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Organotypic cultures of murine brain slices are well-established tools in neuroscience research, including electrophysiology studies, modeling neurodegeneration, and cancer research. Here, we present an optimized ex vivo brain slice invasion assay that models glioblastoma multiforme (GBM) cell invasion into organotypic brain slices. Using this model, human GBM spheroids can be implanted with precision onto murine brain slices and cultured ex vivo to allow tumour cell invasion into the brain tissue. Traditional top-down confocal microscopy allows for imaging of GBM cell migration along the top of the brain slice, but there is limited resolution of tumour cell invasion into the slice. Our novel imaging and quantification technique involves embedding stained brain slices into an agar block, re-sectioning the slice in the Z-direction onto slides, and then using confocal microscopy to image cellular invasion into the brain tissue. This imaging technique allows for the visualization of invasive structures beneath the spheroid that would otherwise go undetected using traditional microscopy approaches. Our ImageJ macro (BraInZ) allows for the quantification of GBM brain slice invasion in the Z-direction. Importantly, we note striking differences in the modes of motility observed when GBM cells invade into Matrigel in vitro versus into brain tissue ex vivo highlighting the importance of incorporating the brain microenvironment when studying GBM invasion. In summary, our version of the ex vivo brain slice invasion assay improves upon previously published models by more clearly differentiating between migration along the top of the brain slice versus invasion into the slice.
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The ability of cancer cells to invade surrounding tissues requires degradation of the extracellular matrix (ECM). Invasive structures, such as invadopodia, form on the plasma membranes of cancer cells and secrete ECM-degrading proteases that play crucial roles in cancer cell invasion. We have previously shown that the protein tyrosine phosphatase alpha (PTPα) regulates focal adhesion formation and migration of normal cells. Here we report a novel role for PTPα in promoting triple-negative breast cancer cell invasion in vitro and in vivo. We show that PTPα knockdown reduces ECM degradation and cellular invasion of MDA-MB-231 cells through Matrigel. PTPα is not a component of TKS5-positive structures resembling invadopodia; rather, PTPα localizes with endosomal structures positive for MMP14, caveolin-1, and early endosome antigen 1. Furthermore, PTPα regulates MMP14 localization to plasma membrane protrusions, suggesting a role for PTPα in intracellular trafficking of MMP14. Importantly, we show that orthotopic MDA-MB-231 tumors depleted in PTPα exhibit reduced invasion into the surrounding mammary fat pad. These findings suggest a novel role for PTPα in regulating the invasion of triple-negative breast cancer cells.
Assuntos
Metaloproteinase 14 da Matriz/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Membrana Celular , Movimento Celular/fisiologia , Matriz Extracelular/fisiologia , Feminino , Humanos , Metaloproteinase 14 da Matriz/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/fisiologia , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/fisiopatologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extrinsic signals that regulate oligodendrocyte maturation and subsequent myelination are essential for central nervous system development and regeneration. Deficiency in the extracellular factor laminin-2 (Lm2, comprising the α2ß1γ1 chains), as occurs in congenital muscular dystrophy, can lead to impaired oligodendroglial development and aberrant myelination, but many aspects of Lm2-regulated oligodendroglial signaling and differentiation remain undefined. We show that receptor-like protein tyrosine phosphatase α (PTPα, also known as PTPRA) is essential for myelin basic protein expression and cell spreading during Lm2-induced oligodendrocyte differentiation. PTPα complexes with the Lm2 receptors α6ß1 integrin and dystroglycan to transduce Fyn activation upon Lm2 engagement. In this way, PTPα mediates a subset of Lm2-induced signals required for differentiation, includeing mTOR-dependent Akt activation but not Erk1/2 activation. We identify N-myc downstream regulated gene-1 (NDRG1) as a PTPα-regulated molecule during oligodendrocyte differentiation, and distinguish Lm2 receptor-specific modes of Fyn-Akt-dependent and -independent NDRG1 phosphorylation. Altogether, this reveals an Lm2-regulated PTPα-Fyn-Akt signaling axis that is critical for key aspects of the gene expression and morphological changes that mark oligodendrocyte maturation.
Assuntos
Laminina/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
CNS myelination defects occur in mice deficient in receptor-like protein tyrosine phosphatase alpha (PTPα). Here, we investigated the role of PTPα in oligodendrocyte differentiation and myelination using cells and tissues from wild-type (WT) and PTPα knockout (KO) mice. PTPα promoted the timely differentiation of neural stem cell-derived oligodendrocyte progenitor cells (OPCs). Compared to WT OPCs, KO OPC cultures had more NG2+ progenitors, fewer myelin basic protein (MBP)+ oligodendrocytes, and reduced morphological complexity. In longer co-cultures with WT neurons, more KO than WT OPCs remained NG2+ and while equivalent MBP+ populations of WT and KO cells formed, the reduced area occupied by the MBP+ KO cells suggested that their morphological maturation was impeded. These defects were associated with reduced myelin formation in KO OPC/WT neuron co-cultures. Myelin formation was also impaired when WT OPCs were co-cultured with KO neurons, revealing a novel role for neuronal PTPα in myelination. Canonical Wnt/ß-catenin signaling is an important regulator of OPC differentiation and myelination. Wnt signaling activity was not dysregulated in OPCs lacking PTPα, but suppression of Wnt signaling by the small molecule XAV939 remediated defects in KO oligodendrocyte differentiation and enhanced myelin formation by KO oligodendrocytes. However, the myelin segments that formed were significantly shorter than those produced by WT oligodendrocytes, raising the possibility of a role for glial PTPα in myelin extension distinct from its pro-differentiating actions. Altogether, this study reveals PTPα as a molecular coordinator of oligodendroglial and neuronal signals that controls multiple aspects of oligodendrocyte development and myelination.
Assuntos
Bainha de Mielina/metabolismo , Neurogênese , Neurônios/metabolismo , Oligodendroglia/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Animais , Antígenos/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/metabolismo , Neurônios/citologia , Oligodendroglia/citologia , Proteoglicanas/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Via de Sinalização WntRESUMO
Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/genética , Humanos , Immunoblotting , Células MCF-7 , Camundongos , Proteínas de Neoplasias/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Proto-Oncogênicas c-abl/genética , Pirimidinas/farmacologia , Interferência de RNA , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Tirosina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
For a time, mast cells were viewed as simple granulocytic effector cells that mediate allergic symptoms. More recent discoveries show that mast cells can also function as potent pro- and anti-inflammatory immune regulators in a plethora of human diseases. Much of the current knowledge about mast cell functions comes from studies on rodent models. The membrane receptors for antigen/IgE and growth factors are the core initiators of signaling cascades that trigger various mast cell responses. Yet, the regulation and multifunctionality of key receptor-proximal protein tyrosine phosphorylation events are still not well understood. The roles of the members of the protein tyrosine phosphatase superfamily of enzymes in regulating mast cell development, survival, and immune activation will be reviewed in this chapter.
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Mastócitos/citologia , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Animais , Regulação Enzimológica da Expressão Gênica , Humanos , Espaço Intracelular/enzimologia , Mastócitos/enzimologia , Proteínas Tirosina Fosfatases/química , Domínios de Homologia de srcRESUMO
The integrin-activated Src-focal adhesion kinase (FAK) kinase complex phosphorylates PTPα at Tyr789, initiating PTPα-mediated signaling that promotes cell migration. Recruitment of the BCAR3-Cas complex by PTPα-phospho-Tyr789 at focal adhesions is one mechanism of PTPα signaling. The adaptor protein Grb2 is also recruited by PTPα-phospho-Tyr789, although the role of the PTPα-Grb2 complex in integrin signaling is unknown. We show that silencing Grb2 expression in fibroblasts abolishes PTPα-Tyr789 phosphorylation and that this is due to two unexpected actions of Grb2. First, Grb2 promotes integrin-induced autophosphorylation of FAK-Tyr397. This is impaired in Grb2-depleted cells and prohibits FAK activation and formation of the Src-FAK complex. Grb2-depleted cells contain less paxillin, and paxillin overexpression rescues FAK-Tyr397 phosphorylation, suggesting that the FAK-activating action of Grb2 involves paxillin. A second distinct role for Grb2 in PTPα-Tyr789 phosphorylation involves Grb2-mediated coupling of Src-FAK and PTPα. This requires two phosphosites, FAK-Tyr925 and PTPα-Tyr789, for Grb2-Src homology 2 (SH2) binding. We propose that a Grb2 dimer links FAK and PTPα, and this positions active Src-FAK in proximity with other, perhaps integrin-clustered, molecules of PTPα to enable maximal PTPα-Tyr789 phosphorylation. These findings identify Grb2 as a new FAK activator and reveal its essential role in coordinating PTPα tyrosine phosphorylation to enable downstream integrin signaling and migration.
Assuntos
Fibroblastos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Proteína Adaptadora GRB2/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Domínios de Homologia de src , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Quinase 1 de Adesão Focal/genética , Proteína Adaptadora GRB2/genética , Immunoblotting , Integrinas/metabolismo , Camundongos , Camundongos Knockout , Paxilina/genética , Paxilina/metabolismo , Fosforilação , Ligação Proteica , Interferência de RNA , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Transdução de Sinais , Tirosina/genética , Tirosina/metabolismoRESUMO
Two-dimensional gel electrophoresis (2-DE) technique is used as a performing technique to assess the variability of protein expression in crops, and especially soybean endogenous food allergens, which are a subset of proteins of interest for assessing whether genetically modified (GM) soybean has a different allergenic profile compared to its non-GM counterpart. On top of the biological variability of the 2-DE, which has already been studied by several laboratories, technical variability has to be evaluated. In this study, several sources of variability (number of gel replicates, protein extracts, study timings and operators) were assessed qualitatively and quantitatively on all detectable polypeptide spots as well as on food allergen spots. Results showed that the major source of variability was the number of gel replicates. Other sources were minor. This has a direct practical impact on the laboratory work as this supports the utilization of three or four gel replicates to get robust results. Furthermore, this implies that the study can be run over several days, and be performed by several trained operators, without impacting its reproducibility. Furthermore, 2-DE could detect a 2-fold change between two samples with an acceptable rate of false positives (below 7%). This level of sensitivity is acceptable in the context of safety assessment of GM soybean as the biological variability of proteins in soybean is higher than the technical variability shown in this study. Overall, the 2-DE technique is suitable for investigating endogenous food allergen variability between several soybean seeds, including GM and non-GM counterpart.
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UNLABELLED: Rhabdomyosarcoma is the most common soft tissue sarcoma in children. Metastatic rhabdomyosarcoma in children has a 5-year event-free survival rate of <30%, and a recent clinical trial with irinotecan, a topoisomerase I inhibitor, failed to improve outcome. Therefore, it was surmised that failure of irinotecan may be the result of overexpression of the DNA repair enzyme tyrosyl-DNA phosphodiesterase (TDP1), which processes topoisomerase I-DNA complexes resulting from topoisomerase I inhibitor treatment. Using human tissue microarrays and gene expression arrays, a marked overexpression of TDP1 protein and mRNA in RMS tumors was observed. Critically, knockdown of TDP1 or inhibition of poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme in the same complex as TDP1, sensitized rhabdomyosarcoma cell lines to analogues of irinotecan. Interestingly, BRCA1/2 mutations or altered expression was not detectable in rhabdomyosarcoma cells; however, TDP1 knockdown and PARP-1 inhibition alone were cytotoxic to a subset of rhabdomyosarcoma cells, suggesting that they harbor genetic lesions in DNA repair components that have synthetic lethal interactions with loss of TDP1 or PARP1 function. Furthermore, culturing embryonal rhabdomyosarcoma cells in serum/nutrient-restricted medium increased cellular cytotoxicity upon PARP-1 inhibition and was intrinsically cytotoxic to alveolar, though not embryonal rhabdomyosarcoma cells. The results of these studies suggest a compensatory role for TDP1 in rhabdomyosarcoma after topoisomerase-I based therapy and further demonstrate that TDP1 knockdown, PARP-1 inhibition, and dietary restriction have therapeutic validity. IMPLICATIONS: Selective targeting of TDP1 and/or PARP-1 in rhabdomyosarcoma induces cytotoxicity and sensitizes to DNA damaging agents.
Assuntos
Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Rabdomiossarcoma/genética , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA2/genética , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Criança , Reparo do DNA , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Glucose/metabolismo , Humanos , Mutação , Mioblastos Esqueléticos/fisiologia , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Soro/metabolismo , Análise Serial de TecidosRESUMO
Integrin-mediated focal adhesions connect the extracellular matrix and cytoskeleton to regulate cell responses, such as migration. Protein tyrosine phosphatase α (PTPα) regulates integrin signaling, focal adhesion formation, and migration, but its roles in these events are incompletely understood. The integrin-proximal action of PTPα activates Src family kinases, and subsequent phosphorylation of PTPα at Tyr789 acts in an unknown manner to promote migration. PTPα-null cells were used in reconstitution assays to distinguish PTPα-Tyr789-dependent signaling events. This showed that PTPα-Tyr789 regulates the localization of PTPα and the scaffolding protein Cas to adhesion sites where Cas interacts with and is phosphorylated by Src to initiate Cas signaling. Linking these events, we identify BCAR3 as a molecular connector of PTPα and Cas, with phospho-Tyr789 PTPα serving as the first defined cellular ligand for the BCAR3 SH2 domain that recruits BCAR3-Cas to adhesions. Our findings reveal a novel role of PTPα in integrin-induced adhesion assembly that enables Src-mediated activation of the pivotal function of Cas in migration.
Assuntos
Proteína Substrato Associada a Crk/metabolismo , Adesões Focais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular , Movimento Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Camundongos , Fosforilação , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
Tightly controlled termination of proliferation determines when oligodendrocyte progenitor cells (OPCs) can initiate differentiation and mature into myelin-forming cells. Protein-tyrosine phosphatase α (PTPα) promotes OPC differentiation, but its role in proliferation is unknown. Here we report that loss of PTPα enhanced in vitro proliferation and survival and decreased cell cycle exit and growth factor dependence of OPCs but not neural stem/progenitor cells. PTPα(-/-) mice have more oligodendrocyte lineage cells in embryonic forebrain and delayed OPC maturation. On the molecular level, PTPα-deficient mouse OPCs and rat CG4 cells have decreased Fyn and increased Ras, Cdc42, Rac1, and Rho activities, and reduced expression of the Cdk inhibitor p27Kip1. Moreover, Fyn was required to suppress Ras and Rho and for p27Kip1 accumulation, and Rho inhibition in PTPα-deficient cells restored expression of p27Kip1. We propose that PTPα-Fyn signaling negatively regulates OPC proliferation by down-regulating Ras and Rho, leading to p27Kip1 accumulation and cell cycle exit. Thus, PTPα acts in OPCs to limit self-renewal and facilitate differentiation.
Assuntos
Proliferação de Células , Células-Tronco Neurais/fisiologia , Oligodendroglia/fisiologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Animais , Apoptose , Encéfalo/citologia , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Ciclo Celular , Sobrevivência Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator 2 de Crescimento de Fibroblastos/fisiologia , Técnicas de Inativação de Genes , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Neuropeptídeos/metabolismo , Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Transdução de Sinais , Esferoides Celulares/fisiologia , Regulação para Cima , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The Y-box binding protein 1 (YB-1) is upregulated in many human malignancies including glioblastoma (GBM). It is also essential for normal brain development, suggesting that YB-1 is part of a neural stem cell (NSC) network. Here, we show that YB-1 was highly expressed in the subventricular zone (SVZ) of mouse fetal brain tissues but not in terminally differentiated primary astrocytes. Conversely, YB-1 knockout mice had reduced Sox-2, nestin, and musashi-1 expression in the SVZ. Although primary murine neurospheres were rich in YB-1, its expression was lost during glial differentiation. Glial tumors often express NSC markers and tend to loose the cellular control that governs differentiation; therefore, we addressed whether YB-1 served a similar role in cancer cells. YB-1, Sox-2, musashi-1, Bmi-1, and nestin are coordinately expressed in SF188 cells and 9/9 GBM patient-derived primary brain tumor-initiating cells (BTIC). Silencing YB-1 with siRNA attenuated the expression of these NSC markers, reduced neurosphere growth, and triggered differentiation via coordinate loss of GSK3-ß. Furthermore, differentiation of BTIC with 1% serum or bone morphogenetic protein-4 suppressed YB-1 protein expression. Likewise, YB-1 expression was lost during differentiation of normal human NSCs. Consistent with these observations, YB-1 expression increased with tumor grade (n = 49 cases). YB-1 was also coexpressed with Bmi-1 (Spearmans 0.80, P > 0.001) and Sox-2 (Spearmans 0.66, P > 0.001) based on the analysis of 282 cases of high-grade gliomas. These proteins were highly expressed in 10/15 (67%) of GBM patients that subsequently relapsed. In conclusion, YB-1 correlatively expresses with NSC markers where it functions to promote cell growth and inhibit differentiation.
Assuntos
Neoplasias Encefálicas/patologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Glioblastoma/patologia , Células-Tronco Neurais/citologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Imuno-Histoquímica , Camundongos , Camundongos Knockout , RNA Interferente PequenoRESUMO
Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Regulação da Expressão Gênica , Neurônios/citologia , Neurônios/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Animais , Células COS , Moléculas de Adesão Celular Neuronais/biossíntese , Membrana Celular/metabolismo , Chlorocebus aethiops , Espaço Extracelular/metabolismo , Fibronectinas/química , Complexo de Golgi/metabolismo , Humanos , Imunoglobulinas/química , Camundongos , Estabilidade Proteica , Transporte Proteico , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/deficiência , Sequências Repetitivas de Aminoácidos , Transdução de Sinais , TransfecçãoRESUMO
Mast cell activation via FcεRI involves activation of the Src family kinases (SFKs) Lyn, Fyn, and Hck that positively or, in the case of Lyn, negatively regulate cellular responses. Little is known of upstream activators of these SFKs in FcεRI-dependent signaling. We investigated the role of receptor protein tyrosine phosphatase (PTP)α, a well-known activator of SFKs in diverse signaling systems, FcεRI-mediated mast cell activation, and IgE-dependent allergic responses in mice. PTPα(-/-) bone marrow-derived mast cells hyperdegranulate and exhibit increased cytokine and cysteinyl leukotriene secretion, and PTPα(-/-) mice display enhanced IgE-dependent anaphylaxis. At or proximal to FcεRI, PTPα(-/-) cells have reduced IgE-dependent activation of Lyn and Fyn, as well as reduced FcεRI and SHIP phosphorylation. In contrast, Hck and Syk activation is enhanced. Syk hyperactivation correlated with its increased phosphorylation at positive regulatory sites and defective phosphorylation at a negative regulatory site. Distal to FcεRI, we observed increased activation of PI3K and MAPK pathways. These findings demonstrate that PTPα activates the FcεRI-coupled kinases Lyn and Fyn and suppresses Hck activity. Furthermore, the findings indicate that hyperactivation of PTPα(-/-) mast cells and enhanced IgE-dependent allergic responses of PTPα(-/-) mice are due to the ablated function of PTPα as a critical regulator of Lyn negative signaling.
Assuntos
Mastócitos/imunologia , Proteínas Proto-Oncogênicas c-fyn/imunologia , Proteínas Proto-Oncogênicas c-hck/imunologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/imunologia , Quinases da Família src/imunologia , Animais , Degranulação Celular/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Proto-Oncogênicas c-hck/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Transdução de Sinais/imunologia , Quinases da Família src/metabolismoRESUMO
Rhabdomyosarcoma, consisting of alveolar (aRMS) and embryonal (eRMS) subtypes, is the most common type of sarcoma in children. Currently, there are no targeted drug therapies available for rhabdomyosarcoma. In searching for new molecular therapeutic targets, we carried out genome-wide small interfering RNA (siRNA) library screens targeting human phosphatases (n = 206) and kinases (n = 691) initially against an aRMS cell line, RH30. Sixteen phosphatases and 50 kinases were identified based on growth inhibition after 72 hours. Inhibiting polo-like kinase 1 (PLK1) had the most remarkable impact on growth inhibition (approximately 80%) and apoptosis on all three rhabdomyosarcoma cell lines tested, namely, RH30, CW9019 (aRMS), and RD (eRMS), whereas there was no effect on normal muscle cells. The loss of PLK1 expression and subsequent growth inhibition correlated with decreased p-CDC25C and Cyclin B1. Increased expression of WEE 1 was also noted. The induction of apoptosis after PLK1 silencing was confirmed by increased p-H2AX, propidium iodide uptake, and chromatin condensation, as well as caspase-3 and poly(ADP-ribose) polymerase cleavage. Pediatric Ewing's sarcoma (TC-32), neuroblastoma (IMR32 and KCNR), and glioblastoma (SF188) models were also highly sensitive to PLK1 inhibition. Finally, based on cDNA microarray analyses, PLK1 mRNA was overexpressed (>1.5 fold) in 10 of 10 rhabdomyosarcoma cell lines and in 47% and 51% of primary aRMS (17 of 36 samples) and eRMS (21 of 41 samples) tumors, respectively, compared with normal muscles. Similarly, pediatric Ewing's sarcoma, neuroblastoma, and osteosarcoma tumors expressed high PLK1. We conclude that PLK1 could be a promising therapeutic target for the treatment of a wide range of pediatric solid tumors including rhabdomyosarcoma.
Assuntos
Terapia Genética/métodos , Monoéster Fosfórico Hidrolases/genética , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Rabdomiossarcoma/enzimologia , Rabdomiossarcoma/terapia , Animais , Apoptose/genética , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/terapia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Criança , Pré-Escolar , Inativação Gênica , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Immunoblotting , Camundongos , Neuroblastoma/enzimologia , Neuroblastoma/genética , Neuroblastoma/terapia , Osteossarcoma/enzimologia , Osteossarcoma/genética , Osteossarcoma/terapia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/administração & dosagem , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Sarcoma de Ewing/enzimologia , Sarcoma de Ewing/genética , Sarcoma de Ewing/terapia , Transfecção , Quinase 1 Polo-LikeRESUMO
The tyrosine kinase Fyn plays a key role in oligodendrocyte differentiation and myelination in the central nervous system, but the molecules responsible for regulating Fyn activation in these processes remain poorly defined. Here we show that receptor-like protein-tyrosine phosphatase alpha (PTPalpha) is an important positive regulator of Fyn activation and signaling that is required for the differentiation of oligodendrocyte progenitor cells (OPCs). PTPalpha is expressed in OPCs and is up-regulated during differentiation. We used two model systems to investigate the role of PTPalpha in OPC differentiation: the rat CG4 cell line where PTPalpha expression was silenced by small interfering RNA, and oligosphere-derived primary OPCs isolated from wild-type and PTPalpha-null mouse embryos. In both cell systems, the ablation of PTPalpha inhibited differentiation and morphological changes that accompany this process. Although Fyn was activated upon induction of differentiation, the level of activation was severely reduced in cells lacking PTPalpha, as was the activation of Fyn effector molecules focal adhesion kinase, Rac1, and Cdc42, and inactivation of Rho. Interestingly, another downstream effector of Fyn, p190RhoGAP, which is responsible for Rho inactivation during differentiation, was not affected by PTPalpha ablation. In vivo studies revealed defective myelination in the PTPalpha(-/-) mouse brain. Together, our findings demonstrate that PTPalpha is a critical regulator of Fyn activation and of specific Fyn signaling events during differentiation, and is essential for promoting OPC differentiation and central nervous system myelination.
Assuntos
Diferenciação Celular , Oligodendroglia/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Transdução de Sinais , Animais , Encéfalo/metabolismo , Linhagem Celular , Células Cultivadas , Quinase 1 de Adesão Focal/metabolismo , Immunoblotting , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Bainha de Mielina/metabolismo , Oligodendroglia/citologia , Fosforilação , Interferência de RNA , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Protein tyrosine phosphatase-alpha (PTPalpha) is a widely expressed receptor-type phosphatase that functions in multiple signaling systems. The actions of PTPalpha can be regulated by its phosphorylation on serine and tyrosine residues, although little is known about the conditions that promote PTPalpha phosphorylation. In this study, we tested the ability of several extracellular factors to stimulate PTPalpha tyrosine phosphorylation. The growth factors IGF-I and acidic FGF induced the highest increase in PTPalpha phosphorylation at tyrosine 789, followed by PMA and lysophosphatidic acid, while EGF had little effect. Further investigation of IGF-I-induced PTPalpha tyrosine phosphorylation demonstrated that this occurs through a novel Src family kinase-independent mechanism that does not require focal adhesion kinase, phosphatidylinositol 3-kinase, or MEK. We also show that PTPalpha physically interacts with the IGF-I receptor. In contrast to IGF-I-induced PTPalpha phosphorylation, this association does not require IGF-I. The interaction of PTPalpha and the IGF-I receptor is independent of PTPalpha catalytic activity, and expression of exogenous PTPalpha does not promote IGF-I receptor tyrosine dephosphorylation, indicating that PTPalpha does not act as an IGF-I receptor phosphatase. However, PTPalpha mediates IGF-I signaling, because IGF-I-stimulated fibroblast migration was reduced by approximately 50% in cells lacking PTPalpha or in cells with mutant PTPalpha lacking the tyrosine 789 phosphorylation site. Our results suggest that PTPalpha tyrosine phosphorylation can occur in response to diverse stimuli and can be mediated by various tyrosine kinases. In the case of IGF-I, we propose that IGF-I-induced tyrosine 789 phosphorylation of PTPalpha, possibly catalyzed by the PTPalpha-associated IGF-I receptor tyrosine kinase, is required for efficient cell migration in response to this growth factor.
Assuntos
Movimento Celular , Fibroblastos/enzimologia , Fator de Crescimento Insulin-Like I/metabolismo , Processamento de Proteína Pós-Traducional , Receptor IGF Tipo 1/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Proto-Oncogênicas c-yes/genética , Proteínas Proto-Oncogênicas c-yes/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/deficiência , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Tirosina , Quinases da Família src/genética , Quinases da Família src/metabolismoAssuntos
Dor/tratamento farmacológico , Dor/metabolismo , Animais , Dor/genética , Peptídeos/uso terapêutico , Ligação Proteica , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
The role of protein-tyrosine phosphatase alpha (PTPalpha) in mast cell function was investigated in tissues and cells from PTPalpha-deficient mice. Bone marrow-derived mast cells (BMMCs) lacking PTPalpha exhibit defective stem cell factor (SCF)-dependent polarization and migration. Investigation of the molecular basis for this reveals that SCF/c-Kit-stimulated activation of the Fyn tyrosine kinase is impaired in PTPalpha(-/-) BMMCs, with a consequent inhibition of site-specific c-Kit phosphorylation at tyrosines 567/569 and 719. Although c-Kit-mediated activation of phosphatidylinositol 3-kinase and Akt is unaffected, profound defects occur in the activation of downstream signaling proteins, including mitogen-activated protein kinases and Rho GTPases. Phosphorylation and interaction of Fyn effectors Gab2 and Shp2, which are linked to Rac/JNK activation in mast cells, are impaired in PTPalpha(-/-) BMMCs. Thus, PTPalpha is required for SCF-induced c-Kit and Fyn activation, and in this way regulates a Fyn-based c-Kit signaling axis (Fyn/Gab2/Shp2/Vav/PAK/Rac/JNK) that mediates mast cell migration. These defective signaling events may underlie the altered tissue-resident mast cell populations found in PTPalpha(-/-) mice.
Assuntos
Regulação Enzimológica da Expressão Gênica , Mastócitos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/fisiologia , Fator de Células-Tronco/metabolismo , Animais , Movimento Celular , Cruzamentos Genéticos , Mastócitos/citologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Aberrant protein tyrosine phosphorylation resulting from the altered activity of protein tyrosine phosphatases (PTPs) is increasingly being implicated in the genesis and progression of human cancer. Accumulating evidence indicates that the dysregulated expression of members of the phosphatase of regenerating liver (PRL) subgroup of PTPs is linked to these processes. Enhanced expression of the PRLs, notably PRL-1 and PRL-3, promotes the acquisition of cellular properties that confer tumorigenic and metastatic abilities. Up-regulation of PRL-3 is associated with the progression and eventual metastasis of several types of human cancer. Indeed, PRL-3 shows promise as a biomarker and prognostic indicator in colorectal, breast, and gastric cancers. However, the substrates and molecular mechanisms of action of the PRLs have remained elusive. Recent findings indicate that PRLs may function in regulating cell adhesion structures to effect epithelial-mesenchymal transition. The identification of PRL substrates is key to understanding their roles in cancer progression and exploiting their potential as exciting new therapeutic targets for cancer treatment.