| PART ONEmTORCl regulates RANKL/OPG and osteoclastogenesis through β-catenin signaling pathwayBackgroundBone remodeling is in a dynamic process where bone resorption and bone formation take responsibilities. Under internal or external stimuli such as mechanical strength, hormones, cytokines, the variational activities of osteoblasts and osteoclasts would maintain coordinated balance in healthy adults.The osteoclast, which takes responsibilities for local bone resorption is derived from hematopoietic stem cells through myeloid progenitor cells to mono/macrophage and into osteoclast lineage. The molecular mechanism of the activation of osteoclast activity is not clear. Osteoclast differentiation and activation are considered to be under the regulation of RANK/RANKL/OPG system since1997. Receptor activator of nuclear factor-kappaB (RANK), receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG) is belongs to tumornecrosis factor receptor super-family (TNFRSF). Osteoblast and bone marrow stromal cells both express RANKL, while they bind to RANK which express on the surface of osteoclast progenitors and mature osteoclast, they will promote the formation and differiation of osteoclast and inhibit the apoptosis through activating the transcriptional factor. OPG is secreted by osteoblast and able to bind to RANKL, which competitively inhibit the combining of RANKL and RANK, as a result of the suppression of osteoclast proliferation, differentiation and bone resorption.At present, several evidences demonstrate that multiple hormone, cytokines can regulate RANK/RANKL/OPG system directly or indirectly, lead to many bone metabolism diseases, including osteoporosis, bone loss induced by glucocorticoids, multiple myeloma, rheumatoid arthritis and so on. Beside these, which target OPG/RANK/RANKL system such as exogenetic OPG, anti-RANKL antibody and RANK-Fc have been confirmed as potent and effectual medicines to prevent and treat osteolytic diseases.Mammalian target of rapamycin (mTOR), is a protein Ser-Thr kinase that functions as a central element in a signaling pathway involved in the control of cell growth and proliferation. Disregulation of mTOR signaling associated to human major diseases, for instance, cancer, cardiovascular diseases, graft rejection and autoimmune diseases, diabetes, adiposity, nerve system diseases. mTOR signaling pathway have been become to a certain target in recent years.mTOR signaling pathway take part in the regulation of bone metaboslism is definitely confirmed:mTOR/S6K1regulates il-6and VEGF to affect bone formation; rapamycin promote mRNA expression of OPN and OCN in osteoblast-like cells, and promote osteogenic differentiation of human embryonic stem cell through inhibit mTOR activity and activate bmp/smad pathways. What’s more, BEZ235as an inhibitor of PI3K/mTOR pathway, is able to stimulate human bone marrow mesenchymal stem cells (hMSC) differentiate to osteoblast. Rapamycin promote osteoblast differentiation is still in argument, but most consideration about mTORC1signaling pathway inhibition can promote osteoblast formation were supported. On the other hand, mTOR activation is necessary for the survival of osteoclast progenitor cells and mature osteoclast. Someone pointed out erythropoletin hematopoietin (EPO) can activate mTOR and up-or down-regulate NFATcl or cathepsin k to enhance osteoclast formation and decrease osteoclast activity, however rapamycin can reverse these. Despite mTOR has been considered as a regulator in bone metabolism, the concrete mechanism is still unclear.Recently published articles have found that the transcriptional factor CCAAT/enhancer binding protein beta (C/EBPβ) plays a key role in bone metabolism. C/EBPβ not only regulates osteoblast activity, but also regulates osteoclast differentiation and activity under the investigation on transgenic mice, cell biology and pharmacology. In view of the importance of RANK/RANKL/OPG system, especially the regulation of osteoclast, we considered that wether mTOR can control osteoblast and its progenitors to secrete RANKL/OPG via C/EBPβ, and modulate osteoclast differentiation and function.Results1mTORCl is involved in OVX-induced osteoclastogenesis and bone resorptionOvariectomized (OVX) female mice will keep low estrogen level and lead to osteoporosis. Firstly, we randomly grouped24C57B6mice into four, respectively, including Sham, OVX, Sham+rapamycin, OVX+rapamycin. Mice were executed then detected and analysed bone samples for bone mineral density (BMD), micro-CT, bone histomorphometry, osteocalcin immunohistochemistry and osteoclast tartaric acid and acid phosphatase (TRAP) staining. These data demonstrated that, osteoclast activity and bore resorption enhanced in OVX mice, and osteoclast activity and bone resorption decreased in rapamycin-treated mice, besides this, rapamycin can dominantly rescure osteoporosis in OVX mice.Alkaline phosphatase (ALP) and tartaric acid and acid phosphatase (TRAP) are respectively the characterized exoenzymes of osteoblast and osteoclast differentiation. We collected serum of four groups mice for biochemical detection and found that mTORC1inhibition will increase ALP/TRAP (data not shown). We further assay mTORC1activity in above samples, as a result of activated mTORC1lead to activated bone resorption progression.2RANKL expression is positively regulated by mTORCl activityTo confirm mTORC1regulate RANKL expression level, we in vitro culture mouse bone marrow stromal cell line OP9, rat primary bone marrow stem cell BMSC and treated cells with mTORCl inhibitor rapamycin, then detected mRNA and protein expression levels of RANKL, the data showed us mTORC1inhibition dominantly down-regulate RANKL expression level(P=0.000).mTORCl upstream suppressor------Tuberous Sclerosis Complex (TSC) is organized with TSC1and TSC2, either of them lost will over-activate mTORCl. In orter to confirm mTORC1take part in RANKL regulation, we cultured mouse embryo fibroblast cell lines MEFTSC2+/+and MEFTSC2-/-, and use TSC2siRNA to knockdown TSC1in OP9cell line, then detected RANKL mRNA and protein expression levels(P=0.000, P=0.024). All data were detected and analysed by statistical method, and with significant differences.3OPG expression is negatively regulated by mTORCl activityOsteoclastogenesis and bone resorption is regulated by RANKL/OPG system.Except for measuring mTORCl regulates RANKL, we have also detected OPG expression on mRNA and protenin level in above cell lines. Data showed us mTORC1activity is suppressed by rapamycin while up-regulating OPG expression(P=0.004, P=0.000), and in embryo fibroblast cell lines or TSC2knockdown OP9cell line, OPG expression is denifitely negatively regulated by mTORC1(P=0.000, P=0.000). All data were detected and analysed by statistical method, and with significant differences.4mTORCl regulates transcriptional activity of RANKL/OPGWe next investigated how mTORC1regulated the expression of RANKL/OPG. Luciferase reporter gene system is a useful system for detecting transcriptional factor activity. To determine the role of mTORC1activity in RANKL/OPG regulation, we use luciferase reporter gene plasmids which have been constructed with RANKL or OPG genes, and companied by rapamycin treatment in OP9cell line. These results suggest that mTORC1activity is required for RANKL and OPG transcriptional activity. All data were detected and analysed by statistical method, and with significant differences (P=0.000, P=0.011).5mTORC1decreases mRNA stability of beta-catenin via inhibition of AktIn previous experiments, a screening test via mRNA chip to find a signifinantly variable transcriptional factor:C/EBPβ (β-catenin), in rapamycin-treated OP9cell line. β-catenin regulated RANKL/OPG has been shown recently, we supposed mTORC1is able to regulate RANKL/OPG through β-catenin signaling pathway while affacting the phosphorylation level of Akt (Ser473), which is a downstream signal of mTORC2.Indeed, we found that mTORC1activity inhibition raised an induction of β-catenin expression, meanwhile, mTORC2downstream signal Akt (Ser473) was phosphorylated enhanced in rapamycin-treated or TSC knockdowning cell lines.To further investigate mTORC1/Akt/β-catenin signaling pathway, we use dominant negtice Akt adenovirus to knockdown Akt activity, then treat cells with rapamycin. The data show us when Akt was totally blocked, β-catenin expression is not affected by mTORCl activity. We proposed that mTORC1may decreases mRNA stability of β-catenin via Akt inhibition. All data were detected and analysed by statistical method, and with significant differences.(P=0.000)6mTORCl regulates RANKL/OPG through beta-catenin signalingAs we known, mTORC1and β-catenin mainly in the regulation of RANKL/OPG system, we performed in vitro measuring the molecular mechanism. Interestingly, β-catenin siRNA knockdown interrupted the regulation of mTORC1to RANKL/OPG expression level.ConclusionIn summary, our study reveals a novel regulatory pathway that involves RANKL/OPG system and bone resorption progress. Through this novel pathway, the key regulator Akt drives β-catenin to regulate RANKL/OPG balance dependent of mTORC1. The activated Akt in turn increases mRNA stability of β-catenin through a mechanism involved mTORC1activation to reduce RANKL secretion and induce OPG expression, finally regulate osteoclastogenesis and bone resorption.mTORCl-Akt-catenin-RANKL/OPG pathway is meaningful to provide a new molecular mechanism to treat osteoporosis patient with rapamycin. At present, we determine the further investigation using inducible systemic-specific knockout TSC1/Raptor transgenic mice and osteoblast-specific knockout TSC1/Raptor mice, to confirm mTORC1as a key regulator in osteoclastogenesis and bone metabolism.PART TWOmTORCl is a target of nordihydroguaiaretic acid to prevent breast tumor growth in vitro and in vivo BackgroundNordihydroguaiaretic acid (NDGA) is a natural phenolic compound isolated from the creosote bush Larrea divaricatta that has anti-tumor activities both in vitro and in vivo. Its analogues are in clinical development for use in refractory solid tumors. But the mechanisms underlying the anti-cancer effect of NDGA are not fully understood. In this study, we identified mammalian target of rapamycin complex1(mTORCl) as a target of NDGA both in cultured breast cancer cells and in xenograft models.Mammalian target of rapamycin (mTOR), is a protein Ser-Thr kinase that functions as a central element in a signaling pathway involved in the control of cell growth and proliferation. Disregulation of mTOR signaling associated to human major diseases, for instance, cancer, cardiovascular diseases, graft rejection and autoimmune diseases, diabetes, adiposity, nerve system diseases. mTOR signaling pathway have been become to a certain target in recent years. In our previous research data we have found that unsaturated fatty acid arachidonic acid (AA) and its metabolites can target mTORC1and promote breast cancers. Lipoxygenase pathway is a critical way in arachidodic acid metabolism, NDGA as a strong antioxidant against to lipoxygenase and dominantly inhibits arachidonic acid production. We suppose that mTORC1can be a potent target of NDGA in prevention of breast cancer growth and carcinogenesis.Together our data provide a novel mechanism for NDGA activity which could help explain its anti-cancer activity. NDGA repressed breast tumor growth and targeted mTORC1and its downstream signaling in vitro and in vivo.Results1NDGA inhibits mTORCl in breast cance cells To investigate the potential effect of NDGA on mTOR signaling in breast cancer cells, we cultured several breast cancer cell lines in vitro and treated them with NDGA, we found that NDGA effectively suppressed basal levels of phosphorylation of S6(S235/236) and4E-BP1(downstream target of S6K1and mTORC1) in a dose-and time-dependent manner in all tested cells. NDGA can particularly target mTORC1but not mTORC2.2NDGA inhibits mTORCl downstream signaling and suppresses proliferation in breast cancer cellsCyclin D1, HIF-α and VEGF are three key mTORC1downstream signaling molecules whose expression are positively regulated by mTORC1and responsible for mTORCl overactivation-related carcinogenesis and progression of cancer. we next examine the effect of NDGA on Cyclin D1, HIF-α and VEGF protein levels in MCF-7cells.Taken together, these results further confirmed mTORC1signaling is a target of NDGA and is involved in prevention of breast cancer proliferation. All data were detected and analysed by statistical method, and with significant differences.(P=0.000)3NDGA inhibits mTORCl in part through activation of AMPK/TSC2AMP-activated protein kinase (AMPK) and tuberous sclerosis complex1/2(TSC1/2) are upstream signals that negatively regulate mTORCl. Energy starvation and various stimuli/drugs have been shown to induce phosphorylation and activation of AMPK, activated AMPK phosphorylates TSC directly and inhibit mTORC1. To understand the mechanism through which NDGA inhibits mTORCl, the roles of AMPK/TSC in this process were examined. Interestingly, NDGA increased phosphorylation of AMPK, and knockdown of AMPK or TSC2by siRNA, partially repressed NDGA-induced mTORC1activity decreased. 4NDGA suppresses amino acid-and insulin-stimulated mTORCl activity and disrupts mTORCl complexWe next investigated how NDGA regulated mTORCl. Our result that deletion of TSC2could not diminish the inhibitory effects NDGA against mTORC1implicates additional mechanisms are involved. We thus examined the effects of NDGA on insulin-and amino acids-stimulated mTORC1, which simulates mTORC1in TSC2-dependent and independent mechanism respectively. Interestingly, both amino acids and insulin-stimulated mTORC1activity were repressed by NDGA, these data implicate NDGA may target mTORC1directly.Assembling and stability of the mTORC1determines its kinase activity. Surprisingly, NDGA did not stabilize mTOR-Raptor interaction under either presence or absence of amino acids conditions, however, it acted like rapamycin to disrupt the mTOR-Raptor complex and inhibited mTORC1in vitro kinase activity, whereas mTOR-Rictor association remained unchanged. Taken together, these results implicate that disruption of mTOR-Raptor complex by NDGA may contribute to its inhibitory effects against mTORC1and mTORC1may be a direct target of NDGA.5NDGA inhibits breast tumor growth and targets mTORCl in xenograftWe next asked whether in vivo administration of NDGA inhibits mTORC1activity and prevents breast tumor growth in xenograft. Nude mice bearing MDA-MB-231breast tumor xenografts were dosed with saline or NDGA in a month, the NDGA therapy as single agent markedly inhibited breast tumor growth, most importantly, in vivo administration of NDGA remarkably reduced the levels of mTORC1downstream signals in the breast tumor xenograft. Suggesting that NDGA could inhibit mTORC1and its downstream signaling in vivo. All data were detected and analysed by statistical method, and with significant differences (P=0.000). These results identified mTORC1as a target of NDGA in vivo. ConclusionIn summary, understanding the mechanisms by which NDGA suppress carcinogenesis and progression of cancer is critical for its clinical application. We made observations regarding effects of NDGA on mTORCl/2signaling in breast cancer cell culture and animal model. Our study suggests that NDGA also functions as direct inhibitor of mTORCl.These data implicate that NDGA may represent a potential new class of agents for the treatment of breast and other cancers where the mTORC1signaling play a role in the oncogenic process. |
PDF Full Text Request