shRNA knockdown of Bmi-1 reveals a critical role for p21-Rb pathway in NSC self-renewal during development

Knockout studies have shown that the polycomb gene Bmi-1 is important for postnatal, but not embryonic, neural stem cell (NSC) self-renewal and have identified the cell-cycle inhibitors p16/p19 as molecular targets. Here, using lentiviral-delivered shRNAs in vitro and in vivo, we determined that Bmi-1 is also important for NSC self-renewal in the embryo. We found that neural progenitors depend increasingly on Bmi-1 for proliferation as development proceeds from embryonic through adult stages. Acute shRNA-mediated Bmi-1 reduction causes defects in embryonic and adult NSC proliferation and self-renewal that, unexpectedly, are mediated by a different cell-cycle inhibitor, p21. Gene array analyses revealed developmental differences in Bmi-1-controlled expression of genes in the p21-Rb cell cycle regulatory pathway. Our data therefore implicate p21 as an important Bmi-1 target in NSCs, potentially with stage-related differences. Understanding stage-related mechanisms underlying NSC self-renewal has important implications for development of stem cell-based therapies.     

Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells

Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is crucial for the maintenance of hematopoietic homeostasis. Here we show that activation of p38 MAPK in response to increasing levels of reactive oxygen species (ROS) limits the lifespan of HSCs in vivo. In Atm(-/-) mice, elevation of ROS levels induces HSC-specific phosphorylation of p38 MAPK accompanied by a defect in the maintenance of HSC quiescence. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity and in the maintenance of HSC quiescence, indicating that the ROS-p38 MAPK pathway contributes to exhaustion of the stem cell population. Furthermore, prolonged treatment with an antioxidant or an inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial transplantation experiments. These data show that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Our characterization of molecular mechanisms that limit HSC lifespan may lead to beneficial therapies for human disease.     

MAPK and mTOR pathways are involved in cadmium-induced neuronal apoptosis

Cadmium (Cd) may be accumulated in human body through long-term exposure to Cd-polluted environment, resulting in neurodegeneration and other diseases. To study the mechanism of Cd-induced neurodegeneration, PC12 and SH-SY5Y cells were exposed to Cd. We observed that Cd-induced apoptosis in the cells in a time- and concentration-dependent manner. Cd rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c -Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2 and JNK, but not p38, partially protected the cells from Cd-induced apoptosis. Consistently, over-expression of dominant negative c- Jun or down-regulation of Erk1/2, but not p38 MAPK, partially prevented Cd-induced apoptosis. To our surprise, Cd also activated mammalian target of rapamycin (mTOR)-mediated signaling pathways. Treatment with rapamycin, an mTOR inhibitor, blocked Cd-induced phosphorylation of S6K1 and eukaryotic initiation factor 4E binding protein 1, and markedly inhibited Cd-induced apoptosis. Down-regulation of mTOR by RNA interference also in part, rescued cells from Cd-induced death. These findings indicate that activation of the signaling network of MAPK and mTOR is associated with Cd-induced neuronal apoptosis. Our results strongly suggest that inhibitors of MAPK and mTOR may have a potential for prevention of Cd-induced neurodegeneration.     

Activation of AMP-activated protein kinase in cerebella of Atm-/- mice is attributable to accumulation of reactive oxygen species

Ataxia telangiectasia (A-T) is an inherited disease, the most prominent feature of which is ataxia caused by degeneration of cerebellar neurons and synapses. The mechanisms underlying A-T neurodegeneration are still unclear, and many factors are likely to be involved. AMP-activated protein kinase (AMPK) is a sensor of energy balance, and research on its function in neural cells has gained momentum in the last decade. The dual roles of AMPK in neuroprotection and neurodegeneration are complex, and they need to be identified and characterized. Using an Atm (ataxia telangiectasia mutated) gene deficient mouse model, we showed here that: (a) upregulation of AMPK phosphorylation and elevation of reactive oxygen species (ROS) coordinately occur in the cerebella of Atm-/- mice; (b) hydrogen peroxide induces AMPK phosphorylation in primary mouse cerebellar astrocytes in an Atm-independent manner; (c) administration of the novel antioxidant monosodium luminol (MSL) to Atm-/- mice attenuates the upregulation of both phosphorylated-AMPK (p-AMPK) and ROS, and corrects the neuromotor deficits in these animals. Together, our results suggest that oxidative activation of AMPK in the cerebellum may contribute to the neurodegeneration in Atm-/- mice, and that ROS and AMPK signaling pathways are promising therapeutic targets for treatment of A-T and other neurodegenerative diseases.     

Investigation of the functional link between ATM and NBS1 in the DNA damage response in the mouse cerebellum

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are related genomic instability syndromes characterized by neurological deficits. The NBS1 protein that is defective in NBS is a component of the Mre11/RAD50/NBS1 (MRN) complex, which plays a major role in the early phase of the complex cellular response to double strand breaks (DSBs) in the DNA. Among others, Mre11/RAD50/NBS1 is required for timely activation of the protein kinase ATM (A-T, mutated), which is missing or inactivated in patients with A-T. Understanding the molecular pathology of A-T, primarily its cardinal symptom, cerebellar degeneration, requires investigation of the DSB response in cerebellar neurons, particularly Purkinje cells, which are the first to be lost in A-T patients. Cerebellar cultures derived from mice with different mutations in DNA damage response genes is a useful experimental system to study malfunctioning of the damage response in the nervous system. To clarify the interrelations between murine Nbs1 and Atm, we generated a mouse strain with specific disruption of the Nbs1 gene in the central nervous system on the background of general Atm deficiency (Nbs1-CNS-Δ//Atm(-/-)). This genotype exacerbated several features of both conditions and led to a markedly reduced life span, dramatic decline in the number of cerebellar granule neurons with considerable cerebellar disorganization, abolishment of the white matter, severe reduction in glial cell proliferation, and delayed DSB repair in cerebellar tissue. Combined loss of Nbs1 and Atm in the CNS significantly abrogated the DSB response compared with the single mutation genotypes. Importantly, the data indicate that Atm has cellular roles not regulated by Nbs1 in the murine cerebellum.     

Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality

The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.     

Iron loading and oxidative stress in the Atm-/- mouse liver

Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder resulting in a myriad of abnormalities, including progressive neurodegeneration and cancer predisposition. At the cellular level, A-T is a disease of chronic oxidative stress (OS) causing damage to proteins, lipids, and DNA. OS is contributed to by pro-oxidative transition metals such as iron that catalyze the conversion of weakly reactive oxygen species to highly reactive hydroxyl radicals. Iron-associated OS has been linked to neurodegeneration in Alzheimer's and Parkinson's diseases and development of lymphoid tumors (which afflict ～30% of A-T patients). To investigate iron regulation in A-T, iron indexes, regulatory genes, and OS markers were studied in livers of wild-type and Ataxia telangiectasia mutated (Atm) null mice on control or high-iron diets. Atm(-/-) mice had increased serum iron, hepatic iron, and ferritin and significantly higher Hepcidin compared with wild-type mice. When challenged with the high-iron diet, Bmp6 and Hfe expression was significantly increased. Atm(-/-) mice had increased protein tyrosine nitration and significantly higher Heme Oxygenase (decycling) 1 levels that were substantially increased by a high-iron diet. Ferroportin gene expression was significantly increased; however, protein levels were unchanged. We demonstrate that Atm(-/-) mice have a propensity to accumulate iron that is associated with a significant increase in hepatic OS. The iron-induced increase in hepcidin peptide in turn suppresses ferroportin protein levels, thus nullifying the upregulation of mRNA expression in response to increased OS. Our results suggest that increased iron status may contribute to the chronic OS seen in A-T patients and development of disease pathology.     

Role of p38γ MAPK in regulation of EMT and cancer stem cells

p38γ is a member of p38 MAPK family which contains four isoforms p38α, p38β, p38γ, and p38δ. p38γ MAPK has unique function and is less investigated. Recent studies revealed that p38γ MAPK may be involved in tumorigenesis and cancer aggressiveness. However, the underlying cellular/molecular mechanisms remain unclear. Epithelial-mesenchymal transition (EMT) is a process that epithelial cancer cells transform to facilitate the loss of epithelial features and gain of mesenchymal phenotype. EMT promotes cancer cell progression and metastasis, and is involved in the regulation of cancer stem cells (CSCs) which have self-renewal capacity and are resistant to chemotherapy and target therapy. We showed that p38γ MAPK significantly increased EMT in breast cancer cells; over-expression of p38γ MAPK enhanced EMT while its down-regulation inhibited EMT. Meanwhile, p38γ MAPK augmented CSC population while knock down of p38γ MAPK decreased CSC ratio in breast cancer cells. MicroRNA-200b (miR-200b) was down-stream of p38γ MAPK and inhibited by p38γ MAPK; miR-200b mimics blocked p38γ MAPK-induced EMT while miR-200b inhibitors promoted EMT. p38γ MAPK regulated miR-200b through inhibiting GATA3. p38γ MAPK induced GATA3 ubiquitination, leading to its proteasome-dependent degradation. Suz12, a Polycomb group protein, was down-stream of miR-200b and involved in miR-200b regulation of EMT. Thus, our study established an important role of p38γ MAPK in EMT and identified a novel signaling pathway for p38γ MAPK–mediated tumor promotion.     

Effect and mechanism of mGluR6 on the biological function of rat embryonic neural stem cells

Here, we investigated the effects and molecular mechanisms of metabotropic glutamate receptor 6 (mGluR6) on rat embryonic neural stem cells (NSCs). Overexpression of mGluR6 significantly promoted the proliferation of NSCs and increased the diameter of neutrospheres after treatment for 24 h, 48 h and 72 h. Overexpression of mGluR6 promoted G1 to S phase transition, with significantly decreased cell ratio in G1/G0 phase but significantly increased cell ratio in S phase. Additionally, mGluR6 overexpression for 48 h decreased the early and late apoptosis significantly. Moreover, overexpression of mGluR6 significantly increased the expression of p-ERK1/2, Cyclin D1 and CDK2, while the expression of p-p38 was significantly decreased. On the contrary, these effects of mGluR6 overexpression were reversed by mGluR6 knockdown. In conclusion, mGluR6 promotes the proliferation of NSCs by activation of ERK1/2-Cyclin D1/CDK2 signaling pathway and inhibits the apoptosis of NSCs by blockage of the p38 MAPK signaling pathway.     

SMAD-Independent Down-Regulation of Caveolin-1 by TGF-β: Effects on Proliferation and Survival of Myofibroblasts

Transforming growth factor-β (TGF-β) mediates growth-inhibitory effects on most target cells via activation of the canonical SMAD signaling pathway. This growth-inhibitory activity may be coupled with cellular differentiation. Our studies demonstrate that TGF-β1 inhibits proliferation of primary, non-transformed human lung fibroblasts in association with the induction of myofibroblast differentiation. Differentiated myofibroblasts maintain the capacity to proliferate in response to exogenous mitogenic stimuli and are resistant to serum deprivation-induced apoptosis. These proliferative and anti-apoptotic properties of myofibroblasts are related, in part, to the down-regulation of caveolin-1 (Cav-1) by TGF-β1. Cav-1 down-regulation is mediated by early activation of p38 MAPK and does not require SMAD signaling. In contrast, myofibroblast differentiation is dependent on activation of the SMAD pathway, but not on p38 MAPK. Thus, combinatorial signaling by TGF-β1 of myofibroblast differentiation and down-regulation of Cav-1 by SMAD and p38 MAPK pathways, respectively, confer proliferative and apoptosis-resistant properties to myofibroblasts. Selective targeting of this SMAD-independent, p38-MAPK/Cav-1-dependent pathway is likely to be effective in the treatment of pathological conditions characterized by TGF-β signaling and myofibroblast activation.     

Role of Smad2/3 and p38 MAP kinase in TGF-β1-induced epithelial-mesenchymal transition of pulmonary epithelial cells

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-β1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-β1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-β1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-β1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-β1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-β1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.     

Impaired functions of neural stem cells by abnormal nitric oxide-mediated signaling in an in vitro model of Niemann-Pick type C disease

Nitric oxide (NO) has been implicated in the promotion of neurodegeneration. However, little is known about the relationship between NO and the self-renewal or differentiation capacity of neural stem cells (NSCs) in neurodegenerative disease. In this study, we investigated the effect of NO on self-renewal of NSCs in an animal model for Niemann-Pick type C (NPC) disease. We found that NO production was significantly increased in NSCs from NPC1-deficient mice (NPC1-/-), which showed reduced NSC self-renewal. The number of nestin-positive cells and the size of neurospheres were both significantly decreased. The expression of NO synthase (NOS) was increased in neurospheres derived from the brain of NPC1-/- mice in comparison to wild-type neurospheres. NO-mediated activation of glycogen synthase kinase-3beta (GSK3beta) and caspase-3 was also observed in NSCs from NPC1-/- mice. The self-renewal ability of NSCs from NPC1-/- mice was restored by an NOS inhibitor, L-NAME, which resulted in the inhibition of GSK3beta and caspase-3. In addition, the differentiation ability of NSCs was partially restored and the number of Fluoro-Jade C-positive degenerating neurons was reduced. These data suggest that overproduction of NO in NPC disease impaired the self-renewal of NSCs. Control of NO production may be key for the treatment of NPC disease.     

The appropriateness of the mouse model for ataxia-telangiectasia: Neurological defects but no neurodegeneration

Patients with ataxia-telangiectasia (A-T) are characterised by genome instability, cancer predisposition and a progressive neurodegeneration. A number of model systems have been developed for A-T but none recapitulate all the phenotype. The majority of these models have been generated in mice. While Atm deficient mouse models exhibit much of the phenotype described in patients with A-T, the broad consensus is that they do not display the most debilitating aspect of A-T, i.e. neurodegeneration. Cerebellar atrophy is one of the neuronal characteristics of A-T patients due to defects in neuronal development and progressive loss of Purkinje and granule cells. This is not evident in Atm-deficient mutants but there are multiple reports on neurological abnormalities in these mice. The focus of this review is to evaluate the appropriateness of Atm mutant mouse models for A-T, particularly with reference to neurological abnormalities and how they might relate to neurodegeneration.     

Interleukin-15 regulates proliferation and self-renewal of adult neural stem cells

The impact of inflammation is crucial for the regulation of the biology of neural stem cells (NSCs). Interleukin-15 (IL-15) appears as a likely candidate for regulating neurogenesis, based on its well-known mitogenic properties. We show here that NSCs of the subventricular zone (SVZ) express IL-15, which regulates NSC proliferation, as evidenced by the study of IL-15-/- mice and the effects of acute IL-15 administration, coupled to 5-bromo-2'-deoxyuridine/5-ethynyl-2'-deoxyuridine dual-pulse labeling. Moreover, IL-15 regulates NSC differentiation, its deficiency leading to an impaired generation of neuroblasts in the SVZ-rostral migratory stream axis, recoverable through the action of exogenous IL-15. IL-15 expressed in cultured NSCs is linked to self-renewal, proliferation, and differentiation. IL-15-/- NSCs presented deficient proliferation and self-renewal, as evidenced in proliferation and colony-forming assays and the analysis of cell cycle-regulatory proteins. Moreover, IL-15-deficient NSCs were more prone to differentiate than wild-type NSCs, not affecting the cell population balance. Lack of IL-15 led to a defective activation of the JAK/STAT and ERK pathways, key for the regulation of proliferation and differentiation of NSCs. The results show that IL-15 is a key regulator of neurogenesis in the adult and is essential to understanding diseases with an inflammatory component.     

The human sef-a isoform utilizes different mechanisms to regulate receptor tyrosine kinase signaling pathways and subsequent cell fate

Negative feedback is among the key mechanisms for regulating receptor tyrosine kinase (RTK) signaling. Human Sef, a recently identified inhibitor of RTK signaling, encodes different isoforms, including a membrane spanning (hSef-a) and a cytosolic (hSef-b) isoform. Previously, we reported that hSef-b inhibited fibroblast proliferation and prevented the activation of mitogen-activated protein kinase (MAPK), without affecting protein kinase B/Akt or p38 MAPK. Conflicting results were reported concerning hSef-a inhibition of MAPK activation, and the effect of hSef-a on other RTK-induced signaling pathways is unknown. Here we show that, in fibroblasts, similar to hSef-b, ectopic expression of hSef-a inhibited fibroblast growth factor-induced cell proliferation. Unlike hSef-b, however, the growth arrest was mediated via a MAPK-independent mechanism, and was accompanied by elevated p38 MAPK phosphorylation and inhibition of protein kinase B/Akt. In addition, hSef-a, but not hSef-b, mediated apoptosis in fibroblast growth factor-stimulated cells. Chemical inhibitor of p38 MAPK abrogated the effect of hSef-a on apoptosis. In epithelial cells, ectopic expression of hSef-a inhibited the activation of MAPK, whereas down-regulation of endogenous hSef-a significantly increased MAPK activation and accelerated growth factor-dependent cell proliferation. These results indicate that hSef-a is a multifunctional negative modulator of RTK signaling and clearly demonstrate that hSef-a can inhibit the activation of MAPK, although in a cell type-specific manner. Moreover, the differences between the activities of hSef-a and hSef-b suggest that hSef isoforms can control signal specificity and subsequent cell fate by utilizing different mechanisms to modulate RTK signaling.     

Inhibition of T cell MAPKs (Erk 1/2, p38) with thermal injury is related to down-regulation of Ca2+ signaling

We evaluated MAPK (Erk 1/2 and p38) signaling mechanisms of altered T-cell-mediated immune responses in thermal injury condition. Rats were subjected to 30% body surface scald burn, and their mesenteric lymph node (MLN) and Peyer's patch (PP) T cells were purified using nylon wool method. Activation of MAPKs, Erk 1/2 and p38 was assessed in T cells by determining its phosphorylation using immunoblot analysis, intracellular immunostaining and confocal microscopy. The results showed a down-regulation of Erk 1/2 and p38 activation in anti-CD3-stimulated T cells from thermally injured animals, compared to Erk 1/2 and p38 in sham rat T cells. The down-regulation of MAPKs in T cells was reversed by treatment of T cells with calcium agonist, ionomycin. These data indicate that attenuated MAPKs (Erk 1/2, p38) activation in thermally injured animals' T cells could result from derangement of Ca(2+) mobilization. This finding suggests that T cell signaling derangements with thermal injury involve an altered cross-talk between Ca(2+) mobilization and MAPK signaling mechanisms.     

Down-regulation of cytokine-induced interleukin-8 requires inhibition of p38 mitogen-activated protein kinase (MAPK) via MAPK phosphatase 1-dependent and -independent mechanisms

Down-regulation of overabundant interleukin (IL)-8 present in cystic fibrosis (CF) airways could ease excessive neutrophil burden and its deleterious consequences for the lung. IL-8 production in airway epithelial cells, stimulated with e.g. inflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α, is regulated by several signaling pathways including nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK). We previously demonstrated that the anti-inflammatory drugs dexamethasone and ibuprofen suppress NF-κB; however, only dexamethasone down-regulates cytokine-induced IL-8, highlighting the importance of non-NF-κB mechanisms. Here, we tested the hypothesis that down-regulation of cytokine-induced IL-8 requires modulation of the MAPK phosphatase (MKP)-1/p38 MAPK/mRNA stability pathway. The effects of dexamethasone (5 nm) and ibuprofen (480 μm) on this pathway and IL-8 were studied in CF (CFTE29o-, CFBE41o-) and non-CF (1HAEo-) airway epithelial cells. We observed that dexamethasone, but not ibuprofen, destabilizes IL-8 mRNA and up-regulates MKP-1 mRNA. Further, siRNA silencing of MKP-1, via p38 MAPK, leads to IL-8 overproduction and diminishes the anti-IL-8 potential of dexamethasone. However, MKP-1 overexpression does not significantly alter IL-8 production. By contrast, direct inhibition of p38 MAPK (inhibitor SB203580) efficiently suppresses IL-8 with potency comparable with dexamethasone. Similar to dexamethasone, SB203580 decreases IL-8 mRNA stability. Dexamethasone does not affect p38 MAPK activation, which excludes its effects upstream of p38 MAPK. In conclusion, normal levels of MKP-1 are necessary for a full anti-IL-8 potential of pharmacological agents; however, efficient pharmacological down-regulation of cytokine-induced IL-8 also requires direct effects on p38 MAPK and mRNA stability independently of MKP-1.     

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.