Margit Rosner

The TSC-mTOR Signaling Pathway Regulates the Innate Inflammatory Response

The innate inflammatory immune response must be tightly controlled to avoid damage to the host. Here, we showed that the tuberous sclerosis complex-mammalian target of rapamycin (TSC-mTOR) pathway regulated inflammatory responses after bacterial stimulation in monocytes, macrophages, and primary dendritic cells. Inhibition of mTOR by rapamycin promoted production of proinflammatory cytokines via the transcription factor NF-kappaB but blocked the release of interleukin-10 via the transcription factor STAT3. Conversely, deletion of TSC2, the key negative regulator of mTOR, diminished NF-kappaB but enhanced STAT3 activity and reversed this proinflammatory cytokine shift. Rapamycin-hyperactivated monocytes displayed a strong T helper 1 (Th1) cell- and Th17 cell-polarizing potency. Inhibition of mTOR in vivo regulated the inflammatory response and protected genetically susceptible mice against lethal Listeria monocytogenes infection. These data identify the TSC2-mTOR pathway as a key regulator of innate immune homeostasis with broad clinical implications for infectious and autoimmune diseases, vaccination, cancer, and transplantation.

In vitro cell migration and invasion assays.

Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.

Cytoplasmic and nuclear distribution of the protein complexes mTORC1 and mTORC2: rapamycin triggers dephosphorylation and delocalization of the mTORC2 components rictor and sin1.

The mammalian target of rapamycin (mTOR) is part of two distinct complexes, mTORC1, containing raptor and mLST8, and mTORC2, containing rictor, mLST8 and sin1. Although great endeavors have already been made to elucidate the function and regulation of mTOR, the cytoplasmic nuclear distribution of the mTOR complexes is unknown. Upon establishment of the proper experimental conditions, we found mTOR, mLST8, rictor and sin1 to be less abundant in the nucleus than in the cytoplasm of non-transformed, non-immortalized, diploid human primary fibroblasts. Although raptor is also high abundant in the nucleus, the mTOR/raptor complex is predominantly cytoplasmic, whereas the mTOR/rictor complex is abundant in both compartments. Rapamycin negatively regulates the formation of both mTOR complexes, but the molecular mechanism of its effects on mTORC2 remained elusive. We describe that in primary cells short-term treatment with rapamycin triggers dephosphorylation of rictor and sin1 exclusively in the cytoplasm, but does not affect mTORC2 assembly. Prolonged drug treatment leads to complete dephosphorylation and cytoplasmic translocation of nuclear rictor and sin1 accompanied by inhibition of mTORC2 assembly. The distinct cytoplasmic and nuclear upstream and downstream effectors of mTOR are involved in many cancers and human genetic diseases, such as tuberous sclerosis, Peutz-Jeghers syndrome, von Hippel-Lindau disease, neurofibromatosis type 1, polycystic kidney disease, Alzheimers disease, cardiac hypertrophy, obesity and diabetes. Accordingly, analogs of rapamycin are currently tested in many different clinical trials. Our data allow new insights into the molecular consequences of mTOR dysregulation under pathophysiological conditions and should help to optimize rapamycin treatment of human diseases.

The mTOR pathway and its role in human genetic diseases

The signalling components upstream and downstream of the protein kinase mammalian target of rapamycin (mTOR) are frequently altered in a wide variety of human diseases. Upstream of mTOR key signalling molecules are the small GTPase Ras, the lipid kinase PI3K, the Akt kinase, and the GTPase Rheb, which are known to be deregulated in many human cancers. Mutations in the mTOR pathway component genes TSC1, TSC2, LKB1, PTEN, VHL, NF1 and PKD1 trigger the development of the syndromes tuberous sclerosis, Peutz-Jeghers syndrome, Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Duclos disease, Proteus syndrome, von Hippel-Lindau disease, Neurofibromatosis type 1, and Polycystic kidney disease, respectively. In addition, the tuberous sclerosis proteins have been implicated in the development of several sporadic tumors and in the control of the cyclin-dependent kinase inhibitor p27, known to be of relevance for several cancers. Recently, it has been recognized that mTOR is regulated by TNF-alpha and Wnt, both of which have been shown to play critical roles in the development of many human neoplasias. In addition to all these human diseases, the role of mTOR in Alzheimers disease, cardiac hypertrophy, obesity and type 2 diabetes is discussed.

The tuberous sclerosis gene products hamartin and tuberin are multifunctional proteins with a wide spectrum of interacting partners.

Mutations in the tumor suppressor genes TSC1 and TSC2, encoding hamartin and tuberin, respectively, cause the tumor syndrome tuberous sclerosis with similar phenotypes. Until now, over 50 proteins have been demonstrated to interact with hamartin and/or tuberin. Besides tuberin, the proteins DOCK7, ezrin/radixin/moesin, FIP200, IKKbeta, Melted, Merlin, NADE(p75NTR), NF-L, Plk1 and TBC7 have been found to interact with hamartin. Whereas Plk1 and TBC7 have been demonstrated not to bind to tuberin, for all the other hamartin-interacting proteins the question, whether they can also bind to tuberin, has not been studied. Tuberin interacts with 14-3-3 beta,epsilon,gamma,eta,sigma,tau,zeta, Akt, AMPK, CaM, CRB3/PATJ, cyclin A, cyclins D1, D2, D3, Dsh, ERalpha, Erk, FoxO1, HERC1, HPV16 E6, HSCP-70, HSP70-1, MK2, NEK1, p27KIP1, Pam, PC1, PP2Ac, Rabaptin-5, Rheb, RxRalpha/VDR and SMAD2/3. 14-3-3 beta,epsilon,gamma,eta,sigma,tau,zeta, Akt, Dsh, FoxO1, HERC1, p27KIP1 and PP2Ac are known not to bind to hamartin. For the other tuberin-interacting proteins this question remains elusive. The proteins axin, Cdk1, cyclin B1, GADD34, GSK3, mTOR and RSK1 have been found to co-immunoprecipitate with both, hamartin and tuberin. The kinases Cdk1 and IKKbeta phosphorylate hamartin, Erk, Akt, MK2, AMPK and RSK1 phosphorylate tuberin, and GSK3 phosphorylates both, hamartin and tuberin. This detailed summary of protein interactions allows new insights into their relevance for the wide variety of different functions of hamartin and tuberin.

Cell size regulation by the human TSC tumor suppressor proteins depends on PI3K and FKBP38

TSC1 and TSC2 are responsible for the tumor suppressor gene syndrome tuberous sclerosis (TSC). Mammalian TSC genes have been shown to be involved in cell cycle regulation. Recently, in Drosophila, these data have been confirmed and TSC genes have further been demonstrated to affect cell size control. Here we provide supporting data for the fact that the latter function is conserved in mammals. Human TSC1 and TSC2 trigger mammalian cell size reduction and a dominant-negative TSC2 mutant induces increased size. These effects occur in all cell cycle phases, are dependent on the activity of the phosphoinositide-3-kinase and are abolished by co-overexpression of a dominant-negative Akt mutant. Two independent naturally occurring and disease-causing mutations within the TSC2 gene eliminate tuberins capacity to affect cell size control, emphasizing the relevance of this function for the development of the disease. The same mutations have earlier been shown not to affect tuberins antiproliferative capacity. That the consequences of modulated TSC gene expression on cell proliferation and on cell size can be assigned to separable functions is further supported by two findings: A mutation within the TSC1 gene, earlier shown to still harbor anti-proliferative effects, was found to eliminate the cell size regulating functions. An important mammalian cell size regulator, c-Myc, was found to inhibit tuberins antiproliferative capacity, but to have no effects on tuberin-dependent cell size control. To obtain further mechanistical insights, microarray screens for genes involved in TSC1- or TSC2-mediated cell size effects were performed. Antisense experiments revealed that the so observed regulation of the FK506-binding protein, FKBP38, plays a role in TSC gene-dependent cell size regulation. These data provide new insights into mammalian cell size regulation and allow a better understanding of the function of human TSC genes.

Inhibition of mTOR blocks the anti-inflammatory effects of glucocorticoids in myeloid immune cells

A central role for the mammalian target of rapamycin (mTOR) in innate immunity has been recently defined by its ability to limit proinflammatory mediators. Although glucocorticoids (GCs) exert potent anti-inflammatory effects in innate immune cells, it is currently unknown whether the mTOR pathway interferes with GC signaling. Here we show that inhibition of mTOR with rapamycin or Torin1 prevented the anti-inflammatory potency of GC both in human monocytes and myeloid dendritic cells. GCs could not suppress nuclear factor-κB and JNK activation, the expression of proinflammatory cytokines, and the promotion of Th1 responses when mTOR was inhibited. Interestingly, long-term activation of monocytes with lipopolysaccharide enhanced the expression of TSC2, the principle negative regulator of mTOR, whereas dexamethasone blocked TSC2 expression and reestablished mTOR activation. Renal transplant patients receiving rapamycin but not those receiving calcineurin inhibitors displayed a state of innate immune cell hyper-responsiveness despite the concurrent use of GC. Finally, mTOR inhibition was able to override the healing phenotype of dexamethasone in a murine lipopolysaccharide shock model. Collectively, these data identify a novel link between the glucocorticoid receptor and mTOR in innate immune cells, which is of considerable clinical importance in a variety of disorders, including allogeneic transplantation, autoimmune diseases, and cancer.

Contribution of human amniotic fluid stem cells to renal tissue formation depends on mTOR

Human amniotic fluid stem cells (hAFSCs) can be grown in large quantities, have a low risk for tumour development and harbour a high differentiation potential. They are a very promising new fetal stem cell type for cell-based therapy approaches and for studying differentiation processes without raising the ethical concerns associated with embryonic stem cells. Recently, a protocol for studies on renal development has been established in which murine embryonic kidneys are dissociated into single-cell suspension and then reaggregated to form organotypic renal structures. Using this approach, we formed chimeric renal structures via mixing murine embryonic kidney cells with monoclonal hAFSCs. We demonstrate here that hAFSCs harbour the potential to contribute to renal tissue formation accompanied by induction of specific renal marker expression. As part of the two kinase complexes mTORC1 and mTORC2, mammalian target of rapamycin (mTOR) is the key component of an important signalling pathway, which is involved in the regulation of differentiation and in the development of a wide variety of human genetic diseases many with characteristic kidney symptoms. Modulating endogenous mTOR activity via specific siRNA approaches revealed that contribution of hAFSCs to renal tissue formation is regulated by mTORC1 and mTORC2. These findings (i) demonstrate renal differentiation potential of hAFSCs, (ii) prove chimeric cultures of mixtures of murine embryonic kidney cells and hAFSCs to be a powerful tool to study the effects of gene knockdowns for renal structure formation and (iii) provide new insights into the role of the mTOR pathway for renal development.

Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle

This protocol describes a method for nucleocytoplasmic protein tracking during normal cell cycle progression using unmanipulated, asynchronous cells. In contrast with prevalent traditional methods, our approach does not require time-consuming, perturbing cell synchronization or separation. To this end, we chose a single-cell approach and developed a flow cytometry assay that is applied to whole cells and isolated nuclei. Our protocol involves a stepwise biochemical fractionation procedure to purify nuclei from whole cells, conventional DNA and indirect immunostaining techniques for the dual labeling of cells and nuclei for DNA and protein, and a refined concept of flow cytometric data processing and calculation: through the specific combination of DNA and cell size analyses, G1, S and G2/M phases of the cell cycle are further dissected to establish a high-resolution map of cell cycle progression, to which protein expression in cells or nuclei is correlated. In a final data analysis step, cell cycle–related, cytoplasmic protein expression is calculated on the basis of results obtained for whole cells and isolated nuclei. A minimum of 8 h is required to complete the procedure. As the approach does not require cell type–restricting pretreatments, numerous cell types of different origin can be readily studied. Human amniotic fluid stem cells, primary human fibroblasts, immortalized mouse fibroblasts and transformed tumor cells are analyzed at comparable efficiencies, demonstrating low intercell assay variability.

Human amniotic fluid stem cells: a new perspective

Summary.The discovery of amniotic fluid stem cells initiated a new and very promising field in stem cell research. In the last four years amniotic fluid stem cells have been shown to express markers specific to pluripotent stem cells, such as Oct-4. Due to their high proliferation potential, amniotic fluid stem cell lineages can be established. Meanwhile, they have been shown to harbor the potential to differentiate into cells of all three embryonic germ layers. It will be a major aim for the future to define the potential of this new source of stem cells for therapies related to specific diseases.