Ad4BP/SF-1稱為腎上腺結合蛋白4或是類固醇合成因子1，是一種轉錄因子，其中主要會影響到類固醇生合成相關器官的生長及發育，以及調控類固醇生合成作用及醣解作用相關基因的表現。在先前的研究中發現，當類固醇生合成細胞受到環境中的壓力時，會透過活化溶酶體而保護類固醇生合成細胞不走向細胞凋亡；除此之外，也發現溶酶體在進行類固醇生合成作用中扮演著重要的角色。然而，目前依然不清楚溶酶體是如何影響類固醇生合成細胞的生長，因此我們想探討溶酶體的活性，是如何影響到類固醇生合成細胞的生長。在我們的研究中發現，溶酶體會透過調控Ad4BP/SF-1蛋白的穩定性，而影響到類固醇生合成細胞的生長。其中，當我們抑制溶酶體的活性時，我們發現會抑制細胞的生長，並且發現自噬作用並不會影響細胞的生長，因此可以知道，溶酶體的活性才是影響類固醇生合成細胞生長的主因。接下來我們去探討溶酶體的活性如何影響類固醇生合成細胞的生長，而我們發現，在抑制溶酶體的活性時，則會降低Ad4BP/SF-1蛋白的穩定性，而導致G1期的停滯以及醣解作用相關基因表現的減少影響細胞的生長；並且我們也發現，Ad4BP/SF-1會隨著細胞週期而調控Ccne 1基因的表現，當細胞進入G1/S轉換期，Ad4BP/SF-1會結合到Ccne 1的啟動子而表現Ccne 1，使細胞能順利進入S期影響類固醇生合成細胞的生長；除此之外，我們也在斑馬魚胚胎中抑制溶酶體活性，可以發現腎上線生長會被抑制，而過度表現FF1b後，斑馬魚的腎上線生長則有回復的現象，因此證明在斑馬魚中溶酶體會透過調控FF1b而影響類固生合成器官的生長。藉由以上的結果，我們證明溶酶體會透過調控Ad4BP/SF-1的穩定性而影響生物的類固醇生合成細胞的生長。

The development and differentiation of steroidogenic organs are controlled by Ad4BP/SF-1 (adrenal 4 binding protein/steroidogenic factor 1). Besides, lysosomal activity is required for steroidogenesis and also enables adrenocortical cell to survive during stress. However, the role of lysosomal activity on steroidogenic cell growth is as yet unknown. Here, we showed that lysosomal activity maintained Ad4BP/SF-1 protein stability for proper steroidogenic cell growth. Treatment of cells with lysosomal inhibitors reduced steroidogenic cell growth in vitro. Suppression of autophagy did not affect cell growth indicating that autophagy was dispensable for steroidogenic cell growth. When lysosomal activity was inhibited, the protein stability of Ad4BP/SF-1 was reduced leading to G1 arrest. Interestingly, treatment of cell with lysosomal inhibitor reduced glycolytic genes expression and supplying the cells with pyruvate alleviated the growth defect. ChIP-sequence/ChIP studies indicated that Ad4BP/SF-1 binds to the upstream region of Ccne1 (cyclin E1) gene during G1/S phase. In addition, treatment of zebrafish embryo with lysosomal inhibitor reduced the growth of the interrenal (adrenal) gland in vivo. Thus lysosomal activity maintains steroidogenic cell growth via stabilizing Ad4BP/SF-1.

Baba, T., Otake, H., Sato, T., Miyabayashi, K., Shishido, Y., Wang, C.Y., Shima, Y., Kimura, H., Yagi, M., Ishihara, Y., et al. (2014). Glycolytic genes are targets of the nuclear receptor Ad4BP/SF-1. Nature communications 5, 3634.
Bangs, F.K., Schrode, N., Hadjantonakis, A.K., and Anderson, K.V. (2015). Lineage specificity of primary cilia in the mouse embryo. Nature cell biology 17, 113-122.
Basciani, S., Mariani, S., Spera, G., and Gnessi, L. (2010). Role of platelet-derived growth factors in the testis. Endocrine reviews 31, 916-939.
Botrugno, O.A., Fayard, E., Annicotte, J.S., Haby, C., Brennan, T., Wendling, O., Tanaka, T., Kodama, T., Thomas, W., Auwerx, J., et al. (2004). Synergy between LRH-1 and beta-catenin induces G1 cyclin-mediated cell proliferation. Molecular cell 15, 499-509.
Brennan, J., Tilmann, C., and Capel, B. (2003). Pdgfr-alpha mediates testis cord organization and fetal Leydig cell development in the XY gonad. Genes & development 17, 800-810.
Chai, C., Liu, Y.W., and Chan, W.K. (2003). Ff1b is required for the development of steroidogenic component of the zebrafish interrenal organ. Developmental biology 260, 226-244.
Chen, T.Y., Syu, J.S., Han, T.Y., Cheng, H.L., Lu, F.I., and Wang, C.Y. (2015a). Cell Cycle-Dependent Localization of Dynactin Subunit p150 glued at Centrosome. Journal of cellular biochemistry 116, 2049-2060.
Chen, T.Y., Syu, J.S., Lin, T.C., Cheng, H.L., Lu, F.L., and Wang, C.Y. (2015b). Chloroquine alleviates etoposide-induced centrosome amplification by inhibiting CDK2 in adrenocortical tumor cells. Oncogenesis 4, e180.
Chen, W.Y., Juan, L.J., and Chung, B.C. (2005). SF-1 (nuclear receptor 5A1) activity is activated by cyclic AMP via p300-mediated recruitment to active foci, acetylation, and increased DNA binding. Molecular and cellular biology 25, 10442-10453.
Chen, W.Y., Lee, W.C., Hsu, N.C., Huang, F., and Chung, B.C. (2004). SUMO modification of repression domains modulates function of nuclear receptor 5A1 (steroidogenic factor-1). The Journal of biological chemistry 279, 38730-38735.
Chen, W.Y., Weng, J.H., Huang, C.C., and Chung, B.C. (2007). Histone deacetylase inhibitors reduce steroidogenesis through SCF-mediated ubiquitination and degradation of steroidogenic factor 1 (NR5A1). Molecular and cellular biology 27, 7284-7290.
Erickson, L.A., Rivera, M., and Zhang, J. (2014). Adrenocortical carcinoma: review and update. Advances in anatomic pathology 21, 151-159.
Gawriluk, T.R., Ko, C., Hong, X., Christenson, L.K., and Rucker, E.B., 3rd (2014). Beclin-1 deficiency in the murine ovary results in the reduction of progesterone production to promote preterm labor. Proceedings of the National Academy of Sciences of the United States of America 111, E4194-4203.
Gawriluk, T.R., and Rucker, E.B. (2015). BECN1, corpus luteum function, and preterm labor. Autophagy 11, 183-184.
Gnessi, L., Basciani, S., Mariani, S., Arizzi, M., Spera, G., Wang, C., Bondjers, C., Karlsson, L., and Betsholtz, C. (2000). Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient mice. The Journal of cell biology 149, 1019-1026.
Goetz, S.C., and Anderson, K.V. (2010). The primary cilium: a signalling centre during vertebrate development. Nature reviews Genetics 11, 331-344.
Goldstein, J.L., Brunschede, G.Y., and Brown, M.S. (1975). Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339. The Journal of biological chemistry 250, 7854-7862.
Gu, Y., Rosenblatt, J., and Morgan, D.O. (1992). Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. The EMBO journal 11, 3995-4005.
Hammer, G.D., Krylova, I., Zhang, Y., Darimont, B.D., Simpson, K., Weigel, N.L., and Ingraham, H.A. (1999). Phosphorylation of the nuclear receptor SF-1 modulates cofactor recruitment: integration of hormone signaling in reproduction and stress. Molecular cell 3, 521-526.
Hoivik, E.A., Lewis, A.E., Aumo, L., and Bakke, M. (2010). Molecular aspects of steroidogenic factor 1 (SF-1). Molecular and cellular endocrinology 315, 27-39.
Hoyer-Hansen, M., and Jaattela, M. (2008). Autophagy: an emerging target for cancer therapy. Autophagy 4, 574-580.
Hsu, H.J., Hsu, N.C., Hu, M.C., and Chung, B.C. (2006). Steroidogenesis in zebrafish and mouse models. Molecular and cellular endocrinology 248, 160-163.
Jacob, A.L., Lund, J., Martinez, P., and Hedin, L. (2001). Acetylation of steroidogenic factor 1 protein regulates its transcriptional activity and recruits the coactivator GCN5. The Journal of biological chemistry 276, 37659-37664.
Komatsu, T., Mizusaki, H., Mukai, T., Ogawa, H., Baba, D., Shirakawa, M., Hatakeyama, S., Nakayama, K.I., Yamamoto, H., Kikuchi, A., et al. (2004). Small ubiquitin-like modifier 1 (SUMO-1) modification of the synergy control motif of Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) regulates synergistic transcription between Ad4BP/SF-1 and Sox9. Molecular endocrinology 18, 2451-2462.
Kroemer, G., and Jaattela, M. (2005). Lysosomes and autophagy in cell death control. Nature reviews Cancer 5, 886-897.
Lai, M.S., Cheng, Y.S., Chen, P.R., Tsai, S.J., and Huang, B.M. (2014). Fibroblast growth factor 9 activates akt and MAPK pathways to stimulate steroidogenesis in mouse leydig cells. PloS one 9, e90243.
Lai, M.S., Wang, C.Y., Yang, S.H., Wu, C.C., Sun, H.S., Tsai, S.J., Chuang, J.I., Chen, Y.C., and Huang, B.M. (2016). The expression profiles of fibroblast growth factor 9 and its receptors in developing mice testes. Organogenesis 12, 61-77.
Lai, P.Y., Wang, C.Y., Chen, W.Y., Kao, Y.H., Tsai, H.M., Tachibana, T., Chang, W.C., and Chung, B.C. (2011). Steroidogenic Factor 1 (NR5A1) resides in centrosomes and maintains genomic stability by controlling centrosome homeostasis. Cell death and differentiation 18, 1836-1844.
Lee, M.B., Lebedeva, L.A., Suzawa, M., Wadekar, S.A., Desclozeaux, M., and Ingraham, H.A. (2005). The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification. Molecular and cellular biology 25, 1879-1890.
Lieberman, A.P., Puertollano, R., Raben, N., Slaugenhaupt, S., Walkley, S.U., and Ballabio, A. (2012). Autophagy in lysosomal storage disorders. Autophagy 8, 719-730.
Lin, Y.M., Tsai, C.C., Chung, C.L., Chen, P.R., Sun, H.S., Tsai, S.J., and Huang, B.M. (2010). Fibroblast growth factor 9 stimulates steroidogenesis in postnatal Leydig cells. International journal of andrology 33, 545-553.
Liu, Y.W. (2007). Interrenal organogenesis in the zebrafish model. Organogenesis 3, 44-48.
Lu, F.I., Sun, Y.H., Wei, C.Y., Thisse, C., and Thisse, B. (2014). Tissue-specific derepression of TCF/LEF controls the activity of the Wnt/beta-catenin pathway. Nature communications 5, 5368.
Luo, X., Ikeda, Y., and Parker, K.L. (1994). A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77, 481-490.
Luo, Z., Wijeweera, A., Oh, Y., Liou, Y.C., and Melamed, P. (2010). Pin1 facilitates the phosphorylation-dependent ubiquitination of SF-1 to regulate gonadotropin beta-subunit gene transcription. Molecular and cellular biology 30, 745-763.
Mitra, K., Wunder, C., Roysam, B., Lin, G., and Lippincott-Schwartz, J. (2009). A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase. Proceedings of the National Academy of Sciences of the United States of America 106, 11960-11965.
Morohashi, K., Zanger, U.M., Honda, S., Hara, M., Waterman, M.R., and Omura, T. (1993). Activation of CYP11A and CYP11B gene promoters by the steroidogenic cell-specific transcription factor, Ad4BP. Molecular endocrinology 7, 1196-1204.
Morohashi, K.I., and Omura, T. (1996). Ad4BP/SF-1, a transcription factor essential for the transcription of steroidogenic cytochrome P450 genes and for the establishment of the reproductive function. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 10, 1569-1577.
Murray, A.W. (2004). Recycling the cell cycle: cyclins revisited. Cell 116, 221-234.
Nygaard, M.B., Almstrup, K., Lindbaek, L., Christensen, S.T., and Svingen, T. (2015). Cell context-specific expression of primary cilia in the human testis and ciliary coordination of Hedgehog signalling in mouse Leydig cells. Scientific reports 5, 10364.
Omura, T., and Morohashi, K. (1995). Gene regulation of steroidogenesis. The Journal of steroid biochemistry and molecular biology 53, 19-25.
Savion, N., Laherty, R., Lui, G.M., and Gospodarowicz, D. (1981). Modulation of low density lipoprotein metabolism in bovine granulosa cells as a function of their steroidogenic activity. The Journal of biological chemistry 256, 12817-12822.
Schmahl, J., Rizzolo, K., and Soriano, P. (2008). The PDGF signaling pathway controls multiple steroid-producing lineages. Genes & development 22, 3255-3267.
Schneider, L., Clement, C.A., Teilmann, S.C., Pazour, G.J., Hoffmann, E.K., Satir, P., and Christensen, S.T. (2005). PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts. Current biology : CB 15, 1861-1866.
Settembre, C., Fraldi, A., Medina, D.L., and Ballabio, A. (2013). Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nature reviews Molecular cell biology 14, 283-296.
Shang, Y., Hu, X., DiRenzo, J., Lazar, M.A., and Brown, M. (2000). Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103, 843-852.
Tang, Z., Lin, M.G., Stowe, T.R., Chen, S., Zhu, M., Stearns, T., Franco, B., and Zhong, Q. (2013). Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites. Nature 502, 254-257.
Thisse, C., and Thisse, B. (2008). High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature protocols 3, 59-69.
Umberger, N.L., and Caspary, T. (2015). Ciliary transport regulates PDGF-AA/alphaalpha signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity. Molecular biology of the cell 26, 350-358.
Umesono, K., Murakami, K.K., Thompson, C.C., and Evans, R.M. (1991). Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell 65, 1255-1266.
Wang, C.Y., Chen, W.Y., Lai, P.Y., and Chung, B.C. (2013a). Distinct functions of steroidogenic factor-1 (NR5A1) in the nucleus and the centrosome. Molecular and cellular endocrinology 371, 148-153.
Wang, C.Y., Huang, E.Y., Huang, S.C., and Chung, B.C. (2015). DNA-PK/Chk2 induces centrosome amplification during prolonged replication stress. Oncogene 34, 1263-1269.
Wang, C.Y., Kao, Y.H., Lai, P.Y., Chen, W.Y., and Chung, B.C. (2013b). Steroidogenic factor 1 (NR5A1) maintains centrosome homeostasis in steroidogenic cells by restricting centrosomal DNA-dependent protein kinase activation. Molecular and cellular biology 33, 476-484.
Wang, C.Y., Lai, P.Y., Chen, T.Y., and Chung, B.C. (2014). NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and beta-catenin accumulation. Cell communication and signaling : CCS 12, 55.
Winnay, J.N., and Hammer, G.D. (2006). Adrenocorticotropic hormone-mediated signaling cascades coordinate a cyclic pattern of steroidogenic factor 1-dependent transcriptional activation. Molecular endocrinology 20, 147-166.
Zhang, J.Y., Wu, Y., Zhao, S., Liu, Z.X., Zeng, S.M., and Zhang, G.X. (2015). Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells. Theriogenology 84, 811-817.
Zhang, S., Wu, W., Wu, Y., Zheng, J., Suo, T., Tang, H., and Tang, J. (2010). RNF152, a novel lysosome localized E3 ligase with pro-apoptotic activities. Protein & cell 1, 656-663.