11:10-12:00, Rm 103

TGF-βSignaling and Its Therapeutic Implications-KSMCB Joint SymposiumS11-3Dopamine D2 receptor-mediated ERK activation: role in physiology ofdopaminergic systemJa-Hyun BaikCollege of Life Sciences and Biotechnology, Korea University, Seoul 136-701, KoreaThe dopamine D2 receptor (D2R) plays an important role in mesencephalic dopaminergicneuronal development, particularly coupled with ERK activation. Wnt5a protein is knownto regulate the development of dopaminergic neurons. We analyzed the effect of Wnt5aon dopaminergic neuron development in mesencaphalic primary cultures from wild-type(WT) and D2R knockout (D2R-/-) mice. Treatment with Wnt5a increased the number andneuritic length of dopamine neurons in mesencephalic neuronal cultures from WT mice,but not from D2R-/- mice. The effect of Wnt5a was completely blocked by treatment withD2R antagonist or inhibitors of MAPK or EGFR. Wnt5a-mediated ERK activation inmesencephalic neuronal cultures was inhibited by treatment of D2R antagonist andEGFR inhibitors in WT mice. However, these regulations were not observed for D2R-/-mice. Co-immunoprecipitation and displacement of [3H]spiperone from D2R by Wnt5ademonstrated that Wnt5a could bind with D2R. These results suggest that the interactionbetween D2R and Wnt5a has an important role in dopamine neuron development inassociation with EGFR and the ERK pathway. Further studies should explore and clarifythe role of EGFR signaling in dopamine-associated regulation of ERK signaling indifferent physiology, including adult brain.S11-4Molecular determinants responsible for interaction between GLP-1peptide and its receptorJae Young SeongLaboratory of GPCRs, Korea University College of Medicine, Seoul 136-705, KoreaGLP-1 and GIP are incretin hormones released in response to food intake and potentiateinsulin secretion from pancreatic beta cells through their distinct yet related G proteincoupledreceptors, GLP1R and GIPR. In addition to its incretin effect, GLP-1 and GIP areimplicated with the regulation of obesity and bone formation. Due to their multiplebeneficial effects, both GLP-1 and GIP have been regarded as potential therapeuticagents for the treatment of diabetes mellitus and obesity. As identification of the molecularmechanisms leading to high-affinity ligand-receptor binding and receptor activation isfundamentally important for development of more potent agonists for GLP1R and GIPR,in this study we investigated amino acid residues in peptides and receptors thatresponsible for ligand-receptor interaction. Using chimeric peptides and receptors weidentified that Tyr/His1 and Ile/Thr7 of GIP/GLP-1 peptides confer differential ligandselectivity toward GIPR and GLP1R and that Ile196 and Lys197 of TMH2, Leu232 andMet233 of ECL1, and Asn302 of ECL2 of GLP1R may confer specific GLP-1 binding andreceptor activation. This study may provide critical clues for development of nonpeptideagonists for the treatment of diabetes mellitus and obesity.S12-2How to achieve cellular-response-selective regulation of TGF-βsignalingKeiji MiyazawaDepartment of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering,University of Yamanashi, JapanTGF-β(transforming growth factor-β) is known to be involved in various physiological aswell as pathological processes. Intriguingly, TGF-βexhibits two opposite faces ontumorigenesis. It has tumor suppressor function in the early phases, but promotes tumorprogression in the advanced stages. Various molecules targeting the TGF-βsignalingpathway have been developed for possible clinical uses, but they comprehensively inhibitTGF-βsignaling; it means that not only negative but also positive faces of TGF-βsignalingare inhibited by these molecules. Therefore, methods to achieve cellular-responseselective regulation of TGF-βsignaling are highly desirable. Recently we identified a novelregulator of TGF-βsignaling, Maid (maternally Id-like molecule). Maid is a helix-loop-helixprotein which lacks a DNA-binding basic region, thus representing a structural feature ofdominant negative helix-loop-helix proteins. Intriguingly, it is a cellular response selectiveregulator of TGF-βsignaling; it inhibits TGF-β-induced cytostasis and cell motility, but notEMT (epithelial-mesenchymal transition). Maid represses only a subset of TGF-βtargetgenes (15% of all target genes) including p21 WAF and p15 INK4B (involved in TGF-β-inducedcytostasis), but not Snail (involved in TGF-βinduced EMT), a finding consistent with theeffects on cytostasis and EMT. Various cellular responses induced by TGF-βareprincipally mediated through Smad proteins. Smad2/3 are phosphorylated by an activatedreceptor kinase upon ligand stimulation, form complexes with Smad4, and translocate intothe nucleus where they regulate expression of target genes. The activated Smadcomplexes usually require DNA-binding transcription factors (so-called Smad cofactors)for its successful regulation of target genes, because the affinity of Smad proteins for thecanonical Smad-binding element is not high enough. Our present findings suggest thatMaid selectively inhibits some of Smad cofactors. To elucidate the mechanism of theselective regulation of TGF-βsignaling by Maid, we searched for Smad cofactors that areinhibited by Maid, and identified Olig1. Olig1 is a basic helix-loop-helix transcription factororiginally identified as a molecule required for maturation of oligodendrocyte progenitors.Olig1 is however, expressed in a wide variety of tumor as well as normal cell lines. Olig1interacts with Smad2/3 in response to TGF-βstimulation, and is recruited to the promoterregion of certain target genes. DNA microarray analysis indicated that Olig1 is involved inregulation of 10% of the TGF-βtarget genes. Remarkably, knockdown of Olig1 resulted inattenuation of TGF-β-induced cell motility, but neither cytostasis nor EMT. Maid was foundto sequester Olig1 and inhibit its association with Smad2/3, which resulted in dissociationof Smad2/3 and Olig1 from the target gene promoters. Specific inhibition of a certainSmad-Smad cofactor complex would be one of the rational ways to achieve cellularresponse selective regulation of TGF-βsignaling. At present, Maid can inhibit Olig1 aswell as other Smad cofactors, thus the specificity is not high enough. We are now workingto develop a method to inhibit the function of the Smad-Olig1 complex specifically.S12-1Innovative in vivo optical imaging for cancer and TGF-βresearchTakeshi Imamura¹Department of Molecular Medicine for Pathogenesis, Ehime University Graduate School ofMedicine, ²Core Research for Evolutional Science and Technology (CREST), ³Division ofBiochemistry, The JFCR Cancer Institute, JapanThere has been a growing interest in applying optical imaging approaches to studydifferent disease process and complex biology in vivo. Recently, we have established invivo bioluminescent imaging systems that effectively labels transforming growth factor(TGF)-βand bone morphogenic protein (BMP) signaling in metastatic phenotypes ofbreast cancer cells in vivo. We generated promoter-reporter luciferase-expressing humanbreast cancer cell lines, MDA-D-TbFluc/Rluc and MDA-D-BREFluc/Rluc cells, andmonitored TGF-βand BMP signaling in breast cancer cells in living mice. The results fromthe present study demonstrated that TGF-βand BMP plays a crucial role at an earlyperiod of bone metastasis of breast cancer. The in vivo optical imaging systems will be apowerful technique in molecular biology.78 Korean Society for Biochemistry and Molecular BiologyS12-3TGF-βsignaling through Smad3 and Smad4 targets Eomesodermin tosuppress immune surveillance by cytotoxic T lymphocytes in melanomaYoung-Ju Kang 1, *, Jeong-Hwan Yoon 1, *, Hyun-Joo Lee 1 , Ji-Woong Shin 1 , Hae-Young Park 1 , Chuxia Deng 2 , Maria H. Festing 3 , Michael Weinstein 3 , Katsuko Sudo 4 ,Ok-Hee Kim 5 , Byung-Chul Oh 5 and Mizuko Mamura 11Laboratory of Immunology, Lee Gil Ya Cancer and Diabetes Institute, Gachon University ofMedicine and Science, Incheon, Korea, 2 Genetics of Development and Disease Branch, NIDDK,National Institutes of Health, Bethesda MD, USA, 3 Department of Molecular Genetics, The OhioUniversity, Columbus, OH, USA, 4 Animal Research Center, Tokyo Medical University, Tokyo,Japan, 5 Laboratory of Molecular and Cellular Biochemistry, Lee Gil Ya Cancer and DiabetesInstitute, Gachon University of Medicine and Science, Incheon, KoreaTransforming growth factor-β(TGF-β) antagonism has been shown to suppress tumorigenesisand metastasis by recovering anti-tumor immunity, which is impaired by abundant expression andactivation of this immunosuppressive cytokinein tumor microenvironment. However, the precisesignaling mechanisms for suppressing T cell-mediated anti-tumor immunity by TGF-βare not fullyunderstood. We found that T cell-specific deficiency of Smad4, a common Smad for TGF-βsignaling, suppressed both primary melanoma progression and lymph node metastasis. Smad4deficiency induced systemic expansion of IFN-γ-producing CD8+ T cells rather than expansion inregional lymph nodes. TGF-βsuppressed the expression of a T-box transcription factor,Eomesodermin (Eomes), but not another functionally related transcription factor, T-bet in CD8+ Tcells. Smad3 was the TGF-βreceptor-activated Smad (R-Smad) to specifically repress the Eomesgene together with Smad4, whereas the other R-Smad, Smad2 reversed the inhibitory effect ofSmad4 on Eomes expression. Smads did not directly regulate the Ifng gene in CD8+ T cells. Ourdata thus show that TGF-βsignaling mediated by Smad3 and Smad4 represses Eomes, whichinhibits systemic immune surveillance by IFN-γ-producing anti-tumor cytotoxic T lymphocytes.