- Email: [email protected]
Regulation of MDR2 p-glycoprotein expression by bile salts and cholesterol in rats and primary cultures of rat hepatocytes
A592 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
3066
3068
REGULATION OF THE HUMAN NA+/GLUCOSE COTRANSPORTER GENE (SGLTl) BY HNF-l AND THE SP FAMILY OF TRANSCRIPTIONAL FACTORS. Martin G. Martin, Jiafang Wang, Sergio Vargas, Jason T. Lam, Eric Turk, Ernest M. Wright, UCLA, Los Angeles, CA. Na +/glucose cotransporter (SGLTl) is expressed primarily by small intestinal epithelial cells, and transports the monosaccharides glucose and galactose across the apical membrane. We describe the isolation and characterization of 5.3 Kb of the 5' -flanking region of the human SGLTl gene by transiently transfecting reporter constructs, containing either truncation or substitution mutations, into a variety of established epithelial cell lines. Deletion analysis of various lengths ( 52951+22 to -271+22) of the promoter cloned upstream of the reporter luciferase suggest that its expression was supported best in the human intestinal cell line Cac02, and that nucleotides 2351+22 represents the gene's minimal promoter. Within this region of the promoter, several DNA-protein complexes were identified by in vitro DNase I footprint analysis. Scanning mutagenesis was performed to define the exact cis-elements within the minimal promoter responsible for basal expression of the promoter and three distinct sites that were responsible for enhancing SGLTl s promoter activity were identified. The three cis-elements included a hepatocyte nuclear factor-I (HNF-l) and two GC boxes sites. Band shift assays of the two GC boxes identified similar protein-DNA complexes; however, the binding affinity for GC box-I with Spl was substantially greater than that of GC box-2. The components of the complexes were defined by supershift analysis and revealed that binding of Spl, Sp2 and Sp3 or a related protein(s) forms portions of the two complexes. Furthermore, EMSA assays of GC box-l revealed that nucleotides immediately downstream of the box (ATTCGCAGGACAGCTC) were critical for complex formation with both crude and recombinant Spl protein. Additional data suggests that the transcriptional factors that bind to the GC box interact with HNF-l to synergistically upregulate transactivation of the heterologous SV40 promoter. In summary, we report the identification and characterization of the human SGLTl minimal promoter, and the critical role that HNF-l and Sp l-multigene members have in enhancing the basal level of its transcription in Cac02 cells.
CDNA ARRAY ANALYSIS OF DIFFERENTIATION-DEPENDENT CACO·2 RESPONSES TO IFNf. Mark J. Ropeleski, Line Dufresne, Chantal Cossette, Alan B. Thomson, Gary E. Wild, McGill Univ IBD Research Program, Montreal, PQ, Canada; Univ of Alberta, Edmonton, AB, Canada. Background: Differential expression of specific genes determines intestinal epithelial phenotype which can be modulated by cytokines such as IFNy. Studies suggest that epithelial differentiation may modulate bi-directional signaling between the epithelial, immune and non-immune cell compartments. The availability of cDNA arrays provides an opportunity to study cytokine-mediated gene responses. We hypothesize that patterns of IFN-y induced gene expression are differentiation-dependent in the Caco-2 model of enterocyte differentiation and have sought to characterize these by cDNA array and immunoblotting. Methods: Pre-confluent, confluent and post-confluent Caco-2 cells were exposed to IFNy(IO U/ml) for 8 hrs. Reverse transcription was carried out using gene specific primers and the resultant probes were hybridized to Atias™ human cDNA Expression Arrays(Clontech, Palo Alto, CA) consisting of 588 cDNA s representing diverse gene families. After normalization, relative differences in gene expression were determined using Atlaslmage" 1.01 software. Gene expression was confirmed by semi-quantitative RT-PCR and characterized further by immunoblotting. Results: Among numerous genes whose expression was modulated by IFNy, we selected those (insulin and epidermal growth factor receptor, early growth response gene-I(egr-l), p105 precursor of p50 of NF-kB and the cytoskeletal anchoring protein, ezrin) whose basal gene expression and responses to IFNy varied with differentiation. All showed upregulation in pre-confluent cells while only the upregulation of pl 05 and ezrin persisted to post-confluence. Responses in cells just achieving confluence showed marked down regulation in most cases. Immunodetectable protein increased with the state of differentiation in all cases, but did not always correlate with altered gene responses detected by array or RT-PCR. Conclusion: The application of cDNA arrays to define patterns of gene expression in in vitro models of intestinal inflammation combined with protein expression data confirms the importance of transcriptional and post-transcriptional mechanisms in the Caco-2 model of differentiation along the crypt-villus axis and in determining responses to IFNy. Though computational strategies for the analysis of array data are evolving, cDNA expression arrays are useful for the identification known and novel gene responses to IFNy such as ezrin, egr-I and pl05.(Support by Crohn s and Colitis Foundation of Canada and Les Fonds de la Recherche en Sante du Quebec).
3067 THE NF-KB MEDIATED EXPRESSION OF INOS IN INTESTINAL EPITHELIAL CELLS IS DEPENDENT ON THE CELLULAR REDOX STATUS. Gerard Dijkstra, Manon Homan, Harry van Goor, Mariska Geuken, Alie D. Jager, Peter L. Jansen, Han Moshage, Div Gastroenterology; Univ Hosp, Groningen, Netherlands; Dept of Pathology, Univ Hosp, Groningen, Netherlands. Background: Inducible Nitric Oxide Synthase (iNOS)is expressed in intestinal epithelial cells of patients with active inflammatory bowel disease. Inflammatory cytokines, like IL-l, TNF-a and interferon(IFN)-y are potent inducers of iNOS. IL-l and TNF-a act via the transcription factor Nuclear Factor-xls (NF-KB). A key determinant in the activation of NF-KB is the cellular redox status, determined mainly by the cellular concentration of reduced glutathione. Aim: To determine the involvement of NF-KB activation and the importance of the cellular redox status in the expression of iNOS in intestinal epithelium. Methods: Cellular redox status was manipulated using the thiol-modifying agent diethylmaleate (DEM) and the glutathione synthesis inhibitor BSO (buthionine sulfoximine). In vivo: Rats were treated with DEM (4mmollkg ip) 0.5 hr before and 3 hrs after LPS injection (5mglkg ip). Rats were sacrificed 6 hr after LPS administration. In vitro: the human colon carcinoma cell-line DLD-I was exposed to a cytokine mix (CM) composed of IL-l(3, TNF-a and IFN-y for 8 hr. DEM (I ruM) and the NF-KB antagonist MG-132 were added 0.5 hr prior to and 4 hr after addition of CM. iNOS expression was evaluated by RT-PCR, Western blot and immunohistology. NF-KB and AP-l activation were determined by EMSA. Results: In vivo: LPS strongly induced iNOS protein and mRNA expression in intestinal epithelial cells of ileum and colon. DEM-treatment completely abolished iNOS expression. In vitro: Cytokines induced iNOS mRNA and protein expression in DLD-l cells. Both DEM and BSO reduced glutathione levels to <10% of control values. DEM treatment but not BSO treatment prevented iNOS induction in CM-exposed DLD-l cells. The NF-KB antagonist MG-132 also prevented iNOS induction in DLD-I cells. iNOS expression correlated with CMinduced NF-KB activation: MG-132 and DEM, but not BSO, prevented CM-induced NF-KB activation. AP-l activation was not influenced by DEM, BSO or MG-132, indicating the specificity for NF-KB of DEM and MG-132. CM-induced degradation of the NF-KB-inhibitory protein IkB-a was not influenced by DEM treatment. Conclusion: iNOS expression in intestinal epithelial cells is dependent on NF-KB activation. iNOS expression can occur in glutathione-depleted cells, but is sensitive to thiolmodifying agents. These appear to interfere down-stream from IkB-a degradation, possibly via direct interaction with essential sulfhydryl groups on NF-KB subunits. Supported by a grant from ASTRA-Zeneca, the Netherlands
3069 REGULATION OF MDR2 P·GLYCOPROTEIN EXPRESSION BY BILE SALTS AND CHOLESTEROL IN RATS AND PRIMARY CULTURES OF RAT HEPATOCYTES. Gupta Seema, Richard T. Stravitz, William M. Pandak, Michael Muller, Zdravko R. Vlahcevic, Phillip B. Hylemon, Virgina Commonwealth Univ, Richmond, VA; Univ Hosp, Groningen, Netherlands. Background: Canalicular secretion of phosphatidylcholine and cholesterol is dependent on the flux of bile salts through the liver. Recently, a canalicular carrier protein (mdr2) responsible for phosphatidylcholine secretion was cloned and identified allowing for the establishment of the molecular basis of these interrelationships. Objective: To define the effects of bile salts with differing hydrophobicity and cholesterol on the expression of mdr2 in primary rat hepatocytes and in in vivo experiments in the rat. Results: In primary rat hepatocyte cultures, addition of 50 JLM of taurocholate, taurodeoxycholate, taurochenodeoxycholate and tauroursodeoxycholate resulted in increases in steady-state mdr2 mRNA levels by 288±369'0, 276±539'0, 216±34% and 175±28%, respectively (p<0.05 for all values) of control. Intraduodenal infusion of taurocholate for 48 h in rats caused a statistically significant increase (p<0.05) in mdr2 mRNA levels of 158±20% of control. In contrast, chronic biliary diversion and cholestyramine decreased mdr2 mRNA levels to 66±99'0 and 63±79'0 of sham operated controls (p<0.05). In rats with intact enterohepatic circulation, cholate feeding for 14 days increased mdr2 transcriptional activity by 4-fold and protein mass by 1.7 fold. A high cholesterol diet (4%) decreased steady-state mRNA levels to 57±29'0 of pair fed controls. Addition of squalestatin (I JLM), an inhibitor of cholesterol synthesis, increased mdr2 mRNA levels by 7-fold in primary rat hepatocyte cultures. In in vivo, squalestatin infusion also increased mdr2 mRNA levels. Conclusion: In vitro and in vivo studies have shown that the coupling of bile salt fluxes through the liver and phosphatidylcholine secretion is due to the direct effects of hydrophobic bile salts on mdr2 steady-state mRNA levels. The up-regulation of mdr2 by bile salts takes place at the level of gene transcription. mdr2 is also down-regulated by excess cholesterol and upregulated by inhibition of cholesterol synthesis.
3070 DUODENAL CALBINDIN-D9K GENE EXPRESSION IN WILDTYPE AND VITAMIN D RECEPTOR KNOCKOUT MICE. Michael D. Sitrin, Merry Bolt, Li Ping Cao, Van Chun Li, U of Chicago, Chicago, IL. Calbindin-Dsj, (CABP) is a duodenal mucosal protein thought to play an important role in calcium absorption. CABP is believed to facilitate diffusion of Ca +2 from the apical to the basolateral cell membrane, but may also
GASTROENTEROLOGY Vol. 118, No.4
3066
3068
REGULATION OF THE HUMAN NA+/GLUCOSE COTRANSPORTER GENE (SGLTl) BY HNF-l AND THE SP FAMILY OF TRANSCRIPTIONAL FACTORS. Martin G. Martin, Jiafang Wang, Sergio Vargas, Jason T. Lam, Eric Turk, Ernest M. Wright, UCLA, Los Angeles, CA. Na +/glucose cotransporter (SGLTl) is expressed primarily by small intestinal epithelial cells, and transports the monosaccharides glucose and galactose across the apical membrane. We describe the isolation and characterization of 5.3 Kb of the 5' -flanking region of the human SGLTl gene by transiently transfecting reporter constructs, containing either truncation or substitution mutations, into a variety of established epithelial cell lines. Deletion analysis of various lengths ( 52951+22 to -271+22) of the promoter cloned upstream of the reporter luciferase suggest that its expression was supported best in the human intestinal cell line Cac02, and that nucleotides 2351+22 represents the gene's minimal promoter. Within this region of the promoter, several DNA-protein complexes were identified by in vitro DNase I footprint analysis. Scanning mutagenesis was performed to define the exact cis-elements within the minimal promoter responsible for basal expression of the promoter and three distinct sites that were responsible for enhancing SGLTl s promoter activity were identified. The three cis-elements included a hepatocyte nuclear factor-I (HNF-l) and two GC boxes sites. Band shift assays of the two GC boxes identified similar protein-DNA complexes; however, the binding affinity for GC box-I with Spl was substantially greater than that of GC box-2. The components of the complexes were defined by supershift analysis and revealed that binding of Spl, Sp2 and Sp3 or a related protein(s) forms portions of the two complexes. Furthermore, EMSA assays of GC box-l revealed that nucleotides immediately downstream of the box (ATTCGCAGGACAGCTC) were critical for complex formation with both crude and recombinant Spl protein. Additional data suggests that the transcriptional factors that bind to the GC box interact with HNF-l to synergistically upregulate transactivation of the heterologous SV40 promoter. In summary, we report the identification and characterization of the human SGLTl minimal promoter, and the critical role that HNF-l and Sp l-multigene members have in enhancing the basal level of its transcription in Cac02 cells.
CDNA ARRAY ANALYSIS OF DIFFERENTIATION-DEPENDENT CACO·2 RESPONSES TO IFNf. Mark J. Ropeleski, Line Dufresne, Chantal Cossette, Alan B. Thomson, Gary E. Wild, McGill Univ IBD Research Program, Montreal, PQ, Canada; Univ of Alberta, Edmonton, AB, Canada. Background: Differential expression of specific genes determines intestinal epithelial phenotype which can be modulated by cytokines such as IFNy. Studies suggest that epithelial differentiation may modulate bi-directional signaling between the epithelial, immune and non-immune cell compartments. The availability of cDNA arrays provides an opportunity to study cytokine-mediated gene responses. We hypothesize that patterns of IFN-y induced gene expression are differentiation-dependent in the Caco-2 model of enterocyte differentiation and have sought to characterize these by cDNA array and immunoblotting. Methods: Pre-confluent, confluent and post-confluent Caco-2 cells were exposed to IFNy(IO U/ml) for 8 hrs. Reverse transcription was carried out using gene specific primers and the resultant probes were hybridized to Atias™ human cDNA Expression Arrays(Clontech, Palo Alto, CA) consisting of 588 cDNA s representing diverse gene families. After normalization, relative differences in gene expression were determined using Atlaslmage" 1.01 software. Gene expression was confirmed by semi-quantitative RT-PCR and characterized further by immunoblotting. Results: Among numerous genes whose expression was modulated by IFNy, we selected those (insulin and epidermal growth factor receptor, early growth response gene-I(egr-l), p105 precursor of p50 of NF-kB and the cytoskeletal anchoring protein, ezrin) whose basal gene expression and responses to IFNy varied with differentiation. All showed upregulation in pre-confluent cells while only the upregulation of pl 05 and ezrin persisted to post-confluence. Responses in cells just achieving confluence showed marked down regulation in most cases. Immunodetectable protein increased with the state of differentiation in all cases, but did not always correlate with altered gene responses detected by array or RT-PCR. Conclusion: The application of cDNA arrays to define patterns of gene expression in in vitro models of intestinal inflammation combined with protein expression data confirms the importance of transcriptional and post-transcriptional mechanisms in the Caco-2 model of differentiation along the crypt-villus axis and in determining responses to IFNy. Though computational strategies for the analysis of array data are evolving, cDNA expression arrays are useful for the identification known and novel gene responses to IFNy such as ezrin, egr-I and pl05.(Support by Crohn s and Colitis Foundation of Canada and Les Fonds de la Recherche en Sante du Quebec).
3067 THE NF-KB MEDIATED EXPRESSION OF INOS IN INTESTINAL EPITHELIAL CELLS IS DEPENDENT ON THE CELLULAR REDOX STATUS. Gerard Dijkstra, Manon Homan, Harry van Goor, Mariska Geuken, Alie D. Jager, Peter L. Jansen, Han Moshage, Div Gastroenterology; Univ Hosp, Groningen, Netherlands; Dept of Pathology, Univ Hosp, Groningen, Netherlands. Background: Inducible Nitric Oxide Synthase (iNOS)is expressed in intestinal epithelial cells of patients with active inflammatory bowel disease. Inflammatory cytokines, like IL-l, TNF-a and interferon(IFN)-y are potent inducers of iNOS. IL-l and TNF-a act via the transcription factor Nuclear Factor-xls (NF-KB). A key determinant in the activation of NF-KB is the cellular redox status, determined mainly by the cellular concentration of reduced glutathione. Aim: To determine the involvement of NF-KB activation and the importance of the cellular redox status in the expression of iNOS in intestinal epithelium. Methods: Cellular redox status was manipulated using the thiol-modifying agent diethylmaleate (DEM) and the glutathione synthesis inhibitor BSO (buthionine sulfoximine). In vivo: Rats were treated with DEM (4mmollkg ip) 0.5 hr before and 3 hrs after LPS injection (5mglkg ip). Rats were sacrificed 6 hr after LPS administration. In vitro: the human colon carcinoma cell-line DLD-I was exposed to a cytokine mix (CM) composed of IL-l(3, TNF-a and IFN-y for 8 hr. DEM (I ruM) and the NF-KB antagonist MG-132 were added 0.5 hr prior to and 4 hr after addition of CM. iNOS expression was evaluated by RT-PCR, Western blot and immunohistology. NF-KB and AP-l activation were determined by EMSA. Results: In vivo: LPS strongly induced iNOS protein and mRNA expression in intestinal epithelial cells of ileum and colon. DEM-treatment completely abolished iNOS expression. In vitro: Cytokines induced iNOS mRNA and protein expression in DLD-l cells. Both DEM and BSO reduced glutathione levels to <10% of control values. DEM treatment but not BSO treatment prevented iNOS induction in CM-exposed DLD-l cells. The NF-KB antagonist MG-132 also prevented iNOS induction in DLD-I cells. iNOS expression correlated with CMinduced NF-KB activation: MG-132 and DEM, but not BSO, prevented CM-induced NF-KB activation. AP-l activation was not influenced by DEM, BSO or MG-132, indicating the specificity for NF-KB of DEM and MG-132. CM-induced degradation of the NF-KB-inhibitory protein IkB-a was not influenced by DEM treatment. Conclusion: iNOS expression in intestinal epithelial cells is dependent on NF-KB activation. iNOS expression can occur in glutathione-depleted cells, but is sensitive to thiolmodifying agents. These appear to interfere down-stream from IkB-a degradation, possibly via direct interaction with essential sulfhydryl groups on NF-KB subunits. Supported by a grant from ASTRA-Zeneca, the Netherlands
3069 REGULATION OF MDR2 P·GLYCOPROTEIN EXPRESSION BY BILE SALTS AND CHOLESTEROL IN RATS AND PRIMARY CULTURES OF RAT HEPATOCYTES. Gupta Seema, Richard T. Stravitz, William M. Pandak, Michael Muller, Zdravko R. Vlahcevic, Phillip B. Hylemon, Virgina Commonwealth Univ, Richmond, VA; Univ Hosp, Groningen, Netherlands. Background: Canalicular secretion of phosphatidylcholine and cholesterol is dependent on the flux of bile salts through the liver. Recently, a canalicular carrier protein (mdr2) responsible for phosphatidylcholine secretion was cloned and identified allowing for the establishment of the molecular basis of these interrelationships. Objective: To define the effects of bile salts with differing hydrophobicity and cholesterol on the expression of mdr2 in primary rat hepatocytes and in in vivo experiments in the rat. Results: In primary rat hepatocyte cultures, addition of 50 JLM of taurocholate, taurodeoxycholate, taurochenodeoxycholate and tauroursodeoxycholate resulted in increases in steady-state mdr2 mRNA levels by 288±369'0, 276±539'0, 216±34% and 175±28%, respectively (p<0.05 for all values) of control. Intraduodenal infusion of taurocholate for 48 h in rats caused a statistically significant increase (p<0.05) in mdr2 mRNA levels of 158±20% of control. In contrast, chronic biliary diversion and cholestyramine decreased mdr2 mRNA levels to 66±99'0 and 63±79'0 of sham operated controls (p<0.05). In rats with intact enterohepatic circulation, cholate feeding for 14 days increased mdr2 transcriptional activity by 4-fold and protein mass by 1.7 fold. A high cholesterol diet (4%) decreased steady-state mRNA levels to 57±29'0 of pair fed controls. Addition of squalestatin (I JLM), an inhibitor of cholesterol synthesis, increased mdr2 mRNA levels by 7-fold in primary rat hepatocyte cultures. In in vivo, squalestatin infusion also increased mdr2 mRNA levels. Conclusion: In vitro and in vivo studies have shown that the coupling of bile salt fluxes through the liver and phosphatidylcholine secretion is due to the direct effects of hydrophobic bile salts on mdr2 steady-state mRNA levels. The up-regulation of mdr2 by bile salts takes place at the level of gene transcription. mdr2 is also down-regulated by excess cholesterol and upregulated by inhibition of cholesterol synthesis.
3070 DUODENAL CALBINDIN-D9K GENE EXPRESSION IN WILDTYPE AND VITAMIN D RECEPTOR KNOCKOUT MICE. Michael D. Sitrin, Merry Bolt, Li Ping Cao, Van Chun Li, U of Chicago, Chicago, IL. Calbindin-Dsj, (CABP) is a duodenal mucosal protein thought to play an important role in calcium absorption. CABP is believed to facilitate diffusion of Ca +2 from the apical to the basolateral cell membrane, but may also