RESUMO
The function of poly(ADP-ribosyl) polymerase 1 (PARP1) in myelination and remyelination of the central nervous system (CNS) remains enigmatic. Here, we report that PARP1 is an intrinsic driver for oligodendroglial development and myelination. Genetic PARP1 depletion impairs the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes and impedes CNS myelination. Mechanistically, PARP1-mediated PARylation activity is not only necessary but also sufficient for OPC differentiation. At the molecular level, we identify the RNA-binding protein Myef2 as a PARylated target, which controls OPC differentiation through the PARylation-modulated derepression of myelin protein expression. Furthermore, PARP1's enzymatic activity is necessary for oligodendrocyte and myelin regeneration after demyelination. Together, our findings suggest that PARP1-mediated PARylation activity may be a potential therapeutic target for promoting OPC differentiation and remyelination in neurological disorders characterized by arrested OPC differentiation and remyelination failure such as multiple sclerosis.
Assuntos
Diferenciação Celular , Sistema Nervoso Central/metabolismo , Bainha de Mielina/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sistema Nervoso Central/crescimento & desenvolvimento , Cuprizona/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/induzido quimicamente , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Bainha de Mielina/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células Precursoras de Oligodendrócitos/citologia , Células Precursoras de Oligodendrócitos/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/deficiência , Fator de Transcrição 2 de Oligodendrócitos/genética , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Oligodendroglia/fisiologia , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , RNA/metabolismo , Remielinização/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme involved in DNA repair and transcription regulation, among other processes. Malignant transformations, tumor progression, the onset of some neuropathies and other disorders have been linked to misregulation of PARP-1 activity. Despite intensive studies during the last few decades, the role of PARP-1 in transcription regulation is still not well understood. In this study, a transcriptomic analysis in Drosophila melanogaster third instar larvae was carried out. A total of 602 genes were identified, showing large-scale changes in their expression levels in the absence of PARP-1 in vivo. Among these genes, several functional gene groups were present, including transcription factors and cytochrome family members. The transcription levels of genes from the same functional group were affected by the absence of PARP-1 in a similar manner. In the absence of PARP-1, all misregulated genes coding for transcription factors were downregulated, whereas all genes coding for members of the cytochrome P450 family were upregulated. The cytochrome P450 proteins contain heme as a cofactor and are involved in oxidoreduction. Significant changes were also observed in the expression of several mobile elements in the absence of PARP-1, suggesting that PARP-1 may be involved in regulating the expression of mobile elements.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Genoma de Inseto , Poli(ADP-Ribose) Polimerase-1/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação para Baixo/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Perfilação da Expressão Gênica , Sequências Repetitivas Dispersas/genética , Larva/genética , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
This study aimed to evaluate the role of FRT in ROS/DNA regulation with or without PARP-1 in radiation-injured thymus cells. The administration of FRT to PARP-1-/- (KO) mice demonstrated that FRT significantly increased the viability of thymus cells and decreased their rate of apoptosis through PARP-1. Radiation increased the levels of ROS, γ-H2AX and 53BP1, and induced DNA double strand breaks. Compared with wild type (WT) mice, levels of ROS, γ-H2AX and 53BP1 in KO mice were much less elevated. The FRT treatment groups also showed little reduction in these indicators in KO mice compared with WT mice. The results of the KO mice study indicated that FRT reduced ROS activation through inhibition of PARP-1. Furthermore, FRT reduced the concentrations of γ-H2AX by decreasing ROS activation. However, we found that FRT did not regulate 53BP1, a marker of DNA damage, because of its elimination of ROS. Levels of apoptosis-inducing factor (AIF), exhibited no significant difference after irradiation in KO mice. To summarize, ROS suppression by PARP-1 knockout in KO mice highlights potential therapeutic target either by PARP-1 inhibition combined with radiation or by treatment with a drug therapy alone. AIF-induced apoptosis could not be activated in KO mice.
Assuntos
Antioxidantes/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Flavonoides/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rosa , Timo/efeitos dos fármacos , Animais , Antioxidantes/isolamento & purificação , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/metabolismo , Células Cultivadas , Flavonoides/isolamento & purificação , Histonas/metabolismo , Camundongos Knockout , Estresse Oxidativo/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Rosa/química , Timo/metabolismo , Timo/patologia , Timo/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.
Assuntos
Testes Genéticos/métodos , Apoptose/genética , Sistemas CRISPR-Cas , Linhagem Celular , Células Clonais , Técnicas de Inativação de Genes , Biblioteca Gênica , Redes Reguladoras de Genes , Humanos , Família Multigênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/deficiência , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Análise de Célula Única , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/deficiência , Proteína bcl-X/genéticaRESUMO
Poly(ADP-ribose) glycohydrolase (Parg) is a central enzyme for poly(ADP-ribose) degradation. We established a Parg+/- mice strain by deletion of a part of exon 1 and around 0.4-kb upstream of sequences of the Parg gene. Parg-/- embryos obtained by intercrossing the Parg+/- mice died in utero between 4.5 and 9.5â¯days postcoitum. We examined whether poly(ADP-ribose) polymerase-1 (Parp-1) deficiency could rescue embryonic lethality of Parg-/- mice. Parg-/-Parp-1-/- mice were born viable at a reduced frequency from the expected mendelian ratio in the intercross progeny of Parg+/-Parp-1-/- mice. The results suggest a possibility that the presence of Parp-1 is responsible for the lethality of Parg-/- embryos, and Parg molecules or Parg activity degrading poly(ADP-ribose) might be important for embryogenesis. In Parg-/-Parp-1-/- mice, Parg protein was not detected in various tissues, and the protein level of Timm23, a 5'-upstream gene of Parg, was reduced compared with that in Parg+/+Parp-1-/- mice. Parg-/-Parp-1-/- mice showed retarded growth compared with Parg+/+Parp-1-/- mice, and died within 3â¯months of age accompanied with severe renal failure. Glomerular sclerosis, tubular dilatation, and hyaline casts in the kidney were observed in Parg-/-Parp-1-/- mice. An increase in blood urea nitrogen (pâ¯<â¯0.05), a marked increase of albumin level in urine (pâ¯<â¯0.01) and its concomitant decrease in serum (pâ¯<â¯0.05) were also detected in Parg-/-Parp-1-/- mice compared with the Parg+/+Parp-1-/- counterpart. The results imply that the combined Parg and Parp-1 loss with a hypomorphic state of Timm23 leads to the development of severe renal failure.
Assuntos
Glicosídeo Hidrolases/deficiência , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Poli(ADP-Ribose) Polimerase-1/deficiência , Insuficiência Renal/metabolismo , Insuficiência Renal/patologia , Animais , Técnicas de Cocultura , Glicosídeo Hidrolases/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Poli(ADP-Ribose) Polimerase-1/genética , Insuficiência Renal/genéticaRESUMO
Poly [ADP-ribose] polymerase 1 (PARP-1) has an inhibitory effect on C-X-C motif chemokine 12 gene (Cxcl12) transcription. We examined whether PARP-1 affects the epigenetic control of Cxcl12 expression by changing its DNA methylation pattern. We observed increased expression of Cxcl12 in PARP-1 knock-out mouse embryonic fibroblasts (PARP1-/-) in comparison to wild-type mouse embryonic fibroblasts (NIH3T3). In the Cxcl12 gene, a CpG island is present in the promoter, the 5' untranslated region (5' UTR), the first exon and in the first intron. The methylation state of Cxcl12 in each cell line was investigated by methylation-specific PCR (MSP) and high resolution melting analysis (HRM). Both methods revealed strong demethylation in PARP1-/- compared to NIH3T3 cells in all four DNA regions. Increased expression of the Ten-eleven translocation (Tet) genes in PARP1-/- cells indicated that TETs could be important factors in Cxcl12 demethylation in the absence of PARP-1, accounting for its increased expression. Our results showed that PARP-1 was a potential upstream player in (de)methylation events that modulated Cxcl12 expression.
Assuntos
Quimiocina CXCL12/genética , Proteínas de Ligação a DNA/genética , DNA/metabolismo , Epigênese Genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas Proto-Oncogênicas/genética , Regiões 5' não Traduzidas , Animais , Quimiocina CXCL12/metabolismo , Ilhas de CpG , DNA/genética , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Éxons , Íntrons , Camundongos , Camundongos Knockout , Células NIH 3T3 , Poli(ADP-Ribose) Polimerase-1/deficiência , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Transdução de SinaisRESUMO
Poly(adenosine diphosphateribose) polymerase (PARP)1 is the prototypical PARP enzyme well known for its role in DNA repair and as a proinflammatory protein. Since PARP1 is an important cofactor of several other proinflammatory proteins, in the present study the possible changes in microbial flora of PARP1 knockout mice were investigated. Samples from the duodenum, cecum and feces from wild type and PARP1 knockout C57BL/6J male mice were collected and 16S ribosomal RNA genes were sequenced. Based on the sequencing results, the microbiome and compared samples throughout the lower part of the gastrointestinal system were reconstructed. The present results demonstrated that the lack of PARP1 enzyme only disturbed the microbial flora of the duodenum, where the biodiversity increased in the knockout animals on the species level but decreased on the order level. The most prominent change was the overwhelming abundance of the family Porphyromonadaceae in the duodenum of PARP1/ animals, which disappeared in the cecum and feces where families were spread out more evenly than in the wild type animals. The findings of the present study may improve current understanding of the role of PARP1 in chronic inflammatory diseases.
Assuntos
Biodiversidade , Microbioma Gastrointestinal , Poli(ADP-Ribose) Polimerase-1/deficiência , Animais , Estudos de Associação Genética , Genótipo , Interações Hospedeiro-Patógeno , Camundongos , Camundongos KnockoutRESUMO
The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1-4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.
Assuntos
Sistemas CRISPR-Cas , Dano ao DNA , Edição de Genes , Neoplasias/genética , Neoplasias/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ribonucleotídeos/genética , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Feminino , Genes BRCA1 , Genoma/genética , Células HeLa , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Ribonuclease H/deficiência , Ribonuclease H/genética , Ribonuclease H/metabolismo , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PARP inhibitors (PARPis) have been used to induce synthetic lethality in BRCA-deficient tumors in clinical trials with limited success. We hypothesized that RAD52-mediated DNA repair remains active in PARPi-treated BRCA-deficient tumor cells and that targeting RAD52 should enhance the synthetic lethal effect of PARPi. We show that RAD52 inhibitors (RAD52is) attenuated single-strand annealing (SSA) and residual homologous recombination (HR) in BRCA-deficient cells. Simultaneous targeting of PARP1 and RAD52 with inhibitors or dominant-negative mutants caused synergistic accumulation of DSBs and eradication of BRCA-deficient but not BRCA-proficient tumor cells. Remarkably, Parp1-/-;Rad52-/- mice are normal and display prolonged latency of BRCA1-deficient leukemia compared with Parp1-/- and Rad52-/- counterparts. Finally, PARPi+RAD52i exerted synergistic activity against BRCA1-deficient tumors in immunodeficient mice with minimal toxicity to normal cells and tissues. In conclusion, our data indicate that addition of RAD52i will improve therapeutic outcome of BRCA-deficient malignancies treated with PARPi.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Animais , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Reparo do DNA/efeitos dos fármacos , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Humanos , Mesilato de Imatinib/farmacologia , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/deficiência , Proteína Rad52 de Recombinação e Reparo de DNA/antagonistas & inibidores , Proteína Rad52 de Recombinação e Reparo de DNA/deficiência , Mutações Sintéticas Letais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
There is a current interest in the development of biodosimetric methods for rapidly assessing radiation exposure in the wake of a large-scale radiological event. This work was initially focused on determining the exposure dose to an individual using biological indicators. Gene expression signatures show promise for biodosimetric application, but little is known about how these signatures might translate for the assessment of radiological injury in radiosensitive individuals, who comprise a significant proportion of the general population, and who would likely require treatment after exposure to lower doses. Using Parp1-/- mice as a model radiation-sensitive genotype, we have investigated the effect of this DNA repair deficiency on the gene expression response to radiation. Although Parp1 is known to play general roles in regulating transcription, the pattern of gene expression changes observed in Parp1-/- mice 24 h postirradiation to a LD50/30 was remarkably similar to that in wild-type mice after exposure to LD50/30. Similar levels of activation of both the p53 and NFκB radiation response pathways were indicated in both strains. In contrast, exposure of wild-type mice to a sublethal dose that was equal to the Parp1-/- LD50/30 resulted in a lower magnitude gene expression response. Thus, Parp1-/- mice displayed a heightened gene expression response to radiation, which was more similar to the wild-type response to an equitoxic dose than to an equal absorbed dose. Gene expression classifiers trained on the wild-type data correctly identified all wild-type samples as unexposed, exposed to a sublethal dose or exposed to an LD50/30. All unexposed samples from Parp1-/- mice were also correctly classified with the same gene set, and 80% of irradiated Parp1-/- samples were identified as exposed to an LD50/30. The results of this study suggest that, at least for some pathways that may influence radiosensitivity in humans, specific gene expression signatures have the potential to accurately detect the extent of radiological injury, rather than serving only as a surrogate of physical radiation dose.
Assuntos
Raios gama/efeitos adversos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Animais , Relação Dose-Resposta à Radiação , CamundongosRESUMO
Strategies to treat cachexia are still at its infancy. Enhanced muscle protein breakdown and ubiquitin-proteasome system are common features of cachexia associated with chronic conditions including lung cancer (LC). Poly(ADP-ribose) polymerases (PARP), which play a major role in chromatin structure regulation, also underlie maintenance of muscle metabolism and body composition. We hypothesized that protein catabolism, proteolytic markers, muscle fiber phenotype, and muscle anabolism may improve in respiratory and limb muscles of LC-cachectic Parp-1-deficient (Parp-1-/- ) and Parp-2-/- mice. In diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing mice (wild type, Parp-1-/- , and Parp-2-/- ), PARP activity (ADP-ribose polymers, pADPr), redox balance, muscle fiber phenotype, apoptotic nuclei, tyrosine release, protein ubiquitination, muscle-specific E3 ligases, NF-κB signaling pathway, markers of muscle anabolism (Akt, mTOR, p70S6K, and mitochondrial DNA) were evaluated along with body and muscle weights, and limb muscle force. Compared to wild type cachectic animals, in both respiratory and limb muscles of Parp-1-/- and Parp-2-/- cachectic mice: cancer induced-muscle wasting characterized by increased PARP activity, protein oxidation, tyrosine release, and ubiquitin-proteasome system (total protein ubiquitination, atrogin-1, and 20S proteasome C8 subunit) were blunted, the reduction in contractile myosin and atrophy of the fibers was attenuated, while no effects were seen in other structural features (inflammatory cells, internal or apoptotic nuclei), and markers of muscle anabolism partly improved. Activation of either PARP-1 or -2 is likely to play a role in muscle protein catabolism via oxidative stress, NF-κB signaling, and enhanced proteasomal degradation in cancer-induced cachexia. Therapeutic potential of PARP activity inhibition deserves attention.
Assuntos
Caquexia/etiologia , Neoplasias Pulmonares/complicações , Fibras Musculares Esqueléticas/enzimologia , Proteínas Musculares/metabolismo , Músculo Esquelético/enzimologia , Estresse Oxidativo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteólise , Animais , Apoptose , Biomarcadores/metabolismo , Caquexia/enzimologia , Caquexia/genética , Caquexia/patologia , Linhagem Celular Tumoral , Diafragma/enzimologia , Diafragma/patologia , Feminino , Genótipo , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , NF-kappa B/metabolismo , Tamanho do Órgão , Fenótipo , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/deficiência , Poli(ADP-Ribose) Polimerases/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Fatores de Tempo , UbiquitinaçãoRESUMO
The maintenance of T-cell homeostasis must be tightly regulated. Here, we have identified a coordinated role of Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 in maintaining T-lymphocyte number and function. Mice bearing a T-cell specific deficiency of PARP-2 in a PARP-1-deficient background showed defective thymocyte maturation and diminished numbers of peripheral CD4+ and CD8+ T-cells. Meanwhile, peripheral T-cell number was not affected in single PARP-1 or PARP-2-deficient mice. T-cell lymphopenia was associated with dampened in vivo immune responses to synthetic T-dependent antigens and virus, increased DNA damage and T-cell death. Moreover, double-deficiency in PARP-1/PARP-2 in T-cells led to highly aggressive T-cell lymphomas with long latency. Our findings establish a coordinated role of PARP-1 and PARP-2 in T-cell homeostasis that might impact on the development of PARP-centred therapies.
Assuntos
Linfoma de Células T/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/genética , Linfócitos T/imunologia , Animais , Morte Celular , Células Cultivadas , Dano ao DNA , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Camundongos , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerases/deficiênciaRESUMO
Cancer cells may acquire drug resistance by activating DNA repair signaling. Poly ADP-ribose polymerase (PARP) plays an important role in DNA repair and it is overexpressed in many cancers including chronic myeloid leukemia (CML). PARP inhibitors have been used either alone or with other drugs to augment cancer cell death. However, whether PARP inhibitors may also augment cell death induced by chemotherapeutic agents in CML cells has not been studied. K562 cells with or without PARP-1 knockdown were treated with cisplatin alone or together with olaparib. The cell death was investigated by propidium iodide staining and apoptosis-related proteins were detected by western blotting. Olaparib suppressed cisplatin-induced cell death in K562 and MEG01 cells. Moreover, PARP-1 knockdown also attenuated cisplatin toxicity in CML cells. Inhibition of PARP decreased cisplatin toxicity by attenuating caspase-3 and caspase-9 activity. These results indicated that the toxicity of cisplatin in CML cells requires PARP activity. Therefore, PARP inhibitors may not be useful with DNA-damaging agents such as cisplatin in CML treatment.
Assuntos
Cisplatino/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Morte Celular/efeitos dos fármacos , Cisplatino/farmacologia , Interações Medicamentosas , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genéticaRESUMO
Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins mediated by PARP family members, such as PARP-1. Although PARylation has been studied extensively, few examples of definitive biological roles for site-specific PARylation have been reported. Here we show that C/EBPß, a key pro-adipogenic transcription factor, is PARylated by PARP-1 on three amino acids in a conserved regulatory domain. PARylation at these sites inhibits C/EBPß's DNA binding and transcriptional activities and attenuates adipogenesis in various genetic and cell-based models. Interestingly, PARP-1 catalytic activity drops precipitously during the first 48 hr of differentiation, corresponding to a release of C/EBPß from PARylation-mediated inhibition. This promotes the binding of C/EBPß at enhancers controlling the expression of adipogenic target genes and continued differentiation. Depletion or chemical inhibition of PARP-1, or mutation of the PARylation sites on C/EBPß, enhances these early adipogenic events. Collectively, our results provide a clear example of how site-specific PARylation drives biological outcomes.
Assuntos
Adipócitos/enzimologia , Adipogenia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Células-Tronco Embrionárias/enzimologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , DNA/genética , DNA/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Células NIH 3T3 , Fenótipo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Domínios Proteicos , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional , TransfecçãoRESUMO
OBJECTIVE: Chronic nonhealing wounds are a major health problem for patients in the United States and worldwide. Diabetes and ischemia are two major risk factors behind impaired healing of chronic lower extremity wounds. Poly-ADP-ribose polymerase (PARP) is found to be overactivated with both ischemic and diabetic conditions. This study seeks a better understanding of the role of PARP in ischemic and diabetic wound healing, with a specific focus on angiogenesis and vasculogenesis. METHODS: Ischemic and diabetic wounds were created in FVB/NJ mice and an in vitro scratch wound model. PARP inhibitor PJ34 was delivered to the animals at 10 mg/kg/d through implanted osmotic pumps or added to the culture medium, respectively. Animal wound healing was assessed by daily digital photographs. Animal wound tissues, peripheral blood, and bone marrow cells were collected at different time points for further analysis with Western blot and flow cytometry. Scratch wound migration and invasion angiogenesis assays were performed using human umbilical vein endothelial cells (HUVECs). Measurements were reported as mean ± standard deviation. Continuous measurements were compared by t-test. P < .05 was considered statistically significant. RESULTS: A significant increase in PARP activity was observed under ischemic and diabetic conditions that correlated with delayed wound healing and slower HUVEC migration. The beneficial effect of PARP inhibition with PJ34 on ischemic and diabetic wound healing was observed in both animal and in vitro models. In the animal model, the percentage of wound healing was significantly enhanced from 43% ± 6% to 71% ± 9% (P < .05) by day 7 with the addition of PJ34. PARP inhibition promoted angiogenesis at the ischemic and diabetic wound beds as evidenced by significantly higher levels of endothelial cell markers (vascular endothelial growth factor receptor 2 [VEGFR2] and endothelial nitric oxide synthase) in mice treated with PJ34 compared with controls. Flow cytometry analysis of peripheral blood mononuclear cells showed that PARP inhibition increased mobilization of endothelial progenitor cells (VEGFR2+/CD133+ and VEGFR2+/CD34+) into the systemic circulation. Furthermore, under in vitro hyperglycemia and hypoxia conditions, PARP inhibition enhanced HUVEC migration and invasion in Boyden chamber assays by 80% and 180% (P < .05), respectively. CONCLUSIONS: Delayed healing in ischemic and diabetic wounds is caused by PARP hyperactivity, and PARP inhibition significantly enhanced ischemic and diabetic wound healing by promoting angiogenesis.
Assuntos
Indutores da Angiogênese/farmacologia , Diabetes Mellitus Experimental/complicações , Angiopatias Diabéticas/tratamento farmacológico , Isquemia/complicações , Neovascularização Fisiológica/efeitos dos fármacos , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Úlcera Cutânea/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Antígeno AC133/metabolismo , Animais , Antígenos CD34/metabolismo , Hipóxia Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/patologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/enzimologia , Glucose/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Isquemia/enzimologia , Isquemia/patologia , Masculino , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/metabolismo , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Úlcera Cutânea/enzimologia , Úlcera Cutânea/etiologia , Úlcera Cutânea/patologia , Fatores de Tempo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND & AIMS: Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). METHODS: We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. RESULTS: PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1-/- animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. CONCLUSION: Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer. LAY SUMMARY: Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.
Assuntos
Fígado Gorduroso Alcoólico/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Fígado Gorduroso Alcoólico/genética , Fígado Gorduroso Alcoólico/metabolismo , Humanos , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo , Estresse Nitrosativo/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenantrenos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Quinolinas/farmacologia , Sirtuína 1/metabolismoRESUMO
Poly (ADP-ribose) polymerase (PARP) plays an important role in endothelial dysfunction, leading to atherogenesis and vascular-related diseases. However, whether PARP regulates nitric oxide (NO), a key regulator of endothelial function, is unclear so far. We investigated whether inhibition of PARP-1, the most abundant PARP isoform, prevents atherogenesis by regulating NO production and tried to elucidate the possible mechanisms involved in this phenomenon. In apolipoprotein E-deficient (apoE-/- ) mice fed a high-cholesterol diet for 12 weeks, PARP-1 inhibition via treatment with 3,4-dihydro-54-(1-piperindinyl) butoxy-1(2H)-isoquinoline (DPQ) or PARP-1 gene knockout reduced aortic atherosclerotic plaque areas (49% and 46%, respectively). Both the groups showed restored NO production in mouse aortas with reduced arginase II (Arg II) expression compared to that in the controls. In mouse peritoneal macrophages and aortic endothelial cells (MAECs), PARP-1 knockout resulted in lowered Arg II expression. Moreover, phosphorylation of endothelial NO synthase (eNOS) was preserved in the aortas and MAECs when PARP-1 was inhibited. Reduced NO production in vitro due to PARP-1 deficiency could be restored by treating the MAECs with oxidized low-density lipoprotein treatment, but this effect could not be achieved with peritoneal macrophages, which was likely due to a reduction in the expression of induced NOS expression. Our findings indicate that PARP-1 inhibition may attenuate atherogenesis by restoring NO production in endothelial cells and thus by reducing Arg II expression and consequently arginase the activity.
Assuntos
Aorta/metabolismo , Arginase/metabolismo , Aterosclerose/metabolismo , Regulação para Baixo/fisiologia , Óxido Nítrico/biossíntese , Poli(ADP-Ribose) Polimerase-1/deficiência , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/prevenção & controle , Células Cultivadas , Colesterol na Dieta/efeitos adversos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease.
Assuntos
Ataxia Cerebelar/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , Poli(ADP-Ribose) Polimerase-1/metabolismo , Adenosina Difosfato Ribose/metabolismo , Alelos , Animais , Apraxias/congênito , Apraxias/genética , Ataxia/genética , Axônios/patologia , Ataxia Cerebelar/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Cromatina/metabolismo , Síndrome de Cogan/genética , Quebras de DNA de Cadeia Simples , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Feminino , Humanos , Interneurônios/metabolismo , Interneurônios/patologia , Masculino , Camundongos , Linhagem , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Repurposing azithromycin has recently emerged as a promising strategy for the acute treatment of ischemic stroke. The mechanism of neuroprotection depends on the ability of this macrolide to promote polarization of microglia/macrophages towards beneficial M2 phenotypes. The immunomodulatory and anti-inflammatory effects of azithromycin, well documented in chronic inflammatory airway diseases, have been ascribed to the inhibition of the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1. Since these inflammatory transcription factors are positively regulated by poly(ADP-ribose) polymerase (PARP)-1, an enzyme actively involved in ischemic brain injury, we have investigated whether the neuroprotective properties of azithromycin in ischemic stroke involve upstream modulation of PARP-1. Administration of a single dose of this macrolide antibiotic upon reperfusion reduced, to a similar extent in wild type and PARP-1 knockout mice, infarct brain damage produced by transient occlusion of the middle cerebral artery. Moreover, we demonstrated the lack of effects of azithromycin on PARP-dependent death of HeLa cells, as well as on activity of purified PARP-1 and PARP-2. Thus, azithromycin protects mice against ischemic stroke injury through a mechanism independent of PARP activation.
Assuntos
Azitromicina/farmacologia , Isquemia Encefálica/complicações , Fármacos Neuroprotetores/farmacologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Azitromicina/uso terapêutico , Morte Celular/efeitos dos fármacos , Técnicas de Inativação de Genes , Imunomodulação/efeitos dos fármacos , Masculino , Camundongos , Fármacos Neuroprotetores/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/patologiaRESUMO
Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2(cko/ko) mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2(ko/ko) cells. PARP1 deficiency does not restore HR in Brca2(ko/ko) cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2(cko/cko) mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.