Activation of CXCL12-CXCR4 signalling induces conversion of immortalised embryonic kidney cells into cancer stem-like cells.

Cancer stem cells (CSCs) have been implicated in the growth and progression of several types of human cancer. The technology to derive and establish CSCs in vitro could be a critical tool for understanding cancer and developing new therapeutic targets. In this study, we derived expandable CD15+ induced CSCs (iCSCs) from immortalised 293FT human epithelial cells by co-culture with human bone marrow-derived mesenchymal stem cells (BM-MSCs) as feeder cells in vitro. The iCSCs converted through an epithelial-mesenchymal transition program acquired mesenchymal traits, the expression of stem cell markers, and epigenetic changes. Moreover, the iCSCs not only efficiently formed tumorspheres in vitro but also initiated tumours in immunocompromised mice injected with only 10 of the iCSCs. Furthermore, we showed that the expression of the chemokine CXCL12 and its receptor CXCR4 by the iCSCs resulted in the activation of the Fut4 gene through CXCR4/ERK/ELK-1-signalling pathways and the maintenance of the iCSCs in the undifferentiated state through CXCR4/AKT/STAT3-signalling. These findings suggest that immortalised 293FT cells may acquire potential oncogenicity through molecular and cellular alteration processes in microenvironments using BM-MSCs, and could represent a valuable in vitro model as a cancer stem cell surrogate for studying the pathophysiological properties of CSCs.

Calcineurin B-like 3 calcium sensor associates with and inhibits 5'-methylthioadenosine nucleosidase 2 in Arabidopsis.

Calcineurin B-like (CBL) proteins constitute a unique family of calcium sensor relays in plants. It is well known that CBLs detect the calcium signals elicited by a variety of abiotic stresses and relay the information to a group of serine/threonine protein kinases called CBL-interacting protein kinases (CIPKs). In this study, we found that a few CBL members can also target another group of enzymes 5'-methylthioadenosine nucleosidases (MTANs), which are encoded by two genes in Arabidopsis, AtMTAN1 and AtMTAN2. In the yeast two-hybrid system, AtMTAN1 interacted with multiple CBL members such as CBL2, CBL3 and CBL6, whereas AtMTAN2 associated exclusively with CBL3. We further demonstrated that the CBL3-AtMTAN2 association occurs in a calcium-dependent manner, which results in a significant decrease in the enzyme activity of the AtMTAN2 protein. Taken together, these results clearly indicate that the CBL family can target at least two distinct groups of enzymes (CIPKs and MTANs), conferring an additional level of complexity on the CBL-mediated signaling networks. In addition, our finding also provides a novel molecular mechanism by which calcium signals are transduced to alter metabolite profiles in plants.

Efficient reprogramming of mouse fibroblasts to neuronal cells including dopaminergic neurons.

Somatic cells were directly converted to functional neurons through the use of a combination of transcription factors, including Ascl1, Brn2, and Myt1l. However, a major limitation is the lack of a reliable source of cell-replacement therapy for neurological diseases. Here, we show that a combination of the transcription factors Ascl1 and Nurr1 (AN) and neurotrophic factors including SHH and FGF8b directly reprogrammed embryonic mouse fibroblasts to induced neuronal (iN) cells: pan-neuronal cells and dopaminergic (DA) neurons under our systematic cell culture conditions. Reprogrammed cells showed the morphological properties of neuronal cells. Additionally, cells were analyzed using various markers, including Tuj1 and Map2 for neuronal cells and Lmx1a, Th, Aadc and Vmat2 for DA neurons in our immunostaining and reverse transcription (RT)-PCR experiments. We found that a combination of transcription factors and neurotrophic factors could directly reprogram fibroblasts to neuronal cells including DA neurons. Various types of reprogrammed cells are promising cell sources for cell-based therapy of neurological disorders like Parkinson's disease and spinal cord injury.

Stem cell therapy and cellular engineering for treatment of neuronal dysfunction in Huntington's disease.

Huntington's disease (HD) is a fatal inherited neurodegenerative disorder characterized by progressive loss of neurons in the striatum, a sub-cortical region of the forebrain. The sub-cortical region of the forebrain is associated with the control of movement and behavior, thus HD initially presents with coordination difficulty and cognitive decline. Recent reprogramming technologies, including induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs), have created opportunities to understand the pathological cascades that underlie HD and to develop new treatments for this currently incurable neurological disease. The ultimate objectives of stem cell-based therapies for HD are to replace lost neurons and to prevent neuronal dysfunction and death. In this review, we examine the current understanding of the molecular and pathological mechanisms involved in HD. We discuss disease modeling with HD-iPSCs derived from the somatic cells of patients, which could provide an invaluable platform for understanding HD pathogenesis. We speculate about the benefits and drawbacks of using iNSCs as an alternative stem cell source for HD treatment. Finally, we discuss cell culture and engineering systems that promote the directed differentiation of pluripotent stem cell-derived NSCs into a striatal DARPP32(+) GABAergic MSN phenotype for HD. In conclusion, this review summarizes the potentials of cell reprogramming and engineering technologies relevant to the development of cell-based therapies for HD.

Neural stem cells inhibit melanin production by activation of Wnt inhibitors.

BACKGROUND: Melanin for skin pigmentation is synthesized from tyrosine via an enzymatic cascade that is controlled by tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase/tyrosinase related protein 2 (Dct/TRP2), which are the targets of microphthalmia-associated transcription factor (MITF). MITF is a master regulator of pigmentation and a target of ß-catenin in Wnt/ß-catenin signaling during melanocyte differentiation. Stem cells have been used in skin pigmentation studies, but the mechanisms were not determined for the conditioned medium (CM)-mediated effects. OBJECTIVES: In this study, the inhibition and mechanisms of melanin synthesis were elucidated in B16 melanoma cells and UV-B irradiated C57/BL-6 mice that were treated with human neural stem cell-conditioned medium (NSC-CM). METHODS: B16-F10 melanoma cells (1.5×10(4)cells/well) and the shaved dorsal skin of mice were pretreated with various amount (5, 10, 20, 50, and 100%) of NSC-CM. Melanin contents and TYR activity were measured by a Spectramax spectrophotometer. The expression of TYR, TRP1, Dct/TRP2, MITF, ß-catenin and Wnt inhibitors were evaluated by RT-PCR and western blot. The dorsal skin samples were analyzed by immunofluorescence with various antibodies and compared with that control of tissues. RESULTS: Marked decreases were evident in melanin content and TYR, TRP1, DCT/TRP2, MITF, and ß-catenin expression in B16 cells and C57/BL-6 mice. NSC-CM negatively regulated Wnt/ß-catenin signaling by decreasing the expression of ß-catenin protein, which resulted from robust expression of Wnt inhibitors Dickkopf-1 (DKK1) and secreted frizzled-related protein 2 (sFRP2). CONCLUSIONS: These results demonstrate that NSC-CM suppresses melanin production in vitro and in vivo, suggesting that factors in NSC-CM may play an important role in deregulation of epidermal melanogenesis.

Technological progress in generation of induced pluripotent stem cells for clinical applications.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is achieved by viral-mediated transduction of defined transcription factors. Generation of iPSCs is of great medical interest as they have the potential to be a source of patient-specific cells. For the eventual goal of clinical application, it is necessary to overcome the limitations of low reprogramming efficiency and chromosomal abnormalities due to viral DNA integration. In this paper, we summarize the current state of reprogramming technology for generation of iPSCs and also discuss potential approaches to the development of safe iPSCs for personalized cell-based replacement therapy.

Biochemical and structural characterization of 5'-methylthioadenosine nucleosidases from Arabidopsis thaliana.

5'-Methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) are important metabolites in all living organisms. Two similar nucleosidases for hydrolyzing MTA in Arabidopsis thaliana (AtMTAN1 and AtMTAN2) exist, but only AtMTAN2 shows markedly broad substrate specificity for hydrolysis of SAH. To examine the biochemical characteristics of AtMTAN2, it was over-expressed in Escherichia coli and purified to homogeneity. Spectroscopic assays confirm AtMTAN2 catalyzes MTA as well as SAH hydrolysis, compared to AtMTAN1 which only hydrolyzes MTA. In addition, crystal structure of the AtMTAN2 enzyme in complex with, adenine was determined at 2.9A resolution. Finally, a structural comparison of AtMTAN2 performed with previously determined structures of AtMTAN1 and an E. coli homolog provides clues for the substrate specificity of MTA nucleosidases in A. thaliana.

The Arabidopsis calcium sensor calcineurin B-like 3 inhibits the 5'-methylthioadenosine nucleosidase in a calcium-dependent manner.

Calcineurin B-like (CBL) proteins represent a unique family of calcium sensors in plant cells. Sensing the calcium signals elicited by a variety of abiotic stresses, CBLs transmit the information to a group of serine/threonine protein kinases (CBL-interacting protein kinases [CIPKs]), which are currently known as the sole targets of the CBL family. Here, we report that the CBL3 member of this family has a novel interaction partner in addition to the CIPK proteins. Extensive yeast two-hybrid screenings with CBL3 as bait identified an interesting Arabidopsis (Arabidopsis thaliana) cDNA clone (named AtMTAN, for 5'-methylthioadenosine nucleosidase), which encodes a polypeptide similar to EcMTAN from Escherichia coli. Deletion analyses showed that CBL3 utilizes the different structural modules to interact with its distinct target proteins, CIPKs and AtMTAN. In vitro and in vivo analyses verified that CBL3 and AtMTAN physically associate only in the presence of Ca(2+). In addition, we empirically demonstrated that the AtMTAN protein indeed possesses the MTAN activity, which can be inhibited specifically by Ca(2+)-bound CBL3. Overall, these findings suggest that the CBL family members can relay the calcium signals in more diverse ways than previously thought. We also discuss a possible mechanism by which the CBL3-mediated calcium signaling regulates the biosynthesis of ethylene and polyamines, which are involved in plant growth and development as well as various stress responses.

Arabidopsis histidine-containing phosphotransfer factor 4 (AHP4) negatively regulates secondary wall thickening of the anther endothecium during flowering.

Cytokinins are essential hormones in plant development. Arabidopsis histidine-containing phosphotransfer proteins (AHPs) are mediators in a multistep phosphorelay pathway for cytokinin signaling. The exact role of AHP4 has not been elucidated. In this study, we demonstrated young flower-specific expression of AHP4, and compared AHP4-overexpressing (Ox) trangenic Arabidopsis lines and an ahp4 knock-out line. AHP4-Ox plants had reduced fertility due to a lack of secondary cell wall thickening in the anther endothecium and inhibition of IRREGURAR XYLEMs (IRXs) expression in young flowers. Conversely, ahp4 anthers had more lignified anther walls than the wild type, and increased IRXs expression. Our study indicates that AHP4 negatively regulates thickening of the secondary cell wall of the anther endothecium, and provides new insight into the role of cytokinins in formation of secondary cell walls via the action of AHP4.

Identification of putative MAPK kinases in Oryza minuta and O. sativa responsive to biotic stresses.

The mitogen-activated protein kinase (MAPK) signaling cascade is critical for regulating plant defense systems against various kinds of pathogen and environmental stresses. One component of this cascade, the MAP kinase kinases (MAPKK), has not yet been shown to be induced in plants following biotic attacks, such as those by insects and fungi. We describe here a gene coding for a blast (Magnaporthe grisea)- and insect (Nilaparvata lugens)-responsive putative MAPK kinase, OmMKK1 (Oryza minuta MAPKK 1), which was identified in a library of O. minuta expressed sequence tags (ESTs). Two copies of OmMKK1 are present in the O. minuta genome. They encode a predicted protein with molecular mass 39 kDa and pI of 6.2. Transcript patterns following imbibition of plant hormones such as methyl jasmonic acid (MeJA), ethephone, salicylic acid (SA) and abscisic acid (ABA), as well as exposure to methyl viologen (MV), revealed that the expression of OmMKK1 is related to defense response signaling pathways. A comparative analysis of OmMKK1 and its O. sativa ortholog OsMKK1 showed that both were induced by stress-related hormones and biotic stresses, but that the kinetics of their responses differed despite their high amino acid sequence identity (96%).