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• W1971603261 abstract "Background & Aims: Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low levels on human colonic circular smooth muscle cells (HCCSMCs) and their expression is increased in patients with Crohn’s disease. We investigated the roles of transcription factors Sp1 and nuclear factor κ B (NF-κB) in the regulation of ICAM-1 expression on HCCSMCs and examined whether ICAM-1 expression mediates the suppression of contractility in response to TNFα. Methods: Experiments were performed on primary cultures of HCCSMCs and fresh human colonic circular muscle strips. Results: TNFα treatment of HCCSMCs induced rapid and prolonged accumulation of ICAM-1 messenger RNA (mRNA) and protein. NF-κB inhibition before, but not after, 1 hour of TNFα-stimulation blocked the expression of ICAM-1. TNFα significantly enhanced Sp1/DNA binding. Sp1 bound to the 3′ flanking region of a variant κB site in the −192/−172 region of ICAM-1 promoter. Mutation of this region abolished the response to TNFα. The treatment of HCCSMCs with Sp1 antisense oligonucleotides (ODNs) blocked the expression of ICAM-1, but sense ODNs had no effect. Protein kinase C ζ (PKCζ) inhibition before or 3 hours after stimulation with TNFα also blocked the expression of ICAM-1. TNFα treatment of circular muscle strips pretreated with ICAM-1 sense ODNs or control medium significantly reduced their response to acetylcholine, whereas pretreatment with antisense ODNs blocked this effect. Conclusions: The expression of ICAM-1 on HCCSMCs in response to TNFα is regulated by transcription factors Sp1 and NF-κB binding independently to the −192/−172 region of the ICAM-1 promoter. The expression of ICAM-1 plays a critical role in the suppression of cell contractility in response to TNFα. Background & Aims: Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low levels on human colonic circular smooth muscle cells (HCCSMCs) and their expression is increased in patients with Crohn’s disease. We investigated the roles of transcription factors Sp1 and nuclear factor κ B (NF-κB) in the regulation of ICAM-1 expression on HCCSMCs and examined whether ICAM-1 expression mediates the suppression of contractility in response to TNFα. Methods: Experiments were performed on primary cultures of HCCSMCs and fresh human colonic circular muscle strips. Results: TNFα treatment of HCCSMCs induced rapid and prolonged accumulation of ICAM-1 messenger RNA (mRNA) and protein. NF-κB inhibition before, but not after, 1 hour of TNFα-stimulation blocked the expression of ICAM-1. TNFα significantly enhanced Sp1/DNA binding. Sp1 bound to the 3′ flanking region of a variant κB site in the −192/−172 region of ICAM-1 promoter. Mutation of this region abolished the response to TNFα. The treatment of HCCSMCs with Sp1 antisense oligonucleotides (ODNs) blocked the expression of ICAM-1, but sense ODNs had no effect. Protein kinase C ζ (PKCζ) inhibition before or 3 hours after stimulation with TNFα also blocked the expression of ICAM-1. TNFα treatment of circular muscle strips pretreated with ICAM-1 sense ODNs or control medium significantly reduced their response to acetylcholine, whereas pretreatment with antisense ODNs blocked this effect. Conclusions: The expression of ICAM-1 on HCCSMCs in response to TNFα is regulated by transcription factors Sp1 and NF-κB binding independently to the −192/−172 region of the ICAM-1 promoter. The expression of ICAM-1 plays a critical role in the suppression of cell contractility in response to TNFα. The inflammatory response in the gut is initiated by the recruitment and activation of infiltrating and resident immunocytes followed by phenotypic changes in nonimmune cells comprising the gut wall. The classic concept of inflammation is that the activated immunocytes release inflammatory mediators in response to injury or infection and nonimmunocytes, including smooth muscle cells, enteric neurons, glia, myofibroblasts, and epithelial cells, are innocent bystanders that simply get hurt in this process. Accumulating evidence over the past decade or so has modified this concept.1Fiocchi C. Intestinal inflammation a complex interplay of immune and nonimmune cell interactions.Am J Physiol. 1997; 273: G739-G775Google Scholar It now appears that nonimmune cells in the gut wall may not be just innocent bystanders; they may participate actively in the overall inflammatory response. When exposed to the inflammatory response mediators secreted by the classic immunocytes, they respond in a precisely regulated manner to alter their function. For example, in circular smooth muscle cells, the tone and phasic contractions are suppressed while the frequency of giant migrating contractions is increased.2Sethi A.K. Sarna S.K. Colonic motor activity in acute colitis in conscious dogs.Gastroenterology. 1991; 100: 954-963PubMed Google Scholar, 3Lu G. Qian X. Berezin I. Telford G.L. Huizinga J.D. Sarna S.K. Inflammation modulates in vitro colonic myoelectric and contractile activity and interstitial cells of Cajal.Am J Physiol. 1997; 273: G1233-G1245PubMed Google Scholar, 4Shi X.Z. Sarna S.K. Impairment of Ca2+ mobilization in circular muscle cells of the inflamed colon.Am J Physiol. 2000; 278: G234-G242Google Scholar, 5Gonzalez A. Sarna S.K. Different types of contractions in rat colon and their modulation by oxidative stress.Am J Physiol. 2001; 280: G546-G554Google Scholar These motility changes are caused by altered gene expression of key signaling molecules, such as α1c subunit of L-type Ca2+ channels,6Liu X. Rusch N.J. Striessnig J. Sarna S.K. Down-regulation of L-type calcium channels in inflamed circular smooth muscle cells of the canine colon.Gastroenterology. 2001; 120: 480-489Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar and contribute to the symptoms of diarrhea, urgency of defecation, and abdominal cramping.7Sarna S.K. Physiology and pathophysiology of colonic motor activity (review article). Part one of two.Dig Dis Sci. 1991; 36: 827-862Crossref PubMed Scopus (184) Google Scholar In addition, some cell types in the gut wall, such as epithelial cells and myofibroblasts, also have been reported to secrete some of the same cytokines and chemokines as those secreted initially by the classic immunocytes.8Powell D.W. Mifflin R.C. Valentich J.D. Crowe S.E. Saada J.I. West A.B. Myofibroblasts. II. Intestinal subepithelial myofibroblasts.Am J Physiol. 1999; 277: C183-C201PubMed Google Scholar, 9Podolsky D.K. Healing the epithelium solving the problem from two sides.J Gastroenterol. 1997; 32: 122-126Crossref PubMed Scopus (141) Google Scholar A few reports have suggested that smooth muscle cells and enteric neurons also may secrete inflammatory response mediators during inflammation.10Khan I. Collins S.M. Expression of cytokines in the longitudinal muscle myenteric plexus of the inflamed intestine of rat.Gastroenterology. 1994; 107: 691-700Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 11Khan I. Blennerhassett M.G. Kataeva G.V. Collins S.M. Interleukin 1 beta induces the expression of interleukin 6 in the rat intestinal smooth muscle cells.Gastroenterology. 1995; 108: 1720-1728Abstract Full Text PDF PubMed Scopus (51) Google Scholar By using gene chip array analysis, Shi et al.12Shi X.-Z. Telford G.L. Sarna S.K. Cytokine/chemokine gene expression in cultured human colon smooth muscle cells mediated by transcription factor NF-κB, but not AP-1.Neurogastroenterol Motil. 2001; 13 (abstr): 43Google Scholar found in a recent study that primary cultures of human colonic circular smooth muscle cells (HCCSMCs) secrete a time-dependent panel of specific cytokines, chemokines, and express cell adhesion molecules in response to treatment with tumor necrosis factor α (TNFα). The role of cytokines and chemokines secreted by circular muscle cells, whose normal function is to contract, is not known. These secretions may contribute to altered motility function in inflammation by acting in autocrine mode and they may enhance the total secreted pool of inflammatory mediators to attain full inflammatory response in muscularis externa. Intercellular adhesion molecule 1 (ICAM-1) is involved directly in the recruitment and trafficking of leukocytes to the areas of tissue injury through its action as a receptor for the leukocyte β2 integrins, such as LFA-1 and Mac-1. The expression of ICAM-1 in colonic muscle layer is significantly up-regulated in the course of active Crohn’s disease.13Bernstein C.N. Sargent M. Gallatin M.W. β2 Integrin/ICAM expression in Crohn’s disease.Clin Immunol Immunopathol. 1998; 86: 147-160Crossref PubMed Scopus (80) Google Scholar The up-regulation of ICAM-1 occurs primarily at the transcriptional level.14Ledebur H.C. Parks T.P. Transcriptional regulation of interecellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells; essential roles of variant NF-κB site and p65 momodimers.J Biol Chem. 1995; 270: 933-943Crossref PubMed Scopus (513) Google Scholar, 15Jahnke A. Johnson J.P. Synergistic activation of intercellular adhesion molecule 1 (ICAM-1) by TNF-α and IFN-γ is mediated by p65/p50 and p65c-Rel and interferon-responsive factor Stat1α (p91) that can be activated by both IFN-γ and IFN-α.FEBS Lett. 1994; 354: 220-226Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 16van de Stolpe A. Caldenhoven E. Stade B.G. Koenderman L. Raaijmakers J.A.M. Johnson J.P. van der Saag P.T. 12-O-tetradecanoylphorbol-13-acetate- and tumor necrosis factor α-mediated induction of intercellular adhesion molecule-1 is inhibited by dexamethasone.J Biol Chem. 1994; 269: 6185-6192Abstract Full Text PDF PubMed Google Scholar Prior studies have identified the −191 to −172 region of ICAM-1 promoter, which includes a variant κB site (TGGAAATTCC), as necessary and sufficient for the expression of ICAM-1 in response to TNFα in endothelial cells.17Paxton L.L. Li L.-J. Secor V. Duff J.L. Naik S.M. Shibagaki N. Caughman S.W. Flanking sequences for the human intercellular adhesion molecule-1 NF-κB response element are necessary for tumor necrosis factor α-induced gene expression.J Biol Chem. 1997; 272: 15928-15935Crossref PubMed Scopus (30) Google Scholar This variant κB site deviates from the consensus κB sequence (GGGRNNYYCC) at the extreme 5′ end, where a thymine residue replaces the conserved guanine residue.18Baeuerle P.A. The inducible transcription activator NF-κ B regulation by distinct protein subunits.Biochim Biophys Acta. 1991; 1072: 63-80PubMed Google Scholar However, the variant κB site (−187/−178) in this region is by itself not sufficient to induce the expression of ICAM-1 in promoter reporter assays, suggesting the involvement of additional nuclear proteins or transcription factors.17Paxton L.L. Li L.-J. Secor V. Duff J.L. Naik S.M. Shibagaki N. Caughman S.W. Flanking sequences for the human intercellular adhesion molecule-1 NF-κB response element are necessary for tumor necrosis factor α-induced gene expression.J Biol Chem. 1997; 272: 15928-15935Crossref PubMed Scopus (30) Google Scholar The identity of the additional transcription factors and whether they compete with nuclear factor κ B (NF-κB) for binding to the variant κB binding site or bind to its 5′ and 3′ flanking regions is not known. The ubiquitous transcription factor Sp1 contains a 3-zinc-finger protein domain and 4 transactivation domains, and binds to GC-rich sequences, including GC boxes, CACCC boxes (also called GT boxes), and basic transcriptional machinery.19Berg J.M. Sp1 and the subfamily of zinc finger proteins with guanine-rich binding sites.Proc Natl Acad Sci U S A. 1992; 89: 11109Crossref PubMed Scopus (164) Google Scholar, 20Black A.R. Black J.D. Azizkhan J. Sp1 and Kruppel-like factor family of transcription factors in cell growth regulation and cancer.J Cell Physiol. 2001; 188: 143-160Crossref PubMed Scopus (574) Google Scholar The potential role of Sp1 in the regulation of gene transcription was highlighted recently by Ainbinder et al.,21Ainbinder E. Revach M. Wolstein O. Moshonow S. Diamant N. Dikstein R. Mechanism of rapid transcriptional induction of tumor necrosis factor alpha-responsive genes by NF-κB.Mol Cell Biol. 2002; 22: 6354-6362Crossref PubMed Scopus (102) Google Scholar who found that constitutive association of Sp1 with transcriptional apparatus was important for the immediate induction by NF-κB of A20 protein after stimulation by TNFα. Consistent with this role, these investigators found that NF-κB activity is directed to the enhancement of the transcription rate, whereas Sp1 is essential for transcription to occur. The precise role of Sp1 in the regulation of ICAM-1 expression, however, is not established. In particular, whether Sp1 is involved also in the long-term maintenance of ICAM-1 expression after its initial induction by NF-κB is not known. The objectives of this study were to investigate the role of Sp1 in the regulation of ICAM-1 gene expression in HCCSMCs and to examine whether this expression participates in the suppression of circular muscle cell contractility in response to TNFα. We investigated also whether protein kinase C ζ (PKCζ) mediates cell signaling between TNFα receptor binding and activation of transcription factors Sp1 and p65 NF-κB. We show here that treatment of primary cultures of HCCSMCs with inhibitors of SP1 expression or inhibitors of SP1-DNA binding prevent TNFα-induced expression of ICAM-1. Because fragment −192/−172 of the ICAM-1 promoter encompassing a variant κB binding site has been shown to be necessary and sufficient for TNFα response, we investigated the model in which activated Sp1 regulates transcription of ICAM-1 through the variant κB site contained in the region and its 5′ and 3′ flanking regions. Our findings suggest a novel role of Sp1 in regulating κB-driven transcription of ICAM-1 gene in HCCSMCs. We found that although both p65 NF-κB subunit and Sp1 are essential for the initial induction of ICAM-1 transcription, Sp1 may sustain transcription after an initial period of 1–3 hours of TNFα stimulation. We show also that the expression of ICAM-1 on HCCSMCs in response to TNFα plays a critical role in the suppression of colonic circular smooth muscle cell contractility. Human colon tissue was obtained from disease-free margins of resected segments from patients undergoing surgery for colon cancer. The circular muscle layer was separated from the taenia coli and lamina propria with a tissue slicer. The circular muscle layer was collected in ice-cold HEPES buffer (in mmol/L: 120 NaCl, 2.6 KH2S04, 4 KCl, 2 CaCl2, 0.6 MgCl2, 25 HEPES, 14 glucose, and 2.1% essential amino acid mixture, pH 7.4). Two successive digestions with papain and collagenase, as described previously by Shi and Sarna,4Shi X.Z. Sarna S.K. Impairment of Ca2+ mobilization in circular muscle cells of the inflamed colon.Am J Physiol. 2000; 278: G234-G242Google Scholar dispersed smooth muscle cells. The cells were cultured in RPMI 1640 (Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum in the presence of 100 U/mL of penicillin G, 100 μg/mL of streptomycin sulfate, and 0.25 μg/mL of amphoteracin B. The media were changed every 3 days. Cells in passages 3–5 were used in all experiments. All cells were cultured in serum-free media for at least 15 hours before experiments to eliminate the possible effects of growth factors. Immunofluoresence imaging showed that more than 95% of the cultured cells stained for smooth muscle-specific α-actin. After 24 hours of starvation or pretreatment with oligonucleotides, cells were stimulated with TNFα for indicated time periods, washed 2 times with ice-cold phosphate-buffered salie (PBS), and placed on ice. Cells were lysed directly on the plate by adding 0.5–1 mL of lysis buffer (1% Nonidet P40, 250 mmol/L NaCl, 50 mmol/L Hepes [pH 7.4], 1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid [EGTA], supplemented with a cocktail protease inhibitor [Sigma, St. Louis, MO], 1 mmol/L phenylmethylsulfonyl fluoride, 50 mmol/L Na3VO4, freshly prepared before use). To ensure complete cell lysis, cells were kept on ice for 15 minutes followed by centrifugation at 14,000 × g at 4°C. Aliquots were stored at −80°C. Nuclear extract was prepared according to the method of Dyer and Herzog22Dyer R.B. Herzog N.K. Isolation of intact nuclei for nuclear extract preparation from a fragile B-lymphocyte cell line.Biotechniques. 1995; 19: 192-195PubMed Google Scholar with modifications. Briefly, after stimulation with TNFα, cells were washed 2 times with cold PBS and suspended in cold sucrose lysis buffer (0.32 mol/L sucrose, 3 mmol/L CaCl2, 2 mmol/L magnesium acetate, 0.1 mmol/L ethylenediaminetetraacetic acid [EDTA], 10 mmol/L Tris-HCl [pH 8.0], 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.2% NP40). After vortexing for 5 seconds to ensure cytoplasmic membrane lysis, samples were centrifuged at 500 × g for 5 minutes at 4°C. Subsequently, 150 μL of supernatant was transferred to a fresh tube containing 50 μL of cytoplasmic extraction buffer (0.15 mmol/L HEPES [pH 7.9], 0.7 mol/L KCl, 0.015 mol/L MgCl2) and centrifuged at 10,000 × g for 15 minutes to obtain cytoplasmic extract. The pellet from the first centrifugation (nuclei) was washed 2 times in 1 mL of sucrose buffer without NP40 (centrifugation 500 × g for 5 minutes). After washing, the pellet was suspended in 20 μL of low-salt buffer (20 mmol/L HEPES [pH 7.9], 25% glycerol, 1.5 mmol/L MgCl2, 0.02 mol/L KCl, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, and 0.5 mmol/L phenylmethylsulfonyl fluoride). After 5 minutes, 5 μL (25% of the volume) of high-salt buffer (20 mmol/L HEPES [pH 7.9], 25% glycerol, 1.5 mmol/L MgCl2, 0.8 mol/L KCl, 0.2 mmol/L EDTA, 1% NP40, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L dithiothreitol) was added and mixed gently. High-salt buffer then was added in 25% increments until equal volumes of low- and high-salt buffers were achieved. After incubation on ice for 15 minutes with gentle shaking, samples were centrifuged at 1200 × g for 15 minutes and aliquots of the supernatant (nuclear extract) were preserved using liquid nitrogen. HCCSMCs, grown on 6-well plates, were incubated in control medium or were stimulated with TNFα (20 ng/mL) for the indicated time period. After trypsinization, cells (50 × 103) were washed twice in fluorescence-activated cell sorter buffer (PBS with 1% fetal calf serum and 0.05% sodium azide) and stained with fluorescein isothiocyanate-conjugated monoclonal antibody against human ICAM-1 or isotype control Ab purchased from R&D Systems, Inc (Minneapolis, MN). After a 1-hour incubation on ice, the cells were washed in the fluorescence-activated cell sorter buffer and fixed with 1% paraformaldehyde. Expression of ICAM-1 was determined by flow cytometry using a FACScan analyzer (Becton Dickinson, San Jose, CA). For detection of ICAM-1 protein or transcription factors by Western blotting, total cell lysates or agarose-probe-conjugated proteins were mixed with equal volume of 2× sodium dodecyl sulfate (SDS) buffer and boiled for 4 minutes at 98°C. After resolving on SDS-polyacrylamide gel electrophoresis, proteins were transferred onto Hybond nitrocellulose membrane (Amersham, Pharmacia Biotech, Piscataway, NJ). Nonspecific binding to the membrane was blocked by immersing the membrane in blocking solution (Tris-buffered saline-Tween 20: 10 mmol/L Tris-HCl [pH 7.6], 150 mmol/L NaCl, 0.05% Tween 20 [vol/vol]) containing 5% skim milk powder for 30 minutes. After overnight incubation with primary antibody (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), bound antibody was coated with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, diluted 1:10,000 in Tris-buffered saline-Tween 20 for 30 minutes) at room temperature. After washing, the proteins were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s protocol. Stripping buffers were 7 mol/L guanidine hydrochloride or 65 mmol/L Tris-HCl, 100 mmol/L 2-ME, and 2% SDS. The sensitivity of subsequent blotting to the stripping agent determined which of these 2 buffers were used. Two micrograms of total RNA extracted from HCCSMCs with the RNeasy Mini isolation kit (Qiagen Inc, Valencia, CA) was subjected to colorimetric quantitation of human ICAM-1 messenger RNA (mRNA) by using hybridization with a specific 2941-bp complementary DNA probe (Genbank Accession number: NM-000201 ) followed by a colorimetric assay using the Quantikine mRNA Base kit according to the manufacturer’s protocol (R&D Systems). The minimum detectable dose of human ICAM-1 mRNA ranged from 1.8 to 6.8 amol/mL in this assay. For in vitro PKCζ kinase assays, 40 μg of nuclear extract was incubated with A/G Plus agarose (Santa Cruz Biotechnology) immobilized with appropriate rabbit anti-PKCζ antibody overnight at 4°C in 500 μL of lysis buffer (50 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 1 mmol/L EGTA, 0.25% sodium deoxycholate, 1 mmol/L Na3VO4, 1 mmol/L NaF, 1% Triton X-100, and aprotinin, leupeptin, and pepstatin at 1 μg/mL each).23Pazdrak K. Olszewska-Pazdrak B. Liu T. Takizawa R. Brasier A.R. Garofalo R.P. Casola A. MAPK activation is involved in posttranscriptional regulation of RSV-induced RANTES gene expression.Am J Physiol. 2002; 283: L364-L372Google Scholar Immunocomplexes were collected by centrifugation and washed twice in 1 mL of the lysis buffer and then twice in kinase buffer (in mmol/L: 10 HEPES [pH 7.4], 50 NaCl, 5 MgCl2, and 0.1 Na3VO4). Kinase assays were performed by resuspension of the samples in 50 μL of kinase buffer containing 0.25 mCi/mL [32P] adenosine triphosphate and substrate for 45 minutes at room temperature. Substrate for PKCζ was purchased from Santa Cruz Biotechnology. Phosphorylation reaction was terminated by spotting 15-μL aliquots of the assay mixture on a 1-×1-cm Whatman phosphocellulose paper. Then filters were washed 5 times for 10 minutes in 0.5% orthophosphoric acid, and the amount of [32P] phosphate transfer was determined by liquid scintillation counting. DNA affinity purification was performed as described previously,24Al-Shami A. Mahanna W. Naccache P.H. Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. Selective activation of Jak2, Stat3, and Stat5b.J Biol Chem. 1998; 273: 1058Crossref PubMed Scopus (103) Google Scholar, 25Miura K. Saini S.S. Gauvreau G. Macglashan Jr, D.W. Differences in functional consequences and signal transduction induced by IL-3, IL-5, and nerve growth factor in human basophils.J Immunol. 2001; 167: 2282-2291PubMed Google Scholar with modifications. The −192/−172 ICAM probe with 3′ terminal biotinylation and its complementary strand were synthesized by BioSource International (Camarelo, CA). After annealing of the 2 single-strand oligonucleotides, the double-stranded oligonucleotide was incubated with streptavidin-conjugated agarose beads (Pierce, Rockford, IL) for 1 hour at 4°C and washed twice with CLB (complete lysis buffer) (20 mmol/L Tris-HCl [pH 7.5], 2 mmol/L EDTA, 2 mmol/L EGTA, 100 μg/mL aprotinin, 10 mmol/L benzamidine, 5 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 100 μg/mL leupeptin, 50 mmol/L NaF, 5 mmol/L Na4P2O7, 1 mmol/L Na3VO4, and 1% Nonidet P-40). Nuclear extract (20 μg) suspended in 300 μL of CLB was precleared with agarose beads for 1 hour at 4°C to remove any nonspecific binding to the beads. The lysates then were incubated with −192/−172 ICAM beads for 1 hour at 4°C. The beads were washed 3 times with CLB buffer and the affinity-adsorbed protein was eluted by boiling in Laemmli buffer for 4 minutes at 95°C and subjected to Western blotting. The 21-bp fragment (−192/−172 ICAM-1) and its 4 relevant mutations were synthesized commercially by Genosys, Sigma (Woodlands, TX) and annealed to their complementary counterparts before subcloning into pNF-κB-secreted alkaline phosphatase (SEAP) vector (Clontech Laboratories, Inc., Palo Alto, CA), containing the secreted alkaline phosphatase reporter gene. This vector originally contained 4 tandem copies of the NF-κB consensus sequence fused to a TATA-like promoter region from the herpes simplex virus thymidine kinase promoter. We removed NF-κB consensus sequences with NheI and Bgl II restriction enzymes (Promega, Madison, WI), followed by ligation with ICAM-1 NF-κB-like sites of 21-bp constructs using T7 DNA polymerase. After endogenous NF-κB nuclear proteins bind to the κB enhancer element within our constructs, transcription is induced and the reporter gene is activated. HCCSMCs were grown in 6-well plates (Costar, Acton, MA). After reaching confluence of 80%, cell cultures were washed 2 times with PBS and transfected with supercoiled plasmid DNA using FuGENE 6 reagent in serum-free, antibiotic-free Opti-minimum essential medium (Gibco/Invitrogen, Carlsbad, CA). Cells were transfected with 2 μg of −192/−172 ICAM and control plasmids. Control plasmid included commercially available basic SEAP vector as well as plasmids containing a consensus NF-κB sequence. Next, cells were stimulated with TNFα for 24 hours, which initiated transcription of SEAP. Preliminary experiments found 24 hours of stimulation resulted in optimal signal-to-noise ratio. SEAP was measured in collected medium by using Great Escape Chemiluminescence Detection kit following the manufacturer’s protocol (Clontech). Sp1 and ICAM-1 were inhibited with antisense oligodeoxynucleotides in HCCSMCs and muscle strips, respectively. A phosphorothioate-modified antisense oligodeoxuynucleotide (5′-ATATTAGGCATCACTCCAGG-3′) directed against the Sp1 transcription start site and its sense control (5′-CCTGGAGTGATGCCTAATAAT-3′) were synthesized commercially (Biosource International). Antisense oligonucleotide against ICAM-1 (5′-GCCCAAGCTGGCATCCGTCA-3′) and its sense control (5′-CCTGGAGTGATGCCTAATAAT-3′) were used for inhibition of ICAM-1. The cells or muscle strips were transfected with 0.125–0.25 μmol/L oligonucleotide by using FuGENE 6 reagent as instructed by the manufacturer. HCCSMCs or muscle strips were stimulated with TNFα 24 hours later in the continued presence of sense or antisense oligonucleotides. All experiments using sense and antisense oligonucleotides were monitored for the expression of target molecule. For verification of antisense oligonucleotide uptake, colonic muscle strips were treated with ICAM-1 antisense oligonucleotides conjugated with fluorescein isothiocyanate (commercially synthesized by Biosource International). After 24 hours of incubation, muscle strips were fixed in 4% paraformaldehyde (in PBS) for 20 minutes at room temperature. Tissues then were washed 3 times in PBS and placed in 20% sucrose with PBS. Tissues were embedded in optimum cutting temperature medium (Miles Laboratories, Naperville, IL) and frozen sections (10 μm) were placed on superfrost plus slides (Calbiochem, La Jolla, CA). Tissue sections then were covered with Fluorsave reagent (Erie Scientific Co., Portsmouth, NH) and examined using an Olympus IX50/FLA fluorescent microscope equipped with a coolsnap-fix camera (Olympus Opticals Co., Melville, NY). The colon samples were stored on ice for no longer than 1 hour and then immersed in warm carbogenated Krebs solution (in mmol/L: 118 NaCl, 4.7 KCl, 2.5 CaC2, 1 NaH2PO4, 1.2 MgCl2, 11 D-glucose, and 25 NaHCO3). The mucosal layer and taenia coli were removed under magnifying glass. Circular muscle strips (2 × 10 mm) were cut along the circumferential axis and placed in RPMI 1640 containing sense or antisense ICAM-1 oligonucleotides or medium controls. After 24 hours of incubation with oligonucleotides, TNFα was added for the next 24 hours and then the strips were mounted in muscle baths (Radnoti Glass, Monrovia, CA) filled with 5 mL of carbogenated Krebs solution at 37°C.26Lu G. Qian X. Berezin I. Telford G.L. Huizinga J.D. Sarna S.K. Inflammation modulates in vitro colonic myoelectric and contractile activity and interstitial cells of Cajal.Am J Physiol. 1997; 273: G1233-G1245PubMed Google Scholar Contractions were measured with Grass isometric force transducers and amplifiers connected to a Biopac data acquisition system (Goleta, CA). The effect of TNFα on acetylcholine (ACh)-induced contractions was tested by obtaining the response to increasing concentrations of ACh in the range of 10−7 to 10−2 mol/L. All sense and antisense oligonucleotide-treated strips were incubated in parallel to monitor changes in contractility over time. The bathing solution was replaced every 15 minutes and also 4 minutes after each dose of ACh. The strips were left to equilibrate for at least 15 minutes before the next dose of ACh. The contractile response of circular muscle was measured as the area under contractions during 3 minutes after addition of ACh to the bath. The comparisons between 2 means were performed by nonparametric Wilcoxon test. Fluorometric analysis indicated little ICAM-1 mRNA in unstimulated cells. TNFα (20 ng/mL) stimulated rapid and prolonged accumulation of ICAM-1 mRNA in HCCSMCs. Significant induction of ICAM-1 message was detected as early as 30 minutes after stimulation and peak levels were attained by 6 hours (Figure 1A). The message level then decreased slowly and by 24 hours it was ∼50% of the maximum value. Flow cytometric analysis of ICAM-1 protein expression showed that activation of HCCSMCs with TNFα for 24 hours increased the surface expression of ICAM-1 by 40- to 50-fold (Figure 1B). In unstimulated cells, the mean fluorescence was 14–19 vs. 7–12 seen with control isotype antibody. Significant induction of ICAM-1 protein by TNFα was observed within 3 hours. It was increasing at 6 and 12 hours, and it began to plateau between 24 and 48 hours. Western blotting confirmed the kinetics of ICAM-1 expression (Figure 2B, top). Kinetic" @default.
• W1971603261 created "2016-06-24" @default.
• W1971603261 creator A5007994290 @default.
• W1971603261 creator A5017533734 @default.
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• W1971603261 date "2004-10-01" @default.
• W1971603261 modified "2023-10-10" @default.
• W1971603261 title "TNFα suppresses human colonic circular smooth muscle cell contractility by SP1- and NF-κB-mediated induction of ICAM-1" @default.
• W1971603261 cites W1508341207 @default.
• W1971603261 cites W1530203607 @default.
• W1971603261 cites W1552340624 @default.
• W1971603261 cites W1594394126 @default.
• W1971603261 cites W1606150020 @default.
• W1971603261 cites W1968363114 @default.
• W1971603261 cites W1981988641 @default.
• W1971603261 cites W1983929891 @default.
• W1971603261 cites W2003081532 @default.
• W1971603261 cites W2004855923 @default.
• W1971603261 cites W2008791808 @default.
• W1971603261 cites W2012080573 @default.
• W1971603261 cites W2018304885 @default.
• W1971603261 cites W2021450731 @default.
• W1971603261 cites W2025768929 @default.
• W1971603261 cites W2027216583 @default.
• W1971603261 cites W2033424777 @default.
• W1971603261 cites W2035088045 @default.
• W1971603261 cites W2035291009 @default.
• W1971603261 cites W2048211446 @default.
• W1971603261 cites W2051831012 @default.
• W1971603261 cites W2061094990 @default.
• W1971603261 cites W2063230936 @default.
• W1971603261 cites W2074011532 @default.
• W1971603261 cites W2074469807 @default.
• W1971603261 cites W2077193261 @default.
• W1971603261 cites W2078889389 @default.
• W1971603261 cites W2085049920 @default.
• W1971603261 cites W2088627897 @default.
• W1971603261 cites W2091109777 @default.
• W1971603261 cites W2091291725 @default.
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