Department of Molecular and Cellular Biochemistry

　The department of molecular and cellular biochemistry was founded by Professor Yoshiro TAKEDA in 1959 and has largely contributed to development of life sciences, medical and dental sciences. The department of molecular and cellular biochemistry has produced many talented and excellent researchers, educators and dentists one after another from starting a course. We are currently focusing on bone and cartilage metabolism and the cancer biology, and leading the fields in Japan and the world.

１．Molecular and cellular mechanism of endochondral bone formation

　The vertebrate skeleton is formed by two modes of bone development: membranous bone formation, in which bone is formed directly by osteoblasts, and endochondral bone formation, which involves chondrocyte formation. With the exception of the skull and some bones, most of bones are formed by endochondral bone formation. Therefore, studies on endochondral bone formation are important for understanding skeletal development, including the human maxillofacial region. In our laboratory, we are intensively studying the cloning and functional analysis of a group of molecules involved in the functional and epigenetic regulation of the transcription factor Sox9, which is essential for the differentiation of chondrocytes from undifferentiated mesenchymal stem cells. We have also revealed that Indian hedgehog (Ihh) plays an important role in the differentiation process from proliferating chondrocytes to hypertrophic chondrocytes. We have also shown that the transcription factor Osterix is essential for the final process of endochondral bone formation.

★ Related literatures
1. Yoshida M, Hata K, Takashima R, Ono K, Nakamura E, Takahata Y, Murakami T, Iseki S, Takano-Yamamoto T, Nishmura R, Yoneda T. The transcription factor Foxc1 is necessary for Ihh-Gli2-regulated endochondral ossification. Nature Commun, 2015, 6:6653 DOI: 10.1038/ncomms7653
2. Hata K, Takashima R, Amano K, Ono K, Nakanishi M, Yoshida M, Wakabayashi M, Matsuda A, Maeda Y, Suzuki Y, Sugano S, Whitson RH, Nishimura R, Yoneda T. Arid5b facilitates chondrogenesis by recruiting the histone demethylase Phf2 to Sox9-regulated genes. Nature Commun. 2013, 4: 2850 DOI: 10.1038/ncomms3850
3. Nishimura R, Wakabayashi M, Hata K, Matsubara T, Honma S, Wakisaka S, Kiyonari H, Shioi G, Yamaguchi A, Tsumaki N, Akiyama H, Yoneda T. Osterix regulates calcification and degradation of chondrogenic matrices through matrix metalloproteinase 13 (MMP13) expression in association with transcription factor Runx2 during endochondral ossification. J Biol Chem. 2012, 287:33179-90

4. Ono K, Hata K, Nakamura E, Ishihara S, Kobayashi S, Nakanishi M, Yoshida M, Takahata Y, Murakami T, Takenoshita S, Komori T, Nishimura R, Yoneda T. Dmrt2 promotes transition of endochondral bone formation by linking Sox9 and Runx2. Commun Biol. 2021, 4:326. DOI: 10.1038/s42003-021-01848-1.

5. Nakamura E, Hata K, Takahata Y, Kurosaka H, Abe M, Abe T, Kihara M, Komori T, Kobayashi S, Murakami T, Inubushi T, Yamashiro T, Yamamoto S, Akiyama H, Kawaguchi M, Sakata N, Nishimura R. Zfhx4 regulates endochondral ossification as the transcriptional platform of Osterix in mice. Commun Biol. 2021, 4:1258. DOI: 10.1038/s42003-021-02793-9.
6. Takigawa Y, Hata K, Muramatsu S, Amano K, Ono K, Wakabayashi M, Matsuda A, Takada K, Nishimura R, Yoneda T.　The transcription factor Znf219 regulates chondrocyte differentiation by assembling a transcription factory with Sox9. J Cell Sci. 2010, 123:3780-8.
7. Amano K, Hata K, Sugita A, Takigawa Y, Ono K, Wakabayashi M, Kogo M, Nishimura R, Yoneda T. Sox9 family members negatively regulate maturation and calcification of chondrocytes through up-regulation of parathyroid hormone-related protein. Mol Biol Cell. 2009, 20: 4541-51.
8. Hata K, Nishimura R, Muramatsu S, Matsuda A, Matsubara T, Amano K, Ikeda F, Harley VR, Yoneda T. Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice. J Clin Invest. 2008, 118: 3098-108.
9. Amano K, Ichida F, Sugita A, Hata K, Wada M, Takigawa Y, Nakanishi M, Kogo M, Nishimura R, Yoneda T. MSX2 stimulates chondrocyte maturation by controlling Ihh expression. J Biol Chem. 2008, 283: 29513-21.

２．Molecular pathogenesis of osteoarthritis and rheumatoid arthritis and establishment of novel therapeutic strategies

　Osteoarthritis and rheumatoid arthritis are estimated to affect 20-30 million and 1 million patients, respectively. Although the causes of osteoarthritis and rheumatoid arthritis are different, both are intractable diseases that result in the destruction of articular cartilage. In order to develop therapeutic strategies for these joint diseases, it is necessary to understand the pathogenesis of the diseases as well as the development and differentiation of articular cartilage. Articular cartilage is permanent cartilage that does not hypertrophy or calcify, a characteristic feature that is very different from growth cartilage, which is involved in endochondral bone formation. We are using genome editing technology to identify transcription factors and signaling molecules involved in articular chondrocyte development and differentiation. We are also investigating the relationship between the inflammation and rheumatoid arthritis. We are also investigating the possible involvement of the transcription factor Arid5a in the pathogenesis of rheumatoid arthritis. In particular, we have recently applied genome editing technology to construct a high-throughput screening system and are working on drug development to promote regeneration or repair of articular cartilage. Furthermore, we are focusing on the development of tissue regeneration therapies using direct programming methods with transcription factors identified by gene cloning methods such as RNA-seq, Microarray, scRNA-seq, mass spectrometry and in vivo cloning approaches.
★ Related literatures
1. Murakami T, Takahata Y, Hata K, Ebina K, Hirose K, Ruengsinpinya L, Nakaminami Y, Yuki Etani Y, Kobayashi S, Maruyama T, Nakano H, Kaneko T, Toyosawa S, Asahara H, Nishimura R. Semaphorin 4D induces articular cartilage destruction and inflammation in joints by transcriptionally reprogramming chondrocytes. (2022) Science Signaling 15 (758) eabl5304. doi: 10.1126/scisignal.abl5304.

2. Takahata Y, Nakamura E, Hata K, Wakabayashi M, Murakami T, Waqkamori K, Yoshikawa H, Matsuda A, Fukui N, Nishimura R. Sox4 is involved in osteoarthritic cartilage deterioration through induction of ADAMTS4 and ADAMTS5. FASEB J 2019, 33: 619-630.
3. Tanaka J, Ogawa M , Hojo H, Kawashima Y, Mabuchi Y, Hata K, Nakamura S, Yasuhara R, Takamatsu K, Irié T, Fukada T, Sakai S, Inoue T, Nishimura R, Prof, Ohara O, Saito I, Ohba S, Tsuji T, Mishima K. Generation of orthotopically functional salivary gland from embryonic stem cells. Nature Commun 2018, 9: 4216 doi.org/10.1038/s41467-018-06469-7
4. Masuda K, Ripley B, Nishimura R, Mino T, Takeuchi O, Shioi G, Kiyonari H, Kishimoto T. Arid5a controls IL-6 mRNA stability, which contributes to elevation of IL-6 level in vivo. Proc Natl Acad Sci USA. 2013, 110: 9409-14.
5. Amano K, Hata K, Muramatsu S, Wakabayashi M, Takigawa Y, Ono K, Nakanishi M, Takashima R, Kogo M, Matsuda A, Nishimura R, Yoneda T. Arid5a cooperates with Sox9 to stimulate chondrocyte-specific transcription. Mol Biol Cell. 2011, 22: 1300-11.

３．Molecular mechanisms of osteoblast development

　To understand membranous bone formation, it is necessary to understand the differentiation mechanism of osteoblasts from undifferentiated mesenchymal stem cells. We have succeeded in identifying and analyzing the function of Smad5 as a signaling molecule of the osteogenic factor BMP2 for the first time in the world, and have clarified the involvement of Runx2 and Osterix as transcription factors that function downstream of Smad molecules. Furthermore, we have identified the target molecules of Runx2 and Osterix, generated knockout mice of the candidate molecules, and are now analyzing the functions of these target molecules. In addition to BMP2, we are also investigating the intracellular signaling mechanisms of cytokines Ihh and Wnt, which have osteogenic effects.
★ Related literatures
1. Takahata Y, Hagino H, Kimura A, Urushizaki M, Kobayashi S, Wakamori K, Fujiwara C, Nakamura E, Yu K, Kiyonari H, Bando K, Murakami T, Komori T, Hata K, Nishimura R. Smoc1 and Smoc2 regulate bone formation as downstream molecules of Runx2. Commun Biol. 2021, 4:1199. DOI: 10.1038/s42003-021-02717-7.

2. Kida J, Hata K, Nakamura E, Yagi H, Takahata Y, Murakami T, Maeda Y, Nishimura R. Interaction of LEF1 with TAZ is necessary for the osteoblastogenic activity of Wnt3a. Sci Rep 2018, 8 :10375. doi: 10.1038/s41598-018-28711-42.
3. Kawane T, Qin X, Jiang Q, Miyazaki T, Komori H, Yoshida CA, dos Santos Matsuura-Kawata VK, Sakane C, Matsuo Y, Nagai K, Maeno T, Date Y, Nishimura R, Komori T. Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation by regulating Fgfr2 and Fgfr3. Sci Rep 2018, 8: doi: 10.1038/s41598-018-31853-0.10.1038/s41598-018-31853-01.
4. Chudnovsky Y, Kim D, Zheng S, Whyte WA, Bansal M, Bray MA, Gopal S, Theisen MA, Bilodeau S, Thiru P, Muffat J, Yilmaz OH, Mitalipova M, Woolard K, Lee J, Nishimura R, Sakata N, Fine HA, Carpenter AE, Silver SJ, Verhaak RG, Califano A, Young RA, Ligon KL, Mellinghoff IK, Root DE, Sabatini DM, Hahn WC, Chheda MG. ZFHX4 interacts with the NuRD core member CHD4 and regulates the glioblastoma tumor-initiating cell state. Cell Reports 2014, 6: 313-24.
5. Matsubara T, Kida K, Yamaguchi A, Hata K, Ichida F, Meguro H, Aburatani H, Nishimura R, Yoneda T. BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation.　J Biol Chem. 2008, 283: 29119-25
6. Shimoyama A, Wada M, Ikeda F, Hata K, Matsubara T, Nifuji A, Noda M, Amano K, Yamaguchi A, Nishimura R, Yoneda T. Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2 expression and function. Mol Biol Cell. 2007,18: 2411-8.

7. Matsubara T, Kida K, Yamaguchi A, Hata K, Ichida F, Meguro H, Aburatani H, Nishimura R, Yoneda T. BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation. J Biol Chem. 2008, 283:29119-25.

４．Molecular mechanism and role of inflammasome and endoplasmic reticulum stress in chronic inflammation and its relationship to bone and joint diseases

　Chronic inflammation and endoplasmic reticulum stress are now known to be involved in the pathogenesis of diseases such as bone and cartilage diseases, obesity, diabetes, atherosclerosis, neurodegenerative diseases, and cancer. However, the molecular mechanisms by which chronic inflammation and endoplasmic reticulum stress are involved in pathogenesis are not well understood. Recently, the involvement of a protein complex called inflammasome in the pathogenesis of chronic inflammation has attracted much attention. The inflammasome is formed in response to infection or various abiotic stresses by the complex of NOD-like receptors (NLRs), adaptor proteins (ASCs), and Caspase-1. This induces continued secretion of proinflammatory cytokines (IL-β, IL-18) and cell death (pyroptosis). While it has been suggested that inflammasomes are involved in the pathogenesis of various diseases, the molecular mechanisms by which inflammasomes are activated and the molecular pathways by which they are involved in pathogenesis are still unknown. We expect that clarifying the molecular mechanisms and roles of inflammasomes in pathogenesis will lead to the elucidation of the pathogenesis mechanisms of various chronic inflammatory diseases such as periodontal disease and rheumatoid arthritis caused by chronic inflammation, and will greatly contribute to the development of novel therapeutic strategies for these diseases.
★ Related literatures
1. Murakami T, Ruengsinpinya L, Nakamura E, Takahata Y, Hata K, Okae H, Taniguchi S, Takahashi M, Nishimura R. G protein subunit beta 1 negatively regulates NLRP3 inflammasome activation. J Immunol (Cutting Edge) 2019, 202: 1942-1947.
2. Yu J, Nagasu H, Murakami T, Hoang H, Broderick L, Hoffman HM, Horng T. Inflammasome activation leads to Caspase-1-dependent mitochondrial damage and block of mitophagy. Proc Natl Acad Sci USA. 2014, 111:15514-15519
3. Murakami T, Ockinger J, Yu J, Byles V, McColl A, Hofer AM, Horng T. Critical role for calcium mobilization in activation of the NLRP3 inflammasome. Proc Natl Acad Sci USA. 2012, 109: 11282-7.
4. Murakami T, Saito A, Hino S, Kondo S, Kanemoto S, Chihara K, Sekiya H, Tsumagari K, Ochiai K, Yoshinaga K, Saitoh M, Nishimura R, Yoneda T, Kou I, Furuichi T, Ikegawa S, Ikawa M, Okabe M, Wanaka A, Imaizumi K. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation. Nature Cell Biol. 2009, 11: 1205-11.
5. Saito A, Hino S, Murakami T, Kanemoto S, Kondo S, Saitoh M, Nishimura R, Yoneda T, Furuichi T, Ikegawa S, Ikawa M, Okabe M, Imaizumi K. Regulation of endoplasmic reticulum stress response by a BBF2H7-mediated Sec23a pathway is essential for chondrogenesis. Nature Cell Biol. 2009, 11: 1197-204.

５. Intracellular signaling and transcription factors in osteoclast differentiation and bone destruction

　Not only osteoporosis and periodontal disease, but also many other bone diseases are caused by progressive bone destruction by osteoclasts. In order to establish effective therapies for these diseases, it is essential to elucidate the differentiation mechanism of osteoclasts and the bone resorption mechanism. We have discovered that c-Jun, a transcription factor activated by the osteoclastogenic factor, RANKL, is essential for osteoclast formation. We have also succeeded in identifying NFAT2/NFATc1 as a transcriptional partner of c-Jun. On the other hand, to clarify the mechanism of bone resorption by osteoclasts, we are also studying the regulatory mechanism of tyrosine kinase, c-Src, which is essential for the bone resorption function of osteoclasts.
★ Related literatures
1. DeSelm CJ, Miller BC, Zou W, Beatty WL, van Meel E, Takahata Y, Klumperman J, Tooze SA, Teitelbaum SL, Virgin HW. Autophagy proteins regulate the secretory component of osteoclastic bone resorption. Dev Cell. 2011, 21: 966-74.
2. Matsubara T, Ikeda F, Hata K, Nakanishi M, Okada M, Yasuda H, Nishimura R, Yoneda T. Cbp recruitment of Csk into lipid rafts is critical to c-Src kinase activity and bone resorption in osteoclasts. J Bone Miner Res. 2010, 25: 1068-76
3. Ikeda F, Matsubara T, Tsurukai T, Hata K, Nishimura R, Yoneda T. JNK/c-Jun signaling mediates an anti-apoptotic effect of RANKL in osteoclasts. J Bone Miner Res. 2008, 23: 907-14.
Ikeda F, Nishimura R, Matsubara T, Hata K, Reddy SV, Yoneda T. Activation of NFAT signal in vivo leads to osteopenia associated with increased osteoclastogenesis and bone-resorbing activity. J Immunol. 2006, 177: 2384-90.
4. Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, Hata K, Yamashita K, Hiraga T, Watanabe T, Kukita T, Yoshioka K, Rao A, Yoneda T. Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J Clin Invest. 2004, 114: 475-84.

６. Reciprocal differentiation of osteoblasts and adipocytes from mesenchymal stem cells

　It has been well-known that adipocytes in bone marrow increase markedly with aging and the progression of osteoporosis. Adipocytes, like osteoblasts, are derived from undifferentiated mesenchymal stem cells. Therefore, the pathogenesis of osteoporosis and other diseases that cause bone loss may be affected not only by an imbalance between bone formation and resorption, but also by an imbalance in the differentiation of osteoblasts and adipocytes from undifferentiated mesenchymal stem cells. Based on these facts, our laboratory is investigating how the balance of differentiation from undifferentiated mesenchymal stem cells to osteoblasts and adipocytes is determined. As a result, we have shown that transcription factors Msx2 and LIP function as switching molecules in the differentiation of undifferentiated mesenchymal stem cells.
★ Related literatures
1. Hata K, Nishimura R, Ueda M, Ikeda F, Matsubara T, Ichida F, Hisada K, Nokubi T, Yamaguchi A, Yoneda T. A CCAAT/enhancer binding protein beta isoform, liver-enriched inhibitory protein, regulates commitment of osteoblasts and adipocytes. Mol Cell Biol. 2005, 25: 1971-9.
2. Ichida F, Nishimura R, Hata K, Matsubara T, Ikeda F, Hisada K, Yatani H, Cao X, Komori T, Yamaguchi A, Yoneda T. Reciprocal roles of MSX2 in regulation of osteoblast and adipocyte differentiation. J Biol Chem. 2004, 279: 34015-22.
3. Hata K, Nishimura R, Ikeda F, Yamashita K, Matsubara T, Nokubi T, Yoneda T. Differential roles of Smad1 and p38 kinase in regulation of peroxisome proliferator-activating receptor gamma during bone morphogenetic protein 2-induced adipogenesis.　Mol Biol Cell. 2003, 14: 545-55.

７．Molecular mechanism of bone metastases of malignancy

　Bone provides a different microenvironment for cancer metastases than other organs because of its constant dynamic remodeling. For example, growth factors such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-β (TGF-β), which are abundant in bone tissue, are released during bone destruction, and cancer cells promote secretion of PTHrP and other bone resorbing factors from bone tissues. In addition, the bone microenvironment has been postulated to induce changes in cellular characteristics such as cancer cell proliferation, chemotherapy resistance, and cancer dormancy. In our laboratory, we have shown that various bone microenvironment factors are involved in the establishment and pathogenesis of bone metastasis. Recently, we have established a gene screening system using a mouse model of bone metastasis in order to elucidate changes in cellular characteristics of metastatic cancer cells induced by the bone microenvironment at the molecular level. Through these studies, we hope to ultimately contribute to the discovery of molecular therapeutic targets and the development of therapeutic strategies for bone metastasis based on molecular mechanisms. We are also developing cancer cell lines that metastasize frequently to the lymph nodes of oral cancer patients and animal models for analysis. We are also studying the mechanism of lymph node metastasis of oral cancer and hope to contribute to the establishment of new treatment strategies and development of diagnostic methods for oral cancer.
★ Related literatures
1. Ishihara S, Hata K, Hirose K, Okui T, Toyosawa S, Nishimura R, Uzawa N, Yoneda T.  The lactate sensor GPR81 regulates glycolysis and tumor growth of breast cancer. Sci Rep 2022, 12: 6261 doi.org/10.1038/s41598-022-10143-w

２. Hiraga T, Myoui A, Hashimoto N, Sasaki A, Hata K, Morita Y, Yoshikawa H, Rosen CJ, Mundy GR, Yoneda T. Bone-derived IGF mediates crosstalk between bone and breast cancer cells in bony metastases. Cancer Res. 2012, 72: 4238-49.
３. Nishisho T, Hata K, Nakanishi M, Morita Y, Sun-Wada GH, Wada Y, Yasui N, Yoneda T.The a3 isoform vacuolar type H⁺-ATPase promotes distant metastasis in the mouse B16 melanoma cells. Mol Cancer Res. 2011, 9: 845-55.