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A novel mitochondrial Ca2+-dependent solute carrier in the liver identified by mRNA differential display
conjugates. Inhibition of PKA and PKC did not have any significant impact on the biological activity of the bile acids. These data obtained at the promoter level were confirmed at the RNA and protein levels when RT-PCR and immunohistochemical experiments were conducted in the same conditions. Conclusion: These results demonstrate that bile acids are important regulators of MUC4 mucin gene expression in esophagus cancer cells and that PI3K is the main signaling pathway triggered by bile acids to activate MUC4 promoters. MAPK pathway acts for some bile acids as a negative control. MUC4 appears thus as a potent marker in esophagus adenocarcinoma derived from abnormal gastroesophageal reflux and bile acids.
Transcription activity of 5' deletions of promoter region was determined by luciferase reporter system. The effects of C/EBPcr and Refl on promoter activity were determined by cotransfection of reporter and C./EBPa or Refl expressing plasmids. Hepatic C/EBPcr and AP1 binding activities in INS-treated rats were determined by EMSA. Hepc mRNA was determined by Northern blots. Results: The region between USF2 and Hepe consists of 1857 bp with a TATA box at -34/-24 (transcription start site = 1). A series of 5' deletions identified ~15-25-fold higher promoter activity in Hepc -113/+ 50 (-113) and -229 than in -603, 705 and -1715 regions in HepG2 cells. In Cos1 cells the promoter activity of -113 was 50% lower than in HepG2. Sequence analysis of the -603 identified numerous putative C../EBPet and AP-1 binding elements. The activity of these elements was tested in cells co-transfected with Hepc -113 or -603 pGL3 and pcDNA-C/EBPcx or pcDNA-Refl. Over expression of C/ EBPa had no effect on -113 in HepG2 and Cos1, but doubled the activity of -603 in HepG2 and reduced activity 70% in Cos1. Refl had no effect on -113 in HepG2, reduced -113 -30% in Cos1, and sigmficantly suppressed -603 in both cell types. In rats INS increased both C/EBPa and AP-1 binding activities at 1 and 2 h and Hepc mRNA expression by 3fold at 2 h, to a peak at 8 h; by 12 h Hepc mRNA returned to normal. Conclusions: The Hepc -113 promoter region contains regulatory elements conferring high levels of cell specific Hepc expression whereas the distal regions 5' of -229 contain elements that suppress Hepc gene transcription. C/EBPa stimulates the region between -229 and -603 in a cell specific manner while AP-1 presumably activated by Ref-1 suppresses Hepc expression. Thus, both C/EBPa and AP-1 are involved in up- and down regulation of Hepc gene expression.
$962 EGF Regulation on the Human Intestinal NaPi-llb Gene Expression Involves e-
Myb Hua Xu, Michael lnouye, Eric Hines, James F. Collins, Fayez K. Ghishan Introduction: The human intestinal sodium-phosphate (hNaPi-llb) cotransporter plays a major role in intestinal Pi absorption. Our previous studies showed that EGF inhibits intestinal NaPi-IIb cotransporter gene transcription and that the EGF response region was between -729 bp and -784 bp of the promoter. Thus, the current study was performed to precisely identify the bases involved in this regulation, and to identify nuclear proteins that bind to this site and participate in EGF regulation of hNaPi-llb gene expression. Additional studies were performed to identify signaling pathways involved in EGF regulation of the hNaPi-llb gene. Methods: Reporter gene assays were performed in transiently transfected CaCo-2 cells. Gel mobility shift assays (GMSAs), western blots and RT-PCR were performed by standard methods. Inhibitors of various proteins involved in EGF signaling were used to identify signal transduction pathways. Results: GMSAs showed that the hNaPi-Ilb promoter region -739 bp to -734 bp (5'-AACTGG-Y) bound a nuclear protein, which was decreased by EGF treatment. Mutation of these bases also abolished the promoter response to EGF in transfected CaCo-2 cells. Supershift analysis with a c-myb antibody indicated that c-myb transcription factor could bind to this sequence. Western blot analyses and RT-PCR experiments confirmed that c-myb is endogenously expressed in Caco-2 cells and that EGF treatment did not alter its expression level. Further studies demonstrated that an EGFreceptor antibody and an EGF receptor tyrosine kinase inhibitor (Tyrnphnstin AG1478) abolished the response of the hNaPi-Ilb promoter to EGF. Furthermore, a protein kinase C inhibitor (H7) and a MAP kinase inhibitor (PD098059) blocked 60% and 30% of the promoter response to EGF, respectively. H7 and PD098059 together completely abolished the promoter response to EGF. Conclusion: We conclude that c-myb may play a role in the EGF-mediated downregulation of hNaPi-llb gene expression, and that PKC and MAPK signal transduction pathways are likely involved in this regulation. This is the first study that demonstrates the involvement of c-myb transcription factor in the regulation of intestinal Pi absorption. This investigation was supported by NIH grant P,37-DK33209.
$965 Stimulation of Hepatic Hepcidin Precedes Inhibition of Hepatic and Intestinal Ferroportin 1 Expression in Rats After LPS Administration Kwo*Yih Yeh, Mary Yeh, Jonathan Glass Background: Decreasing circulating iron is one of the strategies for host innate immunity against infection. Hepcidin, the recently identified gene that produces an anti-microbial peptide in repid response to INS and oxidative stresses. Hepcidin also plays a critical role in the regulation of body iron homeostasis. Hepcidin appears to mediate host iron stores and circulating iron by reducing cellular iron release from fiver iron stores and by decreasing intestinal iron absorption. The sequestration of cellular iron may require down regulation of ferroportin 1 (FPT1), the iron transporter for cellular iron efflux. Aims: To determine 1) after LPS administration time-dependent changes in hepatic hepcidin and FPT1, intestinal FPT1 expression, and serum iron concentration, and 2) the mechanism of inverse relationship between hepcidin and FPT1 expression. Methods: Rats were administered INS (ip, 1 g,g/g) and killed at defined times. In separate experiments, rats were given an iron chelator pyrrolydinedithiocarhamate (PDTC, ip, 100 ~,g/g) lh prior to LPS. The liver and, duodenal mucosa were collected for determinations of hepcidin and FPT1 mRNAs by Northern blots and FPT1 protein by Western blots. The blood was used to determine iron concentration. Results: LPS induced an initial 50% reduction of hepatic hepcidin mRNA levels at 30-60 rain, followed by a 2~3fold increase at 120 min and 6~10 fold increase at 6 h. By 12 h hepcidin mRNA levels had returned to normal. In contrast, INS induced slow changes both in hepatic and intestinal FPT1 mRNA levels, with a 40% decrease at 6-8 h and a 60% decrease at 12 h followed by recovery to normal at 24 h after LPS. Changes in hepatic and intestinal FPT1 mRNA were accompanied by reduced FPT1 protein expression. LPS also induced a gradual decrease of serum iron concentration during the first 6 h; serum iron levels remained low at 24 h. PDTC did not abolish the LPS-induced increase of hepatic hepcidin expression and decrease of serum iron levels, but unexpectedly stimulated FPT1 expression in INS-treated rats. Conclusions: The dynamic changes in hepatic hepcidin, hepatic and intestinal FPT1 expression, and serum iron concentrations after LPS suggest that there is a casual-relationship between hepcidin and FPT1 expression and the reduction of serum iron concentration. The inverse relationship between bepcidin and FPT 1 expression is uncoupled by PDTC indicating differential gene responses to iron and LPS by complex signaling network.
$963 Polyamines Modulate Transcription of the Gene Encoding 4E Binding Protein 1 Alan H. Stephenson, Joseph F. Christian, Edward R. Seidel Cap-dependent translation is controlled by regulating the availability of free elF-4E, the concentration limiting component of the eIF-4F translation initiation complex. This complex is responsible for translation of 80% of all eukaryotic mRNA transcripts, elF-4E is in turn controlled by association with the translational repressor protein 4E binding protein 1 (4EBPl)which binds elF-4E and thereby inhibits cap-dependent translation. Depletion of IEC6 cells of polyammes by 48-hr treatment with difluoromethylomithine (DFMO)produced a 25-fold decrease in the steady state concentration of 4E-BP1 mRNA transcript as measured by tibonuctease protection assay and a 20-fold decrease in 4E-BP1 protein as measured by immnnoblot. The effects were reversed by addition of 10 uM pntreseine. The haff-life (tl/ 2) of the 4E-BP1 mRNA was determined by inhibition of transcription with actinomycin D and following decay of the 4E-BP1 mRNA by slot blot analysis. In control cells the 4E-BP1 tl/2 was 6.9 hrs and was unaffected by either DFMO or putrescine treatment, tl/2 5.9 and 6.4 hrs, respectively. These observations lead us to test the hypothesis that polyamines must control transcription of the gene encoding the 4E-BP1 message. A nuclear run-on assay was performed with nncleii isolated from polyamine-depleted cells. DFMO-induced polyamine depletion induced a 20-fold fall in the rate of 4E-BP1 gene transcription. Conversely, treatment of cells with 10 uM putrescine increased the rate of 4E-BP1 gene transcription by a factor of eight. Neither treatment affected the rate of actin gene transcription. These data suggest that by their effects on 4E-BP1 gene transcription, polyamines should modulate translation of proteins translated by the cap-dependent pathway. As predicted, depletion of intracellufar polyamines with DFMO induced 5 to 10-fold increases in the steady state level of VEGF, Cyclin D1, cMyc, p53, p21, and tubulin as measured by immunoblot. Again, these effects were reversed by treatment of cells with putrescine. Actin, which is not translated in a cap-dependent manner, was not affected by polyamines. These data demonstrate a mechanism by which polyamines may regulate the rate of translation of proteins that are controlled by formation of the elF-4F translation initiation complex.
$966 A Novel Mitoehondrial Ca2+-dependent Solute Carrier in the Liver Identified by mRNA Differential Display Hirosato Mashima, Namiki Ueda, Hideki Ohno, Junko Suzuki, Masao Omata Pancreatic AR42J cells have the feature of pluripntency of the precursor cells of the gut endoderm. Dexamethasone converts them to exocrine cells or liver cells. By using mRNA differential display technique, we have identified a novel Ca2§ member of the mitochondriat solute carrier superfamily, which is expressed during the course of differentiation and designated it MCSC. The corresponding cDNA comprises an open reading frame of 1407 base pairs encoding a polypeptide of 469 amino acids. The carboxyl-terminal half of MCSC has high similarity with other mitochondrial carriers and the amino-terminal half has three canonical elongation factor-hand motifs and has calcinm-binding capacity. The deduced amino acid sequence revealed 79.1% homology to rabbit peroxisomal Ca2§ -dependent member of the mitochondrial superfamily but the subcellular localization of the protein was exclusively mitochondrial, not peroxisomal. Northern blot and Western blot analyses revealed its predominant expression in the liver and the skeletal muscle. In the liver, the expression level of MCSC was higher in the adult stage than in the fetal stage and MCSC was highly upregulated in dexamethasone-treated AR42J ceils before they started to express albumin. Taken together, MCSC may play an important role in regulating the function of hepatocytes rather than in the differentiation in vivo.
$964 OEbp and Ap-1 Sites in Hepcidin Promoter Confer Positive/Negative Regulation of Hepcidin Expression Kwo-Yih Yeh, Mary Yeh, Hong Yin, Jonathan Glass Background: Hepcidin (Hepc) is an anti-microbial peptide and plays a role in the regulation of body iron homeostasis. Hepc is specifically expressed in the liver and its expression is upregulated by bacterial infection and iron. Aims: To determine 1) regulatory dements in the 5' flanking region of Hepc gene, 2) the effect of CCAAT enhancer-binding protein (C/ EBPc0and redox factor 1 (Refl) on the activity of putative regulatory elements, and 3) changes in transcription factor binding activity and Hepc expression in rats after LPS administration. Methods: The Hepc gene lies immediately downstream to stimulation factor 2 (USF2) gene. The intergene region containing rat Hepc promoter was PCR amplified, cloned and sequenced.
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Transcription activity of 5' deletions of promoter region was determined by luciferase reporter system. The effects of C/EBPcr and Refl on promoter activity were determined by cotransfection of reporter and C./EBPa or Refl expressing plasmids. Hepatic C/EBPcr and AP1 binding activities in INS-treated rats were determined by EMSA. Hepc mRNA was determined by Northern blots. Results: The region between USF2 and Hepe consists of 1857 bp with a TATA box at -34/-24 (transcription start site = 1). A series of 5' deletions identified ~15-25-fold higher promoter activity in Hepc -113/+ 50 (-113) and -229 than in -603, 705 and -1715 regions in HepG2 cells. In Cos1 cells the promoter activity of -113 was 50% lower than in HepG2. Sequence analysis of the -603 identified numerous putative C../EBPet and AP-1 binding elements. The activity of these elements was tested in cells co-transfected with Hepc -113 or -603 pGL3 and pcDNA-C/EBPcx or pcDNA-Refl. Over expression of C/ EBPa had no effect on -113 in HepG2 and Cos1, but doubled the activity of -603 in HepG2 and reduced activity 70% in Cos1. Refl had no effect on -113 in HepG2, reduced -113 -30% in Cos1, and sigmficantly suppressed -603 in both cell types. In rats INS increased both C/EBPa and AP-1 binding activities at 1 and 2 h and Hepc mRNA expression by 3fold at 2 h, to a peak at 8 h; by 12 h Hepc mRNA returned to normal. Conclusions: The Hepc -113 promoter region contains regulatory elements conferring high levels of cell specific Hepc expression whereas the distal regions 5' of -229 contain elements that suppress Hepc gene transcription. C/EBPa stimulates the region between -229 and -603 in a cell specific manner while AP-1 presumably activated by Ref-1 suppresses Hepc expression. Thus, both C/EBPa and AP-1 are involved in up- and down regulation of Hepc gene expression.
$962 EGF Regulation on the Human Intestinal NaPi-llb Gene Expression Involves e-
Myb Hua Xu, Michael lnouye, Eric Hines, James F. Collins, Fayez K. Ghishan Introduction: The human intestinal sodium-phosphate (hNaPi-llb) cotransporter plays a major role in intestinal Pi absorption. Our previous studies showed that EGF inhibits intestinal NaPi-IIb cotransporter gene transcription and that the EGF response region was between -729 bp and -784 bp of the promoter. Thus, the current study was performed to precisely identify the bases involved in this regulation, and to identify nuclear proteins that bind to this site and participate in EGF regulation of hNaPi-llb gene expression. Additional studies were performed to identify signaling pathways involved in EGF regulation of the hNaPi-llb gene. Methods: Reporter gene assays were performed in transiently transfected CaCo-2 cells. Gel mobility shift assays (GMSAs), western blots and RT-PCR were performed by standard methods. Inhibitors of various proteins involved in EGF signaling were used to identify signal transduction pathways. Results: GMSAs showed that the hNaPi-Ilb promoter region -739 bp to -734 bp (5'-AACTGG-Y) bound a nuclear protein, which was decreased by EGF treatment. Mutation of these bases also abolished the promoter response to EGF in transfected CaCo-2 cells. Supershift analysis with a c-myb antibody indicated that c-myb transcription factor could bind to this sequence. Western blot analyses and RT-PCR experiments confirmed that c-myb is endogenously expressed in Caco-2 cells and that EGF treatment did not alter its expression level. Further studies demonstrated that an EGFreceptor antibody and an EGF receptor tyrosine kinase inhibitor (Tyrnphnstin AG1478) abolished the response of the hNaPi-Ilb promoter to EGF. Furthermore, a protein kinase C inhibitor (H7) and a MAP kinase inhibitor (PD098059) blocked 60% and 30% of the promoter response to EGF, respectively. H7 and PD098059 together completely abolished the promoter response to EGF. Conclusion: We conclude that c-myb may play a role in the EGF-mediated downregulation of hNaPi-llb gene expression, and that PKC and MAPK signal transduction pathways are likely involved in this regulation. This is the first study that demonstrates the involvement of c-myb transcription factor in the regulation of intestinal Pi absorption. This investigation was supported by NIH grant P,37-DK33209.
$965 Stimulation of Hepatic Hepcidin Precedes Inhibition of Hepatic and Intestinal Ferroportin 1 Expression in Rats After LPS Administration Kwo*Yih Yeh, Mary Yeh, Jonathan Glass Background: Decreasing circulating iron is one of the strategies for host innate immunity against infection. Hepcidin, the recently identified gene that produces an anti-microbial peptide in repid response to INS and oxidative stresses. Hepcidin also plays a critical role in the regulation of body iron homeostasis. Hepcidin appears to mediate host iron stores and circulating iron by reducing cellular iron release from fiver iron stores and by decreasing intestinal iron absorption. The sequestration of cellular iron may require down regulation of ferroportin 1 (FPT1), the iron transporter for cellular iron efflux. Aims: To determine 1) after LPS administration time-dependent changes in hepatic hepcidin and FPT1, intestinal FPT1 expression, and serum iron concentration, and 2) the mechanism of inverse relationship between hepcidin and FPT1 expression. Methods: Rats were administered INS (ip, 1 g,g/g) and killed at defined times. In separate experiments, rats were given an iron chelator pyrrolydinedithiocarhamate (PDTC, ip, 100 ~,g/g) lh prior to LPS. The liver and, duodenal mucosa were collected for determinations of hepcidin and FPT1 mRNAs by Northern blots and FPT1 protein by Western blots. The blood was used to determine iron concentration. Results: LPS induced an initial 50% reduction of hepatic hepcidin mRNA levels at 30-60 rain, followed by a 2~3fold increase at 120 min and 6~10 fold increase at 6 h. By 12 h hepcidin mRNA levels had returned to normal. In contrast, INS induced slow changes both in hepatic and intestinal FPT1 mRNA levels, with a 40% decrease at 6-8 h and a 60% decrease at 12 h followed by recovery to normal at 24 h after LPS. Changes in hepatic and intestinal FPT1 mRNA were accompanied by reduced FPT1 protein expression. LPS also induced a gradual decrease of serum iron concentration during the first 6 h; serum iron levels remained low at 24 h. PDTC did not abolish the LPS-induced increase of hepatic hepcidin expression and decrease of serum iron levels, but unexpectedly stimulated FPT1 expression in INS-treated rats. Conclusions: The dynamic changes in hepatic hepcidin, hepatic and intestinal FPT1 expression, and serum iron concentrations after LPS suggest that there is a casual-relationship between hepcidin and FPT1 expression and the reduction of serum iron concentration. The inverse relationship between bepcidin and FPT 1 expression is uncoupled by PDTC indicating differential gene responses to iron and LPS by complex signaling network.
$963 Polyamines Modulate Transcription of the Gene Encoding 4E Binding Protein 1 Alan H. Stephenson, Joseph F. Christian, Edward R. Seidel Cap-dependent translation is controlled by regulating the availability of free elF-4E, the concentration limiting component of the eIF-4F translation initiation complex. This complex is responsible for translation of 80% of all eukaryotic mRNA transcripts, elF-4E is in turn controlled by association with the translational repressor protein 4E binding protein 1 (4EBPl)which binds elF-4E and thereby inhibits cap-dependent translation. Depletion of IEC6 cells of polyammes by 48-hr treatment with difluoromethylomithine (DFMO)produced a 25-fold decrease in the steady state concentration of 4E-BP1 mRNA transcript as measured by tibonuctease protection assay and a 20-fold decrease in 4E-BP1 protein as measured by immnnoblot. The effects were reversed by addition of 10 uM pntreseine. The haff-life (tl/ 2) of the 4E-BP1 mRNA was determined by inhibition of transcription with actinomycin D and following decay of the 4E-BP1 mRNA by slot blot analysis. In control cells the 4E-BP1 tl/2 was 6.9 hrs and was unaffected by either DFMO or putrescine treatment, tl/2 5.9 and 6.4 hrs, respectively. These observations lead us to test the hypothesis that polyamines must control transcription of the gene encoding the 4E-BP1 message. A nuclear run-on assay was performed with nncleii isolated from polyamine-depleted cells. DFMO-induced polyamine depletion induced a 20-fold fall in the rate of 4E-BP1 gene transcription. Conversely, treatment of cells with 10 uM putrescine increased the rate of 4E-BP1 gene transcription by a factor of eight. Neither treatment affected the rate of actin gene transcription. These data suggest that by their effects on 4E-BP1 gene transcription, polyamines should modulate translation of proteins translated by the cap-dependent pathway. As predicted, depletion of intracellufar polyamines with DFMO induced 5 to 10-fold increases in the steady state level of VEGF, Cyclin D1, cMyc, p53, p21, and tubulin as measured by immunoblot. Again, these effects were reversed by treatment of cells with putrescine. Actin, which is not translated in a cap-dependent manner, was not affected by polyamines. These data demonstrate a mechanism by which polyamines may regulate the rate of translation of proteins that are controlled by formation of the elF-4F translation initiation complex.
$966 A Novel Mitoehondrial Ca2+-dependent Solute Carrier in the Liver Identified by mRNA Differential Display Hirosato Mashima, Namiki Ueda, Hideki Ohno, Junko Suzuki, Masao Omata Pancreatic AR42J cells have the feature of pluripntency of the precursor cells of the gut endoderm. Dexamethasone converts them to exocrine cells or liver cells. By using mRNA differential display technique, we have identified a novel Ca2§ member of the mitochondriat solute carrier superfamily, which is expressed during the course of differentiation and designated it MCSC. The corresponding cDNA comprises an open reading frame of 1407 base pairs encoding a polypeptide of 469 amino acids. The carboxyl-terminal half of MCSC has high similarity with other mitochondrial carriers and the amino-terminal half has three canonical elongation factor-hand motifs and has calcinm-binding capacity. The deduced amino acid sequence revealed 79.1% homology to rabbit peroxisomal Ca2§ -dependent member of the mitochondrial superfamily but the subcellular localization of the protein was exclusively mitochondrial, not peroxisomal. Northern blot and Western blot analyses revealed its predominant expression in the liver and the skeletal muscle. In the liver, the expression level of MCSC was higher in the adult stage than in the fetal stage and MCSC was highly upregulated in dexamethasone-treated AR42J ceils before they started to express albumin. Taken together, MCSC may play an important role in regulating the function of hepatocytes rather than in the differentiation in vivo.
$964 OEbp and Ap-1 Sites in Hepcidin Promoter Confer Positive/Negative Regulation of Hepcidin Expression Kwo-Yih Yeh, Mary Yeh, Hong Yin, Jonathan Glass Background: Hepcidin (Hepc) is an anti-microbial peptide and plays a role in the regulation of body iron homeostasis. Hepc is specifically expressed in the liver and its expression is upregulated by bacterial infection and iron. Aims: To determine 1) regulatory dements in the 5' flanking region of Hepc gene, 2) the effect of CCAAT enhancer-binding protein (C/ EBPc0and redox factor 1 (Refl) on the activity of putative regulatory elements, and 3) changes in transcription factor binding activity and Hepc expression in rats after LPS administration. Methods: The Hepc gene lies immediately downstream to stimulation factor 2 (USF2) gene. The intergene region containing rat Hepc promoter was PCR amplified, cloned and sequenced.
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Abstracts