RIKEN Chemical Genomics Research Group - Molecular Ligand Biology Team

　Team Leader　Soichi Kojima, Ph.D.

The Molecular Ligand Biology Research Team aims to provide unique screening systems and figure out novel biological principles establishing bases for new drug discovery. To this end, we study the regulation of transcription/translation, hormonal reaction, chromatin function, immune reaction, differentiation of stem cells, and organogenesis relating to protein modification as well as epigenetics. We investigate the effects of newly discovered and/or synthesized molecular ligands on biological functions described above utilizing cultured animal and plant cells as well as animal and plant models.

Research Projects

1. Development of unique chemical screening systems
2. Exploration of novel functions and quality control via post-translational modification of proteins
3. Basic studies for establishing new diagnosis and therapy

Specific Research Areas

1. Development of unique chemical screening systems
To develop a screening system for chemicals capable of regulating differentiation and dedifferentiation of stem cells, immortalized human mesenchymal stem cells were derived from bone marrow and their multipotency was confirmed by differentiation to osteocytes, adipocytes, cardiomyocytes, and neuronal cells. They were cultured with chemicals from RIKEN NP depo (RIKEN Natural Products Depositor). We found one chemical that enhanced mineralization and compared its activity with ipriflavone, currently used as a drug for osteoporosis. This year gene expression by the chemical was investigated in vitro; BMP2 expression and signaling were upregulated. Screening chemicals for induction of neurocyte was started. To develop a screening system for chemicals capable of regulating apoptosis through novel molecular mechanisms, we purified the GST fusion protein of Drosophila nucleolar GTP binding protein 1(DNGBP1) and screened chemical compounds that bind to DNGBP1 using a combination of the fusion protein and the chemical array. Six compounds were obtained in the presence of GTP. In addition, we estimated a GTPase activity of DNGBP1 and conducted in vitro assay system. Using Drosophila culture cell, we have established the cell lines that repress the DNGBP1 activity inductively. To develop a screening system for chemicals capable of regulating angiogenesis through novel molecular mechanisms, we demonstrated that the derivative of the newly screened compound, spliceostatin A, has anti-angiogenic activity using in vitro tube formation assay and in vivo assay with tumor cells implanted on chick embryo chorioallantoic membrane. Moreover, we established the screening system to identify CLEC-2-binding small molecules that inhibit CLEC-2-podoplanin binding and investigate the possibility of the molecules as novel anti-metastatic and anti-platelet drugs. At the present, we found that 40 compounds from 4,230 compounds have an inhibitory effect for interaction between CLEC-2 and podoplanin. We are trying to clarify unknown mechanism of plant steroid hormone brassinosteroid signal transduction as a subject for chemical screening. By chemical genetics approach using brassinosteroid biosynthesis inhibitor Brz, brassinosteroid signaling factors were isolated from the Brz-insensitive long hypocotyl mutants. We started chemical library screening based on the bil5 phenotyping that is a trial for BIL5 specific regulatory chemicals. From first 1,000 chemicals, we identified 3 chemicals that can bind to BIL5 or BIL4 specifically.

2. Exploration of novel functions and quality control via post-translational modification of proteins
In order to explore biological significance of protein crosslinking reactions as subjects for chemical screening, we established that the enhanced expression of TG2 crosslinks and inactivates a transcription factor, Sp1 in hepatocyte nuclei of mice injured with alcohol-treatment or free fatty acids as well as patients with alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH), leading to downregulation of c-Met and a caspase-independent hepatic apoptosis. Mechanisms of the induction of TG2 activity and nuclear localization via ER stress were different between ASH and NASH. Nuclear localization of TG2 is dependent on importin-β, but not importin-α. Decreased expression of exportin-1 was also important for nuclear localization of TG2. We verified that acyclic retinoid prevented phosphorylation of the nuclear retinoid receptor, and restored its normal function to transactivate the TG2 gene promoter in hepatocarcinoma cells, leading to activation of caspase3 as well as formation of crosslinked and inactivated Sp1, and thereby causing hepatoma cell apoptosis due to downregulation of EGF receptor. We established the screening system for TG2 inhibitors in alcohol or acetaldehyde-treated hepatocytes. Furthermore, by using the dorsal air sac (DAS) animal model assay, we found that transglutaminase might be involved in angiogenesis. Carcinoma cells were implanted into the dorsal inter-space made in mice. After 7 days, we observed formation of neovessels developed from back skin of mice, which were seen in wild-type B6 mice, but not in TG2 knockout mice. Furthermore, we demonstrated that the tube formation of normal mice endothelial cells were inhibited by TG activity inhibitor (cystamine) by ex vivo aorta ring assay.

Fig. 1. Screening of chemicals targeting crosslinking and inactivation of a transcription factor, Sp1 by a protein crosslinking enzyme, transglutaminase 2 (TG2), which has been revealed to be a cause for hepatic cell death associated with alcoholic steatohepatitis.

3. Basic studies for establishing new diagnosis and therapy
In order to establish a unique screening system and new diagnosis and therapy in future, we studied the proteolytic activation reaction of the transforming growth factor-β (TGF-β), a potent fibrogenic and growth-suppressing cytokine. We made antibodies specifically recognizing plasma kallikrein-cutting edges of C-terminal side LAP degradates (L59 antibody) and established the sELISA system to detect LAP degradates in plasma. In 15-25 % of patients suffered from hepatitis or liver fibrosis, plasma levels of LAP degradates were higher than in healthy volunteers, however plasma levels of LAP degradates did not correlate with existing biomarkers. Plasma LAP degradate levels changed earlier than existing markers in a patient infected with acute hepatitis , and moreover decreased in response to the treatment with PEG-interferon and Ribavilin. These data suggested that the LAP degradates produced during TGF-β activation reaction may be a promising novel biomarker for hepatic fibrosis. In addition, we produced specific antibodies against cutting edges of N-terminal side LAP degradates (R58 antibody) and performed immunohistochemistry with this antibodies. N-terminal side LAP degradates remained in liver tissues in liver fibrosis model animal and patients. We also found that protease-dependent TGF-β activation reaction appeared not to be directly involved in Marfan Syndrome. Furthermore plasma levels of LAP degradates were well correlated with the expression levels of αSMA, which reflects the activation of hepatic stellate cells, in liver tissues.
Two candidate chemicals were identified to regulate the activation of TGF-β as the result of chemical screening. On the other hand, we have analyzed the effect of ethanolic amber extracts on the global gene expression in skin cells using the GeneChip last year and found their inhibitory activity of melanin synthesis. Ethanolic amber extracts decreased the endothelin-1 and SCF mRNA expression in HaCaT cells and reduced melanin synthesis with an organotypic three-dimensional co-culture of human epidermal keratinocytes and human epidermal melanocytes.

Soichi Kojima, Ph.D. (Team Leader)
Hideki Tatsukawa, Ph.D. (Postdoctoral Researcher)
Mitsuko Hara (Postdoctoral Researcher)
Kotaro Sakata (Collaboration Researcher from Industry)
Keisuke Nagatsuma, M.D. (Visiting Scientist)
Tomokazu Matsuura, M.D. (Visiting Scientist)
Akiko Kirita (Technical Staff I)
Yukie Yoshioka (Technical Staff I)
Nozomi Satou (Company-Sponsored Researcher)
Takahiro Aikawa (Company-Sponsored Researcher)
Eri Matsumura (Company-Sponsored Researcher)
Satoshi Eda (Student Trainee)
Yuka Yamamoto (Student Trainee)
Dhana Wolf (Intern)
Lee Eun-Seo (International Program Associate)
Kuo Ting-Fang (International Program Associate)
Kimie Tajima (Assistant)
Yasuyo Takayama (Part Time Staff Ⅲ)


Yoshihiro Ito, Ph.D. (concurrently Chief Scientist of Nano Medical Engineering Laboratory)
Shogo Matsumoto, Ph.D. (concurrently Chief Scientist of Molecular Entomology Laboratory)
Takeshi Nakano, Ph.D. (concurrently Senior Research Scientist of Molecular Membrane Biology Laboratory)
Hiroshi Abe, Ph.D. (concurrently Senior Research Scientist of Nano Medical Engineering Laboratory)
Kazuhide Tsuneizumi, Ph.D. (concurrently Senior Research Scientist of Molecular Entomology Laboratory)
Ayumi Yamagami, Ph.D. (concurrently Postdoctoral Researcher of Molecular Membrane Biology Laboratory)
Setsuko Shimada, Ph.D. (concurrently Postdoctoral Researcher of Molecular Membrane Biology Laboratory)
Takashi Kitajima, Ph.D. (concurrently Contract Researcher of Nano Medical Engineering Laboratory)
Hisao Saneyoshi, Ph.D. (concurrently Postdoctoral Researcher of Nano Medical Engineering Laboratory)

Kuo, T.-F. and Kojima, S.: “New insights into functions and localization of nuclear transglutaminase 2.” FEBS J., in press , 2011.

Watanabe, N., Aizaki, H., Matsuura, T., Kojima, S., Wakita, T., and Suzuki, T.: “Hepatitis C virus RNA replication in human stellate cells regulates gene expression of extracellular matrix-related molecules.” Biochem. Biophys. Res. Commun., 407:135-140, 2011. PMID: 21371436

Kuo, T.-F., Tatsukawa, H., Matsuura, T., Nagatsuma, K., Hirose, S., and Kojima, S.: “Free fatty acids induce transglutaminase 2-dependent apoptosis in hepatocytes via ER stress-stimulated PERK pathways.” J. Cell Physiol., in press, 2011.

Saiki, R., Park, H., Ishii, I., Yoshida, M., Nishimura, K., Toida, T., Tatsukawa, H., Kojima, S., Ikeguchi, Y., Pegg, A. E., Kashiwagi, K., and Igarashi, K.: “Brain infarction correlates more closely with acrolein than with reactive oxygen species.” Biochem. Biophys. Res. Commun., 404:1044-1049, 2011. PMID: 21187074

Tatsukawa, H., Sano, T., Fukaya, Y., Ishibashi, N., Watanabe, M., Okuno, M., Moriwaki, H., and Kojima, S.: “Dual induction of caspase 3- and transglutaminase-dependent apoptosis by acyclic retinoid in hepatocellular carcinoma cells.” Mol. Cancer, 10:4 (11pages), 2011. PMID: 21214951

Itoh, M., Kawamoto, T., Tatsukawa, T., Kojima, S., Yamanishi, K., and Hitomi, K.: “In situ detection of active transglutaminases for keratinocyte type (TGase 1) and tissue type (TGase 2) using fluorescence-labeled highly reactive substrate peptides.” J. Histochem. Cytochem., 59:180-187, 2011. PMID: 20876521

Kojima, S., Kuo, T.-F., Tatsukawa, H., and Hirose, S.: “Induction of crosslinking and silencing of Sp1 by transglutaminase during liver injury in ASH and NASH via different ER stress pathways.” Digestive Diseases., 28: 715-721, 2010. PMID: 21525755

Furumai, R., Uchida, K., Komi, Y., Yoneyama, M., Ishigami, K., Watanabe, H., Kojima, S., and Yoshida, M.: “Spliceostatin A blocks angiogenesis by inhibiting global gene expression including VEGF.” Cancer Sci., 101: 2483-2489, 2010. PMID: 20726856

Seino. J., Ishii, K., Nakano, T., Ishida, N., Tsujimoto, M., Hashimoto, Y., and Takashima, S.: “Characterization of Rice Nucleotide Sugar Transporters Capable of Transporting
UDP-Galactose and UDP-Glucose.” J. Biochem., 148: 35-46, 2010. PMID: 20305274

Komi, Y., Sogabe, Y., Ishibashi, N., Sato, Y., Moriwaki, H., Shimokado, K., and Kojima, S.: "Acyclic retinoid inhibits angiogenesis by suppressing the MAPK pathway." Lab. Invest., 90: 52-60, 2010. PMID: 19841617

Komatsu T., Kawaide H., Saito C., Yamagami A., Shimada S., Nakazawa M., Matsui M., Nakano A., Tsujimoto M., Natsume M., Abe H., Asami T., and Nakano T.: "The chloroplast protein BPG2 functions in brassinosteroid-mediated post-transcriptional accumulation of chloroplast rRNA." Plant J., 61: 409-422, 2010. PMID: 19919572

Tatsukawa, H., and Kojima, S.: “Recent advances in understanding the roles of transglutaminase 2 in alcoholic steatohepatitis .” Cell Biol. Int., 34: 325-334, 2010. PMID: 20192918

Mukhopadhyay, B., Liu, J. Osei-Hyiaman, D., Godlewski, G., Mukhopadhyay, P., Wang, L., Jeong, W., Gao, B., Duester, G., Mackie, K., Kojima, S., and Kunos, G.: “Transcriptional regulation of cannabinoid receptor-1 expression in the liver by retinoic acid acting via retinoic acid receptor-γ. "J. Biol. Chem., 285: 19002-19011, 2010. PMID: 20410309
Mukhopadhyay, B., Liu, J. Osei-Hyiaman, D., Godlewski, G., Mukhopadhyay, P., Wang, L., Jeong, W., Gao, B., Duester, G., Mackie, K., Kojima, S., and Kunos, G.: “Transcriptional regulation of cannabinoid receptor-1 expression in the liver by retinoic acid acting via retinoic acid receptor-γ. "J. Biol. Chem., 285: 19002-19011, 2010. PMID: 20410309

Kitazume, S., Imamaki, R., Ogawa, K., Komi, Y., Futakawa, S., Kojima, S., Hashimoto, Y., Marth, J. D., Paulson, J. C., and Taniguchi, N.: “α2,6-Sialic acid on platelet endothelial cell adhesion molecule (PECAM) regulates its homophilic interactions and downstream antiapoptotic signalling.” J. Biol. Chem., 285: 6515-6521, 2010. PMID: 20048157

Tatsukawa, H., Fukaya, Y., Frampton, G., Martinez-Fuentes, A., Suzuki, K., Kuo, T.-F., Nagatsuma, K., himokado, K., Okuno, M., Wu, J., lismaa, S., Matsuura, T., Tsukamoto, H., Zern, M. A., Graham, R. M., and Kojima, S.: "Role of transglutaminase 2 in liver injury via crosslinking and silencing of transcription factor, Sp1." Gastroenterology, 136: 1783-1795, 2009. PMID: 19208340

Ohnishi A., Hashimoto K., Imai K., and Matsumoto S.: "Functional characterization of the Bombyx mori fatty acid transport protein (BmFATP) within the silkmoth pheromone gland." J. Biol. Chem., 284: 5128-5136, 2009. PMID: 19112106

Tsuzuki, S., Wada, A., and Ito, Y.: "Photo-immobilization of biological components on gold-coated chips for measurements using surface plasmon resonance (SPR) and a quartz crystal microbalance (QCM)." Biotechnol. Bioeng., 102: 700-707, 2009. PMID: 18989902

Murakami M., Kawachi H., Ogawa K., Nishino Y., and Funaba M.: "Receptor expression modulates the specificity of transforming growth factor-beta signaling pathways." Genes Cells., 14: 469-482, 2009. PMID: 19335617

Komi, Y., Suzuki, Y., Shimamura, M., Kajimoto, S., Nakajo, S., Masuda, M., Shibuya, M., Itabe, H., Shimokado, K., Oettgen, P., Nakaya, K., and Kojima, S.: “Mechanism of inhibition of tumor angiogenesis by ß-hydroxyisovalerylshikonin.” Cancer Sci., 100: 269-277, 2009. PMID: 19200258

Botella, L. M., Rodriguez-Sanz, F., Komi, Y., Fernandez-L, A., Varela, E., Garrido-Martin, E. M., Narla, G., Friedman, S. L., and Kojima, S.: “TGF-β regulates expression of KLF6 and its splice variants, and promotes cooperative transactivation of common target genes through a Smad3/Sp1/KLF6 interaction.” Biochem. J., 419: 485-495, 2009. PMID: 19076057

Nakamura A, Fujioka S, Takatsuto S, Tsujimoto M, Kitano H, Yoshida S, Asami T., and Nakano T.: “Involvement of C-22-hydroxylated brassinosteroids in auxin-induced lamina joint bending in rice.” Plant Cell Physiol., 50: 1627-1635, 2009. PMID: 19605414

Yamagami, A., Nakazawa, M., Matsui, M., Tsujimoto, M., Sakuta, M., Asami, T., and Nakano, T.: “Chemical genetics reveal the novel transmembrane protein BIL4, which mediates plant cell elongation on brassinosteroidsignaling.” Biosci. Biotechnol. Biochem., 73: 415-421, 2009. PMID: 19202280

Takashima, S., Seino, J., Nakano, T., Fujiyama, K., Tsujimoto, M., Ishida, N., and Hashimoto, Y.: “Analysis of CMP-sialic acid transporter-like proteins in plants.” Phytochemistry, 70: 1973-1981, 2009. PMID: 19822337

Mashiguchi K., Sasaki E., Shimada Y., Nagae M., Ueno K., Nakano T., Yoneyama K., Suzuki Y.,
and Asami T.: “Feedback-regulation of strigolactone biosynthetic genes and strigolactone-regulated genes in Arabidopsis.” Biosci. Biotechnol. Biochem., 73: 2460-2465, 2009. PMID: 19897913

Kojima, S.: “Regulation of cellular uPA activity and its implication in pathogenesis of diseases.” In Recent Advances in Thrombosis and Hemostasis 2008. (Tanaka, K. and Davie, E.W. eds.), Springer, Tokyo : 301-313, 2008.

Hayashi, M., Tamura, Y., Dohmae, N., Kojima, S., and Shimonaka, M.: “Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration.” Biochem. Biophys. Res. Commun., 369: 635-640, 2008. PMID: 18294956

Asami, Y., Kakeya, H., Komi, Y., Kojima, S., Nishikawa, K., Beebe ,K., Neckers, L., and Osada,H.: “Azaspirene, a fungal product, inhibits angiogenesis by blocking Raf-1 activation.” Cancer Sci., 99: 1853-1858, 2008. PMID: 18637013

Yamauchi, T., Ishidao, T., Nomura, T., Shinagawa, T., Tanaka, Y., Yonemura, S., and Ishii, S.: “A B-Myb complex containing clathrin and filamin is required for mitotic spindle function.” EMBO J., 27: 1852-1862, 2008. PMID: 18548008

Ogawa, K., Funaba, M., and Tsujimoto, M.: “A dual role of activin A in regulating immunoglobulin production of B cells.” J. Leukoc. Biol., 83:1451-1458, 2008. PMID: 18353928

Ogawa, K., Funaba, M., and Tsujimoto, M.: “Suppression of NF-κB and IRF-1-induced transcription of the murine IL-12 p40 by transforming growth factor-β Smad pathway in macrophages” Mol. Cell. Biochem., 308: 9-15, 2008. PMID: 17924060

Nagamine, T., Kawasaki, Y., Abe, A., and Matsumoto, S.: “Nuclear marginalization of host cell chromatin associated with expansion of two discrete virus-induced subnuclear compartments during baculovirus infection” J. Virol., 82: 6409-6418, 2008. PMID: 18434402

Abe, H., Wang, J., Furukawa, K., Oki, K., Uda, M., Tsuneda, S., and Ito, Y.: “A reduction-triggered
fluorescence probe for sensing nucleic acids.” Bioconjug. Chem., 19: 1219-1226, 2008. PMID: 18476727

1.“RIKEN Press Release",“Regulation of angiogenesis via sialic acid synthesis in the endothelial cells: Sialic acid regulates apoptosis of the endothelial cells through localization of PECAM.”
, March 2 (2010)

2.“Asahi Shimbun”, “A master gene of photosynthesis”, January 15 (2010)

3.“RIKEN Press Release”, “Discovery of “BPG2”, a novel controller in the regulation of chloroplast by phytosterol hormone: Chemical biology elucidates a molecular mechanism of brassinosteroid signaling”, December 14 (2009)

4.“Nikkan Kogyo Shimbun”, “Front Line of Inventive Research Group, RIKEN –Novel molecular mechanism of alcoholic liver injury, Discovery of novel mechanism of hepatic cell death, providing a basis for development of a new diagnosis”, September 21 (2009)

5.“Yahoo Japan News”, “Elucidation of molecular mechanism of liver injury due to excessive alcohol consumption”, May 12 (2009)

6.“Nikkei Net”, “RIKEN discovered novel mechanism of alcoholic liver injury”, May 1 (2009)

7.“RIKEN Press Release”, “Protein cross-linking leads to cell death, induces alcohol-related hepatic deficit: Discovery of new mechanism for hepatic cell death in liver damaged by alcohol”, May 1 (2009)

1. Tatsukawa H, Post-doctoral Researcher “CHUGAI Award”, The Japan Society of　Hepatology, CHUGAI PHARMACEUTICAL CO., LTD., October 14 (2010)

2. Tatsukawa H, Post-doctoral Researcher “Young Investigator Award”, Japanese Society for Transglutaminase Research, October 22 (2009)

3. Tatsukawa H, Post-doctoral Researcher “Travel Award”, International Scientific Committee of the 4th International Symposium on Alcoholic Liver and Pancreatic Diseases and Cirrhosis, October 9 (2009)

4. Tatsukawa H, Post-doctoral Researcher “Poster Award”, International Scientific Committee of the 4th International Symposium on Alcoholic Liver and Pancreatic Diseases and Cirrhosis, October 9 (2009)

5. Kojima S, Team Leader “Best Poster Award”, International Scientific Committee of the 4th International Symposium on Alcoholic Liver and Pancreatic Diseases and Cirrhosis, October 9 (2009)

6. Teraoka R, Student Trainee “Young Scientist Encouragement Award”, International Scientific Committee of the 14th International Symposium on Cells of the Hepatic Sinusoid, September 3 (2008)

7. Arai A, Student Trainee “Young Scientist Encouragement Award”, International Scientific Committee of the 14th International Symposium on Cells of the Hepatic Sinusoid, September 3 (2008)

8. Kojima S, Team Leader “Poster Award”, International Scientific Committee of the 3rd International Symposium on Alcoholic Liver and Pancreatic Diseases and Cirrhosis, July 18 (2008)