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1.
Oncotarget ; 11(3): 250-264, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-32076486

RESUMO

INTRODUCTION: The impact of TP53 co-mutations in EGFR mutated patients on PFS and OS is controversial. Different classifications of TP53 mutations with respect to functional and potential clinical impact have been published. Therefore, we retrospectively analyzed the impact of TP53 co-mutations on ORR, PFS and OS in a cohort of EGFR mutated NSCLC IV patients (UICC 7) using different classifications of TP53 mutations. METHODS: 75 EGFR mutated NSCLC IV patients homogeneously treated with 1st line EGFR TKI were analyzed for TP53 co-mutations. TP53 mutations were classified according to three different types of classifications. The endpoints ORR, PFS and OS were investigated. RESULTS: TP53 co-mutations were found in 29/59 patients (49.2%). TP53 co-mutations were a statistically significant independent negative predictive factor for ORR, PFS and OS. TP53 co-mutations were associated with inferior mPFS and mOS: mPFS/mOS 12 vs. 18/24 vs. 42 months for non-disruptive/disruptive mutations vs. WT (p < 0.004)/(p < 0.009), 11 vs. 17/23 vs. 42 months for pathogenic vs. non-pathogenic/WT (p < 0.001)/(p < 0.001), and 7 vs. 12 vs. 18/12 vs. 28 vs. 42 months for exon 8 vs. non-exon 8 vs. WT (p < 0.001)/(p < 0.002). CONCLUSIONS: TP53 co-mutations are frequent in EGFR mt+ NSCLC and have a strong negative impact on all clinical endpoints of TKI therapy.

2.
Gastroenterology ; 156(1): 145-159.e19, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30273559

RESUMO

BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.


Assuntos
Transformação Celular Neoplásica/metabolismo , Células Epiteliais/enzimologia , Instabilidade Genômica , Neoplasias Intestinais/prevenção & controle , Intestino Delgado/enzimologia , Ribonuclease H/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Colite/induzido quimicamente , Colite/enzimologia , Colite/genética , Colite/patologia , Dano ao DNA , Sulfato de Dextrana , Modelos Animais de Doenças , Células Epiteliais/patologia , Feminino , Predisposição Genética para Doença , Humanos , Neoplasias Intestinais/enzimologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Intestino Delgado/patologia , Masculino , Camundongos Knockout , Fenótipo , Ribonuclease H/deficiência , Ribonuclease H/genética , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
3.
Hum Mol Genet ; 26(20): 3960-3972, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-29016854

RESUMO

Hypomorphic mutations in the DNA repair enzyme RNase H2 cause the neuroinflammatory autoimmune disorder Aicardi-Goutières syndrome (AGS). Endogenous nucleic acids are believed to accumulate in patient cells and instigate pathogenic type I interferon expression. However, the underlying nucleic acid species amassing in the absence of RNase H2 has not been established yet. Here, we report that murine RNase H2 knockout cells accumulated cytosolic DNA aggregates virtually indistinguishable from micronuclei. RNase H2-dependent micronuclei were surrounded by nuclear lamina and most of them contained damaged DNA. Importantly, they induced expression of interferon-stimulated genes (ISGs) and co-localized with the nucleic acid sensor cGAS. Moreover, micronuclei associated with RNase H2 deficiency were cleared by autophagy. Consequently, induction of autophagy by pharmacological mTOR inhibition resulted in a significant reduction of cytosolic DNA and the accompanied interferon signature. Autophagy induction might therefore represent a viable therapeutic option for RNase H2-dependent disease. Endogenous retroelements have previously been proposed as a source of self-nucleic acids triggering inappropriate activation of the immune system in AGS. We used human RNase H2-knockout cells generated by CRISPR/Cas9 to investigate the impact of RNase H2 on retroelement propagation. Surprisingly, replication of LINE-1 and Alu elements was blunted in cells lacking RNase H2, establishing RNase H2 as essential host factor for the mobilisation of endogenous retrotransposons.


Assuntos
Doenças Autoimunes do Sistema Nervoso/enzimologia , Micronúcleo Germinativo/enzimologia , Malformações do Sistema Nervoso/enzimologia , Ribonuclease H/deficiência , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/metabolismo , Doenças Autoimunes do Sistema Nervoso/patologia , Autofagia/genética , DNA/genética , Dano ao DNA , Replicação do DNA , Camundongos , Camundongos Knockout , Micronúcleo Germinativo/genética , Micronúcleo Germinativo/imunologia , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/patologia , Ribonuclease H/genética , Ribonuclease H/metabolismo
4.
Arterioscler Thromb Vasc Biol ; 37(2): 247-257, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28062509

RESUMO

OBJECTIVE: ADAM17 (a disintegrin and metalloproteinase 17) is a sheddase releasing different types of membrane-bound proteins, including adhesion molecules, cytokines, and their receptors as well as inflammatory mediators. Because these substrates modulate important mechanisms of atherosclerosis, we hypothesized that ADAM17 might be involved in the pathogenesis of this frequent disease. APPROACH AND RESULTS: Because Adam17-knockout mice are not viable, we studied the effect of Adam17 deficiency on atherosclerosis in Adam17 hypomorphic mice (Adam17ex/ex), which have low residual Adam17 expression. To induce atherosclerosis, mice were crossed onto the low-density lipoprotein receptor (Ldlr)-deficient background. We found that Adam17ex/ex.Ldlr-/- mice developed ≈1.5-fold larger atherosclerotic lesions, which contained more macrophages and vascular smooth muscle cells than wild-type littermate controls (Adam17wt/wt.Ldlr-/-). Reduced Adam17-mediated shedding led to significantly increased protein levels of membrane-resident TNFα (tumor necrosis factor) and TNFR2 (tumor necrosis factor receptor 2), resulting in a constitutive activation of TNFR2 signaling. At the same time, Adam17 deficiency promoted proatherosclerotic cellular functions, such as increased proliferation and reduced apoptosis in cultured macrophages and vascular smooth muscle cells and increased adhesion of macrophages to vascular endothelial cells. Because siRNA (small interfering RNA)-mediated knockdown of Tnfr2 rescued from aberrant proliferation and from misregulation of apoptosis in Adam17-depleted cells, our data indicate that TNFR2 is an important effector of ADAM17 in our mouse model. CONCLUSIONS: Our results provide evidence for an atheroprotective role of ADAM17, which might be mediated by cleaving membrane-bound TNFα and TNFR2, thereby preventing overactivation of endogenous TNFR2 signaling in cells of the vasculature.


Assuntos
Proteína ADAM17/deficiência , Aorta/enzimologia , Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Proteína ADAM17/genética , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apoptose , Aterosclerose/genética , Aterosclerose/patologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Regulação da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Fenótipo , Placa Aterosclerótica , Interferência de RNA , Receptores de LDL/deficiência , Receptores de LDL/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/metabolismo
5.
J Immunol ; 197(9): 3705-3715, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27698010

RESUMO

Neutrophil and mononuclear cell infiltration during inflammatory processes is highly regulated. The first cells at the site of infection or inflammation are neutrophils, followed by mononuclear cells. IL-6 plays an important role during inflammatory states. It has been shown in several models that the soluble form of IL-6R (sIL-6R) is involved in the recruitment of mononuclear cells by a mechanism called IL-6 trans-signaling. It had been speculated that sIL-6R was generated at the site of inflammation by shedding from neutrophils via activation of the metalloprotease ADAM17. Attempts to genetically delete the floxed ADAM17 gene selectively in myeloid cells infiltrating an air pouch cavity upon injection of carrageenan failed because in transgenic mice, LysMcre did not lead to appreciable loss of the ADAM17 protein in these cells. We therefore used ADAM17 hypomorphic mice, which only express ∼5% of ADAM17 wild-type levels in all tissues and show virtually no shedding of all tested ADAM17 substrates, to clarify the role of ADAM17 during local inflammation in the murine air pouch model. In the present study, we demonstrate that although IL-6 and the trans-signaling mechanism is mandatory for cellular infiltration in this model, it is not ADAM17-mediated shedding of IL-6R within the pouch that orchestrates this inflammatory process. Instead, we demonstrate that sIL-6R is infiltrating from the circulation in an ADAM17-independent process. Our data suggest that this infiltrating sIL-6R, which is needed for IL-6 trans-signaling, is involved in the controlled resolution of an acute inflammatory episode.


Assuntos
Proteína ADAM17/metabolismo , Inflamação/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Receptores de Interleucina-6/metabolismo , Proteína ADAM17/genética , Animais , Carragenina , Movimento Celular , Células Cultivadas , Inflamação/induzido quimicamente , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais
6.
Int J Nanomedicine ; 11: 5311-5321, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27789944

RESUMO

Despite aggressive treatment with radiation and combination chemotherapy following tumor resection, the 5-year survival rate for patients with head and neck cancer is at best only 50%. In this study, we examined the therapeutic potential of localized release of diclofenac from electrospun nanofibers generated from poly(D,L-lactide-co-glycolide) polymer. Diclofenac was chosen since anti-inflammatory agents that inhibit cyclooxygenase have shown great potential in their ability to directly inhibit tumor growth as well as suppress inflammation-mediated tumor growth. A mouse resection model of oral carcinoma was developed by establishing tumor growth in the oral cavity by ultrasound-guided injection of 1 million SCC-9 cells in the floor of the mouth. Following resection, mice were allocated into four groups with the following treatment: 1) no treatment, 2) implanted scaffolds without diclofenac, 3) implanted scaffolds loaded with diclofenac, and 4) diclofenac given orally. Small animal ultrasound and magnetic resonance imaging were utilized for longitudinal determination of tumor recurrence. At the end of 7 weeks following tumor resection, 33% of mice with diclofenac-loaded scaffolds had a recurrent tumor, in comparison to 90%-100% of the mice in the other three groups. At this time point, mice with diclofenac-releasing scaffolds showed 89% survival rate, while the other groups showed survival rates of 10%-25%. Immunohistochemical staining of recurrent tumors revealed a near 10-fold decrease in the proliferation marker Ki-67 in the tumors derived from mice with diclofenac-releasing scaffolds. In summary, the local application of diclofenac in an orthotopic mouse tumor resection model of oral cancer reduced tumor recurrence with significant improvement in survival over a 7-week study period following tumor resection. Local drug release of anti-inflammatory agents should be investigated as a therapeutic option in the prevention of tumor recurrence in oral squamous carcinoma.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Diclofenaco/farmacologia , Neoplasias Bucais/tratamento farmacológico , Nanofibras/química , Animais , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Inibidores de Ciclo-Oxigenase/administração & dosagem , Inibidores de Ciclo-Oxigenase/farmacocinética , Inibidores de Ciclo-Oxigenase/farmacologia , Diclofenaco/administração & dosagem , Diclofenaco/farmacocinética , Implantes de Medicamento , Liberação Controlada de Fármacos , Humanos , Inflamação/complicações , Inflamação/tratamento farmacológico , Camundongos Nus , Neoplasias Bucais/mortalidade , Neoplasias Bucais/patologia , Neoplasias Experimentais/tratamento farmacológico , Poliglactina 910/química , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
7.
JCI Insight ; 1(13)2016 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-27642633

RESUMO

Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism, we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of a disintegrin and metalloprotease 17 (ADAM17); of its cleavage-activated proligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG); and of the substrates' receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of proinflammatory and profibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorphic mice, specific ADAM17 inhibitor-treated WT mice, or mice with inducible KO of ADAM17 in proximal tubule (Slc34a1-Cre) were significantly protected against these effects. In vitro, in proximal tubule cells, we show that AREG has unique profibrotic actions that are potentiated by TNFα-induced AREG cleavage. In vivo, in acute kidney injury (AKI) and chronic kidney disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a therapeutic target for human kidney fibrosis.


Assuntos
Proteína ADAM17/metabolismo , Injúria Renal Aguda/patologia , Anfirregulina/farmacologia , Túbulos Renais Proximais/metabolismo , Rim/patologia , Injúria Renal Aguda/metabolismo , Animais , Células Cultivadas , Receptores ErbB/metabolismo , Fibrose , Rim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb
8.
Elife ; 52016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27476853

RESUMO

Cerebral small vessel disease (SVD) is a leading cause of stroke and dementia. CADASIL, an inherited SVD, alters cerebral artery function, compromising blood flow to the working brain. TIMP3 (tissue inhibitor of metalloproteinase 3) accumulation in the vascular extracellular matrix in CADASIL is a key contributor to cerebrovascular dysfunction. However, the linkage between elevated TIMP3 and compromised cerebral blood flow (CBF) remains unknown. Here, we show that TIMP3 acts through inhibition of the metalloprotease ADAM17 and HB-EGF to regulate cerebral arterial tone and blood flow responses. In a clinically relevant CADASIL mouse model, we show that exogenous ADAM17 or HB-EGF restores cerebral arterial tone and blood flow responses, and identify upregulated voltage-dependent potassium channel (KV) number in cerebral arterial myocytes as a heretofore-unrecognized downstream effector of TIMP3-induced deficits. These results support the concept that the balance of TIMP3 and ADAM17 activity modulates CBF through regulation of myocyte KV channel number.


Assuntos
Proteína ADAM17/metabolismo , Encéfalo/fisiologia , CADASIL/fisiopatologia , Hemodinâmica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo
9.
Bone ; 92: 85-93, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27554428

RESUMO

Rankl, the major pro-osteoclastogenic cytokine, is synthesized as a transmembrane protein that can be cleaved by specific endopeptidases to release a soluble form (sRankl). We have previously reported that interleukin-33 (IL-33) induces expression of Tnfsf11, the Rankl-encoding gene, in primary osteoblasts, but we failed to detect sRankl in the medium. Since we also found that PTH treatment caused sRankl release in a similar experimental setting, we directly compared the influence of the two molecules. Here we show that treatment of primary murine osteoblasts with PTH causes sRankl release into the medium, whereas IL-33 only induces Tnfsf11 expression. This difference was not explainable by alternative splicing or by PTH-specific induction of endopeptidases previously shown to facilitate Rankl processing. Since sRankl release after PTH administration was blocked in the presence a broad-spectrum matrix metalloprotease inhibitor, we applied genome-wide expression analyses to identify transcriptional targets of PTH in osteoblasts. We thereby confirmed some of the effects of PTH established in other systems, but additionally identified few PTH-induced genes encoding metalloproteases. By comparing expression of these genes following administration of IL-33, PTH and various other Tnfsf11-inducing molecules, we observed that PTH was the only molecule simultaneously inducing sRankl release and Adamts1 expression. The functional relevance of the putative influence of PTH on Rankl processing was further confirmed in vivo, as we found that daily injection of PTH into wildtype mice did not only increase bone formation, but also osteoclastogenesis and sRankl concentrations in the serum. Taken together, our findings demonstrate that transcriptional effects on Tnfsf11 expression do not generally trigger sRankl release and that PTH has a unique activity to promote the proteolytic processing of Rankl.


Assuntos
Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Hormônio Paratireóideo/farmacologia , Proteólise/efeitos dos fármacos , Ligante RANK/biossíntese , Ligante RANK/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
10.
J Biol Chem ; 291(31): 16186-96, 2016 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-27226573

RESUMO

Soluble forms of the IL-6 receptor (sIL-6R) bind to the cytokine IL-6 with similar affinity as the membrane-bound IL-6R. IL-6·sIL-6R complexes initiate IL-6 trans-signaling via activation of the ubiquitously expressed membrane-bound ß-receptor glycoprotein 130 (gp130). Inhibition of IL-6 trans-signaling has been shown to be favorable in numerous inflammatory diseases. Furthermore, different soluble forms of gp130 (sgp130) exist that, together with the sIL-6R, are thought to form a buffer for IL-6 in the blood. However, a functional role for the different sgp130 forms has not been described to date. Here we demonstrate that the metalloproteases ADAM10 and ADAM17 can produce sgp130 by ectodomain shedding of gp130, even though this mechanism only accounts for a minor proportion of sgp130 in the circulation. We further show that full-length sgp130 and the shorter forms sgp130-rheumatoid arthritis-associated peptide (RAPS) and sgp130-E10 are differentially expressed in a cell type- specific manner. Remarkably, full-length sgp130 is expressed by monocytes, but this expression is completely lost during differentiation into macrophages in vitro Using genetically engineered murine pre-B cells that secrete different forms of sgp130, we found that these secreted sgp130 proteins are able to prevent trans-signaling-driven cell proliferation of the secreting cells, whereas conditioned supernatant from these cells failed to block IL-6 trans-signaling in other cells. Thus, our data suggest that the different sgp130 forms are released from cells into their immediate surroundings and appear to form cell-associated gradients to modulate their own susceptibility for IL-6 trans-signaling.


Assuntos
Receptor gp130 de Citocina/metabolismo , Interleucina-6/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Transdução de Sinais/fisiologia , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Receptor gp130 de Citocina/genética , Células HEK293 , Células Hep G2 , Humanos , Interleucina-6/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos
11.
J Leukoc Biol ; 99(5): 749-60, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26561568

RESUMO

The cytokine IL-6 is part of a regulatory signaling network that controls immune responses. IL-6 binds either to the membrane-bound IL-6 receptor-α (classic signaling) or to the soluble IL-6 receptor-α (trans-signaling) to initiate signal transduction via gp130 activation. Because classic and trans-signaling of IL-6 fulfill different tasks during immune responses, controlled shedding of the membrane-bound IL-6 receptor-α from the surface of immune cells can be considered a central regulator of IL-6 function. The results from cell culture-based experiments have implicated both a disintegrin and metalloprotease 10 and a disintegrin and metalloprotease 17 in IL-6 receptor-α shedding. However, the nature of the protease mediating IL-6 receptor-α release in vivo is not yet known. We used hypomorphic a disintegrin and metalloprotease 17 mice and conditional a disintegrin and metalloprotease 10 knock-out mice to identify the natural protease of the murine IL-6 receptor-α. Circulating homeostatic soluble IL-6 receptor-α levels are not dependent on a disintegrin and metalloprotease 10 or 17 activity. However, during Listeria monocytogenes infection, IL-6 receptor-α cleavage by the α-secretase a disintegrin and metalloprotease 17 is rapidly induced from the surface of different leukocyte populations. In contrast, CD4-Cre-driven a disintegrin and metalloprotease 10 deletion in T cells did not influence IL-6 receptor-α shedding from these cells after L. monocytogenes infection. A disintegrin and metalloprotease 17 was also required for IL-6 receptor-α ectodomain cleavage and release during endotoxemia. These results demonstrate a novel physiologic role for a disintegrin and metalloprotease 17 in regulating murine IL-6 signals during inflammatory processes.


Assuntos
Proteína ADAM17/metabolismo , Membrana Celular/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Leucócitos/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais , Proteína ADAM10/metabolismo , Animais , Células Cultivadas , Endotoxemia/imunologia , Endotoxemia/patologia , Fibroblastos/metabolismo , Inflamação/sangue , Interleucina-6/sangue , Listeria monocytogenes/fisiologia , Listeriose/sangue , Listeriose/imunologia , Listeriose/patologia , Camundongos Endogâmicos C57BL , Receptores de Interleucina-6/sangue , Linfócitos T/metabolismo
13.
J Biol Chem ; 290(43): 26059-71, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26359498

RESUMO

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.


Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Interleucina-6/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Animais , Linhagem Celular , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteólise , Splicing de RNA , Receptores de Interleucina-6/genética , Acetato de Tetradecanoilforbol/farmacologia
14.
Nat Commun ; 6: 7736, 2015 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-26198641

RESUMO

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Interleucina-6/metabolismo , Neoplasias da Próstata/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Linhagem Celular , Progressão da Doença , Genes p16 , Humanos , Interleucina-6/genética , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Fator de Transcrição STAT3/genética , Proteína Supressora de Tumor p53/metabolismo
15.
J Immunol ; 193(6): 2753-63, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25108021

RESUMO

Immune cells regulate cell surface receptor expression during their maturation, activation, and motility. Although many of these receptors are regulated largely at the level of expression, protease-mediated ectodomain shedding represents an alternative means of refashioning the surface of immune cells. Shedding is largely attributed to a family of a disintegrin and metalloprotease domain (ADAM) metalloproteases, including ADAM17. Although ADAM17 is well known to contribute to the innate immune response, mainly by releasing TNF-α, much less is known about whether/how this metalloprotease regulates adaptive immunity. To determine whether ADAM17 contributes to regulating adaptive immune responses, we took advantage of ADAM17 hypomorphic (ADAM17(ex/ex)) mice, in which ADAM17 expression is reduced by 90-95% compared with wild-type littermates. In this study, we show that that ADAM17 deficiency results in spleen and lymph node enlargement, as well as increased levels of Ag-specific class-switched Ig production following immunization with OVA together with anti-CD40 mAbs and polyinosinic-polycytidylic acid. Moreover, we demonstrate that the costimulatory ligand ICOS ligand (ICOSL) is selectively downregulated on the surface of B cells in an ADAM17-specific manner, although it is not proteolitically processed by recombinant ADAM17 in vitro. Finally, we show that higher cell surface levels of ICOSL in ADAM17(ex/ex) mice may contribute to the development of excessive Ab responses. Therefore, our data suggest a functional link between ADAM17 and ICOSL in controlling adaptive immune responses.


Assuntos
Proteínas ADAM/imunologia , Linfócitos B/imunologia , Imunidade Humoral , Ligante Coestimulador de Linfócitos T Induzíveis/imunologia , Proteínas ADAM/genética , Proteína ADAM17 , Animais , Anticorpos Monoclonais/imunologia , Formação de Anticorpos/imunologia , Linfócitos B/transplante , Antígenos CD40/imunologia , Células Cultivadas , Feminino , Switching de Imunoglobulina , Isotipos de Imunoglobulinas/biossíntese , Isotipos de Imunoglobulinas/imunologia , Ligante Coestimulador de Linfócitos T Induzíveis/biossíntese , Linfonodos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Poli I-C/imunologia , Baço/patologia
16.
FASEB J ; 28(11): 4847-56, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25077560

RESUMO

Cellular senescence, a state of persistent cell cycle arrest, has emerged as a potent tumor suppressor mechanism by restricting proliferation of cells at risk for neoplastic transformation. Senescent cells secrete various growth factors, cytokines, and other proteins that can either elicit the clearance of tumor cells or potentially promote tumor progression. In addition, this senescence-associated secretory phenotype (SASP) includes various factors that are synthesized as transmembrane precursors and subsequently converted into their soluble counterparts. Despite the importance of the SASP to tumor biology, it is virtually unknown how transmembrane proteins are released from senescent cancer cells. Here we show in different models of senescence that the metalloprotease A disintegrin and metalloproteinase 17 (ADAM17) is activated and releases the epidermal growth factor receptor ligand amphiregulin and tumor necrosis factor receptor I (TNFRI) from the surface of senescent cells by ectodomain shedding. ADAM17 activation involves phosphorylation of its cytoplasmic tail by mitogen-activated protein kinase (MAPK) p38. Interestingly, unlike amphiregulin and TNFRI, full-length intercellular adhesion molecule 1 (ICAM1) is released from senescent cells by microvesicles independently of ADAM17. Thus, our results suggest that transmembrane proteins can be released by two distinct mechanisms and point to a crucial role for ADAM17 in shaping the secretory profile of senescent cells.


Assuntos
Proteínas ADAM/metabolismo , Senescência Celular , Microvasos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteína ADAM17 , Células Cultivadas , Citocinas/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fosforilação
17.
J Biol Chem ; 289(23): 16336-48, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24790088

RESUMO

A disintegrin and metalloprotease 17 (ADAM17) is a major sheddase involved in the regulation of a wide range of biological processes. Key substrates of ADAM17 are the IL-6 receptor (IL-6R) and TNF-α. The extracellular region of ADAM17 consists of a prodomain, a catalytic domain, a disintegrin domain, and a membrane-proximal domain as well as a small stalk region. This study demonstrates that this juxtamembrane segment is highly conserved, α-helical, and involved in IL-6R binding. This process is regulated by the structure of the preceding membrane-proximal domain, which acts as molecular switch of ADAM17 activity operated by a protein-disulfide isomerase. Hence, we have termed the conserved stalk region "Conserved ADAM seventeen dynamic interaction sequence" (CANDIS). Finally, we identified the region in IL-6R that binds to CANDIS. In contrast to the type I transmembrane proteins, the IL-6R, and IL-1RII, CANDIS does not bind the type II transmembrane protein TNF-α, demonstrating fundamental differences in the respective shedding by ADAM17.


Assuntos
Proteínas ADAM/metabolismo , Receptores de Interleucina-6/metabolismo , Proteínas ADAM/química , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Dicroísmo Circular , Sequência Conservada , Primers do DNA , Células HEK293 , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos
18.
J Biol Chem ; 289(5): 3080-93, 2014 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-24338472

RESUMO

ADAM17 (a disintegrin and metalloprotease 17) controls pro- and anti-inflammatory signaling events by promoting ectodomain shedding of cytokine precursors and cytokine receptors. Despite the well documented substrate repertoire of ADAM17, little is known about regulatory mechanisms, leading to substrate recognition and catalytic activation. Here we report a direct interaction of the acidophilic kinase Polo-like kinase 2 (PLK2, also known as SNK) with the cytoplasmic portion of ADAM17 through the C-terminal noncatalytic region of PLK2 containing the Polo box domains. PLK2 activity leads to ADAM17 phosphorylation at serine 794, which represents a novel phosphorylation site. Activation of ADAM17 by PLK2 results in the release of pro-TNFα and TNF receptors from the cell surface, and pharmacological inhibition of PLK2 leads to down-regulation of LPS-induced ADAM17-mediated shedding on primary macrophages and dendritic cells. Importantly, PLK2 expression is up-regulated during inflammatory conditions increasing ADAM17-mediated proteolytic events. Our findings suggest a new role for PLK2 in the regulation of inflammatory diseases by modulating ADAM17 activity.


Assuntos
Proteínas ADAM/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Fator de Necrose Tumoral alfa/química , Técnicas do Sistema de Duplo-Híbrido
19.
J Biol Chem ; 289(2): 778-88, 2014 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24275664

RESUMO

The haptoglobin-hemoglobin receptor CD163 and proTNF-α are transmembrane macrophage proteins subjected to cleavage by the inflammation-responsive protease ADAM17. This leads to release of soluble CD163 (sCD163) and bioactive TNF-α. Sequence comparison of the juxtamembrane region identified similar palindromic sequences in human CD163 ((1044)Arg-Ser-Ser-Arg) and proTNF-α ((78)Arg-Ser-Ser-Ser-Arg). In proTNF-α the Arg-Ser-Ser-Ser-Arg sequence is situated next to the previously established ADAM17 cleavage site. Site-directed mutagenesis revealed that the sequences harbor essential information for efficient cleavage of the two proteins upon ADAM17 stimulation. This was further evidenced by analysis of mouse CD163 that, like CD163 in other non-primates, does not contain the palindromic CD163 sequence in the juxtamembrane region. Mouse CD163 resisted endotoxin- and phorbol ester-induced shedding, and ex vivo analysis of knock-in of the Arg-Ser-Ser-Arg sequence in mouse CD163 revealed a receptor shedding comparable with that of human CD163. In conclusion, we have identified an essential substrate motif for ADAM17-mediated CD163 and proTNF-α cleavage in macrophages. In addition, the present data indicate that CD163, by incorporation of this motif in late evolution, underwent a modification that allows for an instant down-regulation of surface CD163 expression and inhibition of hemoglobin uptake. This regulatory modality seems to have coincided with the evolution of an enhanced hemoglobin-protecting role of the haptoglobin-CD163 system in primates.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Sítios de Ligação/genética , Células Cultivadas , Endotoxemia/genética , Endotoxemia/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Células HEK293 , Humanos , Immunoblotting , Inflamação/genética , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Proteólise/efeitos dos fármacos , Interferência de RNA , Receptores de Superfície Celular/genética , Homologia de Sequência de Aminoácidos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/genética
20.
FEBS J ; 281(1): 157-74, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24164679

RESUMO

T-cell immunoglobulin and mucin domain (TIM)-2 is expressed on activated B cells. Here, we provide evidence that murine TIM-2 is a target of ADAM10-mediated ectodomain shedding, resulting in the generation of a soluble form of TIM-2. We identified ADAM10 but not ADAM17 as the major sheddase of TIM-2, as shown by pharmacological ADAM10 inhibition and with ADAM10-deficient and ADAM17-deficient murine embryonic fibroblasts. Ionomycin-induced or 2'(3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt-induced shedding of TIM-2 was abrogated by deletion of 10 juxtamembrane amino acids from the stalk region but not by deletion of two further N-terminally located blocks of 10 amino acids, indicating a membrane-proximal cleavage site. TIM-2 lacking the intracellular domain was cleaved after ionomycin or 2' (3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt treatment, indicating that this domain was not involved in the regulation of ectodomain shedding. Moreover, TIM-2 shedding was negatively controlled by calmodulin. Shed and soluble TIM-2 interacted with H-ferritin. In summary, we describe TIM-2 as a novel target for ADAM10-mediated ectodomain shedding, and reveal the involvement of ADAM proteases in cellular iron homeostasis.


Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Apoferritinas/metabolismo , Linfócitos B/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Linfócitos B/citologia , Western Blotting , Células COS , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Proliferação de Células , Células Cultivadas , Chlorocebus aethiops , Citometria de Fluxo , Células HEK293 , Humanos , Imunoprecipitação , Ionomicina/farmacologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação/genética
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