RESUMO
BACKGROUND: Natural killer (NK) cells require a functional lytic granule machinery to mediate effective antitumor responses. Evading the lytic cargo deployed at the immune synapse (IS) could be a critical step for cancer progression through yet unidentified mechanisms. METHODS: NK cell antibody-dependent cellular cytotoxicity (ADCC) is a major determinant of the clinical efficacy of some therapeutic antibodies including the anti-HER2 Trastuzumab. Thus, we screened sera of Trastuzumab-resistant HER2 +patients with breast cancer for molecules that could inhibit NK cell ADCC. We validated our findings in vitro using cytotoxicity assays and confocal imaging of the lytic granule machinery and in vivo using syngeneic and xenograft murine models. RESULTS: We found that sera from Trastuzumab-refractory patients could inhibit healthy NK cell ADCC in vitro. These sera contained high levels of the inflammatory protein chitinase 3-like 1 (CHI3L1) compared with sera from responders and healthy controls. We demonstrate that recombinant CHI3L1 inhibits both ADCC and innate NK cell cytotoxicity. Mechanistically, CHI3L1 prevents the correct polarization of the microtubule-organizing center along with the lytic granules to the IS by hindering the receptor of advanced glycation end-products and its downstream JNK signaling. In vivo, CHI3L1 administration drastically impairs the control of NK cell-sensitive tumors, while CHI3L1 blockade synergizes with ADCC to cure mice with HER2 +xenografts. CONCLUSION: Our work highlights a new paradigm of tumor immune escape mediated by CHI3L1 which acts on the cytotoxic machinery and prevents granule polarization. Targeting CHI3L1 could mitigate immune escape and potentiate antibody and cell-based immunotherapies.
Assuntos
Proteína 1 Semelhante à Quitinase-3/metabolismo , Evasão da Resposta Imune/imunologia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Neoplasias/genética , Animais , Feminino , Humanos , CamundongosRESUMO
High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the 'Fc-load' on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Anticorpos Biespecíficos/química , Anticorpos Monoclonais/química , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/químicaRESUMO
PURPOSE: Genetically engineered T cells are powerful anticancer treatments but are limited by safety and specificity issues. We herein describe an MHC-unrestricted modular platform combining autologous T cells, transduced with a targetable synthetic agonistic receptor (SAR), with bispecific antibodies (BiAb) that specifically recruit and activate T cells for tumor killing. EXPERIMENTAL DESIGN: BiAbs of different formats were generated by recombinant expression. T cells were retrovirally transduced with SARs. T-cell activation, proliferation, differentiation, and T-cell-induced lysis were characterized in three murine and human tumor models in vitro and in vivo. RESULTS: Murine T cells transduced with SAR composed of an extracellular domain EGFRvIII fused to CD28 and CD3ζ signaling domains could be specifically recruited toward murine tumor cells expressing EpCAM by anti-EGFRvIII × anti-EpCAM BiAb. BiAb induced selective antigen-dependent activation, proliferation of SAR T cells, and redirected tumor cell lysis. Selectivity was dependent on the monovalency of the antibody for EGFRvIII. We identified FAS ligand as a major mediator of killing utilized by the T cells. Similarly, human SAR T cells could be specifically redirected toward mesothelin-expressing human pancreatic cancer cells. In vivo, treatment with SAR T cells and BiAb mediated antitumoral activity in three human pancreatic cancer cell xenograft models. Importantly, SAR activity, unlike CAR activity, was reversible in vitro and in vivo. CONCLUSIONS: We describe a novel ACT platform with antitumor activity in murine and human tumor models with a distinct mode of action that combines adoptive T-cell therapy with bispecific antibodies.
Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Receptores ErbB/imunologia , Imunoterapia Adotiva/métodos , Neoplasias Pancreáticas/terapia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/genética , Molécula de Adesão da Célula Epitelial/imunologia , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Mesotelina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias Pancreáticas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development.
Assuntos
Terapia de Alvo Molecular/métodos , Receptores Proteína Tirosina Quinases/imunologia , Anticorpos de Cadeia Única/imunologia , Especificidade de Anticorpos , Apoptose/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Ativação Enzimática , Humanos , Engenharia de Proteínas , Receptores Proteína Tirosina Quinases/metabolismo , Anticorpos de Cadeia Única/genéticaRESUMO
BACKGROUND: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. METHODS: SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40(+) and EpCAM(+)). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met(+) human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. RESULTS: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase(+) melanoma cells by TCR-, as well as of CEA(+) colon cancer cells by chimeric antigen receptor (CAR)-modified T cells. CONCLUSIONS: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT.
Assuntos
Transferência Adotiva , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Receptores ErbB/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/terapia , Linfócitos T/imunologia , Análise de Variância , Animais , Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial , Receptores ErbB/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-met/imunologia , Transdução GenéticaRESUMO
We found that hematopoietic cell-specific Lyn substrate 1 (HCLS1 or HS1) is highly expressed in human myeloid cells and that stimulation with granulocyte colony-stimulating factor (G-CSF) leads to HCLS1 phosphorylation. HCLS1 binds the transcription factor lymphoid-enhancer binding factor 1 (LEF-1), transporting LEF-1 into the nucleus upon G-CSF stimulation and inducing LEF-1 autoregulation. In patients with severe congenital neutropenia, inherited mutations in the gene encoding HCLS1-associated protein X-1 (HAX1) lead to profound defects in G-CSF-triggered phosphorylation of HCLS1 and subsequently to reduced autoregulation and expression of LEF-1. Consistent with these results, HCLS1-deficient mice are neutropenic. In bone marrow biopsies of the majority of tested patients with acute myeloid leukemia, HCLS1 protein expression is substantially elevated, associated with high levels of G-CSF synthesis and, in some individuals, a four-residue insertion in a proline-rich region of HCLS1 protein known to accelerate intracellular signaling. These data demonstrate the importance of HCLS1 in myelopoiesis in vitro and in vivo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Sanguíneas/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Mielopoese , Neutropenia/congênito , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sequência de Bases , Proteínas Sanguíneas/genética , Linhagem Celular , Proliferação de Células , Síndrome Congênita de Insuficiência da Medula Óssea , Feminino , Granulócitos/metabolismo , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Mielopoese/genética , Neutropenia/genética , Neutropenia/fisiopatologia , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Análise de Sequência de DNA , Transdução de SinaisRESUMO
We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.
Assuntos
Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Dissulfetos/química , Expressão Gênica , Humanos , Metaloproteinase 2 da Matriz/imunologia , Metaloproteinase 9 da Matriz/imunologia , Peptídeo Hidrolases/metabolismo , Engenharia de Proteínas , Proteólise , Receptor ErbB-3/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab(CH1-CL) was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.
Assuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/química , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Angiopoietina-2/imunologia , Animais , Anticorpos Biespecíficos/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Humanos , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Modelos Moleculares , Neovascularização Fisiológica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
RanBP2 is a nucleoporin with SUMO E3 ligase activity that functions in both nucleocytoplasmic transport and mitosis. However, the biological relevance of RanBP2 and the in vivo targets of its E3 ligase activity are unknown. Here we show that animals with low amounts of RanBP2 develop severe aneuploidy in the absence of overt transport defects. The main chromosome segregation defect in cells from these mice is anaphase-bridge formation. Topoisomerase IIalpha (Topo IIalpha), which decatenates sister centromeres prior to anaphase onset to prevent bridges, fails to accumulate at inner centromeres when RanBP2 levels are low. We find that RanBP2 sumoylates Topo IIalpha in mitosis and that this modification is required for its proper localization to inner centromeres. Furthermore, mice with low amounts of RanBP2 are highly sensitive to tumor formation. Together, these data identify RanBP2 as a chromosomal instability gene that regulates Topo IIalpha by sumoylation and suppresses tumorigenesis.
Assuntos
Antígenos de Neoplasias/metabolismo , Centrômero/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Anáfase , Aneuploidia , Animais , Carcinógenos , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Mitose , Chaperonas Moleculares/genética , Mutação , Neoplasias/induzido quimicamente , Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Transcriptional regulation by the canonical Wnt pathway involves the stabilization and nuclear accumulation of beta-catenin, which assembles with LEF1/TCF transcription factors and cofactors to activate Wnt target genes. Recently, the nuclear beta-catenin complex has been shown to contain BCL9, which interacts with beta-catenin and recruits Pygopus as a transcriptional coactivator. However, the presumed general functions of Pygopus and BCL9, which has been proposed to act as a scaffolding protein for Pygopus, have been challenged by the rather specific and modest developmental defects of targeted inactivations of both the Pygo1 and the Pygo2 genes. Here, we analyze the function of BCL9 in transcriptional activation by beta-catenin. We find that BCL9 acts in a cell-type-specific manner and, in part, independent of Pygopus. We show that BCL9 itself contains a transcriptional activation domain in the C terminus, which functionally synergizes in lymphoid cells with the C-terminal transactivation domain of beta-catenin. Finally, we identify amino acids in the transactivation domain of beta-catenin that are important for its function and association with the histone acetyltransferases CBP/p300 and TRRAP/GCN5. Thus, BCL9 may serve to modulate and diversify the transcriptional responses to Wnt signaling in a cell-type-specific manner.
Assuntos
Proteínas de Neoplasias/metabolismo , Ativação Transcricional , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Jurkat , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição , Transfecção , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
The SALL4 promoter has not yet been characterized. Animal studies showed that SALL4 is downstream of and interacts with TBX5 during limb and heart development, but a direct regulation of SALL4 by TBX5 has not been demonstrated. For other SAL genes, regulation within the Shh, Wnt, and Fgf pathways has been reported. Chicken csal1 expression can be activated by a combination of Fgf4 and Wnt3a or Wnt7a. Murine Sall1 enhances, but Xenopus Xsal2 represses, the canonical Wnt signaling. Here we describe the cloning and functional analysis of the SALL4 promoter. Within a minimal promoter region of 31bp, we identified a consensus TCF/LEF-binding site. The SALL4 promoter was strongly activated not only by LEF1 but also by TCF4E. Mutation of the TCF/LEF-binding site resulted in decreased promoter activation. Our results demonstrate for the first time the direct regulation of a SALL gene by the canonical Wnt signaling pathway.
Assuntos
Regulação da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologia , Fatores de Transcrição TCF/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Wnt/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/biossíntese , Fator 1 de Ligação ao Facilitador Linfoide/genética , Dados de Sequência Molecular , Fatores de Transcrição/genéticaRESUMO
Cellular senescence and apoptosis have evolved to restrain unwarranted proliferation of potentially tumorigenic cells. Here we show that overexpression of the E3 SUMO ligase PIASy in normal human fibroblasts recruits the p53 and Rb tumor suppressor pathways to provoke a senescence arrest. By contrast, in Rb-deficient fibroblasts, expression of PIASy leads to p53-dependent apoptosis. Induction of senescence requires PIASy E3 activity and is specific for this member of the PIAS ligase family. PIASy stimulates sumoylation and transcriptional activity of p53 and increases Rb-dependent corepression through recruitment to E2F-responsive promoters. Viral oncoprotein E6 suppresses both PIASy-induced senescence and sumoylation of PIASy substrates. Finally, we show that fibroblasts lacking PIASy exhibit a highly reduced propensity to undergo senescence in response to a prosenescence stimulus. Altogether, these data provide the first evidence for a direct role of an E3 SUMO ligase, and by implication of the SUMO pathway, in cellular senescence and apoptosis.
Assuntos
Apoptose/fisiologia , Senescência Celular/fisiologia , Fibroblastos/fisiologia , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteína do Retinoblastoma/metabolismo , Transformação Celular Viral/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Fibroblastos/citologia , Expressão Gênica/genética , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Inibidoras de STAT Ativados/deficiência , Processamento de Proteína Pós-Traducional , Elementos de Resposta/genética , Proteína do Retinoblastoma/genética , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of the Notch pathway in vertebrate somitogenesis. Here, we examine the molecular basis for this presumed hierarchy and show that a targeted mutation of Lef1, which abrogates LEF1 function and impairs the activity of coexpressed TCF factors, affects the patterning of somites and the expression of components of the Notch pathway. LEF1 was found to bind multiple sites in the Dll1 promoter in vitro and in vivo. Moreover, mutations of LEF1-binding sites in the Dll1 promoter impair expression of a Dll1-LacZ transgene in the presomitic mesoderm. Finally, the induced expression of LEF1-beta-catenin activates the expression of endogenous Dll1 in fibroblastic cells. Thus, Wnt signaling can affect the Notch pathway by a LEF1-mediated regulation of Dll1.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana , Proteínas de Membrana/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas/metabolismo , Somitos/fisiologia , Fatores de Transcrição/fisiologia , Animais , Padronização Corporal , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Óperon Lac/fisiologia , Fator 1 de Ligação ao Facilitador Linfoide , Proteínas de Membrana/genética , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células NIH 3T3 , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Receptores Notch , Transdução de Sinais , Somitos/citologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Proteínas Wnt , beta Catenina , beta-Galactosidase/metabolismoRESUMO
Protein inhibitors of activated STATs (PIAS) represent a small family of nuclear proteins that modulate the activity of many transcription factors and act as E3 ligases for covalent modification of proteins with the small ubiquitin-like modifier (SUMO). In particular, PIASy has been shown to inhibit the activation of gene expression by the IFN-responsive transcription factor STAT1 and the Wnt-responsive transcription factor LEF1. To assess the function of PIASy in vivo, we generated and analyzed mice carrying a targeted mutation of the Piasy gene. We find that homozygous mutant mice have no obvious morphological defects and have a normal distribution of lymphocyte populations. Molecular analysis of signaling in response to IFN-gamma and Wnt agonists revealed a modest reduction in the activation of endogenous and transfected target genes. Two-dimensional analysis of total proteins and SUMO-modified proteins in transformed pre-B cells showed no significant differences between wild-type mice and homozygous mutant mice. Taken together, our data indicate that PIASy has a modest effect on cytokine and Wnt signaling, suggesting a redundancy with other members of the family of PIAS proteins.