雄性素(Androgen)已被證實具有刺激造骨細胞的增生的作用。然而截至目前，雄性素刺激細胞增生的分子機制仍有待實驗釐清。在這篇研究中，我們證實雄性素是藉由活化PI(3)K/Akt訊號傳導路徑，進而達到刺激造骨細胞增生的效果。
我們首先在實驗中測試不同類固醇對活化Akt蛋白的能力‚發現只有睪固酮和雌性素與雄性素具有活化Akt蛋白能力，但皮質類固醇和黃體酮卻不能達此之效。當細胞受到雄性素刺激時，15 分鐘後能使Akt被活化的數量到達巔峰，而60分鐘後此Akt被活化的現象會回復到細胞基本量。這個現象並不會受到RNA合成抑制劑(Actinomycin D)而阻斷，但會被雄性素受體競爭抑制劑(Hydroxyflutamide, HF)和轉染RNAi-雄性素(siRNA-AR)所抑制。
當先使用PI(3)K抑制劑(LY294002)或轉染激媒缺陷的突變Akt(dAkt)處理細胞時，皆能有效阻斷雄性素所引起的細胞增生的現象。
接下來，先使用Src激媒抑制劑或Gi蛋白抑制劑或PLC抑制劑處理細胞時，亦可阻斷Akt被雄性素活化的現象，如此證明Src激媒和Gi蛋白和PLC參與在雄性素活化Akt的訊號路徑中。另外，先使用二架離子螯合物(EGTA)或細胞內鈣離子螯合物(BAPTA/AM)處理細胞，同樣可以抑制Akt被雄性素活化的現象，因此證明鈣離子也參與在雄性素活化Akt的訊號路徑中。
利用螢光顯微鏡分析發現，當Akt被雄性素活化之後，會由細胞質移動至細胞核內。但若先將細胞以LY294002或HF處理，便能抑制住被雄性素活化的Akt在細胞內的移動現象。
綜合以上結果，證實雄性素能藉由與其受體結合，進而使造骨細胞內的Akt蛋白活化。而活化的Akt向細胞核內移動的現象，將是對雄性素能刺激細胞增生非常重要的一個指標。

Androgen has been shown to stimulate proliferation of osteoblast-like MC3T3-E1 cells. However, the molecular mechanism responsible for this effect remains to be elucidated. In the present study we demonstrate herein the non-genomic effect of androgen on osteoblast-like MC3T3-E1 cells involving activation of a PI(3)K/Akt signaling pathway and stimulating proliferation.
In studies of steroids signaling, 5a-dihydrotestosterone (DHT), testosterone and 17b-estradiol but not dexamethasone or progesterone induced a rapid and transient phosphorylation of Akt in MC3T3-E1 cells. The androgen-induced Akt activation reached to the climax after 15 min and gradually diminished to baseline after 60 min. This induction of androgen was unaffected by actinomycin D and was specifically blocked by androgen receptor (AR) antagonist hydroxyflutamide (HF) or transfection of siRNA-AR. Treatment of MC3T3-E1 cells with PI(3)K inhibitor LY294002 or transfection with kinase-deficient Akt blocked androgen-induced cells proliferation.
Moreover, androgen-induced activation of Akt was abolished by inhibitors of Src kinase, Gi-protein and phospholipase C showing the involvement of these effectors in androgen signaling pathway. Further, androgen-induced activation of Akt was dependent on intracellular calcium as shown by the effect of EGTA and intracellular calcium chelator BAPTA/AM.
Fluorescence microscopy showed translocation of phospho-Akt from cytosol into nucleus after androgen treatment but no change in the subcellular distribution of phospho-Akt when HF or LY294002 pretreatment was administered to the cells.
These results strongly suggest that phosphorylation of Akt in osteoblast cells is mediated by androgen receptor and the androgen-induced translocation of Akt is an important step in the androgen/AR signaling pathway that mediates osteoblast cells proliferation.

Chinese Abstract--------2
English Abstract--------4
Table of Contents-------6
List of Figures---------7
Abbreviations-----------8
Introduction------------9
Materials and Methods---14
Results-----------------20
Discussion--------------29
References--------------35
Figures-----------------43

Abu, E.O., Horner, A., Kusec, V., Triffitt, J.T., Compston, J.E. (1997). The localization of androgen receptors in human bone. J. Clin. Endocrinol. Metab. 82, 3493-3497.
Ahmed, N.N., Franke, T.F., Bellacosa, A., Datta, K., Gonzalez-Portal, M.E., Taguchi, T., Testa, J.R., Tsichlis, P.N. (1993). The proteins encoded by c-akt and v-akt differ in post-translational modification, subcellular localization and oncogenic potential. Oncogene 8, 1957-1963.
Alessi, D.R. (1996). Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15, 6541-6551.
Astoul, E., Watton, S., Cantrell, D. (1999). The dynamics of protein kinase B regulation during B cell antigen receptor engagement. J. Cell. Biol. 145, 1511-1520.
Auger, K.R., Serunian, L.A., Soltoff, S.P., Libby, P., Cantley, L.C. (1989). PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell 57, 167-175.
Bellacosa, A., Chan, T.O., Ahmed, N.N., Datta, K., Malstrom, S., Stokoe, D., McCormick, F., Feng, J., Tsichlis, P. (1998). Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene 17, 313-325.
Biggs, W.H. 3rd., Meisenhelder, J., Hunter, T., Cavenee, W.K., Arden, K.C. (1999). Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc. Natl. Acad. Sci. USA 96, 7421-7426.
Borgatti, P., Martelli, A.M., Bellacosa, A., Casto, R., Massari, L., Capitani, S., Neri, L.M. (2000). Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors. FEBS Lett. 477, 27-32.
Buitrago, C., Gonzalez, P.V., de Boland, A.R. (2002). Nongenomic action of 1 alpha,25(OH)(2)-vitamin D3. Activation of muscle cell PLC gamma through the tyrosine kinase c-Src and PtdIns 3-kinase. Eur. J. Biochem. 269, 2506-2515.
Canalis, E., Delany, A.M. (2002). Mechanisms of glucocorticoid action in bone. Ann. NY Acad. Sci. 966, 73-81.
Cantley, L.C. (2002). The phosphoinositide 3-kinase pathway. Science 296, 1655-1657.
Carpenter, C.L., Cantley, L.C. (1996). Phosphoinositide 3-kinase and the regulation of cell growth. Biochim. Biophys. Acta. 1288, 11-16.
Chang, C.S., Kokontis, J., Liao, S.T. (1988). Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors. Proc. Natl. Acad. Sci. USA 85, 7211-7215.
Chang, C., Saltzman, A., Yeh, S., Young, W., Keller, E., Lee, H.J., Wang, C., Mizokami, A. (1995). Androgen receptor: an overview. Crit. Rev. Eukaryotic Gene Expression 5, 97-125.
Delcommenne, M., Tan, C., Gray, V., Rue, L., Woodgett, J., Dedhar, S. (1998). Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc. Natl. Acad. Sci. USA 95, 11211-11216.
Derek, P., Brazil, J.P., Brian, A.H. (2002). PKB Binding Proteins: Getting in on the Akt. Cell 111, 293-303.
Diehl, J.A., Cheng, M., Roussel, M.F., Sherr, C.J. (1998). Glycogen synthase kinase-3b regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. 12, 3499-3511.
Endoh, H., Sasaki, H., Maruyama, K., Takeyama, K., Waga, I., Shimizu, T., Kato, S., Kawashima, H. (1997). Rapid activation of MAP kinase by estrogen in the bone cell line. Biochem. Biophys. Res. Commun. 235, 99-102.
Escobedo, J.A., Navankasattusas, S., Kavanaugh, W.M., Milfay, D., Fried, V.A., Williams, L.T. (1991). cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor. Cell 65, 75-82.
Falasca, M., Logan, S.K., Lehto, V.P., Baccante, G., Lemmon, M.A., Schlessinger, J. (1998). Activation of phospholipase C gamma by PI 3-kinase-induced PH domain-mediated membrane targeting. EMBO J. 17, 414-422.
Franke, T.F., Yang, S.I., Chan, T.O., Datta, K., Kazlauskas, A., Morrison, D.K., Kaplan, D.R., Tsichlis, P.N. (1995). The protein kinase encoded by the Akt protooncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81, 727-736.
Goulding, A., Gold, E. (1993). Flutamide-mediated androgen blockade evokes osteopenia in the female rat. J. Bone Miner. Res. 8, 763-769.
Guo, Z., Benten, W.P., Krucken, J., Wunderlich, F. (2002). Nongenomic testosterone calcium signaling. Genotropic actions in androgen receptor-free macrophages. J. Biol. Chem. 277, 29600-29607.
Hisamoto, K., Ohmichi, M., Kurachi, H., Hayakawa, J., Kanda, Y., Nishio, Y., Adachi, K., Tasaka, K., Miyoshi, E., Fujiwara, N., Taniguchi, N., Murata, Y. (2001). Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells. J. Biol. Chem. 276, 3459-3467.
Jones, S.M., Klinghoffer, R., Prestwich, G.D., Toker, A., Kazlauskas, A. (1999). PDGF induces an early and a late wave of PI 3-kinase activity, and only the late wave is required for progression through G1. Curr. Biol. 9, 512-521.
Joshi, S.A., Fan, K.P., Ho, V.W., Wong, Y.H. (1998). Chimeric Galpha(q) mutants harboring the last five carboxy-terminal residues of Galpha(i2) or Galpha(o) are resistant to pertussis toxin-catalyzed ADP-ribosylation. FEBS Lett. 441, 67-70.
Juliet, E. (2001). Sex steroids and bone. Physiological Reviews. 81, 1.
Kops, G.J., Burgering, B.M. (1999). Forkhead transcription factors: new insights into protein kinase B (c-akt) signaling. J. Mol. Med. 77, 656-665.
Kousteni, S., Bellido, T., Plotkin, L.I., O'Brien, C.A., Bodenner, D.L., Han, L. Han, K., DiGregorio, G.B., Katzenellenbogen, J.A., Katzenellenbogen, B.S., Roberson, P.K., Weinstein, R.S., Jilka, R.L., Manolagas, S.C. (2001). Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104, 719-730.
Lee, Y.F., Lin, W.J., Huang, J., Messing, E.M., Chan, F.L., Wilding, G., Chang, C. (2002). Activation of mitogen-activated protein kinase pathway by the antiandrogen hydroxyflutamide in androgen receptor-negative prostate cancer cells. Cancer Res. 62, 6039-6044.
Lieberherr, M., Grosse, B. (1994). Androgens increase intracellular calcium concentration and inositol 1,4,5-trisphosphate and diacylglycerol formation via a pertussis toxin- sensitive G-protein. J. Biol. Chem. 269, 7217-7223.
Luttrell, L.M., Della Rocca, G.J., van Biesen, T., Luttrell, D.K. Lefkowitz, R.J. (1997). Gbetagamma subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation. J. Biol. Chem. 272, 4637-4644.
Ma, Y.C., Huang, J., Ali, S., Lowry, W., Huang, X.Y. (2000). Src tyrosine kinase is a novel direct effector of G proteins. Cell 102, 635-646.
Manolagas, S.C., Kousteni, S., Jilka, R.L. (2002). Sex steroids and bone. Recent Prog. Horm. Res. 57, 385-409.
Marcinkowska, E., Wiedlocha, A., Radzikowski, C. (1997). 1,25-Dihydroxyvitamin D3 induced activation and subsequent nuclear translocation of MAPK is upstream regulated by PKC in HL-60 cells. Biochem. Biophys. Res. Commun. 241, 419-426.
Mathov, I., Plotkin, L.I., Sgarlata, C.L., Leoni, J., Bellido, T. (2001). Extracellular signal-regulated kinases and calcium channels are involved in the proliferative effect of bisphosphonates on osteoblastic cells in vitro. J. Bone. Miner. Res. 16, 2050-2056.
Medema, R.H., Kops, G.J., Bos, J.L., Burgering, B.M. (2000). AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404, 782-787.
Migliaccio, A., Di Domenico, M., Castoria, G., de Falco, A., Bontempo P., Nola E., Auricchio, F. (1996). Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol- receptor complex in MCF-7 cells. EMBO J. 15, 1292-1300.
Migliaccio, A., Piccolo, D., Castoria, G., Di Domenico, M., Bilancio, A., Lombardi, M., Gong, W., Beato, M., Auricchio, F. (1998). Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor. EMBO J. 17, 2008-2018.
Moggs, J.G., Deavall, D.G., Orphanides, G. (2003). Sex steroids, ANGELS and osteoporosis. BioEssays 25, 195-199.
Nawata, H., Tanaka, S., Tanaka, S., Takayanagi, R., Sakai, Y., Yanase, T., Ikuyama, S., Haji, M. (1995). Aromatase in bone cells: association with osteoporosis in postmenopausal women. J. Steroid Biochem. Mol. Biol. 53, 165-174.
Notelovitz, M. (2002). Androgen effects on bone and muscle. Fertil. Steril. 77, 34-41.
Peterziel, H., Mink, S., Schonert, A., Becker, M., Klocker, H., Cato, A.C. (1999). Rapid signalling by androgen receptor in prostate cancer cells. Oncogene 18, 6322-6329.
Rameh, L.E., Cantley, L.C. (1999). The role of phosphoinositide 3-kinase lipid products in cell function. J. Biol. Chem. 274, 8347-8350.
Ruderman, N.B., Kapeller, R., White, M.F., Cantley, L.C. (1990) Activation of phosphatidylinositol 3-kinase by insulin. Proc. Natl. Acad. Sci. USA 87, 1411-1415.
Razandi, M., Pedram, A., Greene, G.L., Levin, E.R. (1999). Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells. Mol. Endocrinol. 13, 307-319.
Schweikert, H.U., Rulf, W., Niederle, N., Schafer, H.E., Keck, E., Kruck, F. (1980). Testosterone metabolism in human bone. Acta. Endocrinol. 95, 258-264.
Simoncini, T., Hafezi-Moghadam, A., Brazil, D.P., Ley, K., Chin, W.W., Liao, J.K. (2000). Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407, 538-541.
Simoncini, T., Fornari, L., Mannella, P., Varone, G., Caruso, A., Liao, J.K., Genazzani, A.R. (2002). Novel non-transcriptional mechanisms for estrogen receptor signaling in the cardiovascular system. Interaction of estrogen receptor alpha with phosphatidylinositol 3-OH kinase. Steroids 67, 935-939.
Stephens, L., Smrcka, A., Cooke, F.T., Jackson, T.R., Sterweis, P.C., Hawkins, P.T. (1994). A novel phosphoinositide 3 kinase activity in myeloid- derived cells is activated by G protein beta gamma subunits. Cell 77, 83-93.
Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G.F., Holmes, A.B., Gaffney, P.R., Reese, C.B., McCormick, F., Tempst, P., Coadwell, J., Hawkins, P.T. (1998). Protein kinase B kinases that mediate phosphatidylinositol 3, 4, 5- trisphosphatedependent activation of protein kinase B. Science 279, 710-714.
Sun, H., Lesche, R., Li, D.M., Liliental, J., Zhang, H., Gao, J., Gavrilova, N., Mueller, B., Liu, X., Wu, H. (1999). PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3, 4, 5, trisphosphate and Akt/protein kinase B signaling pathway. Proc. Natl. Acad. Sci. USA 96, 6169-6204.
Sushma, A.J., Kenneth, P.K.F., Vanessa, W.S.H., Wong, Y.H. (1998). Chimeric Gaq mutants harboring the last five carboxy-terminal residues of Gai2 or Gao are resistant to pertussis toxin-catalyzed ADP-ribosylation. FEBS Letters 441, 67-70.
Testa, J.R., Bellacosa, A. (1997). Membrane translocation and activation of the Akt kinase in growth factor-stimulated hematopoietic cells. Leuk. Res. 21, 1027-1031.
Yano, S., Tokumitsu, H., Soderling, T.R. (1998). Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature 396, 584-587.
Yeh, S., Tsai, M.Y., Xu, Q., Mu, X.M., Lardy, H., Huang, K.E., Lin, H., Yeh, S.D., Altuwaijri, S., Zhou, X., Xing, L., Boyce, B.F., Hung, M.C., Zhang, S., Gan, L., Chang, C., Hung, M.C. (2002). Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues. Proc. Natl. Acad. Sci. USA 99, 13498-13503.