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• W2010336249 abstract "Stem cell factor (SCF)/c-kit plays an important role in the regulation of hematopoiesis, melanogenesis, and spermatogenesis. In the testis, the SCF/c-kit system is believed to regulate germ cell proliferation, meiosis, and apoptosis. Studies with type A spermatogonia in vivo and in vitro have indicated that SCF induces DNA synthesis and proliferation. However, the signaling pathway for this function of SCF/c-kit has not been elucidated. We now demonstrate that SCF activates phosphoinositide 3-kinase (PI3-K) and p70 S6 kinase (p70S6K) and that rapamycin, a FRAP/mammalian target of rapamycin-dependent inhibitor of p70S6K, completely inhibited bromodeoxyuridine incorporation induced by SCF in primary cultures of spermatogonia. SCF induced cyclin D3 expression and phosphorylation of the retinoblastoma protein through a pathway that is sensitive to both wortmannin and rapamycin. Furthermore, AKT, but not protein kinase C-ζ, is used by SCF/c-kit/PI3-K to activate p70S6K. Dominant negative AKT-K179M completely abolished p70S6K phosphorylation induced by the constitutively active PI3-K catalytic subunit p110. Constitutively active v-AKT highly phosphorylated p70S6K, which was totally inhibited by rapamycin. Thus, SCF/c-kit uses a rapamycin-sensitive PI3-K/AKT/p70S6K/cyclin D3 pathway to promote spermatogonial cell proliferation. Stem cell factor (SCF)/c-kit plays an important role in the regulation of hematopoiesis, melanogenesis, and spermatogenesis. In the testis, the SCF/c-kit system is believed to regulate germ cell proliferation, meiosis, and apoptosis. Studies with type A spermatogonia in vivo and in vitro have indicated that SCF induces DNA synthesis and proliferation. However, the signaling pathway for this function of SCF/c-kit has not been elucidated. We now demonstrate that SCF activates phosphoinositide 3-kinase (PI3-K) and p70 S6 kinase (p70S6K) and that rapamycin, a FRAP/mammalian target of rapamycin-dependent inhibitor of p70S6K, completely inhibited bromodeoxyuridine incorporation induced by SCF in primary cultures of spermatogonia. SCF induced cyclin D3 expression and phosphorylation of the retinoblastoma protein through a pathway that is sensitive to both wortmannin and rapamycin. Furthermore, AKT, but not protein kinase C-ζ, is used by SCF/c-kit/PI3-K to activate p70S6K. Dominant negative AKT-K179M completely abolished p70S6K phosphorylation induced by the constitutively active PI3-K catalytic subunit p110. Constitutively active v-AKT highly phosphorylated p70S6K, which was totally inhibited by rapamycin. Thus, SCF/c-kit uses a rapamycin-sensitive PI3-K/AKT/p70S6K/cyclin D3 pathway to promote spermatogonial cell proliferation. stem cell factor phosphoinositide 3-kinase retinoblastoma protein mammalian target of rapamycin bromodeoxyuridine polyacrylamide gel electrophoresis protein kinase C-ζ phosphorylated p70 S6 kinase recombinant mouse stem cell factor Spermatogenesis is a complex process of cellular renewal and differentiation that culminates in the production of spermatozoa (1Dym M. Weiss L. Histology: Cell and Tissue Biology. Elsevier Biomedical, New York1983Google Scholar). Type A spermatogonia, the male germ line stem cells, divide by mitosis to yield other type A stem cells as well as the differentiated type A spermatogonia (2Dym M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11287-11289Crossref PubMed Scopus (152) Google Scholar). The differentiated type A proceed through a series of mitotic divisions to produce meiotic spermatocytes. The early spermatocytes enter the first of two meiotic divisions, producing round spermatids; the round spermatids then differentiate into late spermatids that are released into the lumen of the seminiferous tubules as spermatozoa. Thus far, the molecular mechanisms and signaling pathways for these events remain largely unknown. It has been suggested that the stem cell factor/c-kit system may play a crucial role in regulating proliferation and differentiation of spermatogonial stem cells (3Yoshinaga K. Nishikawa S. Ogawa M. Hayashi S. Kunisada T. Fujimoto T. Development. 1991; 113: 689-699PubMed Google Scholar, 4Packer A.I. Besmer P. Bachvarova R.F. Mol. Reprod. Dev. 1995; 42: 303-310Crossref PubMed Scopus (142) Google Scholar, 5Mauduit C. Hamamah S. Benahmed M. Hum. Reprod. Update. 1999; 5: 535-545Crossref PubMed Scopus (187) Google Scholar). In the testis, Sertoli cells, the supportive elements within the seminiferous epithelium, produce stem cell factor (6Rossi P. Dolci S. Albanesi C. Grimaldi P. Ricca R. Geremia R. Dev. Biol. 1993; 155: 68-74Crossref PubMed Scopus (195) Google Scholar, 7Tajima Y. Sakawaki K. Watanabe D. Koshimizu U. Matsuzawa T. Nishimune Y. J. Reprod. Fertil. 1991; 91: 441-449Crossref PubMed Scopus (17) Google Scholar, 8Loveland K.L. Schlatt S. J. Endocrinol. 1997; 153: 337-344Crossref PubMed Scopus (150) Google Scholar). Stem cell factor (SCF)1 exists in either a soluble form or membrane-bound to Sertoli cells. Type A spermatogonia express the receptor for SCF, c-kit (3Yoshinaga K. Nishikawa S. Ogawa M. Hayashi S. Kunisada T. Fujimoto T. Development. 1991; 113: 689-699PubMed Google Scholar, 9Dym M. Jia M.C. Dirami G. Price J.M. Rabin S.J. Mocchetti I. Ravindranath N. Biol. Reprod. 1995; 52: 8-19Crossref PubMed Scopus (179) Google Scholar). Both the ligand and the receptor are expressed in a stage-specific manner (10Vincent S. Segretain D. Nishikawa S. Nishikawa S.I. Sage J. Cuzin F. Rassoulzadegan M. Development. 1998; 125: 4585-4593PubMed Google Scholar). Studies on the self-renewal and proliferation of spermatogonia have demonstrated that 1) administration of ACK-2, a monoclonal antibody that blocks the binding of SCF to c-kit, disrupts proliferation of mouse spermatogonia and promotes apoptosis (4Packer A.I. Besmer P. Bachvarova R.F. Mol. Reprod. Dev. 1995; 42: 303-310Crossref PubMed Scopus (142) Google Scholar), and 2) recombinant SCF stimulates the division of isolated primary spermatogonia (6Rossi P. Dolci S. Albanesi C. Grimaldi P. Ricca R. Geremia R. Dev. Biol. 1993; 155: 68-74Crossref PubMed Scopus (195) Google Scholar). These studies indicate that SCF/c-kitregulates spermatogonial mitotic progression, a process indispensable for the self-renewal and differentiation of the male germ-line stem cell population. However, the signal pathway triggered by SCF and its downstream cascade to regulate spermatogonial cell mitosis are not known. Recent studies suggest that PI3-K acts as an immediate downstream molecule of growth factor receptors to mediate mitogenic signaling in cells. The activation of PI3-K is sufficient to drive some types of cells to go through G1/S transition and to proliferate (11Klippel A. Escobedo M.A. Wachowicz M.S. Appell G. Brown T.W. Giedlin M.A. Kavanaugh W.M. Williams L.T. Mol. Cell. Biol. 1998; 18: 5699-5711Crossref PubMed Scopus (241) Google Scholar). p70S6K mediates PI3-K signaling to the cell cycle machinery leading to proliferation in a variety of cell types (12Grewe M. Gansauge F. Schmid R.M. Adler G. Seufferlein T. Cancer Res. 1999; 59: 3581-3587PubMed Google Scholar, 13Chou M.M. Blenis J. Curr. Opin. Cell Biol. 1995; 7: 806-814Crossref PubMed Scopus (245) Google Scholar). In the present study, we addressed whether the PI3-K signaling pathway is activated by SCF to regulate the proliferation of spermatogonia. We observed that SCF induced the PI3-K/AKT/p70S6K signaling pathway. Rapamycin completely blocked cyclin D3 expression and the bromodeoxyuridine (BrdUrd) incorporation induced by SCF. Taking these data together, our studies indicted that SCF/c-kitrecruits PI3-K/AKT/p70S6K/cyclin D3 to stimulate proliferation of type A spermatogonia. The testes of 5 day-old mouse pups (Balb/c) were removed and digested with a mixture of enzymes that included collagenase, trypsin, and hyaluronidase; then, type A spermatogonial cells were isolated from the resultant mixture of testicular cells using a bovine serum albumin gradient column as described previously (9Dym M. Jia M.C. Dirami G. Price J.M. Rabin S.J. Mocchetti I. Ravindranath N. Biol. Reprod. 1995; 52: 8-19Crossref PubMed Scopus (179) Google Scholar, 14Dirami G. Ravindranath N. Jia M.C. Dym M. Hansson V. Levy F.O. Taskén K. Ernst Schering Research Foundation Workshop. Signal Transduction in Testicular Cells. Springer-Verlag, Berlin1996: 141-166Google Scholar). Isolated spermatogonial cells were cultured in Ham's F-12 (Irvine Scientific) containing 10% horse serum (Biofluids, Inc.) and antibiotics (Sigma). Long term maintenance of the spermatogonia was achieved by co-culturing the cells with the 15P-1-Sertoli cell line (15Rassoulzadegan M. Paquis-Flucklinger V. Bertino B. Sage J. Jasin M. Miyagawa K. van Heyningen V. Besmer P. Cuzin F. Cell. 1993; 75: 997-1006Abstract Full Text PDF PubMed Scopus (150) Google Scholar). The expanded spermatogonial cells were further purified by differential plating. Plasmids expressing wild type p70S6K and the AKT-K179M were provided by Dr. Xiantao Wang (NIA, National Institutes of Health). pLXSN-v-AKT was provided by Dr. Peter Sabbatini (the Cancer Research Institute, University of California School of Medicine, San Francisco, CA). All transient transfections were performed using Lipofectin reagent following the protocol provided by the manufacturer (Life Technologies, Inc.). Cells were harvested and lysed with the following buffer: 20 mm HEPES, pH 7.4, 50 mm β-glycerol phosphate, 2 mm EGTA, 1 mm dithiothreitol, 10 mm NaF, 1 mmsodium orthovanadate, 1% Triton X-100, 10% glycerol, and protease inhibitors (2 μg/ml leupeptin and 2 mmphenylmethylsulfonyl fluoride). To immunoprecipitate c-kit, whole cell lysates containing 100 μg of total proteins were incubated with 1 μg/ml c-kit polyclonal antibody (Santa Cruz Biotechnology) for 1 h at 4 °C. Immunoprecipitation was facilitated by addition of protein A-Sepharose (Amersham Pharmacia Biotech) for 2 h at 4 °C on a rotating shaker. The immunoprecipitate was washed three times at 4 °C with the lysis buffer. Similarly, the p85 regulatory subunit of PI3-K was immunoprecipitated by incubating 300 μg of total protein with the monoclonal p85 antibody (Upstate Biotechnology) and protein G (Amersham Pharmacia Biotech). The immunoprecipitate was washed three times at 4 °C with lysis buffer. The immunoprecipitated proteins were released from beads by boiling and were loaded onto SDS-PAGE. The separated proteins were blotted onto the nitrocellulose membranes. For immunoblotting, cell lysates were subjected to SDS-PAGE, and proteins were transferred to polyvinylidene difluoride membranes. Membranes were blocked with TBS buffer containing 5% fat-free milk and 0.1% Tween 20 for 1 h and hybridized with the primary antibodies: anti-phosphotyrosine 4G10 (Upstate Biotechnology), c-kitpolyclonal antibody (Santa Cruz Biotechnology), AKT antibody and phospho-AKT antibodies (New England Biolabs, Inc.), p70S6K antibody and phospho-p70S6K antibodies (New England Biolabs, Inc.), Rb antibody and phospho-Rb antibodies (New England Biolabs, Inc.), cyclin D3 antibody (BD Transduction Laboratories), and PKC-ζ antibody (Santa Cruz Biotechnology). After hybridization with secondary antibodies conjugated to horseradish peroxidase, the immunocomplex was detected with the ECL detection reagent (Amersham Pharmacia Biotech). Freshly isolated type A spermatogonial cells were plated on cover slides with serum-free Ham's F-12. The cells were incubated with 50 nm rapamycin for 30 min, and then 100 ng/ml recombinant mouse stem cell factor (mSCF) (R&D Systems, Inc.) and 30 μg/ml BrdUrd (Sigma) were added to the cells. After 18 h of culture, the cells were fixed in 70% ethanol and stained for BrdUrd following the protocol provided by the manufacturer (Zymed Laboratories Inc.). Spermatogonia cells were transfected with active p110 and cultured for 48 h, and then the cells were starved in serum-free medium for 24 h. The cells were lysed in 20 mm Tris, pH 7.5, 10% glycerol, 1% Nonidet P-40, 10 mm EDTA, 150 mm NaCl, 20 mm sodium fluoride, 5 mm sodium pyrophosphate, 1 mmsodium vanadate, 1 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl fluoride at 4 °C. 500 μg of total protein were used to immunoprecipitate PKC-ζ by incubating with 1 μg of anti-PKC-ζ antibody and 50 μl of 50% protein A-Sepharose in a rolling tube overnight at 4 °C. Immunoprecipitates were washed three times with lysis buffer containing 0.5 m NaCl and two times with kinase buffer (35 mm Tris, pH 7.5, 10 mmmagnesium chloride, 1 mm EGTA, 2 mm sodium orthovanadate). PKC-ζ activity was assayed in 30 μl of kinase buffer containing 10 μCi of [γ-32P]ATP (Amersham Pharmacia Biotech) and 4 μg of myelin basic protein for 30 min at room temperature. The reaction was stopped by the addition of sample buffer, and phosphorylated myelin basic protein was resolved by SDS-PAGE. The isolated spermatogonial cells were cultured in serum-free medium for 8 h and then treated with 100 ng/ml mSCF (R&D Systems, Inc.) for 5 or 10 min. 30 μg of whole cell lysates were resolved by SDS-PAGE and blotted with phosphotyrosine antibody (4G10). SCF induced tyrosine phosphorylation of a ∼120-kDa protein (Fig.1 A). To confirm that this protein is c-kit, 100 μg of lysates of the control and SCF-treated (10 min) cells were immunoprecipitated with 1 μg/ml c-kit antibody. The immunoprecipitate was resolved by SDS-PAGE and blotted with the 4G10 antibody. The results showed that the 120-kDa protein was immunoprecipitated by the c-kitantibody and was tyrosine-phosphorylated (Fig. 1 B). Furthermore, 300 μg of cell lysates were immunoprecipitated with the PI3-K (1 μg/ml) p85 subunit monoclonal antibody and blotted with 4G10 phosphotyrosine antibody. The tyrosine phosphorylated c-kitwas co-immunoprecipitated with the p85 subunit of PI3-K (Fig.1 C). These results demonstrated that SCF/c-kittriggers PI3-K signaling in spermatogonia and that the PI3-K signaling pathway may be the pathway for SCF to regulate the proliferation of spermatogonial cells. p70S6K is a downstream regulator of the PI3-K signaling pathway. It has emerged as an important regulator of cell growth, playing a positive role during progression through the G1 phase of the cell cycle (12Grewe M. Gansauge F. Schmid R.M. Adler G. Seufferlein T. Cancer Res. 1999; 59: 3581-3587PubMed Google Scholar). Thus, we next tested whether p70S6K is also activated by SCF/c-kit through PI3-K in type A spermatogonial cells. Isolated spermatogonial cells were cultured in serum-free medium overnight and then the cells were pretreated with 100 nm wortmannin (an inhibitor of PI3-K) or 50 nm rapamycin (an mTOR-dependent inhibitor of p70S6K) for 30 min and then stimulated with 100 ng/ml mSCF (R&D Systems, Inc.) for 45 min or 6 h. 40 μg of protein extracts of treated cells were resolved by SDS-PAGE and blotted with phospho-p70S6K antibodies, including anti-Thr-389 and anti-Thr-421/Ser-424. As shown in Fig. 2, SCF induced phosphorylation of p70S6K strongly at Thr-389 at both the 45-min and 6-h intervals. This phosphorylation was completely inhibited by both wortmannin and rapamycin. Thus, it is clear that SCF/c-kit activates a rapamycin-sensitive PI3-K/p70S6K signaling pathway in spermatogonial cells. The above results suggested that SCF/c-kit may use the PI3-K/p70S6K pathway to cause cell cycle progression in spermatogonia. However, in addition to PI3-K/p70S6K, the cell cycle may be regulated by other multiple mitogenic pathways, including Ras-p42/p44 MAPK (16Gille H. Downward J. J. Biol. Chem. 1999; 274: 22033-22040Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar) and PI3-K-dependent but rapamycin-insensitive pathways (17Takuwa N. Fukui Y. Takuwa Y. Mol. Cell. Biol. 1999; 19: 1346-1358Crossref PubMed Scopus (162) Google Scholar). To confirm that PI3-K/p70S6K, but not other pathways, is recruited by SCF/c-kit to cause spermatogonial cell proliferation, we performed a BrdUrd incorporation study in isolated mouse spermatogonia treated in the presence or absence of SCF and rapamycin. Cells were cultured in serum-free medium and then pretreated with 50 nm rapamycin for 30 min. 100 ng/ml mSCF (R&D Systems, Inc.) and 30 μg/ml BrdUrd were added to the medium. The cells were maintained in culture for another 18 h. Then the cells were stained for BrdUrd. SCF induced a 4-fold increase in BrdUrd-positive cells compared with controls. Rapamycin completely blocked BrdUrd incorporation induced by mSCF (Fig. 3). Thus, the SCF/c-kit-induced DNA synthesis in spermatogonia occurs through the rapamycin-sensitive PI3-K/p70S6K pathway. The above experiments suggested that SCF uses the PI3-K/p70S6K signaling pathway to promote the proliferation of spermatogonial cells. Next, we addressed how SCF/c-kit/PI3-K/p70S6K regulates cell cycle progression in spermatogonial cells. Cell cycle processing through G1/S transition requires the induction of thed-type cyclins. It was suggested that cyclin D3 may play a principal role in regulating the spermatogonial cell cycle through G1/S phase progression (18Zhang Q. Wang X. Wolgemuth D.J. Endocrinology. 1999; 140: 2790-2800Crossref PubMed Scopus (40) Google Scholar). To address whether cyclin D3 is regulated by SCF/c-kit/PI3-K signaling in spermatogonial cells, isolated mouse spermatogonial cells were cultured in serum-free medium overnight. After pretreatment with wortmannin (100 nm) or rapamycin (50 nm) the cells were treated with 100 ng/ml mSCF (R&D Systems, Inc.) for 6 or 18 h. 50 μg of whole lysates of treated cells were resolved by SDS-PAGE and blotted with cyclin D3 antibody. The results indicated that expression of cyclin D3 was down-regulated after serum withdrawal for 18 h in spermatogonial cells (Fig. 4, lanes 1 and 2); addition of 100 ng/ml mSCF restored thisd-type cyclin expression after 6 h (Fig. 4,lanes 3 and 4). Interestingly, the induction of cyclin D3 expression by mSCF was blocked by either wortmannin or rapamycin (Fig. 4, lanes 5–8). Moreover, the same samples were used to analyze the phosphorylation status of RB with phospho-Rb antibodies, including phospho-Rb (Ser-780), phospho-Rb (Ser-249/Ser-252), phospho-Rb (Thr-373), phospho-Rb (Ser-795), and phospho-Rb (Ser-807/Ser-811). Only phospho-Rb (Ser-780) gave a strong phosphorylation signal (Fig. 4, panel A). Furthermore, the phosphorylation at Ser-780 of Rb was also blocked by both wortmannin and rapamycin (Fig. 4, lanes 5–8). These results indicated that SCF increases the expression of cyclin D3 and phosphorylates Rb at Ser-780 through the PI3-K/p70S6K signaling pathway. The signaling between PI3-K and p70S6K is complex. Recently, PKC-ζ and AKT, two important regulators of cell growth, have been demonstrated to mediate the PI3-K signaling to p70S6K. A study with neonatal rat gonocytes, which are the precursors of type A spermatogonia and express c-kit (19Orth J.M. Qiu J. Jester Jr., W.F. Pilder S. Biol. Reprod. 1997; 57: 676-683Crossref PubMed Scopus (63) Google Scholar), demonstrated that PKC-ζ is co-expressed with PI3-K (20Li H. Papadopoulos V. Vidic B. Dym M. Culty M. Endocrinology. 1997; 138: 1289-1298Crossref PubMed Scopus (171) Google Scholar). Western blotting showed that PKC-ζ is expressed in mouse spermatogonia (Fig. 5 A). The constitutively active catalytic p110 subunit of PI3-K activated PKC-ζ, and this activity was suppressed to basal levels by 100 μm PKC-ζ pseudosubstrate, an inhibitor of PKC-ζ, (Fig. 5 B). To test whether PKC-ζ mediates the signal between PI3-K and p70S6K in spermatogonial cells, the cells were cotransfected with the constitutively active catalytic p110 subunit of PI3-K and p70S6K in the presence of 100 μmpseudosubstrate peptide. 20 μg of total protein were resolved by SDS-PAGE and blotted with p-p70S6K (Thr-389). The results showed that the pseudosubstrate peptide did not inhibit the phosphorylation of p70S6K induced by the active p110 catalytic subunit (Fig. 5,C and D). Thus, PKC-ζ is not involved in the SCF/c-kit/PI3-K signaling to activate p70S6K in spermatogonial cells. AKT acts as a downstream kinase of PI3-K, and its activity is stimulated by a variety of growth factors and insulin via a wortmannin-sensitive pathway (21Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar, 22Takata M. Ogawa W. Kitamura T. Hino Y. Kuroda S. Kotani K. Klip A. Gingras A.C. Sonenberg N. Kasuga M. J. Biol. Chem. 1999; 274: 20611-20618Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Activated AKT phosphorylates p70S6K through an indirect pathway, which is not completely understood. To test whether AKT mediates SCF/c-kit/PI3-K signaling to p70S6K in spermatogonial cells, we carried out an experiment analyzing AKT activity and its effect on p70S6K phosphorylation by SCF/c-kit/PI3-K in spermatogonia. Isolated mouse spermatogonia cells were serum-starved for 8 h. The cells were then pretreated or untreated with 100 nm Wortmannin for 20 min, and then they were stimulated with 100 ng/ml mSCF for 30 min. 50 μg of whole cell lysates were resolved by SDS-PAGE and blotted with phospho-AKT (Ser-473) antibody. As shown in Fig. 6 A,phosphorylation of AKT was induced by SCF through a wortmannin-sensitive pathway. We cotransfected the spermatogonial cells with p70S6K plasmid and constitutively active AKT (pLXSN-v-AKT) to determine whether active AKT induces the phosphorylation of p70S6K. After 48 h of transfection, cells were cultured in serum-free medium, with or without 50 nm rapamycin, for 24 h and then harvested for immunoblotting. 20 μg of total proteins were resolved by 10% SDS-PAGE and blotted with p-p70S6K (Thr-389). p70S6K was highly phosphorylated by v-AKT, and rapamycin completely abolished this function of v-AKT (Fig. 6 B). We next cotransfected the spermatogonia with the dominant negative form of AKT-K179M, p70S6K, and constitutively active p110. After 48 h transfection, cells were cultured in serum-free medium for 24 h; they were then harvested for immunoblotting. 20 μg of total proteins were resolved by 10% SDS-PAGE and blotted with p-p70S6K (Thr-389). AKT-K179M markedly inhibited p70S6K phosphorylation induced by the constitutively active catalytic subunit p110 of PI3-K (Fig. 6, C andD). These results suggest that AKT is the principal positive regulator that mediates the signaling of SCF/c-kit/PI3-K to activate p70S6K and that AKT activates p70S6K via FRAP/mTOR kinase in spermatogonia. The molecular mechanisms regulating spermatogonial proliferation by stem cell factor largely remain unknown. We now demonstrate for the first time that the SCF/c-kit system up-regulates cyclin D3 and promotes cell cycle progression in spermatogonia through a rapamycin-sensitive PI3-K/AKT/p70S6K pathway. c-kit belongs to the PDGFR family of receptor tyrosine kinases (23Chabot B. Stephenson D.A. Chapman V.M. Besmer P. Bernstein A. Nature. 1988; 335: 88-89Crossref PubMed Scopus (1110) Google Scholar). It plays a crucial role in regulating hemopoiesis, melanogenesis, and spermatogenesis. In the testes, c-kit is predominantly expressed in type A spermatogonial cells (3Yoshinaga K. Nishikawa S. Ogawa M. Hayashi S. Kunisada T. Fujimoto T. Development. 1991; 113: 689-699PubMed Google Scholar, 9Dym M. Jia M.C. Dirami G. Price J.M. Rabin S.J. Mocchetti I. Ravindranath N. Biol. Reprod. 1995; 52: 8-19Crossref PubMed Scopus (179) Google Scholar). In response to its ligand, SCF, c-kit may trigger multiple signaling pathways to regulate the proliferation and/or differentiation of the spermatogonia. However, the signaling pathways induced by c-kit and how these signals are linked to spermatogonial cell renewal and/or differentiation are poorly understood. Our data indicated that SCF induced the binding of the PI3-K p85 subunit to c-kit. p70S6K is phosphorylated on Thr-389 by SCF/c-kit through the wortmannin and rapamycin-sensitive pathway in this type of cell. Cotransfection experiments showed that constitutively active p110 of PI3-K phosphorylates p70S6K in spermatogonial cells. These data demonstrate that SCF/c-kitrecruits the PI3-K/mTOR pathway to activate p70S6K; the latter interacts with the cell cycle machinery to induce cell proliferation. The cell cycle through the G1/S checkpoint is regulated by multiple mitogenic signaling pathways, including Ras-p42/p44 MAPK (16Gille H. Downward J. J. Biol. Chem. 1999; 274: 22033-22040Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), PI3-K/p70S6K (24Weng Q.P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (211) Google Scholar), and the PI3-K-dependent but rapamycin-insensitive pathway (17Takuwa N. Fukui Y. Takuwa Y. Mol. Cell. Biol. 1999; 19: 1346-1358Crossref PubMed Scopus (162) Google Scholar). Our BrdUrd experiment indicated that the SCF/c-kit-induced spermatogonial cell proliferation is completely abolished by rapamycin (Fig. 3). The mechanisms of rapamycin blocking BrdUrd incorporation induced by SCF is likely caused by the inhibitory function of rapamycin on cyclin D3 expression and phosphorylation of Rb (Fig. 4), both of which are necessary to promote cell cycle progression that is induced by SCF in spermatogonia. The inhibition of rapamycin on cyclin D3 expression and phosphorylation of Rb is consistent with its inhibition of p70S6K phosphorylation by SCF. The SCF-induced cyclin D3 expression and Rb phosphorylation are also sensitive to wortmannin, the inhibitor of PI3-K. Thus, the function of PI3-K to up-regulate cyclin D3 expression and phosphorylate Rb is mediated by p70S6K; SCF/c-kit induces the rapamycin-sensitive PI3-K/p70S6K/cyclin D3 pathway in regulating the cell cycle progression and growth in spermatogonial cells. During the cell cycle through the G1 phase, Rb is phosphorylated at multiple sites, including Ser-249/Ser-252, Ser-780, Ser-795, Ser-807/Ser-811, and Thr-373, by cyclin D/cdk4 (cdk6) and cyclin E/cdk2. Our data demonstrated that in regulating spermatogonial cell proliferation, SCF mainly induces the phosphorylation of Rb at Ser-780, which is phosphorylated only by cyclin D/cdk (25Kitagawa M. Higashi H. Jung H.K. Suzuki-Takahashi I. Ikeda M. Tamai K. Kato J. Segawa K. Yoshida E. Nishimura S. Taya Y. EMBO J. 1996; 15: 7060-7069Crossref PubMed Scopus (534) Google Scholar). Thus, it is suggested that cyclin D3 is the early response gene and the possible main target for SCF that drives G1/S phase progression of spermatogonial cells. This result is consistent with the observation that cyclin D3-associated kinase activity was detected in immature but not adult testes and that cyclin D3 plays an important role in regulating the self-renewal of male germ stem cell (18Zhang Q. Wang X. Wolgemuth D.J. Endocrinology. 1999; 140: 2790-2800Crossref PubMed Scopus (40) Google Scholar). Both PKC-ζ and AKT, downstream kinases of PI3-K signaling, are highly expressed in spermatogonia. Although PI3-K activated PKC-ζ, blocking of PKC-ζ did not suppress the phosphorylation of p70S6K induced by the constitutively active PI3-K catalytic subunit p110 (Fig.5). Thus, PKC-ζ is not involved in PI3-K signaling to activate p70S6K in spermatogonia. AKT is strongly phosphorylated by SCF/c-kit/PI3-K. Cotransfection studies in mouse type A spermatogonial cells showed that constitutively active AKT induced phosphorylation of p70S6K and dominant negative AKT-K179M completely inhibited phosphorylation of p70S6K induced by the constitutively active PI3-K catalytic subunit, p110. Rapamycin totally abolished phosphorylation of p70S6K induced by constitutively active AKT (Fig.6). These results indicate that AKT is the main kinase mediating SCF/c-kit/PI3-K to p70S6K through FRAP/mTOR. A recent study demonstrated that c-kit-induced activation of phosphatidylinositol 3-kinase is essential for spermatogonial proliferation and male fertility (26Blume-Jensen P. Jiang G. Hyman R. Lee K.F. O'Gorman S. Hunter T. Nat Genet. 2000; 24: 157-162Crossref PubMed Scopus (275) Google Scholar). Although c-kit is required for normal hematopoiesis, melanogenesis, and gametogenesis, mutations, which disrupt the PI3-K binding to c-kit and impaired PI3-K signaling pathway, only resulted in male sterility due to blockage of the proliferation of spermatogonial cells. There were no apparent hematopoietic or pigmentation defects in the homozygous mutant mice (26Blume-Jensen P. Jiang G. Hyman R. Lee K.F. O'Gorman S. Hunter T. Nat Genet. 2000; 24: 157-162Crossref PubMed Scopus (275) Google Scholar). Another recent study that disrupted the binding of PI3-K to c-kit resulted in defective oogenesis and spermatogenesis. The males were sterile due to a block in the early stages of spermatogenesis (27Kissel H. Timokhina I. Hardy M.P. Rothschild G. Tajima Y. Soares V. Angeles M. Whitlow S.R. Manova K. Besmer P. EMBO J. 2000; 19: 1312-1326Crossref PubMed Scopus (308) Google Scholar). Thus, PI3-K signaling is particularly important for spermatogonial proliferation and spermatogenesis. Our current data confirm these findings and further elucidate the downstream cascade for SCF/c-kit/PI3-K signaling that regulates spermatogonial proliferation. In summary, upon stimulation with stem cell factor, c-kitrecruits the rapamycin-sensitive PI3-K/p70S6K pathway to induce cyclin D3 expression and phosphorylation of Rb leading to spermatogonial proliferation. AKT is the main transducer that links c-kit/PI3-K to p70S6K and is also important for spermatogonial proliferation (Fig.7). The differential signaling pathway in male germ line stem cells leading to stem cell renewalversus stem cell differentiation remains to be elicited. We thank Dr. Xiantao Wang for providing plasmid AKT-K179M and p70S6K; we also thank Dr. Peter Sabbatini for providing pLXSN-v-AKT. We thank Dr. Chris Taylor (Assistant Professor, Department of Cell Biology, Georgetown University Medical School) for reviewing the manuscript." @default.
• W2010336249 created "2016-06-24" @default.
• W2010336249 creator A5003458854 @default.
• W2010336249 creator A5049983648 @default.
• W2010336249 creator A5060233158 @default.
• W2010336249 date "2000-08-01" @default.
• W2010336249 modified "2023-10-11" @default.
• W2010336249 title "Stem Cell Factor/c-kit Up-regulates Cyclin D3 and Promotes Cell Cycle Progression via the Phosphoinositide 3-Kinase/p70 S6 Kinase Pathway in Spermatogonia" @default.
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