Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy ...for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs). Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE.
•hES-MSCs show increased anti-EAE effects relative to adult human BM-MSCs•hES-MSCs express fewer proinflammatory cytokines than BM-MSCs•hES-MSCs enter the CNS more efficiently than BM-MSCs in EAE
Mesenchymal stem cells (MSCs) offer a potential therapy for multiple sclerosis. Xu, Lanza, and colleagues show that MSCs derived from human embryonic stem cells (hES-MSCs) significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse EAE model of multiple sclerosis, and that the disease-inhibitory effect of hES-MSCs is remarkably greater than that of human bone-marrow-derived MSCs.
Proper control of B cell growth and metabolism is crucial for B-cell-mediated immunity, but the underlying molecular mechanisms remain incompletely understood. In this study, Sin1, a key component of ...mTOR complex 2 (mTORC2), specifically regulates B cell growth and metabolism. Genetic ablation of Sin1 in B cells reduces the cell size at either the transitional stage or upon antigen stimulation and severely impairs metabolism. Sin1 deficiency also severely impairs B-cell proliferation, antibody responses, and anti-viral immunity. At the molecular level, Sin1 controls the expression and stability of the c-Myc protein and maintains the activity of mTORC1 through the Akt-dependent inactivation of GSK3 and TSC1/2, respectively. Therefore, our study reveals a novel and specific role for Sin1 in coordinating the activation of mTORC2 and mTORC1 to control B cell growth and metabolism.
Constitutive activation of the kinases Akt or protein kinase C (PKC) in blood cancers promotes tumor-cell proliferation and survival and is associated with poor patient survival. The mammalian target ...of rapamycin (mTOR) complex 2 (mTORC2) regulates the stability of Akt and conventional PKC (cPKC; PKCα and PKCβ) proteins by phosphorylating the highly conserved turn motif of these proteins. In cells that lack mTORC2 function, the turn motif phosphorylation of Akt and cPKC is abolished and therefore Akt and cPKC protein stability is impaired. However, the chaperone protein HSP90 can stabilize Akt and cPKC, partially rescuing the expression of these proteins. In the present study, we investigated the antitumor effects of inhibiting mTORC2 plus HSP90 in mouse and human leukemia cell models and show that the HSP90 inhibitor 17-allylaminogeldanamycin (17-AAG) preferentially inhibits Akt and cPKC expression and promotes cell death in mTORC2 deficient pre-B leukemia cells. Furthermore, we show that 17-AAG selectively inhibits mTORC2 deficient leukemia cell growth in vivo. Finally, we show that the mTOR inhibitors rapamycin and pp242 work together with 17-AAG to inhibit leukemia cell growth to a greater extent than either drug alone. These studies provide a mechanistic and clinical rationale to combine mTOR inhibitors with chaperone protein inhibitors to treat human blood cancers.
The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. ...Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at Thr 86 and Thr 398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin- or IGF-1-mediated, but also PDGF- or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Finally, a cancer-patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-activation of mTORC2 by bypassing this negative regulation. Together, our results reveal a Sin1-phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1-S6K-Sin1 signalling axis might cause aberrant hyper-activation of the mTORC2-Akt pathway, which facilitates tumorigenesis.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially ...superior cell source for MSCs. However, the generation methods reported so far vary greatly in quality and efficiency. Here, we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast‐like intermediate stage in approximately 11–16 days. We term these cells “T‐MSCs” and show that T‐MSCs express a phenotype and differentiation potential minimally required to define MSCs. T‐MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs, T‐MSCs do not have increased expression of inflammatory mediators in response to IFNγ. Moreover, T‐MSCs constitutively express a high level of the immune inhibitory ligand PD‐L1 and elicit strong and durable efficacy in two distinct animal models of autoimmune disease, dextran sulfate sodium induced colitis, and experimental autoimmune encephalomyelitis, at doses near those approved for clinical trials. Together, we present a simple and fast derivation method to generate MSCs from hESCs, which possess potent immunomodulatory properties in vitro and in vivo and may serve as a novel and ideal candidate for MSC‐based therapies. Stem Cells 2016;34:380–391
The basic helix-loop-helix transcription factor E2A is an essential regulator of B lymphocyte lineage commitment and is required to activate the expression of numerous B lineage-specific genes. ...Studies involving ectopic expression of Id proteins, which inhibit E2A as well as other basic helix-loop-helix proteins such as HEB, suggest additional roles of E2A at later stages of B cell development. We use E2A-deficient and E2A and HEB double-deficient pre-B cell lines to directly assess the function of E2A and HEB in B cell development after lineage commitment. We show that, in contrast to the established role of E2A in lineage commitment, elimination of E2A and HEB in pre-B cell lines has only a modest negative impact on B lineage gene expression. However, E2A single and E2A and HEB double-deficient but not HEB single-deficient cell lines show dramatically enhanced apoptosis upon growth arrest. To address the possible role of E2A in the regulation of B cell survival in vivo, we crossed IFN-inducible Cre-transgenic mice to E2A conditional mice. Cre-mediated E2A deletion resulted in a block in bone marrow B cell development and a significant reduction in the proportion and total number of splenic B cells in these mice. We show that Cre-mediated deletion of E2A in adoptively transferred mature B cells results in the rapid depletion of the transferred population within 24 h of Cre induction. These results reveal that E2A is not required to maintain B cell fate but is essential in promoting pre-B and B cell survival.
The transcription factors encoded by the E2A gene have been shown to play essential roles in the initiation and progression of lymphocyte development. However, there
is still a lack of comprehensive ...understanding of E2A downstream genes in B-cell development. We previously developed a gene
tagging-based chromatin immunoprecipitation (ChIP) system to directly evaluate E2A target genes in B-cell development. Here,
we have improved this ChIP strategy and used it in conjunction with microarray analysis on E2A -deficient pre-B-cell lines to determine E2A target genes in lymphocyte development. Both microarray data and ChIP studies
confirmed that E2A directly controls IgH gene expression. The microarray assay also revealed genes that were significantly
up-regulated after E2A disruption. ChIP analysis showed that E2A was most likely to be directly involved in repression of
some of these target genes such as Nfil3 and FGFR2 . An inducible E2A reconstitution system further demonstrated that E2A-mediated repression of Nfil3 and FGFR2 was reversible. Collectively, these findings indicate that E2A is a positive regulator for one set of genes and a negative
regulator for another set of genes in developing B lymphocytes.
The protein kinase Akt (also known as protein kinase B) is a critical signaling hub downstream of various cellular stimuli such as growth factors that control cell survival, growth, and ...proliferation. The activity of Akt is tightly regulated, and the aberrant activation of Akt is associated with diverse human diseases including cancer. Although it is well documented that the mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of the Akt hydrophobic motif (Ser-473 in Akt1) is essential for full Akt activation, it remains unclear whether this phosphorylation has additional roles in regulating Akt activity. In this study, we found that abolishing Akt Ser-473 phosphorylation stabilizes Akt following agonist stimulation. The Akt Ser-473 phosphorylation promotes a Lys-48-linked polyubiquitination of Akt, resulting in its rapid proteasomal degradation. Moreover, blockade of this proteasomal degradation pathway prolongs agonist-induced Akt activation. These data reveal that mTORC2 plays a central role in regulating the Akt protein life cycle by first stabilizing Akt protein folding through the turn motif phosphorylation and then by promoting Akt protein degradation through the hydrophobic motif phosphorylation. Taken together, this study reveals that the Akt Ser-473 phosphorylation-dependent ubiquitination and degradation is an important negative feedback regulation that specifically terminates Akt activation.
Mammalian target of rapamycin (mTOR) is an important mediator of phosphoinositol-3-kinase (PI3K) signaling. PI3K signaling regulates B cell development, homeostasis, and immune responses. However, ...the function and molecular mechanism of mTOR-mediated PI3K signaling in B cells has not been fully elucidated. Here we show that Sin1, an essential component of mTOR complex 2 (mTORC2), regulates B cell development. Sin1 deficiency results in increased IL-7 receptor (il7r) and RAG recombinase (rag1 and rag2) gene expression, leading to enhanced pro-B cell survival and augmented V(D)J recombinase activity. We further show that Akt2 specifically mediates the Sin1-mTORC2 dependent suppression of il7r and rag gene expression in B cells by regulating FoxO1 phosphorylation. Finally, we demonstrate that the mTOR inhibitor rapamycin induces rag expression and promotes V(D)J recombination in B cells. Our study reveals that the Sin1/mTORC2-Akt2 signaling axis is a key regulator of FoxO1 transcriptional activity in B cells.
Display omitted
► Sin1 maintains mTORC2 integrity and regulates B cell development ► Sin1-mTORC2 utilizes Akt2 but not Akt1 to suppress il7r and rag gene expression ► Akt HM site phosphorylation is required to suppress FoxO1 target gene expression ► Rapamycin inhibits mTORC2 and promotes rag gene expression in B cells
PDF
