- Email: [email protected]
Capsaicin sensitive nervous fibers induce transforming growth factor-alpha and-beta expression in the rat colonic mucosa in vivo
monolayers were incubated with H202 +- pretreatment with EGF or PKC modulators. Other cells were transfected to either stably over-express PKC-,81 or to inhibit its expression, & then pre-treatedwith low or high doses of EGFor PKC activator (OAG) before H202.Effects on BP (fluorometry), actin integrity (laserconfocal microscopy), PKC-~ intracellulartranslocation (immunoblotting), & F-actin & G-actin assembly (western blot) were assessed, n=6/group. In WT monolayers exposed to oxidant, pretreatmentwith EGF or OAG dose-dependently:) depolymerized G-actin; stable F-astin; protected the F-actin cytoskeleton & maintained normal BP. The PKC inhibitor (GF 109203X, but not the inactive analog) abolished the aforementioned protection. Cells stably over-expressing PKC-/31 (~3.1 fold ) ware several fold more sensitive than WT cells to the effects of low doses of EGF or OAG. In these transfected cells exposed to oxidant, pretreatment with even a low dose of EGF or OAG: F-actin & ) G-actin; the integrity of actin cytoskeleton; maintained BP at control levels. This protection by low dose of EGF or OAG did not occur in WT cells. Pretreatmentwith EGF or OAG rapidly shifted the distribution of over-expressed PKC-B1 into the membrane & cytoskeletalassociatedfractions & concomitantly ) its cytosolic fraction, indicating activation of PKC-,81. Stable inhibition of PKC-/31 expression (90%)) by anti-sense transfection abrogated EGF-mediatedprotection as shown by: ) in stable F-actin & in disassembledG-actin; disruption of the actin cytoskeleton; monoleyer disruption. We conclude that EGF protects the dynamic assembly of the F-actin cytoskeleton & of intestinal BP, in large part, through PKC-,81 activation. Accordingly, PKC-,81 signaling may be a candidatetherapeutic target for pharmacological as well as genetic interventions for the treatrneht of a variety of o~ddstive inflammatory disorders such as IBD.
2566 GATA Transcription Factors In Enteroepithelial Differentiation Narashimaswamy Belaguli, David H. Berger, Baylor Coil of Medicine, Houston, TX Introduction: The GATAfamily of zinc finger transcription factors plays an essential role in the process of determination and differentiation of the baematopoietic and cardiovascular systems. During developmentGATA4/5/6are primarily expressedin the cardiovasculartissues and the endoderm-derivedtissues including enternapltheiialcells. Due to the eady lethality of GATA4/6 knockoutsthe role of these factors in enteroepitheliaidifferentiation is not known. in an attempt to begin to understandthe function of these key transcription factors in epithelial differentiation we sought to determine the levels and DNA binding activity of GATA4/5/6 in uninduced and sodium butyrste (NaBT)treated rat intestinal epithelial cells. Methods: Nuclear extracts (NE) were preparedfrom untreated and 5 mM NaBTtreated subcontluent RIE ceils. 25/,~g of NE was analyzed by Western biothng with GATA4 and GATA6 antibodies. For electrophoreticgel mobility shift assays(EMSA), 5#G of NE and the consensusGATAbinding probe were used. Results: GATA4 and -6 were expressed in both uninduced and induced RIEs, as determined by Western blotting analysis. Following induction, GATA4 protein levels increased while GATA6 levels remained relatively constant (Figure). NaBTtreatment resulted in increased GATA binding activity. Supershiff analysis with GATA4 antibody indicated that the enhanced GATA binding activity in NaBT treated cells was primarily due to increased GATA4 binding activity. GATA 5 DNA binding activity was not detected in either cell line. Conclusion: Our results suggest a mutually exclusive regulation of GATA4and GATA6during NaBT induced differentiation of RIE cells.
2564 Figure
Shod Chain Fatty Acids (SCFAs) Protect intestinal Munoca by SstIH;tive and Physiological induction of Epithelial Heat Shod( P~tiin 25 Hongyu Ren, Mark W. Musch, Eugene B. Chang, Univ of Chicago, Chicago, IL
1: E f f e c t o f N a B T o n G A T A 4 / 6
0mM
Background and Aim: SCFAsare products of colonic bacterialfermentation of unabsorbedor poorly digested carbohydrates and are the major luminal anions of the colon. They have numerous trophic effects on colonic mucosaand may enhancemucosal cytoprotection, albeit through undefinedmechanisms.Becauseinducible heatshock proteins (hsps) playa significant role in gut epithelial cytoprotection, we explored the possibility that SCFAs might modulate in vivo and in vitro intestinal epithelial expression of these proteins, thereby enhancing cell survival to oxidant-induced stress. Methods: Rat intestinal IEC18 cells ware treated with butyrate or other GCFAsto determine the time- and dose-dependencyof their effects on inducible hsp72 and hsp25 and constitutive hsc73. Hsp expressionwas determinedby Western blotting and cell protection against oxidant (monochloramine, NH:,Cl)was measured by •Cr release assay. IEC18cells were stably transfected with hsp25 sense and anti-sense cDNA to overexprsss or inhibit induction of hsp25, respectively.To modulate colonic SCFAcontent, rats were fed chow without cellulose or 6% added pectin. Mucosal hsp expression in small and large intestines were quantifatedby Western blot. Results: Butyrate induced a concentration- and time-dependent increase in hsp25, but not hsp72 or hsc73, expression, initially observedby 6 h and peakingby 12 h (EDos5 mM). This effect was cell specific, as no induction was seen in 3T3 fibroblasts. Other SCFAs,including the poorly metabolizedisobutyate, also induced selectiveexpressionof hsp25. Butyratesignificantly improved survival of IEC-18ceils to oxidant-induced stress. This effect was blocked when butyrate's hsp25 induction was inhibited by antisensetranstection. Additionally, sense-transfectedIEC18cells overexpressing hsp25 demonstrated increased resistanceto oxidant. Dietary fiber increased colonic, but not ileal, hsp25 while having no effect on hsp72 or hsc73 expression. Histology was unchanged and mucosal alkaline phosphataseactivity was minimally increased.Conclusions:These data demonstrate an important physiological mechanism mediating the cytoprotactive actions of luminal SCFAs in the gut, i.e. the cell-specific and selective induction of epithelial hsp25. Furthermore, dietary manipulation affecting SCFA bioavailabilify may represent an important mechanism for maintaining colonic mucosal integrity in a relatively hoetile environment.
5rnM
0mM
5ram
!
2567
Iknvoes Rllers induce Transforming Growth Factor-Alpha And Capmicth ~ 8nta ERmmflon in The Rat Colonic Mucosa in Vitro. Peter Hoffmann, Jessica Mazurkiewicz, Univ of Bochum, St Josef Hosp, Bochum Germany; Gerald Holtmenn, Guido Gerken, Div of Gastroenterology,Univ of Essen, Essen Germany; Viktor E. Eysselein, Harbor-UCLAMedical Ctr, Torrance, CA; Harald Goebell, Dept of Medicine, Essen Germany Background: Capsaicin sensitive nervous fibers (CSNF) protect gastrointestinal mucosa in anmial models of mucosal injury presumablyby modulation of mucosel blood flow and mucus secretion. The aim of our study was to evaluatethe effects of CSNF in rat colonic mucosa on eplt~ial cell proliferation and transforming growth factors ~ and/3 expression, which are important for mocossl protection and repair. Methods: Male wistar rats receivedeither a capsaicin enema(CE) with or without giving an antagonist to calcitonine gene related peptlde (CGRP) or Substance P (SP) iv immediately before the CE, a CE after sensory denervation as described previously or a vehicle enema. Animals received 50 mg/kg BrdU iv and were sacrificed at 2, 4, 8,12, 24 and 48 h after CE. In colonic mucosalspecimensBrdU-immunoresctive epithelial cell nuclei and TGFa and ~ -mRNA and -protein expression were evaluated by immenohistochemistry, semi-quantitative RT-PCR and Westarnblots using standardized procedures as described previously. Results: A significant two-fold increase of TGF ~ mRNA and a ten-fold increase of TGF a protein expression was observed 2-12 hours after CE. Concomittantly,a 1,5 and 2-fold increasein TGF,8 mRNAand protein expressionwas detected 4-6 hours after CE. CEs significantly increased the number of BrdU positive nuclei in the mucosal epithelium. This effect was reduced by both CGRP- and SP-antagonistsand was abolished in rats which were previously sensory denervated (Table). Conclusion: Capsaicin sensitive nervous fibers modulate epithelial cell proliferation and TGF ~ and TGF/3expression in colonic muCOse.This effect is mediated by the neurutransmitters CGRPand SP.
2565 Thrombin Enhances Migration Of Intestinal Epithelial Cells Annette Neumann, Marie Yon Depka Prondzinski, Christian Wilhelm, Katja Feigenhauer, Thomas Caspfitz, Ruediger Hoppe, Ingo Lopez Schmidt, Arnold Ganser, Michael P. Manna, Michael N. Goeke, Medicine Hochschule Hannover, HannoverGermany
Changesin BrdUimmunostathednucleiof the colonicepithelium(nuclei/crypt)
Background:Thrombin (coagulationfactor II) is a multifunctiooal serine proteasewhich plays a key role in the coagulationcascade,inflammatory responses,and wound healing. However, its effects on migration(restitution) and growth of intestinal epithelialcells are largely unknown. Therefore, we analyzedthrombin receptor expressionand effects of thrombin on restitution, proliferation, and apoptosis of intestinal epithelial cells in vitro. Methods: Expression of thrombin receptortranscripts in IEC-6and Caco-2cells was assessedby reversetranscriptioncoupled polymerasechain reaction (RT-PCR)and quantitativeTaqMan PCR. Non transformed rat jejunal crypt epithelium-derived IEC-6 cells were stimulated with various concentrations of thrombin (0.01-10 U/ml). Hirudin was used as thrombin inhibitor. Restitutionwas analyzed using the IEC-6 restitution model. Apoptosis of intestinal epithelial cells was determined by a quantitative ELISA detecting histone-associated DNA fragments. Cell proliferation was assessed by [3H]-thymldine incorporation into DNA. Results: Thrombin receptor transcripts were detected in both, IEC-6 and Caco-2 cells. Thrombin stimulated restitution of IEC-6 cells in a dose-dependent manner. Maximal stimulation (3.2-fold; p
Caps. enema •, SP atta9. 4.CGRPantag. • ~n=. dm.
Control 4,3+2,6
24h 17_+5,8" 5,~5,1 ° 2,8±3,5~ 1t0,8"
48h 10,8±2,5" 5,8±4,3* 1,8±2,2"* 1,25~1,5~
Caps.=Capsaicin,antao.=antagonist, sens.den.=aftersensorydenervatJo~(*p~O,O5,"*p
Accelerated intestinal Epithelial Restitution: A Novel Biological Action of GlucagonLike Peptide 2 (GLP-2) Matthew W. Fei, All Tavakkolizadeh,Stanley W. Ashley, Michael J. Zinner, Edward E. Whang, Dept of Surg, Brigham and Women's Hosp, Boston, MA Background:GLP-2 is a potent intestine-specificgrowth factor that amelioratesNSAID-induced intestinal injury in animal models. Whetherthe intestinotrophic actions of GLP-2 alone account for this phenomenonare unknown. In this study we tested the hypothesisthat GLP-2 reverses the deleterious effects of NSAIDs on intestinal epithelial restitution. Methods: Linear wounds were made on monoleyers of serum-starved Caco-2 cells. These wounds were oriented orthogonally to reference lines previously drawn on the outer surface of the culture plates.
A-504
2566 GATA Transcription Factors In Enteroepithelial Differentiation Narashimaswamy Belaguli, David H. Berger, Baylor Coil of Medicine, Houston, TX Introduction: The GATAfamily of zinc finger transcription factors plays an essential role in the process of determination and differentiation of the baematopoietic and cardiovascular systems. During developmentGATA4/5/6are primarily expressedin the cardiovasculartissues and the endoderm-derivedtissues including enternapltheiialcells. Due to the eady lethality of GATA4/6 knockoutsthe role of these factors in enteroepitheliaidifferentiation is not known. in an attempt to begin to understandthe function of these key transcription factors in epithelial differentiation we sought to determine the levels and DNA binding activity of GATA4/5/6 in uninduced and sodium butyrste (NaBT)treated rat intestinal epithelial cells. Methods: Nuclear extracts (NE) were preparedfrom untreated and 5 mM NaBTtreated subcontluent RIE ceils. 25/,~g of NE was analyzed by Western biothng with GATA4 and GATA6 antibodies. For electrophoreticgel mobility shift assays(EMSA), 5#G of NE and the consensusGATAbinding probe were used. Results: GATA4 and -6 were expressed in both uninduced and induced RIEs, as determined by Western blotting analysis. Following induction, GATA4 protein levels increased while GATA6 levels remained relatively constant (Figure). NaBTtreatment resulted in increased GATA binding activity. Supershiff analysis with GATA4 antibody indicated that the enhanced GATA binding activity in NaBT treated cells was primarily due to increased GATA4 binding activity. GATA 5 DNA binding activity was not detected in either cell line. Conclusion: Our results suggest a mutually exclusive regulation of GATA4and GATA6during NaBT induced differentiation of RIE cells.
2564 Figure
Shod Chain Fatty Acids (SCFAs) Protect intestinal Munoca by SstIH;tive and Physiological induction of Epithelial Heat Shod( P~tiin 25 Hongyu Ren, Mark W. Musch, Eugene B. Chang, Univ of Chicago, Chicago, IL
1: E f f e c t o f N a B T o n G A T A 4 / 6
0mM
Background and Aim: SCFAsare products of colonic bacterialfermentation of unabsorbedor poorly digested carbohydrates and are the major luminal anions of the colon. They have numerous trophic effects on colonic mucosaand may enhancemucosal cytoprotection, albeit through undefinedmechanisms.Becauseinducible heatshock proteins (hsps) playa significant role in gut epithelial cytoprotection, we explored the possibility that SCFAs might modulate in vivo and in vitro intestinal epithelial expression of these proteins, thereby enhancing cell survival to oxidant-induced stress. Methods: Rat intestinal IEC18 cells ware treated with butyrate or other GCFAsto determine the time- and dose-dependencyof their effects on inducible hsp72 and hsp25 and constitutive hsc73. Hsp expressionwas determinedby Western blotting and cell protection against oxidant (monochloramine, NH:,Cl)was measured by •Cr release assay. IEC18cells were stably transfected with hsp25 sense and anti-sense cDNA to overexprsss or inhibit induction of hsp25, respectively.To modulate colonic SCFAcontent, rats were fed chow without cellulose or 6% added pectin. Mucosal hsp expression in small and large intestines were quantifatedby Western blot. Results: Butyrate induced a concentration- and time-dependent increase in hsp25, but not hsp72 or hsc73, expression, initially observedby 6 h and peakingby 12 h (EDos5 mM). This effect was cell specific, as no induction was seen in 3T3 fibroblasts. Other SCFAs,including the poorly metabolizedisobutyate, also induced selectiveexpressionof hsp25. Butyratesignificantly improved survival of IEC-18ceils to oxidant-induced stress. This effect was blocked when butyrate's hsp25 induction was inhibited by antisensetranstection. Additionally, sense-transfectedIEC18cells overexpressing hsp25 demonstrated increased resistanceto oxidant. Dietary fiber increased colonic, but not ileal, hsp25 while having no effect on hsp72 or hsc73 expression. Histology was unchanged and mucosal alkaline phosphataseactivity was minimally increased.Conclusions:These data demonstrate an important physiological mechanism mediating the cytoprotactive actions of luminal SCFAs in the gut, i.e. the cell-specific and selective induction of epithelial hsp25. Furthermore, dietary manipulation affecting SCFA bioavailabilify may represent an important mechanism for maintaining colonic mucosal integrity in a relatively hoetile environment.
5rnM
0mM
5ram
!
2567
Iknvoes Rllers induce Transforming Growth Factor-Alpha And Capmicth ~ 8nta ERmmflon in The Rat Colonic Mucosa in Vitro. Peter Hoffmann, Jessica Mazurkiewicz, Univ of Bochum, St Josef Hosp, Bochum Germany; Gerald Holtmenn, Guido Gerken, Div of Gastroenterology,Univ of Essen, Essen Germany; Viktor E. Eysselein, Harbor-UCLAMedical Ctr, Torrance, CA; Harald Goebell, Dept of Medicine, Essen Germany Background: Capsaicin sensitive nervous fibers (CSNF) protect gastrointestinal mucosa in anmial models of mucosal injury presumablyby modulation of mucosel blood flow and mucus secretion. The aim of our study was to evaluatethe effects of CSNF in rat colonic mucosa on eplt~ial cell proliferation and transforming growth factors ~ and/3 expression, which are important for mocossl protection and repair. Methods: Male wistar rats receivedeither a capsaicin enema(CE) with or without giving an antagonist to calcitonine gene related peptlde (CGRP) or Substance P (SP) iv immediately before the CE, a CE after sensory denervation as described previously or a vehicle enema. Animals received 50 mg/kg BrdU iv and were sacrificed at 2, 4, 8,12, 24 and 48 h after CE. In colonic mucosalspecimensBrdU-immunoresctive epithelial cell nuclei and TGFa and ~ -mRNA and -protein expression were evaluated by immenohistochemistry, semi-quantitative RT-PCR and Westarnblots using standardized procedures as described previously. Results: A significant two-fold increase of TGF ~ mRNA and a ten-fold increase of TGF a protein expression was observed 2-12 hours after CE. Concomittantly,a 1,5 and 2-fold increasein TGF,8 mRNAand protein expressionwas detected 4-6 hours after CE. CEs significantly increased the number of BrdU positive nuclei in the mucosal epithelium. This effect was reduced by both CGRP- and SP-antagonistsand was abolished in rats which were previously sensory denervated (Table). Conclusion: Capsaicin sensitive nervous fibers modulate epithelial cell proliferation and TGF ~ and TGF/3expression in colonic muCOse.This effect is mediated by the neurutransmitters CGRPand SP.
2565 Thrombin Enhances Migration Of Intestinal Epithelial Cells Annette Neumann, Marie Yon Depka Prondzinski, Christian Wilhelm, Katja Feigenhauer, Thomas Caspfitz, Ruediger Hoppe, Ingo Lopez Schmidt, Arnold Ganser, Michael P. Manna, Michael N. Goeke, Medicine Hochschule Hannover, HannoverGermany
Changesin BrdUimmunostathednucleiof the colonicepithelium(nuclei/crypt)
Background:Thrombin (coagulationfactor II) is a multifunctiooal serine proteasewhich plays a key role in the coagulationcascade,inflammatory responses,and wound healing. However, its effects on migration(restitution) and growth of intestinal epithelialcells are largely unknown. Therefore, we analyzedthrombin receptor expressionand effects of thrombin on restitution, proliferation, and apoptosis of intestinal epithelial cells in vitro. Methods: Expression of thrombin receptortranscripts in IEC-6and Caco-2cells was assessedby reversetranscriptioncoupled polymerasechain reaction (RT-PCR)and quantitativeTaqMan PCR. Non transformed rat jejunal crypt epithelium-derived IEC-6 cells were stimulated with various concentrations of thrombin (0.01-10 U/ml). Hirudin was used as thrombin inhibitor. Restitutionwas analyzed using the IEC-6 restitution model. Apoptosis of intestinal epithelial cells was determined by a quantitative ELISA detecting histone-associated DNA fragments. Cell proliferation was assessed by [3H]-thymldine incorporation into DNA. Results: Thrombin receptor transcripts were detected in both, IEC-6 and Caco-2 cells. Thrombin stimulated restitution of IEC-6 cells in a dose-dependent manner. Maximal stimulation (3.2-fold; p
Caps. enema •, SP atta9. 4.CGRPantag. • ~n=. dm.
Control 4,3+2,6
24h 17_+5,8" 5,~5,1 ° 2,8±3,5~ 1t0,8"
48h 10,8±2,5" 5,8±4,3* 1,8±2,2"* 1,25~1,5~
Caps.=Capsaicin,antao.=antagonist, sens.den.=aftersensorydenervatJo~(*p~O,O5,"*p
Accelerated intestinal Epithelial Restitution: A Novel Biological Action of GlucagonLike Peptide 2 (GLP-2) Matthew W. Fei, All Tavakkolizadeh,Stanley W. Ashley, Michael J. Zinner, Edward E. Whang, Dept of Surg, Brigham and Women's Hosp, Boston, MA Background:GLP-2 is a potent intestine-specificgrowth factor that amelioratesNSAID-induced intestinal injury in animal models. Whetherthe intestinotrophic actions of GLP-2 alone account for this phenomenonare unknown. In this study we tested the hypothesisthat GLP-2 reverses the deleterious effects of NSAIDs on intestinal epithelial restitution. Methods: Linear wounds were made on monoleyers of serum-starved Caco-2 cells. These wounds were oriented orthogonally to reference lines previously drawn on the outer surface of the culture plates.
A-504