72 results found
Zhang Z, He C, Gao Y, et al., 2021, alpha-ketoglutarate delays age-related fertility decline in mammals, Aging Cell, Vol: 20, Pages: 1-15, ISSN: 1474-9718
The fecundity reduction with aging is referred as the reproductive aging which comes earlier than that of chronological aging. Since humans have postponed their childbearing age, to prolong the reproductive age becomes urgent agenda for reproductive biologists. In the current study, we examined the potential associations of α‐ketoglutarate (α‐KG) and reproductive aging in mammals including mice, swine, and humans. There is a clear tendency of reduced α‐KG level with aging in the follicle fluids of human. To explore the mechanisms, mice were selected as the convenient animal model. It is observed that a long term of α‐KG administration preserves the ovarian function, the quality and quantity of oocytes as well as the telomere maintaining system in mice. α‐KG suppresses ATP synthase and alterations of the energy metabolism trigger the nutritional sensors to down‐regulate mTOR pathway. These events not only benefit the general aging process but also maintain ovarian function and delay the reproductive decline. Considering the safety of the α‐KG as a naturally occurring molecule in energy metabolism, its utility in reproduction of large mammals including humans deserves further investigation.
Ramlaul K, Fu W, Li H, et al., 2021, Architecture of the Tuberous Sclerosis protein complex, Journal of Molecular Biology, Vol: 433, ISSN: 0022-2836
The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream of the mammalian target of rapamycin complex 1 (mTORC1). The TSCC negatively regulates mTORC1 by acting as a GTPase-activating protein (GAP) towards the small GTPase Rheb. Both human TSC1 and TSC2 are important tumour suppressors, and mutations in them underlie the disease tuberous sclerosis.We used single-particle cryo-EM to reveal the organisation and architecture of the complete human TSCC. We show that TSCC forms an elongated scorpion-like structure, consisting of a central “body”, with a “pincer” and a “tail” at the respective ends. The “body” is composed of a flexible TSC2 HEAT repeat dimer, along the surface of which runs the TSC1 coiled-coil backbone, breaking the symmetry of the dimer. Each end of the body is structurally distinct, representing the N- and C-termini of TSC1; a “pincer” is formed by the highly flexible N-terminal TSC1 core domains and a barbed “tail” makes up the TSC1 coiled-coil-TBC1D7 junction. The TSC2 GAP domain is found abutting the centre of the body on each side of the dimerisation interface, poised to bind a pair of Rheb molecules at a similar separation to the pair in activated mTORC1.Our architectural dissection reveals the mode of association and topology of the complex, casts light on the recruitment of Rheb to the TSCC, and also hints at functional higher order oligomerisation, which has previously been predicted to be important for Rheb-signalling suppression.
Wang X, Yang S, Li S, et al., 2020, Aberrant gut microbiota alters host metabolome and impacts renal failure in humans and rodents, Gut, Vol: 69, Pages: 2131-2142, ISSN: 0017-5749
OBJECTIVE: Patients with renal failure suffer from symptoms caused by uraemic toxins, possibly of gut microbial origin, as deduced from studies in animals. The aim of the study is to characterise relationships between the intestinal microbiome composition, uraemic toxins and renal failure symptoms in human end-stage renal disease (ESRD). DESIGN: Characterisation of gut microbiome, serum and faecal metabolome and human phenotypes in a cohort of 223 patients with ESRD and 69 healthy controls. Multidimensional data integration to reveal links between these datasets and the use of chronic kidney disease (CKD) rodent models to test the effects of intestinal microbiome on toxin accumulation and disease severity. RESULTS: A group of microbial species enriched in ESRD correlates tightly to patient clinical variables and encode functions involved in toxin and secondary bile acids synthesis; the relative abundance of the microbial functions correlates with the serum or faecal concentrations of these metabolites. Microbiota from patients transplanted to renal injured germ-free mice or antibiotic-treated rats induce higher production of serum uraemic toxins and aggravated renal fibrosis and oxidative stress more than microbiota from controls. Two of the species, Eggerthella lenta and Fusobacterium nucleatum, increase uraemic toxins production and promote renal disease development in a CKD rat model. A probiotic Bifidobacterium animalis decreases abundance of these species, reduces levels of toxins and the severity of the disease in rats. CONCLUSION: Aberrant gut microbiota in patients with ESRD sculpts a detrimental metabolome aggravating clinical outcomes, suggesting that the gut microbiota will be a promising target for diminishing uraemic toxicity in those patients. TRIAL REGISTRATION NUMBER: This study was registered at ClinicalTrials.gov (NCT03010696).
Lv C, Li F, Li X, et al., 2020, Author Correction: MiR-31 promotes mammary stem cell expansion and breast tumorigenesis by suppressing Wnt signaling antagonists., Nat Commun, Vol: 11
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Sheng X, Lin Z, Lv C, et al., 2020, Cycling Stem Cells Are Radioresistant and Regenerate the Intestine., Cell Reports, Vol: 32, Pages: 107952-107952, ISSN: 2211-1247
A certain number of epithelial cells in intestinal crypts are DNA damage resistant and contribute to regeneration. However, the cellular mechanism underlying intestinal regeneration remains unclear. Using lineage tracing, we show that cells marked by an Msi1 reporter (Msi1+) are right above Lgr5high cells in intestinal crypts and exhibit DNA damage resistance. Single-cell RNA sequencing reveals that the Msi1+ cells are heterogeneous with the majority being intestinal stem cells (ISCs). The DNA damage-resistant subpopulation of Msi1+ cells is characterized by low-to-negative Lgr5 expression and is more rapidly cycling than Lgr5high radiosensitive crypt base columnar stem cells (CBCs). This enables an efficient repopulation of the intestinal epithelium at early stage when Lgr5high cells are not emerging. Furthermore, relative to CBCs, Msi1+ cells preferentially produce Paneth cells during homeostasis and upon radiation repair. Together, we demonstrate that the DNA damage-resistant Msi1+ cells are cycling ISCs that maintain and regenerate the intestinal epithelium.
Tan GC, Wong YP, Cui W, et al., 2020, Construction of a doxycycline inducible lentivirus that expresses stem cell-specific miR-302 cluster, MALAYSIAN JOURNAL OF PATHOLOGY, Vol: 42, Pages: 91-97, ISSN: 0126-8635
Zhao R, Du S, Liu Y, et al., 2020, Mucoadhesive-to-penetrating controllable peptosomes-in-microspheres co-loaded with anti-miR-31 oligonucleotide and Curcumin for targeted colorectal cancer therapy, THERANOSTICS, Vol: 10, Pages: 3594-3611, ISSN: 1838-7640
Diksin M, Rowlinson J, Kondrashov A, et al., 2019, CHARACTERISATION OF THE POST-SURGICAL INVASIVE TUMOUR NICHE USING ASTROCYTE-GLIOBLASTOMA ORGANOIDS AND DECELLULARISED HUMAN BRAIN, 24th Annual Scientific Meeting and Education Day of the Society-for-Neuro-Oncology (SNO) / 3rd SNO-SCIDOT Joint Conference on Therapeutic Delivery to the CNS, Publisher: OXFORD UNIV PRESS INC, Pages: 264-265, ISSN: 1522-8517
Li G, Tian X, Lv D, et al., 2019, NLRP7 is expressed in ovine ovary and associated with in vitro preimplantation embryo development, Reproduction, Vol: 158, Pages: 415-427, ISSN: 0022-4251
NLRP (NACHT, LRR and PYD domains-containing proteins) family plays the pivotal roles in mammalian reproduction. Mutation of NLRP7 is often associated with human recurrent hydatidiform moles. Few studies regarding the functions of NLRP7 have been performed in another mammalian species rather than humans. In the current study, for the first time, the function of NLRP7 has been explored in ovine ovary. NLRP7 protein was mainly located in ovarian follicles and in the in vitro pre-implantation embryos. To identify its origin, 763bp partial CDS of NLRP7 deriving from sheep cumulus oocyte complexes (COCs) was cloned, it showed a great homology with Homo sapiens. The high levels of mRNA and protein of NLRP7 were steadily expressed in oocytes, parthenogenetic embryos or IVF embryos. NLRP7 knockdown by the combination of siRNA and shRNA jeopardized both the parthenogenetic and IVF embryo development. The results strongly suggest that NLRP7 plays an important role in ovine reproduction. The potential mechanisms of NLRP7 will be fully investigated in the future.
Xu W, Hua H, Chiu Y, et al., 2019, CD146 Regulates Growth Factor-Induced mTORC2 Activity Independent of PI3K and mTORC1 Pathways, Cell Reports, Vol: 29, Pages: 1311-1322.E5, ISSN: 2211-1247
The mechanistic target of rapamycin complex 2(mTORC2) coordinates cell proliferation, survival,and metabolism with environmental inputs, yet howextracellular stimuli such as growth factors (GFs)activate mTORC2 remains enigmatic. Here wedemonstrate that in human endothelial cells,activation of mTORC2 signaling by GFs is mediatedby transmembrane cell adhesion protein CD146.Upon GF stimulation, the cytoplasmic tail of CD146is phosphorylated, which permits its positivelycharged, juxtamembrane KKGK motif to interactwith Rictor, the defining subunit of mTORC2. The formation of the CD146-Rictor/mTORC2 complexprotects Rictor from ubiquitin-proteasome-mediated degradation, thereby specifically upregulatingmTORC2 activity with no intervention of thePI3K and mTORC1 pathways. This CD146-mediatedmTORC2 activation in response to GF stimulationpromotes cell proliferation and survival. Therefore,our findings identify a molecular mechanism bywhich extracellular stimuli regulate mTORC2 activity,linking environmental cues with mTORC2 regulation.
Diksin M, Rowlinson J, Kondrashov A, et al., 2019, CHARACTERISATION OF THE INVASIVE TUMOUR NICHE USING ASTROCYTE-GLIOBLASTOMA ORGANOIDS AND DECELLULARISED HUMAN BRAIN, Meeting of the British-Neuro-Oncology-Society (BNOS), Publisher: OXFORD UNIV PRESS INC, Pages: 7-7, ISSN: 1522-8517
Xu W, Hua H, Chiu Y-H, et al., 2019, CD146 Regulates Growth Factor-Induced mTORC2 Activity Independent of the PI3K and mTORC1 Pathways, Cell Reports, Vol: 29, Pages: 1311-1322.e5, ISSN: 2211-1247
Tian Y, Xu J, Li Y, et al., 2019, MicroRNA-31 reduces inflammatory signaling and promotes regeneration in colon epithelium, and delivery of mimics in microspheres reduces colitis in mice, Gastroenterology, Vol: 156, Pages: 2281-2296.e6, ISSN: 0016-5085
BACKGROUND & AIMS: Levels of microRNA 31 (MIR31) are increased in intestinal tissues from patients with inflammatory bowel diseases and colitis-associated neoplasias. We investigated the effects of this microRNA on intestinal inflammation by studying mice with colitis. METHODS: We obtained colon biopsy samples from 82 patients with ulcerative colitis (UC), 79 patients with Crohn's disease (CD), and 34 healthy individuals (controls) at Shanghai Tenth People's Hospital. MIR31- knockout mice and mice with conditional disruption of Mir31 specifically in the intestinal epithelium (MIR31 conditional knockouts) were given dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS) to induce colitis. We performed chromatin immunoprecipitation (ChIP) and luciferase assays to study proteins that regulate expression of MIR31, including STAT3 and p65, in LOVO colorectal cancer cells and organoids derived from mouse colon cells. Partially hydrolyzed alpha-lactalbumin (alpha-La) was used to generate peptosome nanoparticles, and MIR31 mimics were loaded onto their surface using electrostatic adsorption. Peptosome-MIR31 mimic particles were encapsulated into oxidized konjac glucomannan (OKGM) microspheres, which were administered by enema into the large intestines of mice with DSS-induced colitis. Intestinal tissues were collected and analyzed by histology and immunohistochemistry. RESULTS: Levels of MIR31 were increased in inflamed mucosa from patients with CD or UC, and from mice with colitis, compared with controls. STAT3 and NF-κB activated transcription of MIR31 in colorectal cancer cells and organoids in response to TNF and IL6. MIR31-knockout and conditional-knockout mice developed more severe colitis in response to DSS and TNBS, with increased immune responses, compared to control mice. MIR31 bound to 3' untranslated regions of Il17ra and Il7r mRNAs (which encode receptors for the inflammatory cytokine IL17) and Il16st mRNA (which encodes GP130, a c
Cui W, Zhang S, Bell E, et al., 2019, OCT4 and PAX6 determine the dual function of SOX2 in human ESCs as a key pluripotent or neural factor, Stem Cell Research and Therapy, Vol: 10, ISSN: 1757-6512
BackgroundSox2 is a well-established pluripotent transcription factor that plays an essential role in establishing and maintaining pluripotent stem cells (PSCs). It is also thought to be a linage specifier that governs PSC neural lineage specification upon their exiting the pluripotent state. However, the exact role of SOX2 in human PSCs was still not fully understood. In this study, we studied the role of SOX2 in human embryonic stem cells (hESCs) by gain- and loss-of-function approaches and explored the possible underlying mechanisms.ResultsWe demonstrate that knockdown of SOX2 induced hESC differentiation to endoderm-like cells, whereas overexpression of SOX2 in hESCs enhanced their pluripotency under self-renewing culture conditions but promoted their neural differentiation upon replacing the culture to non-self-renewal conditions. We show that this culture-dependent dual function of SOX2 was probably attributed to its interaction with different transcription factors predisposed by the culture environments. Whilst SOX2 interacts with OCT4 under self-renewal conditions, we found that, upon neural differentiation, PAX6, a key neural transcription factor, is upregulated and shows interaction with SOX2. The SOX2-PAX6 complex has different gene regulation pattern from that of SOX2-OCT4 complex.ConclusionsOur work provides direct evidence that SOX2 is necessarily required for hESC pluripotency; however, it can also function as a neural factor, depending on the environmental input. OCT4 and PAX6 might function as key SOX2-interacting partners that determine the function of SOX2 in hESCs.
Luo Y, Xu W, Li G, et al., 2018, Weighing in on mTOR complex 2 signaling: The expanding role in cell metabolism, Oxidative Medicine and Cellular Longevity, Vol: 2018, ISSN: 1942-0900
In all eukaryotes, the mechanistic target of rapamycin (mTOR) signaling emerges as a master regulator of homeostasis, which integrates environmental inputs, including nutrients, energy, and growth factors, to regulate many fundamental cellular processes such as cell growth and metabolism. mTOR signaling functions through two structurally and functionally distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which correspond to two major branches of signal output. While mTORC1 is well characterized for its structure, regulation, and function in the last decade, information of mTORC2 signaling is only rapidly expanding in recent years, from structural biology, signaling network, to functional impact. Here we review the recent advances in many aspects of the mTORC2 signaling, with particular focus on its involvement in the control of cell metabolism and its physiological implications in metabolic diseases and aging.
Ramasamy TS, Ong ALC, Cui W, 2018, Impact of Three-Dimentional Culture Systems on Hepatic Differentiation of Puripotent Stem Cells and Beyond., Adv Exp Med Biol, Vol: 1077, Pages: 41-66, ISSN: 0065-2598
Generation of functional hepatocytes from human pluripotent stem cells (hPSCs) is a vital tool to produce large amounts of human hepatocytes, which hold a great promise for biomedical and regenerative medicine applications. Despite a tremendous progress in developing the differentiation protocols recapitulating the developmental signalling and stages, these resulting hepatocytes from hPSCs yet achieve maturation and functionality comparable to those primary hepatocytes. The absence of 3D milieu in the culture and differentiation of these hepatocytes may account for this, at least partly, thus developing an optimal 3D culture could be a step forward to achieve this aim. Hence, review focuses on current development of 3D culture systems for hepatic differentiation and maturation and the future perspectives of its application.
Shi J, Ma X, Su Y, et al., 2018, MiR-31 mediates inflammatory signaling to promote re-epithelialization during skin wound healing, Journal of Investigative Dermatology, Vol: 138, Pages: 2253-2263, ISSN: 0022-202X
Wound healing is essential for skin repair after injury, and consists of hemostasis, inflammation, re-epithelialization and remodeling phases. Successful re-epithelialization, which relies on proliferation and migration of epidermal keratinocytes, requires reduction in tissue inflammation. Therefore, understanding the molecular mechanism underlying the transition from inflammation to re-epithelialization will help to better understand the principles of wound healing. Currently, the in vivo functions of specific microRNAs in wound healing are not fully understood. We observed that miR-31 expression is strongly induced in wound edge keratinocytes, and is directly regulated by the activity of NF-κB and STAT3 signaling pathways during inflammation phase. We utilized miR-31 loss-of-function mouse models to demonstrate that miR-31 promotes keratinocyte proliferation and migration. Mechanistically, miR-31 activates the RAS/MAPK signaling by directly targeting Rasa1, Spred1, Spred2 and Spry4, which are negative regulators of the RAS/MAPK pathway. Knockdown of these miR-31 targets at least partially rescues the delayed scratch wound re-epithelialization phenotype observed in vitro in miR-31 knockdown keratinocytes. Taken together, these findings identify miR-31 as an important cell-autonomous mediator during the transition from inflammation to re-epithelialization phases of wound healing, suggesting a therapeutic potential for miR-31 in skin injury repair.
Li Y, Wang J, Ren F, et al., 2018, Lactoferrin Promotes Osteogenesis through TGF-ss Receptor II Binding in Osteoblasts and Activation of Canonical TGF-ss Signaling in MC3T3-E1 Cells and C57BL/6J Mice, JOURNAL OF NUTRITION, Vol: 148, Pages: 1285-1292, ISSN: 0022-3166
BackgroundLactoferrin (LF), as a major functional protein in dairy products, is known to modulate bone anabolic effects. However, the underlying molecular mechanisms remain unclear; the receptor of LF in osteoblast differentiation has not been identified.ObjectiveThe aims of the study were to 1) illuminate whether the receptor of LF in osteoblast differentiation is transforming growth factor-β (TGF-β) receptor (TβR) II and 2) determine whether the TGF-β signaling pathway is activated by LF in promoting osteogenesis in vitro and in vivo, in addition to P38 and extracellular signal-regulated kinase (ERK) pathways.MethodsWe utilized co-immunoprecipitation to detect any binding of LF to TβRII. Subsequently, the role of the TGF-β signaling pathway involved in LF-induced osteoblast proliferation and differentiation was determined by inhibition of TβRI activity by inhibition and knockout of TβRII expression by small guide RNA (sgRNAs) in MC3T3-E1 cells. In addition, 4-wk-old male C57BL/6J mice were orally administered 100 mg LF/kg body weight for 16 wk, after which any activation of the TGF-β signaling pathway in vivo was measured by Western blots.ResultsLF was shown to directly interact with the TβRII protein and to activate the TGF-β signaling pathway in MC3T3-E1 cells. Inhibition of TβRI activity and knockout TβRII expression both attenuated the stimulation of LF in osteoblast proliferation and differentiation by 30–50%. LF-induced activation of TGF-β canonical signaling resulted in upregulation of osteogenic factors. Moreover, the expression of phosphorylated-drosophila mothers against decapentaplegic protein 2 (SMAD2) was increased by 1-fold after LF treatment in the femoral tissue of mice.ConclusionsThis study provides evidence identifying TβRII as an LF receptor in LF-induced osteoblast differentiation. In addition, the TβRII-dependent TGF-β canonical signaling pathways were p
Lv C, Li F, Li X, et al., 2017, MiR-31 promotes mammary stem cell expansion and breast tumorigenesis by suppressing Wnt signaling antagonists, Nature Communications, Vol: 8, ISSN: 2041-1723
MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFβ and Wnt/β-catenin. Particularly, it activates Wnt/β-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.
Tian Y, Ma X, Lv C, et al., 2017, Stress responsive miR-31 is a major modulator of mouse intestinal stem cells during regeneration and tumorigenesis, eLife, Vol: 6, Pages: 1-30, ISSN: 2050-084X
Intestinal regeneration and tumorigenesis are believed to be driven by intestinal stemcells (ISCs). Elucidating mechanisms underlying ISC activation during regeneration andtumorigenesis can help uncover the underlying principles of intestinal homeostasis and diseaseincluding colorectal cancer. Here we show that miR-31 drives ISC proliferation, and protects ISCsagainst apoptosis, both during homeostasis and regeneration in response to ionizing radiationinjury. Furthermore, miR-31 has oncogenic properties, promoting intestinal tumorigenesis.Mechanistically, miR-31 acts to balance input from Wnt, BMP, TGFb signals to coordinate control ofintestinal homeostasis, regeneration and tumorigenesis. We further find that miR-31 is regulated bythe STAT3 signaling pathway in response to radiation injury. These findings identify miR-31 as acritical modulator of ISC biology, and a potential therapeutic target for a broad range of intestinalregenerative disorders and cancers.
Zhu J, Cui W, Dai Y-F, 2017, Production of inbred offspring by intracytoplasmic sperm injection of oocytes from juvenile female mice, Reproduction, Fertility and Development, Vol: 30, Pages: 451-458, ISSN: 1031-3613
The aim of the present study was to determine whether the use of oocytes from juvenile female mice would improve the efficiency of intracytoplasmic sperm injection (ICSI). In the present study, 15 adult and 14 juvenile C57BL6/J female mice were superovulated, with 17.8 oocytes per mouse harvested from adults, significantly lower than the 40.2 harvested from juveniles (P<0.01). Sixty and 233 oocytes were harvested from C57BL/6J adult and juvenile mice respectively, activated in 10mM SrCl2+5μgmL-1 cytochalasin B for 5-6h and cultured in potassium simplex optimisation medium (KSOM) for 3.5 days, with no differences in morula and blastocyst rates between groups (91.7% vs 96.6%; P>0.05). Twelve hours after injection of human chorionic gonadotrophin, oocytes were harvested from C57BL/6J juvenile mice into KSOM, randomly divided into groups and activated with the same method mentioned above at 0, 2, 4 or 6h and then cultured in KSOM for 3.5 days. There was no significant difference in morula and blastocyst rates among the different groups (P>0.05). Oocytes from juvenile mice activated in 10mM SrCl2 for 2h were subjected to ICSI and the rates of pronuclear formation and Day 1 cleavage were significantly improved compared with the control group (P<0.01). ICSI combined with activation of oocytes from inbred mouse strains (C57BL/6J, C57BL/6N and 129Svev) successfully produced pups. The fertility of some these mice resulting from ICSI was tested, and the animals proved fertile. In conclusion, superovulated juvenile mice can yield more useable oocytes than adult mice, but additional activation is essential for full development of ICSI oocytes harvested from juvenile inbred mice.
Imran SAM, Cui W, 2017, The Role of TRF2 in Maintaining and Protecting Neural Cell Properties., 64th Annual Scientific Meeting of the Society-for-Reproductive-Investigation (SRI), Publisher: SAGE PUBLICATIONS INC, Pages: 235A-235A, ISSN: 1933-7191
Cui W, Yu J, 2016, Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination, Development, Vol: 143, Pages: 3050-3060, ISSN: 0950-1991
Phosphatidylinositide 3 kinases (PI3Ks) and their downstream mediators AKT and mammaliantarget of rapamycin (mTOR) constitute the core components of the PI3K/AKT/mTORsignalling cascade, regulating cell proliferation, survival and metabolism. Although thesefunctions are well defined in the context of tumorigenesis, recent studies – in particular thoseusing pluripotent stem cells – have highlighted the importance of this pathway to developmentand cellular differentiation. Here we review the recent in vitro and in vivo evidence for the rolePI3K/Akt/mTOR signalling plays in the control of pluripotency and differentiation, with aparticular focus on the molecular mechanisms underlying these functions.
Ovando-Roche P, Cui W, 2015, Identification of Factors in Regulating a Protein Ubiquitination by Immunoprecipitation: a Case Study of TRF2 on Human REST4 Ubiquitination., Bio-protocol, Vol: 5, ISSN: 2331-8325
Ubiquitination is the first step of the ubiquitin-proteasome pathway that regulates cells for their homeostatic functions and is an enzymatic, protein post-translational modification process in which ubiquitin is transferred to a target protein substrate by a set of three ubiquitin enzymes (Weissman et al., 2011; Bhattacharyya et al., 2014; Ristic et al., 2014). Given the importance of this process, it is plausible that ubiquitination is under strict control by many factors and that the regulatory machineries are protein-specific. An assay for the detection of a specific protein ubiquitination will enable us to examine whether a factor has a function to regulate the ubiquitination of this protein. Here we describe a protocol that detects the ubiquitination status of the human REST4 protein in cultured cells, a neural alternative splicing isoform of REST (RE-1 silencing transcription factor), that antagonizes the repressive function of REST on neural differentiation and neuron formation. Using this protocol, we show that the telomere binding protein TRF2 stabilizes the expression of the human REST4 by inhibiting its ubiquitination. This indicates that TRF2 plays a positive role in neural differentiation (Ovando-Roche et al., 2014). This protocol is also useful for the detection of ubiquitination of other proteins of interest.
Yu JS, Ramasamy TS, Murphy N, et al., 2015, PI3K/mTORC2 regulates TGF-β/Activin signalling by modulating Smad2/3 activity via linker phosphorylation, Nature Communications, Vol: 6, ISSN: 2041-1723
Crosstalk between the phosphatidylinositol 3-kinase (PI3K) and the transforming growth factor-β signalling pathways play an important role in regulating many cellular functions. However, the molecular mechanisms underpinning this crosstalk remain unclear. Here, we report that PI3K signalling antagonizes the Activin-induced definitive endoderm (DE) differentiation of human embryonic stem cells by attenuating the duration of Smad2/3 activation via the mechanistic target of rapamycin complex 2 (mTORC2). Activation of mTORC2 regulates the phosphorylation of the Smad2/3-T220/T179 linker residue independent of Akt, CDK and Erk activity. This phosphorylation primes receptor-activated Smad2/3 for recruitment of the E3 ubiquitin ligase Nedd4L, which in turn leads to their degradation. Inhibition of PI3K/mTORC2 reduces this phosphorylation and increases the duration of Smad2/3 activity, promoting a more robust mesendoderm and endoderm differentiation. These findings present a new and direct crosstalk mechanism between these two pathways in which mTORC2 functions as a novel and critical mediator.
Noisa P, Raivio T, Cui W, 2015, Neural Progenitor Cells Derived from Human Embryonic Stem Cells as an Origin of Dopaminergic Neurons, Stem Cells International, Vol: 2015, ISSN: 1687-9678
Human embryonic stem cells (hESCs) are able to proliferate in vitro indefinitely without losing their ability to differentiate into multiple cell types upon exposure to appropriate signals. Particularly, the ability of hESCs to differentiate into neuronal subtypes is fundamental to develop cell-based therapies for several neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In this study, we differentiated hESCs to dopaminergic neurons via an intermediate stage, neural progenitor cells (NPCs). hESCs were induced to neural progenitor cells by Dorsomorphin, a small molecule that inhibits BMP signalling. The resulting neural progenitor cells exhibited neural bipolarity with high expression of neural progenitor genes and possessed multipotential differentiation ability. CBF1 and bFGF responsiveness of these hES-NP cells suggested their similarity to embryonic neural progenitor cells. A substantial number of dopaminergic neurons were derived from hES-NP cells upon supplementation of FGF8 and SHH, key dopaminergic neuron inducers. Importantly, multiple markers of midbrain neurons were detected, including NURR1, PITX3, and EN1, suggesting that hESC-derived dopaminergic neurons attained the midbrain identity. Altogether, this work underscored the generation of neural progenitor cells that retain the properties of embryonic neural progenitor cells. These cells will serve as an unlimited source for the derivation of dopaminergic neurons, which might be applicable for treating patients with Parkinson’s disease.
Tan GC, Chan E, Molnar A, et al., 2014, 5 ' isomiR variation is of functional and evolutionary importance, Nucleic Acids Research, Vol: 42, Pages: 9424-9435, ISSN: 1362-4962
We have sequenced miRNA libraries from human embryonic,neural and foetal mesenchymal stem cells.We report that the majority of miRNA genes encodemature isomers that vary in size by one ormore bases at the 3 and/or 5 end of the miRNA.Northern blotting for individual miRNAs showed thatthe proportions of isomiRs expressed by a singlemiRNA gene often differ between cell and tissuetypes. IsomiRs were readily co-immunoprecipitatedwith Argonaute proteins in vivo and were active inluciferase assays, indicating that they are functional.Bioinformatics analysis predicts substantial differencesin targeting between miRNAs with minor 5differences and in support of this we report that a 5isomiR-9–1 gained the ability to inhibit the expressionof DNMT3B and NCAM2 but lost the ability toinhibit CDH1 in vitro. This result was confirmed bythe use of isomiR-specific sponges. Our analysis ofthe miRGator database indicates that a small percentageof human miRNA genes express isomiRs asthe dominant transcript in certain cell types and analysisof miRBase shows that 5 isomiRs have replacedcanonical miRNAs many times during evolution. Thisstrongly indicates that isomiRs are of functional importanceand have contributed to the evolution ofmiRNA genes.INT
Zhang S, Cui W, 2014, Sox2, a key factor in the regulation of pluripotency and neural differentiation., World Journal of Stem Cells, Vol: 6, Pages: 305-311, ISSN: 1948-0210
Sex determining region Y-box 2 (Sox2), a member of the SoxB1 transcription factor family, is an important transcriptional regulator in pluripotent stem cells (PSCs). Together with octamer-binding transcription factor 4 and Nanog, they co-operatively control gene expression in PSCs and maintain their pluripotency. Furthermore, Sox2 plays an essential role in somatic cell reprogramming, reversing the epigenetic configuration of differentiated cells back to a pluripotent embryonic state. In addition to its role in regulation of pluripotency, Sox2 is also a critical factor for directing the differentiation of PSCs to neural progenitors and for maintaining the properties of neural progenitor stem cells. Here, we review recent findings concerning the involvement of Sox2 in pluripotency, somatic cell reprogramming and neural differentiation as well as the molecular mechanisms underlying these roles.
Ovando-Roche P, Yu JSL, Testori S, et al., 2014, TRF2-Mediated Stabilization of hREST4 Is Critical for the Differentiation and Maintenance of Neural Progenitors, Stem Cells, Vol: 32, Pages: 2111-2122, ISSN: 1549-4918
Telomere repeat binding factor 2 (TRF2) is a component of the shelterin complex that is knownto bind and protect telomeric DNA, yet the detection of TRF2 in extra-telomeric regions of chromosomessuggests other roles for TRF2 besides telomere protection. Here, we demonstrate thatTRF2 plays a critical role in antagonizing the repressive function of neuron-restrictive silencerfactor, also known as repressor element-1 silencing transcription factor (REST), during the neuraldifferentiation of human embryonic stem cells (hESCs) by enhancing the expression of a truncatedREST splice isoform we term human REST4 (hREST4) due to its similarity to rodent REST4.We show that TRF2 is specifically upregulated during hESC neural differentiation concordantlywith an increase in the expression of hREST4 and that both proteins are highly expressed inNPCs. Overexpression of TRF2 in hESCs increases hREST4 levels and induces their neural differentiation,whereas TRF2 knockdown in hESCs and NPCs reduces hREST4 expression, hinderingtheir ability to differentiate to the neural lineage. Concurrently, we show that TRF2 directlyinteracts with the C-terminal of hREST4 through its TRF2 core binding motif [F/Y]xL, protectinghREST4 from ubiquitin-mediated proteasomal degradation and consequently furthering neuralinduction. Thus, the TRF2-mediated counterbalance between hREST4 and REST is vital for boththe generation and maintenance of NPCs, suggesting an important role for TRF2 in both neurogenesisand function of the central nervous system.
Ramasamy TS, Yu JSL, Selden C, et al., 2013, Application of Three-Dimensional Culture Conditions to Human Embryonic Stem Cell-Derived Definitive Endoderm Cells Enhances Hepatocyte Differentiation and Functionality, TISSUE ENGINEERING PART A, Vol: 19, Pages: 360-367, ISSN: 1937-3341
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