Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 57
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
J Transl Med ; 20(1): 521, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36348405

RESUMO

BACKGROUND: We reported that PARP-1 regulates genes whose products are crucial for asthma, in part, by controlling STAT6 integrity speculatively through a calpain-dependent mechanism. We wished to decipher the PARP-1/STAT6 relationship in the context of intracellular trafficking and promoter occupancy of the transcription factor on target genes, its integrity in the presence of calpains, and its connection to autophagy. METHODS: This study was conducted using primary splenocytes or fibroblasts derived from wild-type or PARP-1-/- mice and Jurkat T cells to mimic Th2 inflammation. RESULTS: We show that the role for PARP-1 in expression of IL-4-induced genes (e.g. gata-3) in splenocytes did not involve effects on STAT6 phosphorylation or its subcellular trafficking, rather, it influenced its occupancy of gata-3 proximal and distal promoters in the early stages of IL-4 stimulation. At later stages, PARP-1 was crucial for STAT6 integrity as its inhibition, pharmacologically or by gene knockout, compromised the fate of the transcription factor. Calpain-1 appeared to preferentially degrade JAK-phosphorylated-STAT6, which was blocked by calpastatin-mediated inhibition or by genetic knockout in mouse fibroblasts. The STAT6/PARP-1 relationship entailed physical interaction and modification by poly(ADP-ribosyl)ation independently of double-strand-DNA breaks. Poly(ADP-ribosyl)ation protected phosphorylated-STAT6 against calpain-1-mediated degradation. Additionally, our results show that STAT6 is a bonafide substrate for chaperone-mediated autophagy in a selective and calpain-dependent manner in the human Jurkat cell-line. The effects were partially blocked by IL-4 treatment and PARP-1 inhibition. CONCLUSIONS: The results demonstrate that poly(ADP-ribosyl)ation plays a critical role in protecting activated STAT6 during Th2 inflammation, which may be synthetically targeted for degradation by inhibiting PARP-1.


Assuntos
Poli ADP Ribosilação , Poli(ADP-Ribose) Polimerases , Humanos , Camundongos , Animais , Poli(ADP-Ribose) Polimerases/metabolismo , Calpaína/genética , Calpaína/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Interleucina-4/farmacologia , Interleucina-4/metabolismo , Autofagia , Inflamação , Fator de Transcrição STAT6/metabolismo
2.
Respirology ; 24(7): 675-683, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30747487

RESUMO

BACKGROUND AND OBJECTIVE: Endothelial microparticles (EMP) are submicron vesicles released from endothelial cells. We aimed to determine the utility of EMP as biomarkers of pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc) patients and the pathogenic role of microparticles (MP) in vascular inflammation. METHODS: Levels of EMP (CD144+, CD31+, CD62E+ and CD143+) were compared between three groups (10 SSc patients with PAH, 10 SSc patients without pulmonary hypertension (no-PH) and 10 healthy age- and sex-matched controls). Human pulmonary artery endothelial cells (HPAEC) were exposed in vitro to MP obtained from SSc patients or healthy controls, and levels of cytokines and inflammatory adhesion molecules were compared. RESULTS: CD144+ EMP were significantly higher in the SSc-PAH group compared to either the SSc-no PH or healthy controls (diagnostic accuracy 80%, P = 0.02). Compared to controls, SSc patients had higher CD31+/CD62E+ ratios, indicating larger contributions of apoptosis to EMP release (P = 0.04). Patients with limited SSc had significantly higher levels of CD143+ EMP compared to those with diffuse subtype (P = 0.008). When HPAEC were exposed to MP from SSc patients, there was a significant increase in inflammatory cytokines and adhesion molecules. Interestingly, exposure to healthy control MP caused a reduction in inflammatory markers. CONCLUSION: EMP (particularly CD144+) are promising biomarkers of PAH in SSc but require further study. MP isolated from SSc patients induced an increase in endothelial cell inflammation and may be an important pathogenic factor in SSc.


Assuntos
Micropartículas Derivadas de Células , Células Endoteliais/metabolismo , Hipertensão Pulmonar/metabolismo , Escleroderma Sistêmico/metabolismo , Biomarcadores/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Artéria Pulmonar/patologia , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/patologia
3.
Mediators Inflamm ; 2019: 1656484, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178661

RESUMO

Dendritic cells (DCs) are critical in asthma and many other immune diseases. We previously demonstrated a role for PARP-1 in asthma. Evidence on PARP-1 playing a role in Th2-associated DC function is not clear. In this study, we examined whether PARP-1 is critical for DC differentiation and function using bone marrow progenitors and their migration to the lung in an ovalbumin-based mouse model of asthma. Results show that changes in PARP-1 levels during GM-CSF-induced DC differentiation from bone marrow progenitors were cyclic and appear to be part of an array of changes that included STAT3/STAT5/STAT6/GRAIL/RAD51. Interestingly, PARP-1 gene deletion affected primarily STAT6 and γH2AX. PARP-1 inhibition significantly reduced the migration of DCs to the lungs of ovalbumin-challenged mice, which was associated with a concomitant reduction in lung levels of the adhesion molecule VCAM-1. The requirement of PARP-1 for VCAM-1 expression was confirmed using endothelial and lung smooth muscle cells. PARP-1 expression and activity were also required for VCAM-1 in differentiated DCs. An assessment of CD11b+/CD11c+/MHCIIhigh DCs in spleens and lymph nodes of OVA-sensitized mice revealed that PARP-1 inhibition genetically or by olaparib exerted little to no effect on DC differentiation, percentage of CD80+/CD86+/CD40+-expressing cells, or their capacity to promote proliferation of ovalbumin-primed (OTII) CD4+ T cells. These findings were corroborated using GM-CSF-induced differentiation of DCs from the bone marrow. Surprisingly, the PARP-1-/- DCs exhibited a higher intrinsic capacity to induce OTII CD4+ T cell proliferation in the absence of ovalbumin. Overall, our results show that PARP-1 plays little to no role in DC differentiation and function and that the protective effect of PARP-1 inhibition against asthma is associated with a prevention of DC migration to the lung through a reduction in VCAM-1 expression. Given the current use of PARP inhibitors (e.g., olaparib) in the clinic, the present results may be of interest for the relevant therapies.


Assuntos
Asma/metabolismo , Células Dendríticas/metabolismo , Pulmão/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Animais , Citometria de Fluxo , Camundongos , Camundongos Mutantes , Poli(ADP-Ribose) Polimerase-1/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT6/metabolismo
4.
J Transl Med ; 16(1): 243, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30172259

RESUMO

BACKGROUND: The efficacy of heparins and low-MW-heparins (LMWH) against human asthma has been known for decades. However, the clinical utility of these compounds has been hampered by their anticoagulant properties. Much effort has been put into harnessing the anti-inflammatory properties of LMWH but none have been used as therapy for asthma. Sulfated-non-anticoagulant heparin (S-NACH) is an ultra-LMWH with no systemic anticoagulant effects. OBJECTIVE: The present study explored the potential of S-NACH in blocking allergic asthma and examined the potential mechanism by which it exerts its effects. METHODS: Acute and chronic ovalbumin-based mouse models of asthma, splenocytes, and a lung epithelial cell line were used. Mice were challenged with aerosolized ovalbumin and administered S-NACH or saline 30 min after each ovalbumin challenge. RESULTS: Sulfated-non-anticoagulant heparin administration in mice promoted a robust reduction in airway eosinophilia, mucus production, and airway hyperresponsiveness even after chronic repeated challenges with ovalbumin. Such effects were linked to suppression of Th2 cytokines IL-4/IL-5/IL-13/GM-CSF and ovalbumin-specific IgE without any effect on IFN-γ. S-NACH also reduced lung fibrosis in mice that were chronically-exposed to ovalbumin. These protective effects of S-NACH may be attributed to modulation of the IL-4/JAK1 signal transduction pathway through an inhibition of STAT6 phosphorylation and a subsequent inhibition of GATA-3 and inducible NO synthase expression. The effect of the drug on STAT6 phosphorylation coincided with a reduction in JAK1 phosphorylation upon IL-4 treatment. The protective effects of S-NACH treatment was associated with reduction of the basal expression of the two isoforms of arginase ARG1 and ARG2 in lung epithelial cells. CONCLUSIONS: Our study demonstrates that S-NACH constitutes an opportunity to benefit from the well-known anti-asthma properties of heparins/LMWH while bypassing the risk of bleeding. Our results show, for the first time, that such anti-asthma effects may be associated with reduction of the IL-4/JAK1/STAT6 pathway.


Assuntos
Asma/tratamento farmacológico , Heparina/efeitos adversos , Interleucina-4/metabolismo , Janus Quinase 1/metabolismo , Fator de Transcrição STAT6/metabolismo , Células Th2/metabolismo , Células A549 , Animais , Anti-Inflamatórios/química , Anticoagulantes/química , Asma/complicações , Linhagem Celular , Separação Celular , Modelos Animais de Doenças , Citometria de Fluxo , Hemorragia/tratamento farmacológico , Humanos , Hipersensibilidade/complicações , Hipersensibilidade/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/química , Transdução de Sinais , Baço/citologia
5.
J Hepatol ; 66(3): 589-600, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27984176

RESUMO

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/metabolismo
6.
Am J Physiol Lung Cell Mol Physiol ; 310(7): L630-8, 2016 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-26851260

RESUMO

Pulmonary endothelial prostacyclin appears to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The effect of treatment with a prostacyclin analog in animal models of previously established COPD is unknown. We evaluated the short- and long-term effect of iloprost on inflammation and airway hyperresponsiveness (AHR) in a murine model of COPD. Nineteen mice were exposed to LPS/elastase, followed by either three doses of intranasal iloprost or saline. In the long-term treatment experiment, 18 mice were exposed to LPS/elastase and then received 6 wk of iloprost or were left untreated as controls. In the short-term experiment, iloprost did not change AHR but significantly reduced serum IL-5 and IFN-γ. Long-term treatment with iloprost for both 2 and 6 wk significantly improved AHR. After 6 wk of iloprost, there was a reduction in bronchoalveolar lavage (BALF) neutrophils, serum IL-1ß (30.0 ± 9.2 vs. 64.8 ± 7.4 pg/ml, P = 0.045), IL-2 (36.5 ± 10.6 vs. 83.8 ± 0.4 pg/ml, P = 0.01), IL-10 (75.7 ± 9.3 vs. 96.5 ± 3.5 pg/ml, P = 0.02), and nitrite (15.1 ± 5.4 vs. 30.5 ± 10.7 µmol, P = 0.01). Smooth muscle actin (SMA) in the lung homogenate was also significantly reduced after iloprost treatment (P = 0.02), and SMA thickness was reduced in the small and medium blood vessels after iloprost (P < 0.001). In summary, short- and long-term treatment with intranasal iloprost significantly reduced systemic inflammation in an LPS/elastase COPD model. Long-term iloprost treatment also reduced AHR, serum nitrite, SMA, and BALF neutrophilia. These data encourage future investigations of prostanoid therapy as a novel treatment for COPD patients.


Assuntos
Anti-Inflamatórios/administração & dosagem , Iloprosta/administração & dosagem , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Administração Intranasal , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Doença Pulmonar Obstrutiva Crônica/imunologia , Hipersensibilidade Respiratória/tratamento farmacológico , Hipersensibilidade Respiratória/imunologia
7.
Mediators Inflamm ; 2016: 1984703, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27524861

RESUMO

Although expression of inducible NO synthase (iNOS) in the lungs of asthmatics and associated nitrosative damage are established, iNOS failed as a therapeutic target for blocking airway hyperresponsiveness (AHR) and inflammation in asthmatics. This dichotomy calls for better strategies with which the enzyme is adequately targeted. Here, we confirm iNOS expression in the asthmatic lung with concomitant protein nitration and poly(ADP-ribose) polymerase (PARP) activation. We show, for the first time, that iNOS is highly expressed in peripheral blood mononuclear cells (PBMCs) of asthmatics with uncontrolled disease, which did not correspond to protein nitration. Selective iNOS inhibition with L-NIL protected against AHR upon acute, but not chronic, exposure to ovalbumin or house dust mite (HDM) in mice. Supplementation of NO by nitrite administration significantly blocked AHR in chronically HDM-exposed mice that were treated with L-NIL. Protection against chronic HDM exposure-induced AHR by olaparib-mediated PARP inhibition may be associated with the partial but not the complete blockade of iNOS expression. Indeed, L-NIL administration prevented olaparib-mediated protection against AHR in chronically HDM-exposed mice. Our study suggests that the amount of iNOS and NO are critical determinants in the modulation of AHR by selective iNOS inhibitors and renews the potential of iNOS as a therapeutic target for asthma.


Assuntos
Alérgenos/imunologia , Asma/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase/imunologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Hipersensibilidade Respiratória/metabolismo , Animais , Asma/imunologia , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II/genética , Nitritos/farmacologia , Óxidos de Nitrogênio/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/imunologia , Transdução de Sinais/efeitos dos fármacos
8.
J Allergy Clin Immunol ; 135(2): 425-40, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25441643

RESUMO

BACKGROUND: We reported that DNA-dependent protein kinase (DNA-PK) is critical for the expression of nuclear factor κB-dependent genes in TNF-α-treated glioblastoma cells, suggesting an involvement in inflammatory diseases. OBJECTIVE: We sought to investigate the role of DNA-PK in asthma. METHODS: Cell culture and ovalbumin (OVA)- or house dust mite-based murine asthma models were used in this study. RESULTS: DNA-PK was essential for monocyte adhesion to TNF-α-treated endothelial cells. Administration of the DNA-PK inhibitor NU7441 reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness, and OVA-specific IgE production in mice prechallenged with OVA. Such effects correlated with a marked reduction in lung vascular cell adhesion molecule 1 expression and production of several cytokines, including IL-4, IL-5, IL-13, eotaxin, IL-2, and IL-12 and the chemokines monocyte chemoattractant protein 1 and keratinocyte-derived chemokine, with a negligible effect on IL-10/IFN-γ production. DNA-PK inhibition by gene heterozygosity of the 450-kDa catalytic subunit of the kinase (DNA-PKcs(+/-)) also prevented manifestation of asthma-like traits. These results were confirmed in a chronic model of asthma by using house dust mite, a human allergen. Remarkably, such protection occurred without causing severe combined immunodeficiency. Adoptive transfer of TH2-skewed OT-II wild-type CD4(+) T cells reversed IgE and TH2 cytokine production but not airway hyperresponsiveness in OVA-challenged DNA-PKcs(+/-) mice. DNA-PK inhibition reduced IL-4, IL-5, IL-13, eotaxin, IL-8, and monocyte chemoattractant protein 1 production without affecting IL-2, IL-12, IFN-γ, and interferon-inducible protein 10 production in CD3/CD28-stimulated human CD4(+) T cells, potentially by blocking expression of Gata3. These effects occurred without significant reductions in T-cell proliferation. In mouse CD4(+) T cells in vitro DNA-PK inhibition severely blocked CD3/CD28-induced Gata3 and T-bet expression in CD4(+) T cells and prevented differentiation of TH1 and TH2 cells under respective TH1- and TH2-skewing conditions. CONCLUSION: Our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.


Assuntos
Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Mucosa Respiratória/imunologia , Transferência Adotiva , Alérgenos/imunologia , Animais , Asma/metabolismo , Asma/patologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/patologia , Adesão Celular , Citocinas/metabolismo , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/metabolismo , Células Epiteliais/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Expressão Gênica , Heterogeneidade Genética , Humanos , Imunoglobulina E/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão , Ovalbumina/efeitos adversos , Ovalbumina/imunologia , Fenótipo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Pyroglyphidae/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Imunodeficiência Combinada Severa , Baço/anatomia & histologia , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
9.
J Transl Med ; 13: 233, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26183824

RESUMO

BACKGROUND: We and others have extensively investigated the role of PARP-1 in cell growth and demise in response to pathophysiological cues. Most of the clinical trials on PARP inhibitors are targeting primarily estrogen receptor (ER) negative cancers with BRCA-deficiency. It is surprising that the role of the enzyme has yet to be investigated in ER-mediated cell growth. It is noteworthy that ER is expressed in the majority of breast cancers. We recently showed that the scaffolding protein PDZK1 is critical for 17ß-estradiol (E2)-induced growth of breast cancer cells. We demonstrated that E2-induced PDZK1 expression is indirectly regulated by ER and requires IGF-1 receptor (IGF-1R). METHODS: The breast cancer cell lines MCF-7 and BT474 were used as ER(+) cell culture models. Thieno[2,3-c]isoquinolin-5-one (TIQ-A) and olaparib (AZD2281) were used as potent inhibitors of PARP. PARP-1 knockdown by shRNA was used to show specificity of the effects to PARP-1. RESULTS: In this study, we aimed to determine the effect of PARP inhibition on estrogen-induced growth of breast cancer cells and examine whether the potential effect is linked to PDZK1 and IGF-1R expression. Our results show that PARP inhibition pharmacologically by TIQ-A or olaparib or by PARP-1 knockdown blocked E2-dependent growth of MCF-7 cells. Such inhibitory effect was also observed in olaparib-treated BT474 cells. The effect of PARP inhibition on cell growth coincided with an efficient reduction in E2-induced PDZK1 expression. This effect was accompanied by a similar decrease in the cell cycle protein cyclin D1. PARP appeared to regulate E2-induced PDZK1 at the mRNA level. Such regulation may be linked to a modulation of IGF-1R as PARP inhibition pharmacologically or by PARP-1 knockdown efficiently reduced E2-induced expression of the receptor at the protein and mRNA levels. CONCLUSIONS: Overall, our results show for the first time that PARP regulates E2-mediated cell growth by controlling the ER/IGF-1R/PDZK1 axis. These findings suggest that the relationship between ER, PDZK1, and IGF-1R may be perturbed by blocking PARP function and that PARP inhibitors may be considered in clinical trials on ER(+) cancers.


Assuntos
Proteínas de Transporte/metabolismo , Estradiol/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Transporte/genética , Proliferação de Células/efeitos dos fármacos , Ciclina D1 , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Proteínas de Membrana , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
10.
J Transl Med ; 13: 225, 2015 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-26169874

RESUMO

BACKGROUND: An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. METHODS: We used ovalbumin (OVA)-based mouse models of asthma and primary CD4(+) T cells. C57BL/6J WT or PARP-1(-/-) mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. RESULTS: Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-γ. These traits were associated with a decrease in splenic CD4(+) T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1(-/-) mice. Adoptive transfer of Th2-skewed OT-II-WT CD4(+) T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-γ, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1(-/-)mice suggesting a role for PARP-1 in CD4(+) T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4(+) T cells while causing a moderate elevation in t-bet and ifn-γ expression in Th1-skewed CD4(+) T cells. CONCLUSIONS: Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials.


Assuntos
Asma/tratamento farmacológico , Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Técnicas de Inativação de Genes , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Transferência Adotiva , Animais , Antígenos CD/metabolismo , Asma/complicações , Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Eosinofilia/complicações , Eosinofilia/tratamento farmacológico , Eosinofilia/imunologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Humanos , Imunoglobulina E/biossíntese , Camundongos Endogâmicos C57BL , Muco/metabolismo , Ovalbumina/imunologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Baço/imunologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/metabolismo
11.
Hepatology ; 59(5): 1998-2009, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24089324

RESUMO

UNLABELLED: Poly (ADP-ribose) polymerase 1 (PARP-1) is a constitutive enzyme, the major isoform of the PARP family, which is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. Pharmacological inhibitors of PARP provide significant therapeutic benefits in various preclinical disease models associated with tissue injury and inflammation. However, our understanding the role of PARP activation in the pathophysiology of liver inflammation and fibrosis is limited. In this study we investigated the role of PARP-1 in liver inflammation and fibrosis using acute and chronic models of carbon tetrachloride (CCl4 )-induced liver injury and fibrosis, a model of bile duct ligation (BDL)-induced hepatic fibrosis in vivo, and isolated liver-derived cells ex vivo. Pharmacological inhibition of PARP with structurally distinct inhibitors or genetic deletion of PARP-1 markedly attenuated CCl4 -induced hepatocyte death, inflammation, and fibrosis. Interestingly, the chronic CCl4 -induced liver injury was also characterized by mitochondrial dysfunction and dysregulation of numerous genes involved in metabolism. Most of these pathological changes were attenuated by PARP inhibitors. PARP inhibition not only prevented CCl4 -induced chronic liver inflammation and fibrosis, but was also able to reverse these pathological processes. PARP inhibitors also attenuated the development of BDL-induced hepatic fibrosis in mice. In liver biopsies of subjects with alcoholic or hepatitis B-induced cirrhosis, increased nitrative stress and PARP activation was noted. CONCLUSION: The reactive oxygen/nitrogen species-PARP pathway plays a pathogenetic role in the development of liver inflammation, metabolism, and fibrosis. PARP inhibitors are currently in clinical trials for oncological indications, and the current results indicate that liver inflammation and liver fibrosis may be additional clinical indications where PARP inhibition may be of translational potential.


Assuntos
Hepatite/etiologia , Cirrose Hepática Experimental/etiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Células Estreladas do Fígado/fisiologia , Hepatite/tratamento farmacológico , Humanos , Cirrose Hepática Experimental/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases
12.
Clin Sci (Lond) ; 129(11): 951-62, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26205779

RESUMO

Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-γ or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice.


Assuntos
Antialérgicos/farmacologia , Antiasmáticos/farmacologia , Antígenos de Dermatophagoides , Asma/prevenção & controle , Pulmão/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Asma/enzimologia , Asma/imunologia , Asma/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/fisiopatologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/deficiência , Poli(ADP-Ribose) Polimerases/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Células Th2/efeitos dos fármacos , Células Th2/enzimologia , Células Th2/imunologia
13.
J Biol Chem ; 288(5): 3460-8, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23229555

RESUMO

Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca(2+)-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca(2+) chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca(2+) independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca(2+) chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca(2+)-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis.


Assuntos
Apoptose , Núcleo Celular/enzimologia , Fragmentação do DNA , Desoxirribonucleases/metabolismo , Endodesoxirribonucleases/metabolismo , Retículo Endoplasmático/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Pareamento de Bases , Cálcio/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Cisteína Proteases/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Etoposídeo , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Humanos , Camundongos , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Inibidores de Proteases/farmacologia , Transporte Proteico/efeitos dos fármacos , Serina Proteases/metabolismo
14.
J Biol Chem ; 288(3): 1458-68, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23184953

RESUMO

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.


Assuntos
Asma/tratamento farmacológico , Fator de Transcrição GATA3/genética , Fatores Imunológicos/uso terapêutico , Inflamação/tratamento farmacológico , Interleucina-4/genética , Minociclina/uso terapêutico , NF-kappa B/genética , Receptores de Antígenos de Linfócitos T/genética , Animais , Asma/complicações , Asma/genética , Asma/imunologia , Fator de Transcrição GATA3/agonistas , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Fatores Imunológicos/farmacologia , Inflamação/complicações , Inflamação/genética , Inflamação/imunologia , Interleucina-4/antagonistas & inibidores , Interleucina-4/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Minociclina/farmacologia , NF-kappa B/agonistas , NF-kappa B/imunologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/imunologia , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos
15.
Mol Med ; 20: 270-9, 2014 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-24869908

RESUMO

PDZ domain containing 1 (PDZK1) is a scaffold protein that plays a role in the fate of several proteins. Estrogen can induce PDZK1 gene expression; however, our recent report showed that PDZK1 expression in the breast cancer cell line MCF-7 is indirect and involves insulin-like growth factor (IGF)-1 receptor function. Such a relationship was established in cell culture systems and human breast cancer tissues. Here we show that overexpression of PDZK1 promoted an increase in cyclin D1 and enhanced anchorage-independent growth of MCF-7 cells in the absence of 17ß-estradiol, suggesting that PDZK1 harbors oncogenic activity. Indeed, PDKZ1 overexpression enhanced epidermal growth factor receptor (EGFR)-stimulated MEK/ERK1/2 signaling and IGF-induced Akt phosphorylation. PDZK1 appeared to play this role, in part, by stabilizing the integrity of the growth promoting factors Akt, human epidermal growth factor receptor 2 (Her2/Neu) and EGFR. Increased Akt levels occurred via a decrease in the ubiquitination of the kinase. PDZK1 overexpression was associated with resistance to paclitaxel/5-fluorouracil/etoposide only at low concentrations. Although the increased stability of Akt was sensitive to heat shock protein 90 (HSP90) inhibition, increased levels of the cochaperone cell division cycle 37 (Cdc37), as well as its ability to bind PDZK1, appear to play a larger role in kinase stability. Using human tissue microarrays, we show strong positive correlation between PDZK1, Akt and Cdc37 protein levels, and all correlated with human breast malignancy. There were no positive correlations between PDZK1 and Cdc37 at the mRNA levels, confirming our in vitro studies. These results demonstrate a relationship between PDZK1, Akt and Cdc37, and potentially Her2/Neu and EGFR, in breast cancer, representing a new axis that can be targeted therapeutically to reduce the burden of human breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proliferação de Células , Feminino , Humanos , Células MCF-7 , Proteínas de Membrana
16.
Nat Cell Biol ; 9(10): 1175-83, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17891139

RESUMO

Poly(ADP-ribose) polymerase 1 (PARP-1) and p53 are two key proteins in the DNA-damage response. Although PARP-1 is known to poly(ADP-ribosyl)ate p53, the role of this modification remains elusive. Here, we identify the major poly(ADP-ribosyl)ated sites of p53 by PARP-1 and find that PARP-1-mediated poly(ADP-ribosyl)ation blocks the interaction between p53 and the nuclear export receptor Crm1, resulting in nuclear accumulation of p53. These findings molecularly link PARP-1 and p53 in the DNA-damage response, providing the mechanism for how p53 accumulates in the nucleus in response to DNA damage. PARP-1 becomes super-activated by binding to damaged DNA, which in turn poly(ADP-ribosyl)ates p53. The nuclear export machinery is unable to target poly(ADP-ribosyl)ated p53, promoting accumulation of p53 in the nucleus where p53 exerts its transactivational function.


Assuntos
Núcleo Celular/metabolismo , Carioferinas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Cães , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Carioferinas/genética , Luciferases/genética , Luciferases/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Exportina 1
17.
J Inflamm (Lond) ; 21(1): 14, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38689261

RESUMO

BACKGROUND: The DNA-dependent protein kinase (DNA-PK) complex comprises a catalytic (PRKDC) and two requisite DNA-binding (Ku70/Ku80) subunits. The role of the complex in repairing double-stranded DNA breaks (DSBs) is established, but its role in inflammation, as a complex or individual subunits, remains elusive. While only ~ 1% of PRKDC is necessary for DNA repair, we reported that partial inhibition blocks asthma in mice without causing SCID. METHODS: We investigated the central role of PRKDC in inflammation and its potential association with DNA repair. We also elucidated the relationship between inflammatory cytokines (e.g., TNF-α) and PRKDC by analyzing its connections to inflammatory kinases. Human cell lines, primary human endothelial cells, and mouse fibroblasts were used to conduct the in vitro studies. For animal studies, LPS- and oxazolone-induced mouse models of acute lung injury (ALI) and delayed-type hypersensitivity (DHT) were used. Wild-type, PRKDC+/-, or Ku70+/- mice used in this study. RESULTS: A ~ 50% reduction in PRKDC markedly blocked TNF-α-induced expression of inflammatory factors (e.g., ICAM-1/VCAM-1). PRKDC regulates Th1-mediated inflammation, such as DHT and ALI, and its role is highly sensitive to inhibition achieved by gene heterozygosity or pharmacologically. In endothelial or epithelial cells, TNF-α promoted rapid PRKDC phosphorylation in a fashion resembling that induced by, but independent of, DSBs. Ku70 heterozygosity exerted little to no effect on ALI in mice, and whatever effect it had was associated with a specific increase in MCP-1 in the lungs and systemically. While Ku70 knockout blocked VP-16-induced PRKDC phosphorylation, it did not prevent TNF-α - induced phosphorylation of the kinase, suggesting Ku70 dispensability. Immunoprecipitation studies revealed that PRKDC transiently interacts with p38MAPK. Inhibition of p38MAPK blocked TNF-α-induced PRKDC phosphorylation. Direct phosphorylation of PRKDC by p38MAPK was demonstrated using a cell-free system. CONCLUSIONS: This study presents compelling evidence that PRKDC functions independently of the DNA-PK complex, emphasizing its central role in Th1-mediated inflammation. The distinct functionality of PRKDC as an individual enzyme, its remarkable sensitivity to inhibition, and its phosphorylation by p38MAPK offer promising therapeutic opportunities to mitigate inflammation while sparing DNA repair processes. These findings expand our understanding of PRKDC biology and open new avenues for targeted anti-inflammatory interventions.

18.
Mol Med ; 19: 253-62, 2013 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-23821363

RESUMO

Although a relationship between PDZK1 expression and estrogen receptor (ER)-α stimulation has been suggested, the nature of such a connection and the function of PDZK1 in breast cancer remain unknown. Human tissue microarrays (cancer tissue: 262 cores; normal tissue: 87 cores) and breast cancer cell lines were used to conduct the study. We show that PDZK1 protein expression is tightly correlated with human breast malignancy, is negatively correlated with age and had no significant correlation with ER-α expression levels. PDZK1 exhibited an exclusive epithelial expression with mostly cytosolic subcellular localization. Additionally, 17ß-estradiol induced PDZK1 expression above its basal level more than 24 h after treatment in MCF-7 cells. PDZK1 expression was indirectly regulated by ER-α stimulation, requiring insulinlike growth factor 1 receptor (IGF-1R) expression and function. The molecular link between PDZK1 and IGF-1R was supported by a significant correlation between protein and mRNA levels (r = 0.591, p < 0.001, and r = 0.537, p < 0.001, respectively) of the two factors in two different cohorts of human breast cancer tissues. Interestingly, PDZK1 knockdown in MCF-7 cells blocked ER-dependent growth and reduced c-Myc expression, whereas ectopic expression of PDZK1 enhanced cell proliferation in the presence or absence of 17ß-estradiol potentially through an increase in c-Myc expression, suggesting that PDZK1 has oncogenic activity. PDKZ1 also appeared to interact with the Src/ER-α/epidermal growth factor receptor (EGFR) complex, but not with IGF-1R and enhanced EGFR-stimulated MEK/ERK1/2 signaling. Collectively, our results clarify the relationship between ER-α and PDZK1, propose a direct relationship between PDZK1 and IGF-1R, and identify a novel oncogenic activity for PDZK1 in breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor IGF Tipo 1/metabolismo , Linhagem Celular Tumoral , Estrogênios/farmacologia , Feminino , Humanos , Proteínas de Membrana , Análise Serial de Tecidos
19.
J Immunol ; 185(5): 3076-85, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20668217

RESUMO

The role of inducible NO synthase (iNOS) in allergic airway inflammation remains elusive. We tested the hypothesis that iNOS plays different roles during acute versus chronic airway inflammation. Acute and chronic mouse models of OVA-induced airway inflammation were used to conduct the study. We showed that iNOS deletion was associated with a reduction in eosinophilia, mucus hypersecretion, and IL-5 and IL-13 production upon the acute protocol. Such protection was completely abolished upon the chronic protocol. Interestingly, pulmonary fibrosis observed in wild-type mice under the chronic protocol was completely absent in iNOS(-/-) mice despite persistent IL-5 and IL-13 production, suggesting that these cytokines were insufficient for pulmonary fibrosis. Such protection was associated with reduced collagen synthesis and indirect but severe TGF-beta modulation as confirmed using primary lung smooth muscle cells. Although activation of matrix metalloproteinase-2/-9 exhibited little change, the large tissue inhibitor of metalloproteinase-2 (TIMP-2) increase detected in wild-type mice was absent in the iNOS(-/-) counterparts. The regulatory effect of iNOS on TIMP-2 may be mediated by peroxynitrite, as the latter reversed TIMP-2 expression in iNOS(-/-) lung smooth muscle cells and fibroblasts, suggesting that the iNOS-TIMP-2 link may explain the protective effect of iNOS-knockout against pulmonary fibrosis. Analysis of lung sections from chronically OVA-exposed iNOS(-/-) mice revealed evidence of residual but significant protein nitration, prevalent oxidative DNA damage, and poly(ADP-ribose) polymerase-1 activation. Such tissue damage, inflammatory cell recruitment, and mucus hypersecretion may be associated with substantial arginase expression and activity. The results in this study exemplify the complexity of the role of iNOS in asthma and the preservation of its potential as a therapeutic a target.


Assuntos
Alérgenos/administração & dosagem , Mediadores da Inflamação/fisiologia , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/fisiologia , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , Doença Aguda , Alérgenos/toxicidade , Animais , Células Cultivadas , Galinhas , Eosinofilia/imunologia , Eosinofilia/prevenção & controle , Deleção de Genes , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Interleucina-13/antagonistas & inibidores , Interleucina-13/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muco/imunologia , Muco/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Ovalbumina/administração & dosagem , Ovalbumina/toxicidade , Fibrose Pulmonar/enzimologia
20.
J Immunol ; 185(3): 1894-902, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20610652

RESUMO

The role of NF-kappaB in the expression of inflammatory genes and its participation in the overall inflammatory process of chronic diseases and acute tissue injury are well established. We and others have demonstrated a critical involvement of poly(ADP-ribose) polymerase (PARP)-1 during inflammation, in part, through its relationship with NF-kappaB. However, the mechanism by which PARP-1 affects NF-kappaB activation has been elusive. In this study, we show that PARP-1 inhibition by gene knockout, knockdown, or pharmacologic blockade prevented p65 NF-kappaB nuclear translocation in smooth muscle cells upon TLR4 stimulation, NF-kappaB DNA-binding activity, and subsequent inducible NO synthase and ICAM-1 expression. Such defects were reversed by reconstitution of PARP-1 expression. PARP-1 was dispensable for LPS-induced IkappaBalpha phosphorylation and subsequent degradation but was required for p65 NF-kappaB phosphorylation. A perinuclear p65 NF-kappaB localization in LPS-treated PARP-1(-/-) cells was associated with an export rather an import defect. Indeed, whereas PARP-1 deficiency did not alter expression of importin alpha3 and importin alpha4 and their cytosolic localization, the cytosolic levels of exportin (Crm)-1 were increased. Crm1 inhibition promoted p65 NF-kappaB nuclear accumulation as well as reversed LPS-induced p65 NF-kappaB phosphorylation and inducible NO synthase and ICAM-1 expression. Interestingly, p65 NF-kappaB poly(ADP-ribosyl)ation decreased its interaction with Crm1 in vitro. Pharmacologic inhibition of PARP-1 increased p65 NF-kappaB-Crm1 interaction in LPS-treated smooth muscle cells. These results suggest that p65 NF-kappaB poly(ADP-ribosyl)ation may be a critical determinant for the interaction with Crm1 and its nuclear retention upon TLR4 stimulation. These results provide novel insights into the mechanism by which PARP-1 promotes NF-kappaB nuclear retention, which ultimately can influence NF-kappaB-dependent gene regulation.


Assuntos
Núcleo Celular/metabolismo , Carioferinas/fisiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptor 4 Toll-Like/fisiologia , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Animais , Linhagem Celular , Núcleo Celular/enzimologia , Núcleo Celular/imunologia , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/imunologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Carioferinas/antagonistas & inibidores , Lipopolissacarídeos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/deficiência , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/fisiologia , Proteína Exportina 1
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA