Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling

The effects of mesenchymal stem cells (MSCs) on proliferation and cell fate determination of neural stem cells (NSCs) have been investigated. NSCs were co-cultured with MSCs or NIH3T3 cells using an in vitro transwell system. After 4 days, immunofluorescence staining showed that the number of cells positive for the cell proliferation antigen, ki-67, in neurospheres in MSCs was greater than in NIH3T3 cells. In some experiments, the top-layers of MSCs and NIH3T3 cells were removed to induce NSCs differentiation. Seven days after initiating differentiation, the levels of the neuronal marker, NSE, were higher in NSCs in MSCs co-culture group, and those of glial fibrillary acidic protein (GFAP) were lower, compared with NIH3T3 cells co-culture group. These were confirmed by immunofluorescence. The role of the Notch signaling pathway analyzed with the specific inhibitor, DAPT, and by examining the expression of Notch-related genes using RT-PCR showed that after co-culturing with MSCs for 24 h, NSCs expressed much higher levels of ki-67, Notch1, and Hes1 than did NSCs co-cultured with NIH3T3 cells. Treatment with DAPT decreased ki-67, Notch1 and Hes1 expression in NCSs, and increased Mash1 expression. The data indicate that the interactions between MSCs and NSCs promote NSCs proliferation and are involved in specifying neuronal fate, mediated in part by Notch signaling.     

Endoplasmin regulates differentiation of tonsil-derived mesenchymal stem cells into chondrocytes through ERK signaling

It is well-known that some species of lizard have an exceptional ability known as caudal autotomy (voluntary self-amputation of the tail) as an anti-predation mechanism. After amputation occurs, they can regenerate their new tails in a few days. The new tail section is generally shorter than the original one and is composed of cartilage rather than vertebrae bone. In addition, the skin of the regenerated tail distinctly differs from its original appearance. We performed a proteomics analysis for extracts derived from regenerating lizard tail tissues after amputation and found that endoplasmin (ENPL) was the main factor among proteins up-regulated in expression during regeneration. Thus, we performed further experiments to determine whether ENPL could induce chondrogenesis of tonsil-derived mesenchymal stem cells (T-MSCs). In this study, we found that chondrogenic differentiation was associated with an increase of ENPL expression by ER stress. We also found that ENPL was involved in chondrogenic differentiation of T-MSCs by suppressing extracellular signal-regulated kinase (ERK) phosphorylation.     

Protein signaling pathways in differentiation of neural stem cells

Neural stem cells (NSC) capable of differentiating into neurons, astrocytes and oligodendrocytes are a promising source of cells for the treatment of central nervous system diseases. Access to signaling proteins present in such cells in low copies and with specific regulatory functions has been very restrictive until now as judged by classical proteomic approaches and limitations due to scarcity of stem cell populations. Hence, we utilized the Kinex Antibody Microarray analysis where profiles of the proliferating porcine NSC and differentiated counterparts were compared and selected changes were verified by immunoblotting. Differentiated neural cells exhibited an increased level of RafB proto-oncogene-encoded protein-serine kinase, MAP kinase protein-serine kinase 3, heme oxygenase 2 (HO2) and protein phosphatase 4 catalytical subunit. On the other hand, relatively high level of G protein-coupled receptor-serine kinase 2 and enhanced phosphorylations of alphaB-crystallin (S45), protein-serine kinase C gamma (T655), protein-serine kinase D (PKCmu; S738+S742) together with eukaryotic translation initiation factor 2 alpha (eIF2alpha) (S51) raised intriguing questions as regards their potential functionality within stem cells. In-depth study of HO2 and phospho-S45 alphaB-crystallin confirmed expression profiles and intense cytoplasmic localization of HO2 in neurons but a weaker signal in glial cells. Phospho-S45 alphaB-crystallin was localized in nuclei of differentiated neural cells. Computer simulation of possible interaction network connecting regulated proteins, exposed additional relationships including direct interactions of HO2 with amyloid precursor protein or huntingtin-associated protein 1.     

EphrinB-EphB signalling regulates clathrin-mediated endocytosis through tyrosine phosphorylation of synaptojanin 1

Recent studies show that Eph receptors act mainly through the regulation of actin reorganization. Here, we show a novel mode of action for EphB receptors. We identify synaptojanin 1 - a phosphatidylinositol 5'-phosphatase that is involved in clathrin-mediated endocytosis - as a physiological substrate for EphB2. EphB2 causes tyrosine phosphorylation in the proline-rich domain of synaptojanin 1, and inhibits both the interaction with endophilin and the 5'-phosphatase activity of synaptojanin 1. Treatment with the EphB ligand, ephrinB2, elevates the cellular level of phosphatidylinositol 4,5-bisphosphate and promotes transferrin uptake. A kinase inactive mutant of EphB2 and a phosphorylation site mutant of synaptojanin 1 both neutralize the increase of transferrin uptake after ephrinB2 treatment. These mutants also inhibit AMPA glutamate receptor endocytosis in hippocampal neurons. Interestingly, incorporated transferrin does not reach endosomes, suggesting dual effects of EphB signalling on the early and late phases of clathrin-mediated endocytosis. Our results indicate that ephrinB-EphB signalling regulates clathrin-mediated endocytosis in various cellular contexts by influencing protein interactions and phosphoinositide turnover through tyrosine phosphorylation of synaptojanin 1.     

Autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway

Autophagy plays important roles in self-renewal and differentiation of stem cells. Hepatic progenitor cells (HPCs) are thought to have the ability of self-renewal as well as possess a bipotential capacity, which allows them to differentiate into both hepatocytes and bile ductular cells. However, how autophagy contributes to self-renewal and differentiation of hepatic progenitor cells is not well understood. In this study, we use a well-established rat hepatic progenitor cell lines called WB-F344, which is treated with 3.75 mM sodium butyrate (SB) to promote the differentiation of WB-F344 along the biliary phenotype. We found that autophagy was decreased in the early stage of biliary differentiation, and maintained a low level at the late stage. Activation of autophagy by rapamycin or starvation suppressed the biliary differentiation of WB-F344. Further study reported that autophagy inhibited Notch1 signaling pathway, which contributed to biliary differentiation and morphogenesis. In conclusions, autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway.     

Delta-like 1-Lysine613 regulates notch signaling

Delta ligands are important for regulating Notch signaling through transcellular stimulation of Notch receptors. The cytoplasmic tails of Delta ligands have multiple potential regulatory sites including several lysine residues that are putative targets for ubiquitination by the E3 ubiquitin ligases, Mind Bomb and Neuralized. To identify possible roles for specific lysine residues in the cytoplasmic tail of the Notch ligand Dll1 a mutational and functional analysis was performed. Examination of a panel of individual or clustered lysine mutants demonstrated that lysine 613 (K613) in the cytoplasmic tail of Dll1 is a key residue necessary for transcellular activation of Notch signaling. Multi-ubiquitination of the Dll1 mutant Dll1-K613R was altered compared to wild type Dll1, and the K613R mutation blocked the ability of Dll1 to interact with Notch1. Finally, mutation of K613 did not affect the stability of Dll1 or its ability to traffic to recycle to the plasma membrane, but did enhance the fraction associated with lipid rafts. Collectively these results suggest that the transcellular defect in Notch signaling attributed to residue K613 in cytoplasmic tail of Dll1 may result from altering its multi-ubiquitination and increasing its retention in lipid rafts.     

Radiation-induced glioblastoma signaling cascade regulates viability,apoptosis and differentiation of neural stem cells (NSC)

Ionizing radiation alone or in combination with chemotherapy is the main treatment modality for brain tumors including glioblastoma. Adult neurons and astrocytes demonstrate substantial radioresistance; in contrast, human neural stem cells (NSC) are highly sensitive to radiation via induction of apoptosis. Irradiation of tumor cells has the potential risk of affecting the viability and function of NSC. In this study, we have evaluated the effects of irradiated glioblastoma cells on viability, proliferation and differentiation potential of non-irradiated (bystander) NSC through radiation-induced signaling cascades. Using media transfer experiments, we demonstrated significant effects of the U87MG glioblastoma secretome after gamma-irradiation on apoptosis in non-irradiated NSC. Addition of anti-TRAIL antibody to the transferred media partially suppressed apoptosis in NSC. Furthermore, we observed a dramatic increase in the production and secretion of IL8, TGFβ1 and IL6 by irradiated glioblastoma cells, which could promote glioblastoma cell survival and modify the effects of death factors in bystander NSC. While differentiation of NSC into neurons and astrocytes occurred efficiently with the corresponding differentiation media, pretreatment of NSC for 8 h with medium from irradiated glioblastoma cells selectively suppressed the differentiation of NSC into neurons, but not into astrocytes. Exogenous IL8 and TGFβ1 increased NSC/NPC survival, but also suppressed neuronal differentiation. On the other hand, IL6 was known to positively affect survival and differentiation of astrocyte progenitors. We established a U87MG neurosphere culture that was substantially enriched by SOX2⁺ and CD133⁺ glioma stem-like cells (GSC). Gamma-irradiation up-regulated apoptotic death in GSC via the FasL/Fas pathway. Media transfer experiments from irradiated GSC to non-targeted NSC again demonstrated induction of apoptosis and suppression of neuronal differentiation of NSC. In summary, intercellular communication between glioblastoma cells and bystander NSC/NPC could be involved in the amplification of cancer pathology in the brain.     

hCINAP regulates the differentiation of embryonic stem cells by regulating NEDD4 liquid-liquid phase-separation-mediated YAP1 activation

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Serine phosphorylation regulates disabled-1 early isoform turnover independently of Reelin

The Reelin-Disabled 1 (Dab1) signaling pathway plays an important role in neuronal cell migration during brain development. Dab1, an intracellular adapter protein which is tyrosine phosphorylated upon Reelin stimulation, has been directly implicated in the transmission and termination of Reelin-mediated signaling. Two main forms of Dab1 have been identified in the developing chick retina, an early isoform (Dab1-E) expressed in progenitor cells and a late isoform (Dab1-L, a.k.a. Dab1) expressed in differentiated cells. Dab1-E is missing two Src family kinase (SFK) phosphorylation sites that are critical for Reelin-Dab1 signaling and is not tyrosine phosphorylated. We have recently demonstrated a role for Dab1-E in the maintenance of retinal progenitor cells. Here, we report that Dab1-E is phosphorylated at serine/threonine residues independent of Reelin. Cdk2, highly expressed in retinal progenitor cells, mediates Dab1-E phosphorylation at serine 475 which in turn promotes ubiquitination-triggered proteasome degradation of Dab1-E. Inhibition of protein phosphatase 1 and/or protein phosphatase 2A leads to increased Dab1-E instability. We propose that Dab1 turnover is regulated by both Reelin-independent serine/threonine phosphorylation and Reelin-dependent tyrosine phosphorylation.     

Annexin1 regulates the erythroid differentiation through ERK signaling pathway

K562 cells can be used as a model of erythroid differentiation on being induced by hemin. We found that the level of annexin1 gene expression was notably increased during this indicated process. To test the hypothesis that annexin1 can regulate erythropoiesis, K562 cell clones in which annexin1 was stably increased and was knocked down by RNAi were established, respectively. With analysis by hemoglobin quantification, benzidine staining, and marker gene expression profile determination, we confirmed that hemin-induced erythroid differentiation of K562 cells was modestly stimulated by overexpression of annexin1 while it was significantly blocked by knock down of annexin1. Further studies revealed that the mechanisms of annexin1 regulation of the erythroid differentiation was partially related to the increased ERK phosphorylation and expression of p21(cip/waf), since specific inhibitor of MEK blocked the function of annexin1 in erythroid differentiation. We concluded that annexin1 exerted its erythropoiesis regulating effect by ERK pathway.     

Reelin induces a radial glial phenotype in human neural progenitor cells by activation of Notch-1

Background  

Reelin and Notch-1 signaling pathways have been recently found to be necessary to induce the expression of brain lipid binding protein (BLBP) and to promote the process extension and the maturation of the neuronal progenitors, the radial glial cells. In this study, we report the cross talk between these two pathways.     

Beta-catenin signaling is required for neural differentiation of embryonic stem cells

Culture of embryonic stem (ES) cells at high density inhibits both beta-catenin signaling and neural differentiation. ES cell density does not influence beta-catenin expression, but a greater proportion of beta-catenin is targeted for degradation in high-density cultures. Moreover, in high-density cultures, beta-catenin is preferentially localized to the membrane further reducing beta-catenin signaling. Increasing beta-catenin signaling by treatment with Wnt3a-conditioned medium, by overexpression of beta-catenin, or by overexpression of a dominant-negative form of E-cadherin promotes neurogenesis. Furthermore, beta-catenin signaling is sufficient to induce neurogenesis in high-density cultures even in the absence of retinoic acid (RA), although RA potentiates the effects of beta-catenin. By contrast, RA does not induce neurogenesis in high-density cultures in the absence of beta-catenin signaling. Truncation of the armadillo domain of beta-catenin, but not the C terminus or the N terminus, eliminates its proneural effects. The proneural effects of beta-catenin reflect enhanced lineage commitment rather than proliferation of neural progenitor cells. Neurons induced by beta-catenin overexpression either alone or in association with RA express the caudal neuronal marker Hoxc4. However, RA treatment inhibits the beta-catenin-mediated generation of tyrosine hydroxylase-positive neurons, suggesting that not all of the effects of RA are dependent upon beta-catenin signaling. These observations suggest that beta-catenin signaling promotes neural lineage commitment by ES cells, and that beta-catenin signaling may be a necessary co-factor for RA-mediated neuronal differentiation. Further, enhancement of beta-catenin signaling with RA treatment significantly increases the numbers of neurons generated from ES cells, thus suggesting a method for obtaining large numbers of neural species for possible use in for ES cell transplantation.     

Ginsenoside Rg1 facilitates neural differentiation of mouse embryonic stem cells via GR-dependent signaling pathway

Ginsenoside Rg1, a steroidal saponin of high abundance in ginseng, possesses the neuroprotective effects. In this study, we tried to explore the effect of Rg1 on promoting differentiation of mouse embryonic stem (ES) cells towards the neuronal lineage and its potential role involved in glucocorticoid receptor (GR) activation. Rg1 treatment induced a remarkable increase in the population of neuron-like cells in a time-dependent manner. More than 80% of Rg1-treated embryoid bodies (EBs) differentiated into neuron-like cells on d 8 + 10. Furthermore, the gradually increased protein expression of neurofilament (NEFM) and β-tubulin III (a neuronal specific protein) was determined. GR expression gradually increased during the differentiation course. RU486, an antagonist of GR, could efficiently block the neurogenesis-promoting activity of Rg1. On the other side, Rg1 stimulated the phosphorylation of ERK1/2 and Akt at different time points through GR activation-dependent mechanisms. Treatment of both U0126 (an inhibitor of MEK) and LY294002 (an inhibitor of PI3 K), hampered the neuronal differentiation induced by Rg1. Meantime, U0126 further decreased Rg1-induced p-Akt expression. In conclusion, Rg1 possesses the effects on inducing differentiation of mouse ES cells into neurons in vitro via the GR-MEK-ERK1/2-PI3 K-Akt signaling pathway.     

A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.     

Notch信号在盐酸法舒地尔诱导大鼠骨髓间充质干细胞向神经元分化过程中的作用

目的:探讨Notch信号通路在盐酸法舒地尔诱导大鼠骨髓间充质干细胞(MSCs)向神经元分化中的作用。方法:实验分为未转染组、转染组(转染Rn-Notch1-siRNA)、阳性对照组(转染Rn-MAPK-1 Control siRNA)及阴性对照组(转染Negative Control siRNA)等4组。采用盐酸法舒地尔诱导大鼠MSCs分化为神经元。倒置荧光显微镜下观察MSCs转染后荧光表达情况;RT-PCR检测Notch1、Hes1和MAPK1 mRNA的表达变化;免疫细胞化学法检测Notch1、神经元烯醇化酶(NSE)、神经微丝蛋白亚单位(NF-M)和胶质纤维酸性蛋白(GFAP)的表达变化;MTT方法检测细胞存活率。结果:①siRNA转染72h,MSCs荧光表达最强,转染率可达91.3%±4.2%;同时,转染组MSCs的Notch1和Hes1 mRNA转录下降(P0.05);MTT提示转染组细胞存活率也显著减少(P0.05)。②盐酸法舒地尔可以诱导MSCs向神经元分化,其中以转染组诱导效果最佳,NSE、NF-M的表达率显著的高于其它各组(P0.05)。结论:盐酸法舒地尔在诱导大鼠MSCs向神经元分化过程中,可能存在Notch信号通路与RhoA/Rho激酶通路信号的协同作用,共同促进MSCs向神经元分化。