RESUMO
Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/metabolismo , Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Osteossarcoma/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Indóis/farmacologia , Receptor beta de Linfotoxina/antagonistas & inibidores , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Propionatos/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma , Transdução de Sinais , Receptor de TWEAK/antagonistas & inibidores , Receptor de TWEAK/genética , Receptor de TWEAK/metabolismo , Células Tumorais Cultivadas , Quinase Induzida por NF-kappaBRESUMO
Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD.
RESUMO
Despite the important role of apolipoprotein E (apoE) secretion from astrocytes in brain lipid metabolism and the strong association of apoE4, one of the human apoE isoforms, with sporadic and late onset forms of Alzheimer's disease (AD) little is known about the regulation of astrocytic apoE. Utilizing annotated chemical libraries and a phenotypic screening strategy that measured apoE secretion from a human astrocytoma cell line, inhibition of pan class I histone deacetylases (HDACs) was identified as a mechanism to increase apoE secretion. Knocking down select HDAC family members alone or in combination revealed that inhibition of the class I HDAC family was responsible for enhancing apoE secretion. Knocking down LXRα and LXRß genes revealed that the increase in astrocytic apoE in response to HDAC inhibition occurred via an LXR-independent pathway. Collectively, these data suggest that pan class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.
Assuntos
Apolipoproteínas E/metabolismo , Histona Desacetilases/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteínas E/análise , Apolipoproteínas E/genética , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Genótipo , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Hidrocarbonetos Fluorados/farmacologia , Receptores X do Fígado/antagonistas & inibidores , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Ligação Proteica , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Sulfonamidas/farmacologiaRESUMO
Specificity of neurotrophin factor signaling is dictated through the action of Trk receptor tyrosine kinases. Once activated, Trk receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin receptors are differentially regulated by ubiquitination to modulate the survival of neurons.
Assuntos
Sobrevivência Celular/fisiologia , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Receptor trkA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Western Blotting , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte , Imunofluorescência , Humanos , Dados de Sequência Molecular , Ubiquitina-Proteína Ligases Nedd4 , RNA Interferente Pequeno , Ratos , Receptor trkA/genética , Receptores de Fator de Crescimento Neural/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Ubiquitina/metabolismoRESUMO
Prostate cancer is the most common solid tumor in men, and it shares with all cancers the hallmark of elevated, nonhomeostatic cell proliferation. Here we have tested the hypothesis that the SONIC HEDGEHOG (SHH)-GLI signaling pathway is implicated in prostate cancer. We report expression of SHH-GLI pathway components in adult human prostate cancer, often with enhanced levels in tumors versus normal prostatic epithelia. Blocking the pathway with cyclopamine or anti-SHH antibodies inhibits the proliferation of GLI1+/PSA+ primary prostate tumor cultures. Inversely, SHH can potentiate tumor cell proliferation, suggesting that autocrine signaling may often sustain tumor growth. In addition, pathway blockade in three metastatic prostate cancer cell lines with cyclopamine or through GLI1 RNA interference leads to inhibition of cell proliferation, suggesting cell-autonomous pathway activation at different levels and showing an essential role for GLI1 in human cells. Our data demonstrate the dependence of prostate cancer on SHH-GLI function and suggest a novel therapeutic approach.