial Cells" @default.
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