RESUMO
Mesenchymal Stromal Cells (MSCs) represent promising tools for cellular therapy owing to their multipotentiality and ability to localize to injured, inflamed sites and tumor. Various approaches to manipulate expression of MSC surface markers, including adhesion molecules and chemokine receptors, have been explored to enhance homing of MSCs. Recently, Neural Cell Adhesion Molecule (NCAM) has been found to be expressed on MSCs yet its function remains largely elusive. Herein, we show that bone marrow-derived MSCs from NCAM deficient mice exhibit defective migratory ability and significantly impaired adipogenic and osteogenic differentiation potential. We further explore the mechanism governing NCAM mediated migration of MSCs by showing the interplay between NCAM and Fibroblast Growth Factor Receptor (FGFR) induces activation of MAPK/ERK signaling, thereby the migration of MSCs. In addition, re-expression of NCAM180, but not NCAM140, could restore the defective MAPK/ERK signaling thereby the migration of NCAM deficient MSCs. Finally, we demonstrate that NCAM180 expression level could be manipulated by pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α treatment. Overall, our data reveal the vital function of NCAM in MSCs migration and differentiation thus raising the possibility of manipulating NCAM expression to enhance homing and therapeutic potential of MSCs in cellular therapy.
Assuntos
Antígeno CD56/fisiologia , Diferenciação Celular , Movimento Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Western Blotting , Adesão Celular/fisiologia , Feminino , Citometria de Fluxo , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteogênese , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND AIMS: The widespread NG2-expressing neural progenitors in the central nervous system (CNS) are considered to be multifunctional cells with lineage plasticity, thereby possessing the potential for treating CNS diseases. Their lineages and functional characteristics have not been completely unraveled. The present study aimed to disclose the lineage potential of clonal NG2(+) populations in vitro and in vivo. METHODS: Twenty-four clones from embryonic cerebral cortex-derived NG2(+) cells were induced for oligodendrocyte, astrocyte, neuronal and chondrocyte differentiation. The expression profiles of neural progenitor markers chondroitin sulfate proteoglycan 4 (NG2), platelet-derived growth factor-α receptor (PDGFαR); nestin and neuronal cell surface antigen (A2B5) were subsequently sorted on cells with distinct differentiation capacity. Transplantation of these NG2(+) clones into the spinal cord was used to examine their lineage potential in vivo. RESULTS: In vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and seven of them were bipotent (oligodendrocytes and astrocytes). Amazingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal-glial lineages but also chondrocytes. These distinct subtypes were further found to exhibit phenotypic heterogeneity based on the examination of a spectrum of neural progenitor markers. Transplanted clones survived, migrated extensively and differentiated into oligodendrocytes, astrocytes or even neurons to integrate with the host spinal cord environment. CONCLUSIONS: These results suggest that NG2(+) cells contain heterogeneous progenitors with distinct differentiation capacities, and the immortalized clonal NG2(+) cell lines might provide a cell source for treating spinal cord disorders.
Assuntos
Antígenos/metabolismo , Diferenciação Celular , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Neurônios/citologia , Proteoglicanas/metabolismo , Medula Espinal/transplante , Células-Tronco , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Linhagem da Célula , Células Cultivadas , Sistema Nervoso Central/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Receptor A2B de Adenosina/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Telomerase/metabolismoRESUMO
Calcium carbonate (CaCO(3)) is one of the most abundant and important biominerals in nature. Due to its biocompatibility, biodegradability and nontoxicity, CaCO(3) has been investigated extensively in recent years for various fundamental properties and technological applications. Inspired by basic wall structures of cells, we report a protein-assisted approach to synthesize CaCO(3) into a double-shelled structural configuration. Due to varying reactivities of outer and inner shells, the CaCO(3) microcapsules exhibit different sorption capacities and various resultant structures toward different kinds of heavy metal ions, analogical to biologically controlled mineralization (BCM) processes. Surprisingly, three mineralization modes resembling those found in BCM were found with these bacterium-like "CaCO(3) cells". Our investigation of the cytotoxicity (MTT assay protocol) also indicates that the CaCO(3) microcapsules have almost no cytotoxicity against HepG2 cells, and they might be useful for future application of detoxifying heavy metal ions after further study.
Assuntos
Carbonato de Cálcio/síntese química , Cápsulas/síntese química , Metais Pesados/química , Proteínas/química , Animais , Cádmio/análise , Carbonato de Cálcio/química , Cápsulas/química , Células HeLa , Humanos , Íons , Chumbo/análise , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Mercúrio/análise , Modelos BiológicosRESUMO
Parkinson's disease (PD) is primarily caused by severe degeneration and loss of dopamine neurons in the substantia nigra pars compacta. Thus, preventing the death of dopaminergic neurons is thought to be a potential strategy to interfere with the development of PD. In the present work, we studied the effect of insulin-like growth factor-1 (IGF-1) on 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis in human neuroblastoma SH-EP1 cells. We found that the PI3K/AKT pathway plays a central role in IGF-mediated cell survival against MPP+ neurotoxicity. Furthermore, we demonstrated that the protective effect of AKT is largely dependent on the inactivation of GSK-3ß, since inhibition of GSK-3ß by its inhibitor, BIO, could mimic the protective effect of IGF-1 on MPP+-induced cell death in SH-EP1 cells. Interestingly, the IGF-1 potentiated PI3K/AKT activity is found to negatively regulate the JNK related apoptotic pathway and this negative regulation is further shown to be mediated by AKT-dependent GSK-3ß inactivation. Thus, our results demonstrated that IGF-1 protects SH-EP1 cells from MPP+-induced apoptotic cell death via PI3K/AKT/GSK-3ß pathway, which in turn inhibits MPP+-induced JNK activation.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Apoptose , Quinase 3 da Glicogênio Sintase/metabolismo , Fator de Crescimento Insulin-Like I , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Neuroblastoma/metabolismo , Neurônios/fisiologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Locked-in syndrome (LIS) characterizes individuals who have experienced pontine lesions, who have limited motor output but with preserved cognitive abilities. Despite their severe physical impairment, individuals with LIS self-profess a higher quality of life than generally expected. Such third-person expectations about LIS are shaped by personal and cultural factors in western countries. OBJECTIVE: We sought to investigate whether such opinions are further influenced by the cultural background in East Asia. We surveyed attitudes about the ethics of life-sustaining treatment in LIS in a cohort of medical and non-medical Chinese participants. RESULTS: The final study sample included 1545 respondents: medical professionals (n=597, 39%), neurologists (n=303, 20%), legal professionals (n=276, 18%) and other professionals (n=369, 24%), including 180 family members of individuals with LIS. Most of the participants (70%), especially neurologists, thought that life-sustaining treatment could not be stopped in individuals with LIS. It might be unnecessary to withdraw life-sustaining treatment, because the condition involved is not terminal and irreversible, and physical treatment can be beneficial for the patient. A significant proportion (59%) of respondents would like to be kept alive if they were in that condition; however, older people thought the opposite. Families experience the stress of caring for individuals with LIS. The mean (SD) quality of life score for relatives was 0.73 (2.889) (on a -5, +5 scale), which was significantly lower than that of non-relatives, 1.75 (1.969) (P<0.001). CONCLUSIONS: Differences in opinions about end of life in LIS are affected by personal characteristics. The current survey did not identify a dissociation between personal preferences and general opinions, potentially because of a social uniformity in China where individualism is less pronounced. Future open-ended surveys could identify specific needs of caregivers so that strategic interventions to reduce ethical debasement are designed.
Assuntos
Ética Médica , Cuidados para Prolongar a Vida/ética , Cuidados para Prolongar a Vida/psicologia , Síndrome do Encarceramento/psicologia , Síndrome do Encarceramento/reabilitação , Adulto , Povo Asiático/psicologia , Atitude do Pessoal de Saúde , China , Características Culturais , Família/etnologia , Família/psicologia , Feminino , Pessoal de Saúde/ética , Pessoal de Saúde/psicologia , Humanos , Individualidade , Advogados/psicologia , Síndrome do Encarceramento/etnologia , Masculino , Pessoa de Meia-Idade , Neurologistas/ética , Neurologistas/psicologia , Qualidade de Vida/psicologia , Inquéritos e Questionários , Adulto JovemRESUMO
The NG2 chondroitin sulfate proteoglycan-expressing neural cells (NG2 cells) have originally been considered as oligodendrocyte progenitor cells (OPCs). However, recent findings on their diverse functions and lineage heterogeneity demonstrated that the NG2 cells contain various sub-populations whose concrete features and therapeutic potential yet remained elucidated. In the present study, we characterized a Nestin(+) 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)(+) NG2(+) subpopulation from embryonic rat cerebral cortex. The Nestin(+) CNP(+) NG2(+) cells exhibited remarkable progenitor characteristics. Having been immortalized by human telomerase reverse transcriptase (hTERT), the life span of Nestin(+) CNP(+) NG2(+) cells was extended to 230 population doublings (PDs). With immortalized NG2 cells, we demonstrated their differentiation capacities to oligodendrocytes, astrocytes and neurons. Furthermore, transplanted into injured spinal cord of a mouse model, they were able to promote remyelination and neuronal regeneration, thereby enhancing the functional recovery. Our findings suggest that the Nestin(+) CNP(+) NG2(+) progenitor cells could be a good alternative cell source of cell therapy for neurological disorders.
Assuntos
Diferenciação Celular/fisiologia , Neurônios/citologia , Oligodendroglia/citologia , Traumatismos da Medula Espinal/patologia , Células-Tronco/citologia , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/metabolismo , Animais , Antígenos/metabolismo , Astrócitos/citologia , Linhagem da Célula/fisiologia , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Regeneração Nervosa/fisiologia , Nestina/metabolismo , Proteoglicanas/metabolismo , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapiaRESUMO
Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma.
Assuntos
Proteínas de Transporte/genética , Movimento Celular/genética , Inativação Gênica , Melanoma Experimental , Animais , Proteínas de Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Proteínas Proto-Oncogênicas c-akt , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Neural cell adhesion molecule (NCAM) has been implicated in tumor metastasis yet its function in melanoma progression remains unclear. Here, we demonstrate that stably silencing NCAM expression in mouse melanoma B16F0 cells perturbs their cellular invasion and metastatic dissemination in vivo. The pro-invasive function of NCAM is exerted via dual mechanisms involving both cAMP-dependent protein kinase (PKA) and phosphatidylinositol 3-kinase (PI3K) pathways. Pharmacologic inhibition of PKA and PI3K leads to impaired cellular invasion. In contrast, forced expression of constitutively activated Akt, the major downstream target of PI3K, restores the defective cellular invasiveness of NCAM knock-down (KD) B16F0 cells. Furthermore, attenuation of either PKA or Akt activity in NCAM KD cells is shown to affect their common downstream target, transcription factor cAMP response element binding protein (CREB), which in turn down-regulates mRNA expression of matrix metalloproteinase-2 (MMP-2), thus contributes to impaired cellular invasion and metastasis of melanoma cells. Together, these findings indicate that NCAM potentiates cellular invasion and metastasis of melanoma cells through stimulation of PKA and PI3K signaling pathways thus suggesting the potential implication of anti-NCAM strategy in melanoma treatment.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Melanoma Experimental/patologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Movimento Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Progressão da Doença , Regulação para Baixo , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Metaloproteinase 2 da Matriz/genética , Melanoma Experimental/genética , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Moléculas de Adesão de Célula Nervosa/deficiência , Moléculas de Adesão de Célula Nervosa/genética , FosforilaçãoRESUMO
Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons in substantia nigra with unknown etiology. Neuropathology seen in the brains of PD patients can be closely mimicked by MPP(+)-induced neurotoxicity in vitro. In this study, we used an S-type human neuroblastoma cell line (SH-EP1) as a model to investigate the involvement of NF-kappaB and JNK pathways in MPP(+)-induced neurotoxicity. We show that NF-kappaB was activated by MPP(+) as evidenced by NF-kappaB p65 nuclear translocation, the increased DNA binding activity and a rapid phosphorylation of NF-kappaB inhibitor (IkappaBalpha). NF-kappaB partially mediated the neurotoxicity of MPP(+), as suggested by the reduction of MPP(+)-induced cell death by both a specific IkappaB kinase (IKK) inhibitor and a dominant negative form of IkappaBalpha (IkappaBalpha-M). Besides NF-kappaB, JNK and c-Jun/AP-1 were also activated upon MPP(+) stimulation. Inhibition of JNK activation with a specific JNK inhibitor partially reduced the MPP(+)-mediated cell death. Similarly, inhibition of c-Jun/AP-1 activation, either by a dominant negative c-Jun or c-Jun/AP-1 inhibitor, significantly attenuated MPP(+)-mediated cell death. These results suggest that both JNK and c-Jun/AP-1 activation are pro-apoptotic. Furthermore, we provide clear evidence for the existence of a crosstalk between the NF-kappaB and JNK signaling as MPP(+)-induced activation of JNK and c-Jun/AP-1 was strongly down-regulated in IkappaBalpha-M cells. In conclusion, we demonstrate that in SH-EP1 cells MPP(+)-induced neurotoxicity is partially mediated by NF-kappaB which in turn acts on the activation of JNK and c-Jun/AP-1. These results may point to a combined inhibition of NF-kappaB and JNK as a new approach to PD therapy.
Assuntos
Apoptose/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Degeneração Neural/metabolismo , Transtornos Parkinsonianos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , 1-Metil-4-fenilpiridínio/toxicidade , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Herbicidas/toxicidade , Humanos , Proteínas I-kappa B/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Degeneração Neural/induzido quimicamente , Degeneração Neural/fisiopatologia , Neuroblastoma , Transtornos Parkinsonianos/fisiopatologia , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
It has been reported recently that bone marrow stromal cells (BMSCs) are able to differentiate into various neural cells both in vivo and in vitro (Egusa, H., Schweizer, F. E., Wang, C. C., Matsuka, Y., and Nishimura, I. (2005) J. Biol. Chem. 280, 23691-23697). However, the underlying mechanisms remain largely unknown. In this report, we have demonstrated that basic fibroblast growth factor (bFGF) alone effectively induces mouse BMSC neuronal differentiation. These differentiated neuronal cells exhibit characteristic electrophysiological properties and elevated levels of the neuronal differentiation marker, growth-associated protein-43 (GAP-43). To explore possible signaling pathways, we first analyzed the expression of various FGF receptors in mouse BMSCs. FGF receptor-1, -2, and -3 were detected, but only FGFR-1 was shown to be activated by bFGF. Small interfering RNA knock down of FGFR-1 in BMSCs significantly inhibited neuronal differentiation. Moreover, we have shown that the mitogen-activated protein kinase (ERK1/2) is persistently activated and blockage of ERK activity with the ERK-specific inhibitor U0126 prevents neuronal differentiation. It appears that activation of ERK cascade and neuronal differentiation of BMSCs induced by bFGF are independent of Ras activity but require functions of phospholipase C-gamma pathway. Lastly, we examined the role of the immediate-early transcription factors AP-1 and NF-kappaB and have found that phospholipase C-gamma-dependent c-Jun and ERK-dependent c-fos, but not the NF-kappaB, are strongly activated by bFGF, which in turn regulates the neuronal differentiation of BMSCs.