W1761 PKD Regulates the Synergistic Expression of COX-2 By Bradykinin and TNF-α in Colonic...
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AGA Abstracts use of In Vitro models employing monolayers of intestinal epithelial cells. However, the molecular mechanisms that mediate migration of intestinal epithelial cells in response to wounding and external stimuli remain incompletely understood. Recent studies in fibroblasts indicated that PKD, the founding member of a novel family of serine/threonine protein kinases, regulates directional migration but the role of PKD in the migration of intestinal epithelial cells remains unknown. Aim: To test the hypothesis that catalytic activation of PKD is one of the earliest signals in response to wound-induced migration in intestinal epithelial cells. Methods: IEC18 and IEC6 cells, growing in monolayer culture, were wounded by applying a razor blade to the dish and scraping perpendicularly to the plane of the blade. Alternatively wounds were made by aspiration. After periods of times ranging from minutes to several hours, the cells were fixed in 4% paraformaldehyde in PBS, fixation was followed by permeablization with 0.4% Triton-X. Primary antibodies against phosphorylated PKD on Ser-916, a major autophosphorylation site, were applied overnight, followed by exposure to secondary goat anti-rabbit antibodies conjugated to Qdot 655 (Invitrogen). In other experiments, PKD catalytic activity was measured by In Vitro kinase assays after immunopreci- pitation. Results: In IEC18 and IEC6 monolayers, wounding produced a rapid (within 2- 3 min) increase in PKD phosphorylated on Ser-916 in cells located at the edge of the wound rather than in the whole cell monolayer. We also determined PKD catalytic activation in response to wounding using In Vitro kinase assays. In order to maximize the number of cells at or near a wound, cell monolayers were injured by multiple parallel scrapes. PKD activity in lysates of IEC-18 cells harvested at different times after epithelial injury was assessed by In Vitro kinase assays after immunoprecipitation. A marked increase in PKD activity in wounded IEC-18 monolayers was evident within 5 min after injury. Conclusion: Taken together, the results of both approaches show that PKD activation is one of the early signaling events initiated by wounding monolayers of intestinal epithelial cells. W1760 Rapid FAK Phosphorylation On Ser-910 and Ser-843 in Intestinal Epithelial Cells Xiaohua Jiang, James Sinnett-Smith, Enrique Rozengurt Bacground: A rapid increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase FAK is a prominent early event in fibroblasts stimulated by a variety of signaling molecules. However, a variety of epithelial cells, including intestinal epithelial cells, show a high basal level of tyrosine phosphorylated FAK that is only slightly further increased by addition of G protein-coupled receptors (GPCR) agonists or growth factors. Aim: To deter- mine whether GPCRs could elicit FAK phosphorylation at serine residues, including Ser- 910 and Ser-843. Methods: Cultures of IEC-18 or T84 cells were washed twice with serum- free DMEM or DMEM/Ham's F12, equilibrated in the same medium at 37°C for 3 h, and then treated with ANG II, LPA, carbachol, or other factors as described in the individual experiments. The stimulation was terminated by aspirating the medium and solubilaizing the cells in 200ml of 2x SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. The samples were then analyzed by SDS-PAGE and western blotting using one of the following phospho specific antibodies: anti-FAK-Ser(P)-843 Ab (0.1 mg/ml), anti-FAK-Ser(P)-910 Ab (0.1 mg/ml), anti-phospho-p44/42 MAP kinase(0.1 mg/ml) or anti-ERK2 (0.1 mg/ml) as indicated. Results: Our results show that multiple agonists including angiotensin II (ANGII), lysophosphatidic acid (LPA), phorbol esters and EGF induced a striking stimulation of FAK phosphorylation at Ser-910 in rat intestinal epithelial IEC-18 cells via an ERK-dependent pathway. In striking contrast, none of these stimuli promoted a significant further increase in FAK phosphorylation at Tyr-397 in these cells. These results were extended using cultures of polarized human colonic epithelial T84 cells. We found that either carbachol or EGF promoted a striking ERK-dependent phosphorylation of FAK at Ser-910, but these agonists caused only slight stimulation of FAK at Tyr-397 in T84 cells. In addition, we demonstrated that GPCR agonists also induced a dramatic increase of FAK phosphorylation at Ser-843 in either IEC-18 or T84 cells. Our results indicate that Ser-910 and Ser-843, rather than Tyr- 397, are prominent sites differentially phosphorylated in response to neurotransmitters, bioactive lipids, tumor promoters and growth factors in intestinal epithelial cells. W1761 PKD Regulates the Synergistic Expression of COX-2 By Bradykinin and TNF-α in Colonic Myofibroblasts James Yoo, Christine Chung, Lee W. Slice, James Sinnett-Smith, Enrique Rozengurt Introduction: Colonic myofibroblasts are an important source of COX-2-derived prosta- glandins that contribute to key biological processes, including mucosal repair and epithelial cell proliferation. Increased numbers of myofibroblasts, in parallel with elevated levels of COX-2, have also been identified in areas of intestinal inflammation and neoplasia. However, the cell signaling pathways regulating COX-2 expression in myofibroblasts have not been completely characterized. Protein kinase D (PKD), the founding member of a new family of protein kinases that includes PKD2 and PKD3, has been linked to tumor-promoting processes, but its role in myofibroblast cell signaling and COX-2 expression has not been explored. Methods: Human diploid 18Co cells were grown to confluence on 35x10mm cell culture dishes and used from passages 8-14. 18Co cells were stimulated with bradykinin (BK, 100nM) and tumor necrosis factor-α (TNF, 8.33ng/ml). COX-2 expression was assessed by Western Blot analysis. Small interfering RNA (siRNA) specific to PKD was used for transfection experiments. Results: There is no detectable COX-2 protein in unstimulated 18Co cells as assessed by Western blot analysis. The G-protein-coupled-receptor agonist and inflammatory mediator BK stimulated PKD activation after 30 minutes and led to expression of COX-2 after 2 hours of treatment. TNF also induced COX-2 expression after 2 hours, though this effect was less pronounced. However, simultaneous treatment with BK and TNF synergistically augmented COX-2 expression beginning at 3 hours. This effect was dose-dependent, sustained over 24 hrs, and not affected by pre-treatment with indomethacin. This synergistic increase in COX-2 expression was completely inhibited by the p38 MAPK inhibitor SB202190 (10mM), by the PKC inhibitor Ro318220 (2.5mM), and by Go6976 (10mM), an inhibitor of conventional PKC's and PKD. Knockdown of PKD using siRNA strikingly inhibited synergistic COX-2 expression induced by BK and TNF. Conclusions: BK and TNF synergistically enhance COX-2 expression in 18Co cells, an effect that is A-710 AGA Abstracts mediated via p38 MAPK- and PKC/PKD-dependent pathways. Unique cross talk signaling between inflammatory mediators may activate a novel PKD phosphorylation cascade that amplifies COX-2 expression in colonic myofibroblasts. PKD may regulate COX-2 expression in colonic myofibroblasts to promote an inflammatory microenvironment that supports tumor growth. W1762 Basal Prostaglandin Levels Are Required for Pyk2 Expression and Ang II- Dependent MAPK Phosphorylation in Intestinal Epithelial Cells H. Pham, Steven S. Wu, Romina Vincenti, Enrique Rozengurt, Lee W. Slice Objective. Angiotensin (Ang) II rapidly induces proline-rich protein tyrosine kinase 2 (PYK2) phosphorylation on Tyr 402 along with increased prostaglandin (PG) production in epithelial cells suggesting possible feedback regulation of Ang II signaling by PGs. We investigated the role of PGs in PYK2 signaling in cells over-expressing 15-hydroxyprostaglandin dehydro- genase (PGDH), an enzyme that inactivates PGs. Methods. Intestinal epithelial cells (IEC- 18) were transduced using lentivirus to express green fluorescent protein (18.GFP) or PGDH (18.PGDH). Phospho-specific antibodies were used in western blots. PGs were measured using immunoassays. Results. Ang II induced PYK2 Tyr 402 phosphorylation in IEC-18 and 18.GFP cells. Both cell lines produced low basal levels of PGs that were increased by stimulation with Ang II or EGF. These agonists induced ERK phophorylation, which was blocked by the selective EGFR kinase inhibitor, AG1478. PG levels were undetectable in 18.PGDH cells and did not increase with agonist stimulation. PYK2 expression was absent in 18.PGDH cells. Ang II did not induce ERK phosphorylation. Conversely, EGF induced ERK phosphorylation. Ang II-dependent ERK phosphorylation was rescued by expression of wt PYK2 but not by expression of an inactive PYK2 mutant. Reduction of PYK2 expression by siRNA in IEC-18 cells correlated with a decrease in Ang II-dependent ERK phosphoryl- ation. Conclusions. Basal PG levels are required to maintain PYK2 expression in IEC-18 cells. In turn, PYK2 plays a critical role in mediating ERK signaling by Ang II. Supported by RO1 DK061485 W1763 New Critical Residues in the Predicted Intracellular Loop Domains of the Human Motilin Receptor Bunzo Matsuura, Teruhisa Ueda, Maoqing Dong, Laurence J. Miller, Teruki Miyake, Shinya Furukawa, Morikazu Onji The motilin receptor belongs to a recently recognized family of Class I G protein-coupled receptors that also includes growth hormone secretagogue receptors. Their potentially unique structure and the molecular basis of their binding and activation are not yet clear. We previously reported that the perimembranous residues in the predicted extracellular loops and amino-terminal tail of the motilin receptor, Gly36, Pro103, Leu109, Val179, Leu245, Arg246, and Phe332, were functionally important for responses to the natural peptide agonist, motilin, while these residues were not critical for responses to a non-peptidyl agonist, erythromycin (J Biol Chem 277:9834, 2002, and J Biol Chem 281: 12390, 2006). In the current work, we have focused on the predicted intracellular loop domains of the motilin receptor, and studied functional responses to motilin and erythromycin. For this purpose, we prepared motilin receptor constructs that included sequential deletions ranging from two to six amino acid residues throughout the predicted first, second and third intracellular loops. Each construct was expressed in COS cells and characterized for motilin binding and motilin- and erythromycin-stimulated intracellular calcium responses. Alanine or phenylalan- ine or histidine replacements for each of the potentially important amino acid residues in the relevant segments revealed that residues Tyr66, Arg136, and Val299 were responsible for negative impact on motilin and erythromycin biological activity. These data support important roles of new regions in the intracellular loop domains of the motilin receptor for motilin and erythromycin action. W1764 Relevance of Functional Promoter Variant for the Regulation of the Extrahepatic UDP-Glucuronosyltransferase Ugt1a7 By the Hepatocyte Nuclear Factors Hnf1alpha and Hnf4 Alpha Ursula Ehmer, Thomas J. Erichsen, Tim O. Lankisch, Sandra Kalthoff, Nicole Freiberg, Michael P. Manns, Christian P. Strassburg Introduction: Glucuronidation through UDP-glucuronosyltransferases (UGT) is a major path- way for metabolism of various exogenous and endogenous substrates including numerous drugs and carcinogens. Hepatocyte nuclear factors (HNF) are involved in the regulation of hepatic (UGT1A1, UGT1A3, UGT1A4, UGT1A9) and extrahepatic UGTs (UGT1A8, UGT1A10). Differential transcriptional regulation represents an interesting feature underlying the tissue-specific expression and metabolism of the UGT1A gene locus. UGT1A7 is exclus- ively expressed extrahepaticly and low activity alleles of UGT1A7 characterized by variations within coding and non-coding sequence are associated with carcinoma predisposition and drug toxicity. The transcriptional regulation of this gene and influences of single nucleotide polymorphisms are incompletely characterized. Aim of this study was to identify transcription factors relevant for UGT1A7 induction and to elucidate the impact of the functional UGT1A7 promoter polymorphism -57T>G on inducibility. Methods: Luciferase reporter gene con- structs of the UGT1A7 5'non-coding promoter region were analyzed in CaCo2 and HepG2 cells. Transcription factor binding motifs were identified by site-directed mutagenesis and electrophoretic mobility shift assay (EMSA). Results: Reporter gene assays showed high (up to 75-fold) UGT1A7 inducibility by hepatocyte nuclear factor 1alpha (HNF1alpha), and 5- fold inducibility by hepatocyte nuclear factor (HNF4alpha). The -57T>G promoter poly- morphism significantly reduced HNF4alpha induction but not HNF1alpha induction. Two DNA binding motifs for each HNF were identified, and mutagenesis of the respective sites led to a near to complete loss of inducibility. Binding was confirmed by EMSA. Discussion: Hepatocyte nuclear factors play an important role as regulatory transcription factors not
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suse of In Vitro models employing monolayers of intestinal epithelial cells. However, themolecular mechanisms that mediate migration of intestinal epithelial cells in response towounding and external stimuli remain incompletely understood. Recent studies in fibroblastsindicated that PKD, the founding member of a novel family of serine/threonine proteinkinases, regulates directional migration but the role of PKD in the migration of intestinalepithelial cells remains unknown. Aim: To test the hypothesis that catalytic activation ofPKD is one of the earliest signals in response to wound-induced migration in intestinalepithelial cells. Methods: IEC18 and IEC6 cells, growing in monolayer culture, were woundedby applying a razor blade to the dish and scraping perpendicularly to the plane of the blade.Alternatively wounds were made by aspiration. After periods of times ranging from minutesto several hours, the cells were fixed in 4% paraformaldehyde in PBS, fixation was followedby permeablization with 0.4% Triton-X. Primary antibodies against phosphorylated PKD onSer-916, a major autophosphorylation site, were applied overnight, followed by exposureto secondary goat anti-rabbit antibodies conjugated to Qdot 655 (Invitrogen). In otherexperiments, PKD catalytic activity was measured by In Vitro kinase assays after immunopreci-pitation. Results: In IEC18 and IEC6 monolayers, wounding produced a rapid (within 2-3 min) increase in PKD phosphorylated on Ser-916 in cells located at the edge of the woundrather than in the whole cell monolayer. We also determined PKD catalytic activation inresponse to wounding using In Vitro kinase assays. In order to maximize the number ofcells at or near a wound, cell monolayers were injured by multiple parallel scrapes. PKDactivity in lysates of IEC-18 cells harvested at different times after epithelial injury wasassessed by In Vitro kinase assays after immunoprecipitation. A marked increase in PKDactivity in wounded IEC-18 monolayers was evident within 5 min after injury. Conclusion:Taken together, the results of both approaches show that PKD activation is one of the earlysignaling events initiated by wounding monolayers of intestinal epithelial cells.
W1760
Rapid FAK Phosphorylation On Ser-910 and Ser-843 in Intestinal EpithelialCellsXiaohua Jiang, James Sinnett-Smith, Enrique Rozengurt
Bacground: A rapid increase in the tyrosine phosphorylation of the non-receptor tyrosinekinase FAK is a prominent early event in fibroblasts stimulated by a variety of signalingmolecules. However, a variety of epithelial cells, including intestinal epithelial cells, showa high basal level of tyrosine phosphorylated FAK that is only slightly further increased byaddition of G protein-coupled receptors (GPCR) agonists or growth factors. Aim: To deter-mine whether GPCRs could elicit FAK phosphorylation at serine residues, including Ser-910 and Ser-843. Methods: Cultures of IEC-18 or T84 cells were washed twice with serum-free DMEM or DMEM/Ham's F12, equilibrated in the same medium at 37°C for 3 h, andthen treated with ANG II, LPA, carbachol, or other factors as described in the individualexperiments. The stimulation was terminated by aspirating the medium and solubilaizingthe cells in 200ml of 2x SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. Thesamples were then analyzed by SDS-PAGE and western blotting using one of the followingphospho specific antibodies: anti-FAK-Ser(P)-843 Ab (0.1 mg/ml), anti-FAK-Ser(P)-910 Ab(0.1 mg/ml), anti-phospho-p44/42 MAP kinase(0.1 mg/ml) or anti-ERK2 (0.1 mg/ml) asindicated. Results: Our results show that multiple agonists including angiotensin II (ANGII),lysophosphatidic acid (LPA), phorbol esters and EGF induced a striking stimulation of FAKphosphorylation at Ser-910 in rat intestinal epithelial IEC-18 cells via an ERK-dependentpathway. In striking contrast, none of these stimuli promoted a significant further increasein FAK phosphorylation at Tyr-397 in these cells. These results were extended using culturesof polarized human colonic epithelial T84 cells. We found that either carbachol or EGFpromoted a striking ERK-dependent phosphorylation of FAK at Ser-910, but these agonistscaused only slight stimulation of FAK at Tyr-397 in T84 cells. In addition, we demonstratedthat GPCR agonists also induced a dramatic increase of FAK phosphorylation at Ser-843 ineither IEC-18 or T84 cells. Our results indicate that Ser-910 and Ser-843, rather than Tyr-397, are prominent sites differentially phosphorylated in response to neurotransmitters,bioactive lipids, tumor promoters and growth factors in intestinal epithelial cells.
W1761
PKD Regulates the Synergistic Expression of COX-2 By Bradykinin and TNF-αin Colonic MyofibroblastsJames Yoo, Christine Chung, Lee W. Slice, James Sinnett-Smith, Enrique Rozengurt
Introduction: Colonic myofibroblasts are an important source of COX-2-derived prosta-glandins that contribute to key biological processes, including mucosal repair and epithelialcell proliferation. Increased numbers of myofibroblasts, in parallel with elevated levels ofCOX-2, have also been identified in areas of intestinal inflammation and neoplasia. However,the cell signaling pathways regulating COX-2 expression in myofibroblasts have not beencompletely characterized. Protein kinase D (PKD), the founding member of a new familyof protein kinases that includes PKD2 and PKD3, has been linked to tumor-promotingprocesses, but its role in myofibroblast cell signaling and COX-2 expression has not beenexplored. Methods: Human diploid 18Co cells were grown to confluence on 35x10mm cellculture dishes and used from passages 8-14. 18Co cells were stimulated with bradykinin(BK, 100nM) and tumor necrosis factor-α (TNF, 8.33ng/ml). COX-2 expression was assessedby Western Blot analysis. Small interfering RNA (siRNA) specific to PKD was used fortransfection experiments. Results: There is no detectable COX-2 protein in unstimulated18Co cells as assessed by Western blot analysis. The G-protein-coupled-receptor agonistand inflammatory mediator BK stimulated PKD activation after 30 minutes and led toexpression of COX-2 after 2 hours of treatment. TNF also induced COX-2 expression after2 hours, though this effect was less pronounced. However, simultaneous treatment with BKand TNF synergistically augmented COX-2 expression beginning at 3 hours. This effect wasdose-dependent, sustained over 24 hrs, and not affected by pre-treatment with indomethacin.This synergistic increase in COX-2 expression was completely inhibited by the p38 MAPKinhibitor SB202190 (10mM), by the PKC inhibitor Ro318220 (2.5mM), and by Go6976(10mM), an inhibitor of conventional PKC's and PKD. Knockdown of PKD using siRNAstrikingly inhibited synergistic COX-2 expression induced by BK and TNF. Conclusions:BK and TNF synergistically enhance COX-2 expression in 18Co cells, an effect that is
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mediated via p38 MAPK- and PKC/PKD-dependent pathways. Unique cross talk signalingbetween inflammatory mediators may activate a novel PKD phosphorylation cascade thatamplifies COX-2 expression in colonic myofibroblasts. PKD may regulate COX-2 expressionin colonic myofibroblasts to promote an inflammatory microenvironment that supportstumor growth.
W1762
Basal Prostaglandin Levels Are Required for Pyk2 Expression and Ang II-Dependent MAPK Phosphorylation in Intestinal Epithelial CellsH. Pham, Steven S. Wu, Romina Vincenti, Enrique Rozengurt, Lee W. Slice
Objective. Angiotensin (Ang) II rapidly induces proline-rich protein tyrosine kinase 2 (PYK2)phosphorylation on Tyr402 along with increased prostaglandin (PG) production in epithelialcells suggesting possible feedback regulation of Ang II signaling by PGs. We investigatedthe role of PGs in PYK2 signaling in cells over-expressing 15-hydroxyprostaglandin dehydro-genase (PGDH), an enzyme that inactivates PGs. Methods. Intestinal epithelial cells (IEC-18) were transduced using lentivirus to express green fluorescent protein (18.GFP) or PGDH(18.PGDH). Phospho-specific antibodies were used in western blots. PGs were measuredusing immunoassays. Results. Ang II induced PYK2 Tyr402 phosphorylation in IEC-18 and18.GFP cells. Both cell lines produced low basal levels of PGs that were increased bystimulation with Ang II or EGF. These agonists induced ERK phophorylation, which wasblocked by the selective EGFR kinase inhibitor, AG1478. PG levels were undetectable in18.PGDH cells and did not increase with agonist stimulation. PYK2 expression was absentin 18.PGDH cells. Ang II did not induce ERK phosphorylation. Conversely, EGF inducedERK phosphorylation. Ang II-dependent ERK phosphorylation was rescued by expressionof wt PYK2 but not by expression of an inactive PYK2 mutant. Reduction of PYK2 expressionby siRNA in IEC-18 cells correlated with a decrease in Ang II-dependent ERK phosphoryl-ation. Conclusions. Basal PG levels are required to maintain PYK2 expression in IEC-18cells. In turn, PYK2 plays a critical role in mediating ERK signaling by Ang II. Supportedby RO1 DK061485
W1763
New Critical Residues in the Predicted Intracellular Loop Domains of theHuman Motilin ReceptorBunzo Matsuura, Teruhisa Ueda, Maoqing Dong, Laurence J. Miller, Teruki Miyake,Shinya Furukawa, Morikazu Onji
The motilin receptor belongs to a recently recognized family of Class I G protein-coupledreceptors that also includes growth hormone secretagogue receptors. Their potentially uniquestructure and the molecular basis of their binding and activation are not yet clear. Wepreviously reported that the perimembranous residues in the predicted extracellular loopsand amino-terminal tail of the motilin receptor, Gly36, Pro103, Leu109, Val179, Leu245,Arg246, and Phe332, were functionally important for responses to the natural peptideagonist, motilin, while these residues were not critical for responses to a non-peptidyl agonist,erythromycin (J Biol Chem 277:9834, 2002, and J Biol Chem 281: 12390, 2006). In thecurrent work, we have focused on the predicted intracellular loop domains of the motilinreceptor, and studied functional responses to motilin and erythromycin. For this purpose,we prepared motilin receptor constructs that included sequential deletions ranging fromtwo to six amino acid residues throughout the predicted first, second and third intracellularloops. Each construct was expressed in COS cells and characterized for motilin binding andmotilin- and erythromycin-stimulated intracellular calcium responses. Alanine or phenylalan-ine or histidine replacements for each of the potentially important amino acid residues inthe relevant segments revealed that residues Tyr66, Arg136, and Val299 were responsiblefor negative impact on motilin and erythromycin biological activity. These data supportimportant roles of new regions in the intracellular loop domains of the motilin receptor formotilin and erythromycin action.
W1764
Relevance of Functional Promoter Variant for the Regulation of theExtrahepatic UDP-Glucuronosyltransferase Ugt1a7 By the Hepatocyte NuclearFactors Hnf1alpha and Hnf4 AlphaUrsula Ehmer, Thomas J. Erichsen, Tim O. Lankisch, Sandra Kalthoff, Nicole Freiberg,Michael P. Manns, Christian P. Strassburg
Introduction: Glucuronidation through UDP-glucuronosyltransferases (UGT) is a major path-way for metabolism of various exogenous and endogenous substrates including numerousdrugs and carcinogens. Hepatocyte nuclear factors (HNF) are involved in the regulationof hepatic (UGT1A1, UGT1A3, UGT1A4, UGT1A9) and extrahepatic UGTs (UGT1A8,UGT1A10). Differential transcriptional regulation represents an interesting feature underlyingthe tissue-specific expression and metabolism of the UGT1A gene locus. UGT1A7 is exclus-ively expressed extrahepaticly and low activity alleles of UGT1A7 characterized by variationswithin coding and non-coding sequence are associated with carcinoma predisposition anddrug toxicity. The transcriptional regulation of this gene and influences of single nucleotidepolymorphisms are incompletely characterized. Aim of this study was to identify transcriptionfactors relevant for UGT1A7 induction and to elucidate the impact of the functional UGT1A7promoter polymorphism -57T>G on inducibility. Methods: Luciferase reporter gene con-structs of the UGT1A7 5'non-coding promoter region were analyzed in CaCo2 and HepG2cells. Transcription factor binding motifs were identified by site-directed mutagenesis andelectrophoretic mobility shift assay (EMSA). Results: Reporter gene assays showed high (upto 75-fold) UGT1A7 inducibility by hepatocyte nuclear factor 1alpha (HNF1alpha), and 5-fold inducibility by hepatocyte nuclear factor (HNF4alpha). The -57T>G promoter poly-morphism significantly reduced HNF4alpha induction but not HNF1alpha induction. TwoDNA binding motifs for each HNF were identified, and mutagenesis of the respective sitesled to a near to complete loss of inducibility. Binding was confirmed by EMSA. Discussion:Hepatocyte nuclear factors play an important role as regulatory transcription factors not
