S1705 Corticotropin-Releasing Factor (CRF) and Urocortin 1 (UCN1) Differentially Regulate...
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levels returned to control levels in ambient air. Intestinal NF-κB protein levels were elevated significantly. Lung apelin mRNA levels were unaffected. Exp 2: In Vitro hyperoxia reduced ileal cell apelin mRNA levels by 50% (P < 0.05). Exp 3: Overexpression of NF-κB reduced rat apelin core promoter activity by 60-80%. Mutation of putative NF-κB binding sites impaired NF-κB-induced reduction in apelin core promoter activity. Conclusions and Discussion. Our findings demonstrate that hyperoxia decreases intestinal apelin expression in the rat. NF-κB may play a role in the hyperoxia-induced reduction in intestinal apelin expression since NF-κB is activated by hyperoxia and overexpression of NF-κB down- regulates apelin transcriptional activity. More importantly, these findings suggest that a reduced apelin expression may influence organogenesis in infants exposed to postnatal hyperoxia. It is well known that hypoxia upregulates apelin expression though HIF. This is the first demonstration of down-regulated apelin expression by hyperoxia. S1703 The Mechanism of Transfomer 2-β Gene Expression in Response to Oxidative Stress in Human Colon Epithelial Cells Yuki Kuwano, Yuzuru Satake, Kensei Nishida, Kiyoshi Masuda, Ken Kurokawa, Shigetada Teshima-kondo, Toshihito Tanahashi, Kazuhito Rokutan Background and Aims: Alternative splicing system is an important biological process for diversity of gene expression and at least 75% of human genes undergo this regulation. The serine-/arginine-rich (SR)-protein family contains the RS domain and participates in constitutive/alternative pre-mRNA splicing and post-splicing activities. Transformer 2-β (tra2β, SFRS10) is a member of the SR-like protein family, known as an alternative splicing regulator of several genes including CD44, FGFR2, and tau. Moreover, the tra2β gene itself generates five alternative spliced forms (tra2β1-tra2β5). We previously reported that exposure to arsenite transiently produced tra2β4, followed by enhanced expression of tra2β1 in gastric epithelial cells. Here, we examined oxidative stress-induced expression of SR protein family members and focused on the mechanisms of regulation of tra2β gene expression in colon epithelial cells.Results: First we tested whether arsenite changed mRNA isoform expression of the SR protein family (SFRS1-11) in a colon cancer cell line (HCT-116). Among the family members, SFRS3, 6, and 7 as well as SFRS10 responded to arsenite and transiently increased mRNA variants containing premature stop codon (PTC)-encoded exons with a peak around 2 h. We also found that a specific inhibitor for p38 MAPK or for JNKs partially blocked arsenite-induced tra2β4 isoform, a PTC variant of the tra2β gene. To characterize the mechanism of expression of the tra2β gene, we subcloned 1066 bp-upstream of the tra2β 5'-flanking region and the promoter activity was measured by the dual-luciferase assay system. Serially truncated regions of the 1066 bp-upstream showed that -128 to -268 bp of the upstream of the tra2β gene possessed the basal promoter activity. From sequencing results, this region contains the putative binding sites for the transcription factors, SP-1, Ets, HSF-1, and NRF-2. To identify crucial cis element(s) and trans activator(s), we generated the sites-mutated luciferase reporter vectors for these transcription factors. The luciferase assay and chromatin immunoprecipitation showed that Ets (from -172 to -163 bp) was crucial for basal transcription, whereas three HSEs (from -250 to -202 bp) regulated oxidative stress-induced gene expression of the tra2β gene. Conclusion: Alternative splicing is regu- lated by the cooperative action of multiple splicing factors, including tra2β. Our results suggest that arsenite exposure stimulate alternative splicing of tra2β pre-mRNA possibly through activation of p38 MAP and/or JNKs. Ets and HSF-1 may participate in the regulation of basal and oxidative stress-induced transcription of the tra2β gene, respectively. S1704 The Molecular Basis Responsible for High Affinity of All Human Bombesin Receptor (BnR) Family Members for a Universal Ligand (Un.Lig.) Hirotsugu Uehara, Nieves Gonzalez, Samuel A. Mantey, Tomoo Nakagawa, Tatsuro Katsuno, Robert T. Jensen BKG: The synthetic Bn analog, [D-Tyr6, β-Ala11, Phe13, Nle14]Bn (6-14) [Un.Lig] has the unique property of functioning as a high affinity agonist at all 3 human BnRs [neuromedin B receptor (NMB-R), gastrin-releasing peptide receptor (GRPR) and orphan receptor (BRS- 3)]. However, the molecular basis for this unique property is unknown. AIM: To determine the molecular basis for the high affinity of the universal ligand (Un.Lig) for all human Bn receptors. METHODS: A combined chimeric and site-directed mutagenesis approach was used. The sequences of all human BnR's [all = high affinity for Un.Lig] and human CCKA- R [low affinity for Un.Lig] were aligned and chimeric receptors of the extracellular membrane (EC) domains were made. Chimeric receptors were made by substitutive amino acids of hCCKA-R for the corresponding ones in hGRPR in the extracellular (EC) of the important area. Single amino acid mutations were made with Quikchange. All Bn receptors were expressed in CHOP cells. Receptor affinities were assessed using binding with radiolabeled Un.lig. or I125 [Tyr4]Bn. RESULTS: The chimeric studies suggested some amino acids in EC4, and to a less extent EC2 and EC3, were important for Un.lig high affinity. 40 point mutations were made of possible important amino acids in EC4, EC3 and EC2 by replacing amino acids of hGRPR by the comparable amino acid of hCCKA-R. Ten single amino acid GRPR mutations showed greater than >3 hold decrease in affinity for Un.lig (IC50>0.032 nM). Differences of at least 5-hold in affinity were seen with the mutated hGRP receptors at D97N, G112V, R287N and H300S. D97N and G112V are in the 2nd-extracellular (EC2) region, whereas R287N and H300S are in EC4. The substitution of arginine by asparagine in hGRPR at position 287 [R287N] had the greatest effect causing a >200-fold decrease in affinity for Un.lig. All hBnRs with high affinity for Un.lig had a positive charged amino acid at position 287 (i.e. R, H). Therefore additional mutations at this site were made. Complete loss of binding was seen with R287D or R287A suggesting a positive charged amino acid at position 287 in EC4 is important for Un.lig. binding. CONCLUSIONS: These results show high affinity for the Un.lig, by all human Bn receptors is dependent particularly an amino acids in extracellular region 4 and to a less extent EC2 and EC3. Particularly important is the presence of a positive charged amino acid in EC4 (positive 287) suggesting charge- charge interactions may be particularly important for determining the general high affinity of this ligand. S-257 AGA Abstracts S1705 Corticotropin-Releasing Factor (CRF) and Urocortin 1 (UCN1) Differentially Regulate Trafficking and Signaling of Corticotropin-Releasing Factor Receptor 1 (CRF-R1) Burcu Hasdemir, Min Liao, Nigel W. Bunnett, Aditi Bhargava BACKGROUND. Like many receptors, CRF-R1 binds to several endogenous agonists, albeit with graded affinity. Although CRF-R1 binds to Ucn1 with a 40-fold higher affinity than to CRF, it is not known whether Ucn1 or CRF differentially affect trafficking or signaling of CRF-R1. By degrading internalized neuropeptides in endosomes, the metalloendopeptidase endothelin-converting enzyme-1 (ECE-1) induces receptor dissociation from β-arrestins to promote recycling and resensitization. We investigated whether Ucn1 and CRF induce endocytosis, recycling and resensitization of CRF-R1, and whether ECE-1 similarly controls post-endocytic trafficking and signaling of CRF-R1 activated with Ucn1 or CRF. METHODS AND RESULTS. Human CRF-R1 with an extracellular HA11 epitope was stably expressed in HEK-FLP cells. To examine trafficking, HEK-CRF-R1 cells were incubated with HA11 antibody to label cell-surface receptors, and cells were stimulated with CRF or Ucn1 (100 nM, 0-30 min). Trafficking of antibody-labeled CRF-R1 from the cell-surface was studied by immunofluorescence and confocal microscopy. In unstimulated cells, CRF-R1 was local- ized to the plasma membrane. Within 30 min of CRF or Ucn1 stimulation, CRF-R1 was detected in endosomes, where it co-localized with early endosomal antigen 1, the endosomal GTPases Rab4 and Rab11, and ECE-1. After 2 h, CRF-R1 recycled to the plasma membrane. Co-expression of a dominant negative mutant of Rab11, but not Rab4, impaired CRF-R1 recycling. Inhibition of ECE-1 with the selective inhibitor SM-19712 did not affect receptor endocytosis in cells stimulated with CRF or Ucn1. However, ECE-1 inhibition prevented CRF- R1 recycling in cells stimulated with Ucn1, but not with CRF. To examine the resensitization of CRF-R1 signaling, [Ca2+]i was measured using fura-2 in response to repeated stimulation with CRF or Ucn1. Exposure of cells to CRF or Ucn1 (100 nM, 30 min) caused a similar increase in [Ca2+]i. A second challenge after 0 h of recovery resulted in a minimal [Ca2+]i response (Ucn1, 11±5%; CRF, 21±6% of controls), indicating desensitization. After 4 h of recovery, responses to Ucn1 were recovered to 76±3% of control and CRF recovered to 94±5% of control, indicating resensitization. ECE-1 inhibition strongly inhibited resensitization to Ucn1 (27±9% of control), whereas CRF-mediated resensitization was unaffected (82±8% of control). CONCLUSIONS. CRF and Ucn1 similarly target CRF-R1 to endosomes and a Rab11- dependent recycling pathway. Ucn1- but not CRF-mediated receptor recycling and resensitization are ECE-1 dependent. Thus, ECE-1 may regulate differential trafficking and signaling of CRF-R1 upon binding different ligands. S1706 Activation of β-Catenin, Downstream of NfκB, is Required for Measuring Growth Effects of Autocrine Progastrin: Critical Role of Extracellular Annexin2 Shubhashish Sarkar, Pomila Singh Background and Aims. Progastrin (PG) exerts anti-apoptotic/proliferative effects on normal and cancerous GI epithelial cells, and increases risk of colon carcinogenesis (Cancer 2004; Oncogene 2007; CanRes 2007; Oncogene 2008). Extracellular, membrane associated, Annexin2 (ANX2) binds PG with high affinity and is required for mediating proliferative effects of PG (Oncogene 2007; CanRes 2007). Activation of NFκBp65 is critically required for measuring hyperproliferative/anti-apoptotic effects of PG, In Vitro and In Vivo (CanRes 2007; Oncogene 2008; JBC 2009). In Vivo inhibition of p65 with pharmacological agents down-regulated β-catenin activation in response to PG (JBC 2009). In the current studies we examined role of Anx2/PG binding in dual activation of NFκB/β-catenin, In Vitro and In Vivo, and further examined possible two-way cross-talk between NFκB and β-catenin. Methods and Results Mutant-hGAS-HEK293 clones (HEK-PG) were generated and confirmed to over-express full-length (9KDa) PG. HEK293 clones transfected with empty vector (HEK- C) served as controls. A significant increase In Vitro and In Vivo proliferation of HEK-PG clones, associated with activation/nuclear translocation of p65/β-catenin, and up-regulation of target genes (c-MYC, COX-2, iNOS, Cyclin D1) was measured. Down-regulation of ANX2 (with specific siRNA), resulted in loss of activation of both β-catenin/p65, strongly implicating a critical role of ANX2 in mediating up-regulation of the two transcriptional factors, in response to PG. Cells, treated with control or specific siRNA, against p65/β-catenin were confirmed for loss of p65/β-catenin expression. Down-regulation of β-catenin had no effect on activation of p65, while down-regulation of p65, attenuated nuclear translocation of β- catenin; these results confirm a novel cross-talk between p65 and β-catenin in response to PG. However, both transcriptional factors were required for measuring full spectrum of growth effects in response to PG. Since NFκB activation was upstream of β-catenin activation, loss of p65 attenuated growth effects of PG; loss of β-catenin partially negated growth effects of PG. Conclusions. β-catenin activation is down-stream of NFκB activation, in response to PG/ANX2 binding; however, the reverse is not true. Importantly, down-regulation of either ANX2/NFκB/or β-catenin had no effect on growth of HEK-C cells, suggesting that targeting either autocrine PG/extracellular ANX2/or NFκB/β-catenin, may completely attenuate growth of PG-dependent cancers, without collateral damage to normal epithelial cells. Supported by NIH grants R01CA114264 and R01CA097959 to P. Singh S1707 Assessment of a New, Novel, Putative High Affinity and Selective Ligand for the Hbrs3 Receptor and Comparison to Older Less Selective Ligands Veronica Sancho, Samuel A. Mantey, Terry W. Moody, Alessia Di Florio, David H. Coy, Robert T. Jensen BKG: Bombesin (Bn)-receptor subtype-3 (BRS3) is an orphan receptor widely expressed in the GI tract and CNS which shares 50% homology with the human Bn receptors for gastrin-releasing peptide(GRPR) and neuromedin B(NMBR). It is receiving increased attention because BRS3 knockout animals develop obesity, diabetes and hypertension. Furthermore, recent studies show it is overexpressed by various tumors, is a tumor growth factor, regulates AGA Abstracts
Transcript of S1705 Corticotropin-Releasing Factor (CRF) and Urocortin 1 (UCN1) Differentially Regulate...
levels returned to control levels in ambient air. Intestinal NF-κB protein levels were elevatedsignificantly. Lung apelin mRNA levels were unaffected. Exp 2: In Vitro hyperoxia reducedileal cell apelin mRNA levels by 50% (P < 0.05). Exp 3: Overexpression of NF-κB reducedrat apelin core promoter activity by 60-80%. Mutation of putative NF-κB binding sitesimpaired NF-κB-induced reduction in apelin core promoter activity. Conclusions andDiscussion. Our findings demonstrate that hyperoxia decreases intestinal apelin expressionin the rat. NF-κB may play a role in the hyperoxia-induced reduction in intestinal apelinexpression since NF-κB is activated by hyperoxia and overexpression of NF-κB down-regulates apelin transcriptional activity. More importantly, these findings suggest that areduced apelin expression may influence organogenesis in infants exposed to postnatalhyperoxia. It is well known that hypoxia upregulates apelin expression though HIF. Thisis the first demonstration of down-regulated apelin expression by hyperoxia.
S1703
The Mechanism of Transfomer 2-β Gene Expression in Response to OxidativeStress in Human Colon Epithelial CellsYuki Kuwano, Yuzuru Satake, Kensei Nishida, Kiyoshi Masuda, Ken Kurokawa, ShigetadaTeshima-kondo, Toshihito Tanahashi, Kazuhito Rokutan
Background and Aims: Alternative splicing system is an important biological process fordiversity of gene expression and at least 75% of human genes undergo this regulation.The serine-/arginine-rich (SR)-protein family contains the RS domain and participates inconstitutive/alternative pre-mRNA splicing and post-splicing activities. Transformer 2-β(tra2β, SFRS10) is a member of the SR-like protein family, known as an alternative splicingregulator of several genes including CD44, FGFR2, and tau. Moreover, the tra2β gene itselfgenerates five alternative spliced forms (tra2β1-tra2β5). We previously reported that exposureto arsenite transiently produced tra2β4, followed by enhanced expression of tra2β1 in gastricepithelial cells. Here, we examined oxidative stress-induced expression of SR protein familymembers and focused on the mechanisms of regulation of tra2β gene expression in colonepithelial cells.Results: First we tested whether arsenite changed mRNA isoform expressionof the SR protein family (SFRS1-11) in a colon cancer cell line (HCT-116). Among thefamily members, SFRS3, 6, and 7 as well as SFRS10 responded to arsenite and transientlyincreased mRNA variants containing premature stop codon (PTC)-encoded exons with apeak around 2 h. We also found that a specific inhibitor for p38 MAPK or for JNKs partiallyblocked arsenite-induced tra2β4 isoform, a PTC variant of the tra2β gene. To characterizethe mechanism of expression of the tra2β gene, we subcloned 1066 bp-upstream of thetra2β 5'-flanking region and the promoter activity was measured by the dual-luciferase assaysystem. Serially truncated regions of the 1066 bp-upstream showed that -128 to -268 bpof the upstream of the tra2β gene possessed the basal promoter activity. From sequencingresults, this region contains the putative binding sites for the transcription factors, SP-1,Ets, HSF-1, and NRF-2. To identify crucial cis element(s) and trans activator(s), we generatedthe sites-mutated luciferase reporter vectors for these transcription factors. The luciferaseassay and chromatin immunoprecipitation showed that Ets (from -172 to -163 bp) wascrucial for basal transcription, whereas three HSEs (from -250 to -202 bp) regulated oxidativestress-induced gene expression of the tra2β gene. Conclusion: Alternative splicing is regu-lated by the cooperative action of multiple splicing factors, including tra2β. Our resultssuggest that arsenite exposure stimulate alternative splicing of tra2β pre-mRNA possiblythrough activation of p38 MAP and/or JNKs. Ets and HSF-1 may participate in the regulationof basal and oxidative stress-induced transcription of the tra2β gene, respectively.
S1704
The Molecular Basis Responsible for High Affinity of All Human BombesinReceptor (BnR) Family Members for a Universal Ligand (Un.Lig.)Hirotsugu Uehara, Nieves Gonzalez, Samuel A. Mantey, Tomoo Nakagawa, TatsuroKatsuno, Robert T. Jensen
BKG: The synthetic Bn analog, [D-Tyr6, β-Ala11, Phe13, Nle14]Bn (6-14) [Un.Lig] has theunique property of functioning as a high affinity agonist at all 3 human BnRs [neuromedinB receptor (NMB-R), gastrin-releasing peptide receptor (GRPR) and orphan receptor (BRS-3)]. However, the molecular basis for this unique property is unknown. AIM: To determinethe molecular basis for the high affinity of the universal ligand (Un.Lig) for all human Bnreceptors. METHODS: A combined chimeric and site-directed mutagenesis approach wasused. The sequences of all human BnR's [all = high affinity for Un.Lig] and human CCKA-R [low affinity for Un.Lig] were aligned and chimeric receptors of the extracellular membrane(EC) domains were made. Chimeric receptors were made by substitutive amino acids ofhCCKA-R for the corresponding ones in hGRPR in the extracellular (EC) of the importantarea. Single amino acid mutations were made with Quikchange. All Bn receptors wereexpressed in CHOP cells. Receptor affinities were assessed using binding with radiolabeledUn.lig. or I125 [Tyr4]Bn. RESULTS: The chimeric studies suggested some amino acids inEC4, and to a less extent EC2 and EC3, were important for Un.lig high affinity. 40 pointmutations were made of possible important amino acids in EC4, EC3 and EC2 by replacingamino acids of hGRPR by the comparable amino acid of hCCKA-R. Ten single amino acidGRPR mutations showed greater than >3 hold decrease in affinity for Un.lig (IC50>0.032nM). Differences of at least 5-hold in affinity were seen with the mutated hGRP receptorsat D97N, G112V, R287N and H300S. D97N and G112V are in the 2nd-extracellular (EC2)region, whereas R287N and H300S are in EC4. The substitution of arginine by asparaginein hGRPR at position 287 [R287N] had the greatest effect causing a >200-fold decrease inaffinity for Un.lig. All hBnRs with high affinity for Un.lig had a positive charged amino acidat position 287 (i.e. R, H). Therefore additional mutations at this site were made. Completeloss of binding was seen with R287D or R287A suggesting a positive charged amino acidat position 287 in EC4 is important for Un.lig. binding. CONCLUSIONS: These resultsshow high affinity for the Un.lig, by all human Bn receptors is dependent particularly anamino acids in extracellular region 4 and to a less extent EC2 and EC3. Particularly importantis the presence of a positive charged amino acid in EC4 (positive 287) suggesting charge-charge interactions may be particularly important for determining the general high affinityof this ligand.
S-257 AGA Abstracts
S1705
Corticotropin-Releasing Factor (CRF) and Urocortin 1 (UCN1) DifferentiallyRegulate Trafficking and Signaling of Corticotropin-Releasing Factor Receptor1 (CRF-R1)Burcu Hasdemir, Min Liao, Nigel W. Bunnett, Aditi Bhargava
BACKGROUND. Like many receptors, CRF-R1 binds to several endogenous agonists, albeitwith graded affinity. Although CRF-R1 binds to Ucn1 with a 40-fold higher affinity thanto CRF, it is not known whether Ucn1 or CRF differentially affect trafficking or signalingof CRF-R1. By degrading internalized neuropeptides in endosomes, the metalloendopeptidaseendothelin-converting enzyme-1 (ECE-1) induces receptor dissociation from β-arrestins topromote recycling and resensitization. We investigated whether Ucn1 and CRF induceendocytosis, recycling and resensitization of CRF-R1, and whether ECE-1 similarly controlspost-endocytic trafficking and signaling of CRF-R1 activated with Ucn1 or CRF. METHODSAND RESULTS. Human CRF-R1 with an extracellular HA11 epitope was stably expressedin HEK-FLP cells. To examine trafficking, HEK-CRF-R1 cells were incubated with HA11antibody to label cell-surface receptors, and cells were stimulated with CRF or Ucn1 (100nM, 0-30 min). Trafficking of antibody-labeled CRF-R1 from the cell-surface was studiedby immunofluorescence and confocal microscopy. In unstimulated cells, CRF-R1 was local-ized to the plasma membrane. Within 30 min of CRF or Ucn1 stimulation, CRF-R1 wasdetected in endosomes, where it co-localized with early endosomal antigen 1, the endosomalGTPases Rab4 and Rab11, and ECE-1. After 2 h, CRF-R1 recycled to the plasma membrane.Co-expression of a dominant negative mutant of Rab11, but not Rab4, impaired CRF-R1recycling. Inhibition of ECE-1 with the selective inhibitor SM-19712 did not affect receptorendocytosis in cells stimulated with CRF or Ucn1. However, ECE-1 inhibition prevented CRF-R1 recycling in cells stimulatedwithUcn1, but not with CRF. To examine the resensitization ofCRF-R1 signaling, [Ca2+]i was measured using fura-2 in response to repeated stimulationwith CRF or Ucn1. Exposure of cells to CRF or Ucn1 (100 nM, 30 min) caused a similarincrease in [Ca2+]i. A second challenge after 0 h of recovery resulted in a minimal [Ca2+]iresponse (Ucn1, 11±5%; CRF, 21±6% of controls), indicating desensitization. After 4 h ofrecovery, responses to Ucn1were recovered to 76±3%of control andCRF recovered to 94±5%of control, indicating resensitization. ECE-1 inhibition strongly inhibited resensitization toUcn1 (27±9% of control), whereas CRF-mediated resensitization was unaffected (82±8% ofcontrol). CONCLUSIONS. CRF and Ucn1 similarly target CRF-R1 to endosomes and aRab11- dependent recycling pathway. Ucn1- but not CRF-mediated receptor recycling andresensitization are ECE-1 dependent. Thus, ECE-1 may regulate differential trafficking andsignaling of CRF-R1 upon binding different ligands.
S1706
Activation of β-Catenin, Downstream of NfκB, is Required for MeasuringGrowth Effects of Autocrine Progastrin: Critical Role of ExtracellularAnnexin2Shubhashish Sarkar, Pomila Singh
Background and Aims. Progastrin (PG) exerts anti-apoptotic/proliferative effects on normaland cancerous GI epithelial cells, and increases risk of colon carcinogenesis (Cancer 2004;Oncogene 2007; CanRes 2007; Oncogene 2008). Extracellular, membrane associated,Annexin2 (ANX2) binds PG with high affinity and is required for mediating proliferativeeffects of PG (Oncogene 2007; CanRes 2007). Activation of NFκBp65 is critically requiredfor measuring hyperproliferative/anti-apoptotic effects of PG, In Vitro and In Vivo (CanRes2007; Oncogene 2008; JBC 2009). In Vivo inhibition of p65 with pharmacological agentsdown-regulated β-catenin activation in response to PG (JBC 2009). In the current studieswe examined role of Anx2/PG binding in dual activation of NFκB/β-catenin, In Vitro andIn Vivo, and further examined possible two-way cross-talk between NFκB and β-catenin.Methods and Results Mutant-hGAS-HEK293 clones (HEK-PG) were generated and confirmedto over-express full-length (9KDa) PG. HEK293 clones transfected with empty vector (HEK-C) served as controls. A significant increase In Vitro and In Vivo proliferation of HEK-PGclones, associated with activation/nuclear translocation of p65/β-catenin, and up-regulationof target genes (c-MYC, COX-2, iNOS, Cyclin D1) was measured. Down-regulation of ANX2(with specific siRNA), resulted in loss of activation of both β-catenin/p65, strongly implicatinga critical role of ANX2 in mediating up-regulation of the two transcriptional factors, inresponse to PG. Cells, treated with control or specific siRNA, against p65/β-catenin wereconfirmed for loss of p65/β-catenin expression. Down-regulation of β-catenin had no effecton activation of p65, while down-regulation of p65, attenuated nuclear translocation of β-catenin; these results confirm a novel cross-talk between p65 and β-catenin in response toPG. However, both transcriptional factors were required for measuring full spectrum ofgrowth effects in response to PG. Since NFκB activation was upstream of β-catenin activation,loss of p65 attenuated growth effects of PG; loss of β-catenin partially negated growth effectsof PG. Conclusions. β-catenin activation is down-stream of NFκB activation, in response toPG/ANX2 binding; however, the reverse is not true. Importantly, down-regulation of eitherANX2/NFκB/or β-catenin had no effect on growth of HEK-C cells, suggesting that targetingeither autocrine PG/extracellular ANX2/or NFκB/β-catenin, may completely attenuate growthof PG-dependent cancers, without collateral damage to normal epithelial cells. Supportedby NIH grants R01CA114264 and R01CA097959 to P. Singh
S1707
Assessment of a New, Novel, Putative High Affinity and Selective Ligand forthe Hbrs3 Receptor and Comparison to Older Less Selective LigandsVeronica Sancho, Samuel A. Mantey, Terry W. Moody, Alessia Di Florio, David H. Coy,Robert T. Jensen
BKG: Bombesin (Bn)-receptor subtype-3 (BRS3) is an orphan receptor widely expressed inthe GI tract and CNS which shares 50% homology with the human Bn receptors forgastrin-releasing peptide(GRPR) and neuromedin B(NMBR). It is receiving increased attentionbecause BRS3 knockout animals develop obesity, diabetes and hypertension. Furthermore,recent studies show it is overexpressed by various tumors, is a tumor growth factor, regulates
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