探討小鼠骨骼肌中FoxO6基因的特性

Forkhead box O (FoxO)轉錄因子成員包含FoxO1、FoxO3、FoxO4、FoxO6。已知其在細胞中調控著各種生理功能，如:細胞週期、葡萄糖代謝、DNA的修復、細胞的分化及凋亡等。FoxO家族中的FoxO6是近期才被發現，它不同於家族其他成員缺少了C端保守的PKB磷酸位，這使得他的進出細胞核的能力受到影響，因此相較其它成員它偏好留在細胞核內。目前已知FoxO6主要表現在大腦組織，但在肌肉組織中也發現到FoxO6的表現，過去文獻報導指出FoxO3會去活化Atrogen-1與MuRF1等ubiquitin ligase的表現，進而促使肌肉走向萎縮，由於目前對FoxO6在骨骼肌中所扮演的角色尚未釐清，因此我們想探討FoxO6對骨骼肌的影響，其中在小鼠後腿缺血的實驗中，我們觀察到FoxO6在缺血的狀態下蛋白質的表現量會上升。另外在C2C12肌纖維母細胞FoxO6 knock down細胞株中，發現到FoxO6遭受到抑制時Atrogen-1及MuRF1基因的表現量上升，這與FoxO家族其它成員似乎是相反的結果。另外我們想藉由Tet Off系統，探討當FoxO6被大量表達時對肌纖維母細胞的影響，所以我們目標建立一株可以藉由Tetracycline(Tet)調控FoxO6表達的細胞株。另一方面，由Schmidt團隊公布的FoxO6 CDS與IMAGE clone的CDS，其FoxO6蛋白質大小分別為559胺基酸及640胺基酸，但這與我們所觀察到的大小有所差異，所以我們合理的懷疑FoxO6在5’UTR，是否存在一段未曾被揭露的轉錄起始序列。我們從FoxO6 5’RACE的實驗中成功地找到FoxO6新的轉錄起始序列。未來我們希望能從肌纖維母細胞中，藉由大量表達FoxO6及抑制FoxO6的方式，分別探討對肌肉細胞代謝基因的影響及肌肉細胞分化情形。

Characterizing FoxO6 gene in mouse skeletal muscle

Forkhead box O (FoxO) transcription factor family that membrane, including FoxO1, FoxO3, FoxO4 and FoxO6 are they crucial for the regulation of metabolism, cell cycle, cell death, and cell survival. Among these FoxOs, the recently discovered FoxO6 is differs from other by lacking C-terminal PKB sites and which impairs its subcellular shuttling ability. FoxO6 is not only majorly expressed in the tissue of brain, but also expressed in the muscle. In the past, the reported that FoxO could activated Atrogen-1and MuRF1, which ubiquitin ligase expressed lead to muscle atrophy. However, the function of FoxO6 in skeletal muscle and the subcellular and fiber-type specific localization during myogenesis in vitro and in vivo are largely unknown. In addition, whether FoxO6 participates in muscle ischemia has not been reported before. We find out FoxO6 protein has higly expression in the muscle ischemia. In addition, we also discover both of the Atrogen-1 and MuRF1 are higly expression in the C2C12 FoxO6 knock down cell line. This result is obvisouly different to other FoxO members. On the other hand, we want to set up a Tet Off FoxO6 cell line system that could swithch gene on and off by tetracycline. In another aspect the size of FoxO6 is 559 and 640 amino acids that FoxO6 has reported by team of Schmidt and IMAGE clone. But the size of FoxO6 is different from them by our observating. So we guess the pass reported sequence that is not FoxO6 whole sequence. It may have another undiscover sequenc in FoxO6 5’ UTR. We successful to discover a new FoxO6 sequence with transcription start site. In the future we hope to clarify the character of FoxO6 in the muscle cell by overexpression and knock its expression.

Anderson, M.J., Viars, C.S., Czekay, S., Cavenee, W.K., and Arden, K.C. (1998). Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics 47, 187-199.
Andersson, U., and Scarpulla, R.C. (2001). Pgc-1-related coactivator, a novel, serum-inducible coactivator of nuclear respiratory factor 1-dependent transcription in mammalian cells. Molecular and cellular biology 21, 3738-3749.
Brault, J.J., Jespersen, J.G., and Goldberg, A.L. (2010). Peroxisome proliferator-activated receptor gamma coactivator 1alpha or 1beta overexpression inhibits muscle protein degradation, induction of ubiquitin ligases, and disuse atrophy. The Journal of biological chemistry 285, 19460-19471.
Bulfield, G., Siller, W.G., Wight, P.A., and Moore, K.J. (1984). X chromosome-linked muscular dystrophy (mdx) in the mouse. Proceedings of the National Academy of Sciences of the United States of America 81, 1189-1192.
Chamberlain, J.S., Metzger, J., Reyes, M., Townsend, D., and Faulkner, J.A. (2007). Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 21, 2195-2204.
Clark, K.L., Halay, E.D., Lai, E., and Burley, S.K. (1993). Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature 364, 412-420.
Finck, B.N., and Kelly, D.P. (2006). PGC-1 coactivators: inducible regulators of energy metabolism in health and disease. The Journal of clinical investigation 116, 615-622.
Galili, N., Davis, R.J., Fredericks, W.J., Mukhopadhyay, S., Rauscher, F.J., 3rd, Emanuel, B.S., Rovera, G., and Barr, F.G. (1993). Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nature genetics 5, 230-235.
Greer, E.L., and Brunet, A. (2005). FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24, 7410-7425.
Guerfali, I., Manissolle, C., Durieux, A.C., Bonnefoy, R., Bartegi, A., and Freyssenet, D. (2007). Calcineurin A and CaMKIV transactivate PGC-1alpha promoter, but differentially regulate cytochrome c promoter in rat skeletal muscle. Pflugers Archiv : European journal of physiology 454, 297-305.
Handschin, C., Rhee, J., Lin, J., Tarr, P.T., and Spiegelman, B.M. (2003). An autoregulatory loop controls peroxisome proliferator-activated receptor gamma coactivator 1alpha expression in muscle. Proceedings of the National Academy of Sciences of the United States of America 100, 7111-7116.
Hribal, M.L., Nakae, J., Kitamura, T., Shutter, J.R., and Accili, D. (2003). Regulation of insulin-like growth factor-dependent myoblast differentiation by Foxo forkhead transcription factors. The Journal of cell biology 162, 535-541.
Ikeya, M., and Takada, S. (1998). Wnt signaling from the dorsal neural tube is required for the formation of the medial dermomyotome. Development (Cambridge, England) 125, 4969-4976.
Jacobs, F.M., van der Heide, L.P., Wijchers, P.J., Burbach, J.P., Hoekman, M.F., and Smidt, M.P. (2003). FoxO6, a novel member of the FoxO class of transcription factors with distinct shuttling dynamics. The Journal of biological chemistry 278, 35959-35967.
Johnson, R.L., Laufer, E., Riddle, R.D., and Tabin, C. (1994). Ectopic expression of Sonic hedgehog alters dorsal-ventral patterning of somites. Cell 79, 1165-1173.
Kamei, Y., Miura, S., Suzuki, M., Kai, Y., Mizukami, J., Taniguchi, T., Mochida, K., Hata, T., Matsuda, J., Aburatani, H., et al. (2004). Skeletal muscle FOXO1 (FKHR) transgenic mice have less skeletal muscle mass, down-regulated Type I (slow twitch/red muscle) fiber genes, and impaired glycemic control. The Journal of biological chemistry 279, 41114-41123.
Kitamura, T., Kitamura, Y.I., Funahashi, Y., Shawber, C.J., Castrillon, D.H., Kollipara, R., DePinho, R.A., Kitajewski, J., and Accili, D. (2007). A Foxo/Notch pathway controls myogenic differentiation and fiber type specification. The Journal of clinical investigation 117, 2477-2485.
Knutti, D., Kressler, D., and Kralli, A. (2001). Regulation of the transcriptional coactivator PGC-1 via MAPK-sensitive interaction with a repressor. Proceedings of the National Academy of Sciences of the United States of America 98, 9713-9718.
Lara-Pezzi, E., Winn, N., Paul, A., McCullagh, K., Slominsky, E., Santini, M.P., Mourkioti, F., Sarathchandra, P., Fukushima, S., Suzuki, K., et al. (2007). A naturally occurring calcineurin variant inhibits FoxO activity and enhances skeletal muscle regeneration. The Journal of cell biology 179, 1205-1218.
Lees, S.J., Childs, T.E., and Booth, F.W. (2008). Age-dependent FOXO regulation of p27Kip1 expression via a conserved binding motif in rat muscle precursor cells. American journal of physiology Cell physiology 295, C1238-1246.
Leone, T.C., Lehman, J.J., Finck, B.N., Schaeffer, P.J., Wende, A.R., Boudina, S., Courtois, M., Wozniak, D.F., Sambandam, N., Bernal-Mizrachi, C., et al. (2005). PGC-1alpha deficiency causes multi-system energy metabolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis. PLoS biology 3, e101.
Liu, Z.P., Wang, Z., Yanagisawa, H., and Olson, E.N. (2005). Phenotypic modulation of smooth muscle cells through interaction of Foxo4 and myocardin. Developmental cell 9, 261-270.
Matsuzaki, H., Daitoku, H., Hatta, M., Tanaka, K., and Fukamizu, A. (2003). Insulin-induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation. Proceedings of the National Academy of Sciences of the United States of America 100, 11285-11290.
Musch, B.C., Papapetropoulos, T.A., McQueen, D.A., Hudgson, P., and Weightman, D. (1975). A comparison of the structure of small blood vessels in normal, denervated and dystrophic human muscle. Journal of the neurological sciences 26, 221-234.
Nakae, J., Park, B.C., and Accili, D. (1999). Insulin stimulates phosphorylation of the forkhead transcription factor FKHR on serine 253 through a Wortmannin-sensitive pathway. The Journal of biological chemistry 274, 15982-15985.
Nakatani, T., Nakashima, T., Kita, T., Hirofuji, C., Itoh, K., Itoh, M., and Ishihara, A. (1999). Succinate dehydrogenase activities of fibers in the rat extensor digitorum longus, soleus, and cardiac muscles. Archives of histology and cytology 62, 393-399.
Ordahl, C.P., and Le Douarin, N.M. (1992). Two myogenic lineages within the developing somite. Development (Cambridge, England) 114, 339-353.
Pourquie, O., Fan, C.M., Coltey, M., Hirsinger, E., Watanabe, Y., Breant, C., Francis-West, P., Brickell, P., Tessier-Lavigne, M., and Le Douarin, N.M. (1996). Lateral and axial signals involved in avian somite patterning: a role for BMP4. Cell 84, 461-471.
Puigserver, P., Adelmant, G., Wu, Z., Fan, M., Xu, J., O'Malley, B., and Spiegelman, B.M. (1999). Activation of PPARgamma coactivator-1 through transcription factor docking. Science (New York, NY) 286, 1368-1371.
Rena, G., Woods, Y.L., Prescott, A.R., Peggie, M., Unterman, T.G., Williams, M.R., and Cohen, P. (2002). Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion. The EMBO journal 21, 2263-2271.
Roberts, R.G. (2001). Dystrophins and dystrobrevins. Genome biology 2, REVIEWS3006.
Sadana, P., and Park, E.A. (2007). Characterization of the transactivation domain in the peroxisome-proliferator-activated receptor gamma co-activator (PGC-1). The Biochemical journal 403, 511-518.
Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Picard, A., Walsh, K., Schiaffino, S., Lecker, S.H., and Goldberg, A.L. (2004). Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117, 399-412.
Sienko Thomas, S., Buckon, C.E., Nicorici, A., Bagley, A., McDonald, C.M., and Sussman, M.D. (2010). Classification of the gait patterns of boys with Duchenne muscular dystrophy and their relationship to function. Journal of child neurology 25, 1103-1109.
Stern, H.M., Brown, A.M., and Hauschka, S.D. (1995). Myogenesis in paraxial mesoderm: preferential induction by dorsal neural tube and by cells expressing Wnt-1. Development (Cambridge, England) 121, 3675-3686.
Suzuki, N., Motohashi, N., Uezumi, A., Fukada, S., Yoshimura, T., Itoyama, Y., Aoki, M., Miyagoe-Suzuki, Y., and Takeda, S. (2007). NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS. The Journal of clinical investigation 117, 2468-2476.
van der Heide, L.P., Jacobs, F.M., Burbach, J.P., Hoekman, M.F., and Smidt, M.P. (2005). FoxO6 transcriptional activity is regulated by Thr26 and Ser184, independent of nucleo-cytoplasmic shuttling. The Biochemical journal 391, 623-629.
Wallace, G.Q., and McNally, E.M. (2009). Mechanisms of muscle degeneration, regeneration, and repair in the muscular dystrophies. Annual review of physiology 71, 37-57.
Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A., Cinti, S., Lowell, B., Scarpulla, R.C., et al. (1999). Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98, 115-124.
Yun, K., and Wold, B. (1996). Skeletal muscle determination and differentiation: story of a core regulatory network and its context. Current opinion in cell biology 8, 877-889.