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Thyroid hormone positively regulates an enterocyte differentiation marker gene through an atypical response element
mediate this effect in vitro (Gastroenterology 123:2005). The following studies highlight the physiologic relevance of our previous investigations. Methods: Transgenic mice were denved to express a human growth hormone (HGH) reporter under control of nt -218 to + 118 of the rat ASBT promoter, c-los antisense oligonucleotides were used to generate clos negative Caco-2 cells, c-los knock out (ko) mice were bred from commercially available heterozygotes. Results: Nucleotides -218 to + 118 of the rat ASBT promoter directed HGH expression ubiquitously throughout multiple tissues from five different transgenic mouse lines, lndomethacin Induced.acute ileitis in these transgenic mice resulted in repression of both ASBT (-49%) and HGH (-52%). ASBT promoter activity In the c-los negative Caco-2 cells was enhanced (+ 51%) compared to standard Caco-2 cells. 1L-1B treatment of Caco2 cells resulted in reduced ASBT promoter activity (-80%), while c-los antisense treatment abrogated the cytokine mediated response, c-los expression was absent from c-los ko mice Indomethacin induced ileitis was associated with up-regulation (700%) of c-jun in both ~ld type and ko mice. Steady-state ASBT mRNA levels were repressed (-56%) in indomethatin treated wild type mice, while ASBT was induced (+ 80%) in ko mice. Conclusion: Basal activityof the ASBT promoter is dependent upon the balance of negative (c-los) and positive (ojun) AP-1 elements. Cis elements located between -218 and + 118 are sufficient to mediate the inflammatory response of ASBT. ASBT transcription is enhanced when negative tone is removed (i.e. c-los antisense or knock-out). Anomalous inflammatory mediated induction at ASBTexpression is observed in o-los knock-out mice, since up-regulated c-jun is unopposed by c-los.
axis, with greatest expression in the villus tip. Expression of this unique isoform may contribute to the phenotypic switch as enterocytes migrate from crypt to villus. 695 IL-6 Induces NF-/CB Activation That is Down-Regulated By s a c s - 3 in Intestinal Epithelial Cells Lixin Wang, John Evans, Baljit Wafia, Didier Merlin, Shanthi V. Sitaraman Background and Significance: IL-6 is a pro-inflammatory cytokine that is shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is up-regulated in patients with IBD and its levels correlate with the disease activity. We have previously shown that IL-6 secretion is polarized to the luminal compartment of model intestinal epithelial cells and IL-6 is present in high concentrations in the luminal fluid of patient with active IBD (J. Clin. Invest. 107:861). The putative subsequent effects of IL-6 on epithelial cells are not known. Methods: The model intestinal epithelial cell lines, Caco2-BBE and T84 cells, were used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling ( s a c s ) proteins play a role in the negative regulation of IL-6 signaling. Results: We show that IL-6 receptors are present in intestinal epithelia, apical (ap) and basolateral (bs) IL-6 induces a dose- and time-dependent increase in phospho- STAT-3, a classical signaling pathway for IL-6 Interestingly, IL-6 also induces the activation of NF-kB pathway, an atypical signaling pathway for IL-6. While resting cells have no detectable NF-kB, stimulation with ap or bs IL-6 results in a maximal induction of NF-kB activation and NF-kB p 65 nuclear translocation at 2 hours after apical or basolateral stimulation and a marked decrease by four hours after IL-6 stimulation. IL-6 Induces an apically polarized expression of Intercellular adhesion molecule-1 (ICAM-1 ), an adhesion molecule shown to be important in neutrophil-epithelial interaction in IBD. Using various deletion constructs of ICAM-1 promoter, we show that the ICAM-1 induction by IL-6 requires both STAT-3 and NF-kB elements. Overexpression of s a c s - 3 abolishes NF-kB activation and ICAM-1 expression Induced by IL-6 but not the NF-kB or ICAM1 Induced by TNF-alpha. Conclusions: This is the first study to show the activation of NF-kB by IL-6 in intestinal epithelia and the down-regulation of NF-kB induction by s a c s - 3 , a protein known to inhibit STAT activation by various cytokines. This study demonstrates that IL-6 may activate multiple pro-inflammatory pathways and more importantly, is able to down-regulate these pathways by the induction of s a c s - 3 .
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Thyroid Hormone Positively Regulates an Enterocyte Differentiation Marker Geae through an Atypical Response Element Madhu S. Malo, Wenying Zhang, Maria A. Abedrapo, Joseph W. Henderson, Aleem Siddique, Moushumi Mozumder, Richard A. Hodin BACKGROUND: Thyroid hormone (T3) is an important regulator of intestinal epithelial development and homeostasis, and specifically induces the transcription of the enterocyte differentiation marker gene, intestinal alkaline phosphatase (tAP) The present studies were undertaken to elucidate the precise mechanism by which T3 activates the lAP gene within intestinal epithelia. METHODS: RT-PCR was performed to determine whether TRal was expressed in human colon carcinoma derived Caco-2 cells. Total RNA was isolated from Caco-2 cells treated +/- T3 (100 nM), and lAP mRNA expression determined by northern blotting Firefly luciferase reporter plasmids (plAPs) were constructed carrying different fragments of the human IAP gene promoter Caco-2 cells were transiently transfected with the plAP plasmids along with the plasmids expressing TRal and Renilla luciferase (control), and the cells then treated +/- T3 (100 nM). Electrophoretie mobility shift assays (EMSA) were performed to more precisely define the T3-response element (TRE). Site-directed mutagenesis was performed on the TRE to assess its biological role in lAP gene regulation. RESULTS: RT-PCR confirmed a low level expression of TRal in Caco-2 cells. Northem analyses showed ~3 fold IAP induction within 24 hr of T3 treatment. In the presence of TJ, the plAP plasmid carrying the full-length tAP promoter (2.5 kb) showed a 25-fold activation.Analyses of 5' and internal deletion constructs localized a putative TRE in between 750 and -616 (90 % d~crease in luciferase activity when this region was deleted). EMSA studiesusing in vitro-synthesized TRn 1 and RXR, along with TR antibodies for super-shifting, confirmed the existence of a TRE (5'-TGAACTCAgccTGAGGTTA-3') located between -631 and -613. This DNA element shares ~ 80% homology with the consensus TRE sequence, but is novel in that it has only three nucleotides separating its octa-nucleotide half-sites. Site-directed mutagenesis studies confirmed that this cis-elemem functions as a biologically active TRE in Caco-2 cells. CONCLUSION: The enterocyte differentiation marker tAP gene transcnption is activated by thyroid hormone via an atypical response element located between -631 and -613 bp upstream of the ATG start codon.
696 FGFR3 Regulates Crypt Regeneration in the Mouse Intestine Following, Radiation Injury Alda Vidrich, Jenny Buzan, Chibuzo flo, Leigh Bradley, Kirstin Skaar, Steven M. Cobn Fibroblast growth factor receptors are a family of tyrosine kinases that mediate cellular growth and differentiation in many cell types during normal development and during mjuryrepair. We previously found that FGF receptor-3 (FGFR3) is expressed in crypt epithelial cells during normal intestinal development and that this receptor is necessary for formation of normal numbers of crypts during gut development. The goal of this study is to define whether FGFR3 also regulates crypt regeneration following intestinal injury and to determine which FGFR3 ligands may be involved in this process. METHODS: Expression of specific FGFR3 isoforms which differ in ligand binding affinity were determined by rtPCR of RNA from regenerating crypts isolated by laser capture microdisection at 96 hours after irradiation. The microcolony assay was used to determine the crypt stem cell survival in FGFR3 -/mice and wild-type (vat) littermates following "/-irradiation. Levels of FGF mRNAs (FGF1FGF10) were determined at various times after irradiation by real-time PCR. RESULTS: Immunohistochemical analysis showed that FGFR3 was expressed in all epithelial cells within regenerating crypts at 96 hours after ~-irradiation mRNAs encoding both FGFR-3 II[b and IIlc isoforms were expressed in isolated regenerating crypt epithelial cells. Fractional crypt survival following t4 Gy 7-irradiation was decreased in FGFR3 -/- mice compared with wt littermates in both the proximal jejunum (1.6_+ 0.7% vs.7.9-- 1.2%~ p<0.01) and ileum (0.8 "4"0.3% vs. 5.5 +- 1.4%, p<0.05). Of the ten FGFs where receptor binding specificity has been fully characterized only FGF2 and FGF10 mRNAs increased significantly following 14 Gy "/-irradiation. FGF2 mRNA was increased by 24 hrs after irradiation and maximal induction of FGF2 (~9.6 fold) occurred at 72 hours. Induction of FGF10 mRNA was delayed compared with FGF2; levels of FGF10 start to increase at 48 hrs and induction was maximum (-67 fold) at 144 hrs after irradiation, a time point corresponding to rapid expansion of crypt numbers through crypts fission. Recombinant FGF10 enhanced fractional crypt stem cell survival compared to animals treated with vehicle alone when given 24 hrs prior to 14 Gy 7-irradiation (26.1 -+ 3.6% vs. 13.9 _+1.6%, p<0.01) but not when given 1 hour prior to injury. CONCLUSIONS: These data suggest that signaling through FGFR3 regulates stem cell turnover and/or crypt morphogenesis following intestinal injury. Candidate ligands mediating this effect include FGF2 and FGF10.
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Myosin Light Chain Kinase Isoform Expression Is Regulated During Crypt-Villus Maturation Daniel R. Clayburgh, Shari Rosen, Sara Palkon, John A. Alverdy, Jerrold R. Tumer Multiple splice variants of non-muscla myosin light chain kinase (MYLK) have been found, but to date the physiological role of these isoforms is unknown. MYLK1 is the full length transcript and includes a 69 amino acid internal sequence that contains p60Src phosphorylaclan sites. MYLK2 is a splice variant of MYLK1 that lacks the 69 amino acid domain and is not a substrate for p6OSrc. The activities of MYLK1 and MYLK2 are similar when MYLK is not phosphorylated by p60Src, but MYI_K1 phosphorylation leads to a 2-3-fold increase in Vmax We hypothesized that differential expression of MYLKI along the crypt-villus axis might,in part, explain the enhanced phosphoryfation of myosin light chain in villus, relative to crypt, enterocytes. METHODS: Caco-2 ceils were cultured on semipermeable supports and harvested as they differentiated as an in vitro model of intestinal epithelial maturation. CD'osections of normal human jejunum were laser microdissected to yield pure samples of cg?t, lower villus, and upper villus enterocytes. RNA was extracted from both specimen I)pes and MYLK isoforms were detected by RT-PCR. Polyclonal antisera were generated against a peptide derived from the MLYKl-specific 69 amino acid domain. RESULTS: DifferentiatedCaco-2 monolayers expressed MYLK1 and MYLK2 transcripts, but RNA encoding other MYLK splice variants was not detected. RT-PCR analysis using primers flanking the splice sites showed that MYLK2 is the dominant isoform in subconfluent and newly confluent monolayers (MYLKI:MYLK2 1:1.7 and 1:2.1, respectively). However, as monalayersmatured, MYLK1 expression increased and MYLK2 expression decreased, with MYLK] becomingthe principal isoform expressed by 8 days post-confluence (ratio of 1.7:1). Analysis of human jejunal enterocytes showed that MYLK2 was heavily expressed throughout the crypt-villusaxis. Thus, isoform-specific primers were used to determine MYLK1 expression, which was normalized to keratin K8. MYLK1 mRNA expression was lowest in the crypt and increased 1.6-fold and 3.7- fold in lower and upper villus samples, respectively. Immunofluorescence of normal human jejunum with MYLKl-specific antisera confirmed the crypt~nllusexpression gradient and showed that MYLK1 is localized within the cortical actin web of villus enterocytes CONCLUSION: MYLK1 expression is regulated along the crypt-villus
697
Epidermal Growth Factor Receptor (EGFR) and NF-KB Regulate Intestinal Epithelial Restitution in a Novel Motogenic Cascade Laurence Egan, Evan Lehrman, Ann De Lecea, Gennet Myhre, Jars Eckmann, Martin F. Kagnoff Epithelial restitution, the movement of wound-edge epithelial cells into areas of cell denudation, is an important early step in wound healing. Aithough intestinal epithelial restitution is promoted by growth factors, little is known about the downstream effectors that translate those stimuli into cell migration. The transcription factor nuclear factor kappa B (NF-KB) regulates cellular responses that are essential for inflammation and tissue protection. Since signaling through the EGFR was recently shown to activate NF-KB, we tested the hypothesis that NF-KB functions as a downstream regulator of intestinal epithelial restitution. Methods. Epithelial restitution was modeled using scrape-wounded monolayers of the non-transformed rat intestinal epithelial cell (IEC) line, RIE-1 and RIE-1 cells stably transfected with a
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AGA Abstracts
axis, with greatest expression in the villus tip. Expression of this unique isoform may contribute to the phenotypic switch as enterocytes migrate from crypt to villus. 695 IL-6 Induces NF-/CB Activation That is Down-Regulated By s a c s - 3 in Intestinal Epithelial Cells Lixin Wang, John Evans, Baljit Wafia, Didier Merlin, Shanthi V. Sitaraman Background and Significance: IL-6 is a pro-inflammatory cytokine that is shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is up-regulated in patients with IBD and its levels correlate with the disease activity. We have previously shown that IL-6 secretion is polarized to the luminal compartment of model intestinal epithelial cells and IL-6 is present in high concentrations in the luminal fluid of patient with active IBD (J. Clin. Invest. 107:861). The putative subsequent effects of IL-6 on epithelial cells are not known. Methods: The model intestinal epithelial cell lines, Caco2-BBE and T84 cells, were used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling ( s a c s ) proteins play a role in the negative regulation of IL-6 signaling. Results: We show that IL-6 receptors are present in intestinal epithelia, apical (ap) and basolateral (bs) IL-6 induces a dose- and time-dependent increase in phospho- STAT-3, a classical signaling pathway for IL-6 Interestingly, IL-6 also induces the activation of NF-kB pathway, an atypical signaling pathway for IL-6. While resting cells have no detectable NF-kB, stimulation with ap or bs IL-6 results in a maximal induction of NF-kB activation and NF-kB p 65 nuclear translocation at 2 hours after apical or basolateral stimulation and a marked decrease by four hours after IL-6 stimulation. IL-6 Induces an apically polarized expression of Intercellular adhesion molecule-1 (ICAM-1 ), an adhesion molecule shown to be important in neutrophil-epithelial interaction in IBD. Using various deletion constructs of ICAM-1 promoter, we show that the ICAM-1 induction by IL-6 requires both STAT-3 and NF-kB elements. Overexpression of s a c s - 3 abolishes NF-kB activation and ICAM-1 expression Induced by IL-6 but not the NF-kB or ICAM1 Induced by TNF-alpha. Conclusions: This is the first study to show the activation of NF-kB by IL-6 in intestinal epithelia and the down-regulation of NF-kB induction by s a c s - 3 , a protein known to inhibit STAT activation by various cytokines. This study demonstrates that IL-6 may activate multiple pro-inflammatory pathways and more importantly, is able to down-regulate these pathways by the induction of s a c s - 3 .
669
Thyroid Hormone Positively Regulates an Enterocyte Differentiation Marker Geae through an Atypical Response Element Madhu S. Malo, Wenying Zhang, Maria A. Abedrapo, Joseph W. Henderson, Aleem Siddique, Moushumi Mozumder, Richard A. Hodin BACKGROUND: Thyroid hormone (T3) is an important regulator of intestinal epithelial development and homeostasis, and specifically induces the transcription of the enterocyte differentiation marker gene, intestinal alkaline phosphatase (tAP) The present studies were undertaken to elucidate the precise mechanism by which T3 activates the lAP gene within intestinal epithelia. METHODS: RT-PCR was performed to determine whether TRal was expressed in human colon carcinoma derived Caco-2 cells. Total RNA was isolated from Caco-2 cells treated +/- T3 (100 nM), and lAP mRNA expression determined by northern blotting Firefly luciferase reporter plasmids (plAPs) were constructed carrying different fragments of the human IAP gene promoter Caco-2 cells were transiently transfected with the plAP plasmids along with the plasmids expressing TRal and Renilla luciferase (control), and the cells then treated +/- T3 (100 nM). Electrophoretie mobility shift assays (EMSA) were performed to more precisely define the T3-response element (TRE). Site-directed mutagenesis was performed on the TRE to assess its biological role in lAP gene regulation. RESULTS: RT-PCR confirmed a low level expression of TRal in Caco-2 cells. Northem analyses showed ~3 fold IAP induction within 24 hr of T3 treatment. In the presence of TJ, the plAP plasmid carrying the full-length tAP promoter (2.5 kb) showed a 25-fold activation.Analyses of 5' and internal deletion constructs localized a putative TRE in between 750 and -616 (90 % d~crease in luciferase activity when this region was deleted). EMSA studiesusing in vitro-synthesized TRn 1 and RXR, along with TR antibodies for super-shifting, confirmed the existence of a TRE (5'-TGAACTCAgccTGAGGTTA-3') located between -631 and -613. This DNA element shares ~ 80% homology with the consensus TRE sequence, but is novel in that it has only three nucleotides separating its octa-nucleotide half-sites. Site-directed mutagenesis studies confirmed that this cis-elemem functions as a biologically active TRE in Caco-2 cells. CONCLUSION: The enterocyte differentiation marker tAP gene transcnption is activated by thyroid hormone via an atypical response element located between -631 and -613 bp upstream of the ATG start codon.
696 FGFR3 Regulates Crypt Regeneration in the Mouse Intestine Following, Radiation Injury Alda Vidrich, Jenny Buzan, Chibuzo flo, Leigh Bradley, Kirstin Skaar, Steven M. Cobn Fibroblast growth factor receptors are a family of tyrosine kinases that mediate cellular growth and differentiation in many cell types during normal development and during mjuryrepair. We previously found that FGF receptor-3 (FGFR3) is expressed in crypt epithelial cells during normal intestinal development and that this receptor is necessary for formation of normal numbers of crypts during gut development. The goal of this study is to define whether FGFR3 also regulates crypt regeneration following intestinal injury and to determine which FGFR3 ligands may be involved in this process. METHODS: Expression of specific FGFR3 isoforms which differ in ligand binding affinity were determined by rtPCR of RNA from regenerating crypts isolated by laser capture microdisection at 96 hours after irradiation. The microcolony assay was used to determine the crypt stem cell survival in FGFR3 -/mice and wild-type (vat) littermates following "/-irradiation. Levels of FGF mRNAs (FGF1FGF10) were determined at various times after irradiation by real-time PCR. RESULTS: Immunohistochemical analysis showed that FGFR3 was expressed in all epithelial cells within regenerating crypts at 96 hours after ~-irradiation mRNAs encoding both FGFR-3 II[b and IIlc isoforms were expressed in isolated regenerating crypt epithelial cells. Fractional crypt survival following t4 Gy 7-irradiation was decreased in FGFR3 -/- mice compared with wt littermates in both the proximal jejunum (1.6_+ 0.7% vs.7.9-- 1.2%~ p<0.01) and ileum (0.8 "4"0.3% vs. 5.5 +- 1.4%, p<0.05). Of the ten FGFs where receptor binding specificity has been fully characterized only FGF2 and FGF10 mRNAs increased significantly following 14 Gy "/-irradiation. FGF2 mRNA was increased by 24 hrs after irradiation and maximal induction of FGF2 (~9.6 fold) occurred at 72 hours. Induction of FGF10 mRNA was delayed compared with FGF2; levels of FGF10 start to increase at 48 hrs and induction was maximum (-67 fold) at 144 hrs after irradiation, a time point corresponding to rapid expansion of crypt numbers through crypts fission. Recombinant FGF10 enhanced fractional crypt stem cell survival compared to animals treated with vehicle alone when given 24 hrs prior to 14 Gy 7-irradiation (26.1 -+ 3.6% vs. 13.9 _+1.6%, p<0.01) but not when given 1 hour prior to injury. CONCLUSIONS: These data suggest that signaling through FGFR3 regulates stem cell turnover and/or crypt morphogenesis following intestinal injury. Candidate ligands mediating this effect include FGF2 and FGF10.
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Myosin Light Chain Kinase Isoform Expression Is Regulated During Crypt-Villus Maturation Daniel R. Clayburgh, Shari Rosen, Sara Palkon, John A. Alverdy, Jerrold R. Tumer Multiple splice variants of non-muscla myosin light chain kinase (MYLK) have been found, but to date the physiological role of these isoforms is unknown. MYLK1 is the full length transcript and includes a 69 amino acid internal sequence that contains p60Src phosphorylaclan sites. MYLK2 is a splice variant of MYLK1 that lacks the 69 amino acid domain and is not a substrate for p6OSrc. The activities of MYLK1 and MYLK2 are similar when MYLK is not phosphorylated by p60Src, but MYI_K1 phosphorylation leads to a 2-3-fold increase in Vmax We hypothesized that differential expression of MYLKI along the crypt-villus axis might,in part, explain the enhanced phosphoryfation of myosin light chain in villus, relative to crypt, enterocytes. METHODS: Caco-2 ceils were cultured on semipermeable supports and harvested as they differentiated as an in vitro model of intestinal epithelial maturation. CD'osections of normal human jejunum were laser microdissected to yield pure samples of cg?t, lower villus, and upper villus enterocytes. RNA was extracted from both specimen I)pes and MYLK isoforms were detected by RT-PCR. Polyclonal antisera were generated against a peptide derived from the MLYKl-specific 69 amino acid domain. RESULTS: DifferentiatedCaco-2 monolayers expressed MYLK1 and MYLK2 transcripts, but RNA encoding other MYLK splice variants was not detected. RT-PCR analysis using primers flanking the splice sites showed that MYLK2 is the dominant isoform in subconfluent and newly confluent monolayers (MYLKI:MYLK2 1:1.7 and 1:2.1, respectively). However, as monalayersmatured, MYLK1 expression increased and MYLK2 expression decreased, with MYLK] becomingthe principal isoform expressed by 8 days post-confluence (ratio of 1.7:1). Analysis of human jejunal enterocytes showed that MYLK2 was heavily expressed throughout the crypt-villusaxis. Thus, isoform-specific primers were used to determine MYLK1 expression, which was normalized to keratin K8. MYLK1 mRNA expression was lowest in the crypt and increased 1.6-fold and 3.7- fold in lower and upper villus samples, respectively. Immunofluorescence of normal human jejunum with MYLKl-specific antisera confirmed the crypt~nllusexpression gradient and showed that MYLK1 is localized within the cortical actin web of villus enterocytes CONCLUSION: MYLK1 expression is regulated along the crypt-villus
697
Epidermal Growth Factor Receptor (EGFR) and NF-KB Regulate Intestinal Epithelial Restitution in a Novel Motogenic Cascade Laurence Egan, Evan Lehrman, Ann De Lecea, Gennet Myhre, Jars Eckmann, Martin F. Kagnoff Epithelial restitution, the movement of wound-edge epithelial cells into areas of cell denudation, is an important early step in wound healing. Aithough intestinal epithelial restitution is promoted by growth factors, little is known about the downstream effectors that translate those stimuli into cell migration. The transcription factor nuclear factor kappa B (NF-KB) regulates cellular responses that are essential for inflammation and tissue protection. Since signaling through the EGFR was recently shown to activate NF-KB, we tested the hypothesis that NF-KB functions as a downstream regulator of intestinal epithelial restitution. Methods. Epithelial restitution was modeled using scrape-wounded monolayers of the non-transformed rat intestinal epithelial cell (IEC) line, RIE-1 and RIE-1 cells stably transfected with a
A-87
AGA Abstracts