Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.657
Filtrar
1.
Nat Commun ; 11(1): 4820, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973160

RESUMO

Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.


Assuntos
Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo , Tirosina/análogos & derivados , Tirosina/genética , Tirosina/metabolismo , Animais , Sistemas CRISPR-Cas , Quimiocinas/metabolismo , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Tirosina-tRNA Ligase/química
2.
PLoS Pathog ; 16(8): e1008230, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797076

RESUMO

Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.


Assuntos
Eritrócitos/imunologia , Armadilhas Extracelulares/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Malária/imunologia , Neutrófilos/imunologia , Plasmodium/imunologia , Receptores CXCR4/metabolismo , Animais , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/parasitologia , Humanos , Malária/metabolismo , Malária/parasitologia , Malária/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/parasitologia , Parasitemia/imunologia , Parasitemia/metabolismo , Parasitemia/parasitologia , Parasitemia/patologia
3.
Virology ; 548: 49-58, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838946

RESUMO

Human cytomegalovirus (HCMV) is a widespread herpesvirus that establishes latency in myeloid cells and persists by manipulating immune signaling. Chemokine receptor CXCR4 and its ligand CXCL12 regulate movement of myeloid progenitors into bone marrow and out into peripheral tissues. HCMV amplifies CXCL12-CXCR4 signaling through viral chemokine receptor US27 and cmvIL-10, a viral cytokine that binds the cellular IL-10 receptor (IL-10R), but precisely how these viral proteins influence CXCR4 is unknown. We used the proximity ligation assay (PLA) to examine association of CXCR4, IL-10R, and US27 in both transfected and HCMV-infected cells. CXCR4 and IL-10R colocalized to discrete clusters, and treatment with CXCL12 and cmvIL-10 dramatically increased receptor clustering and calcium flux. US27 was associated with CXCR4 and IL-10R in PLA clusters and further enhanced cluster formation and calcium signaling. These results indicate that CXCR4, IL-10R, and US27 form a novel virus-host signaling complex that enhances CXCL12 signaling during HCMV infection.


Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/metabolismo , Receptores CXCR4/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores de Interleucina-10/metabolismo , Proteínas Virais/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Receptores CXCR4/genética , Receptores de Quimiocinas/genética , Receptores de Interleucina-10/genética , Transdução de Sinais , Proteínas Virais/genética
4.
Cancer Sci ; 111(9): 3327-3337, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32639651

RESUMO

Tirabrutinib is a second-generation Bruton's tyrosine kinase inhibitor with greater selectivity than ibrutinib. Here, we conducted a multicenter, phase II study of tirabrutinib in patients with treatment-naïve (Cohort A) or with relapsed/refractory (Cohort B) Waldenström's macroglobulinemia (WM). Patients were treated with tirabrutinib 480 mg once daily. The primary endpoint was major response rate (MRR; ≥ partial response). Secondary endpoints included overall response rate (ORR; ≥ minor response), time to major response (TTMR), progression-free survival (PFS), overall survival (OS), and safety. In total, 27 patients (18 in Cohort A; 9 in Cohort B) were enrolled. The median age was 71 y, and the median serum immunoglobulin M level was 3600 mg/dL. Among the patients, 96.2% had the MYD88L265P mutation. MRR and ORR were 88.9% and 96.3%, respectively (Cohort A: MRR, 88.9%; ORR, 94.4%; Cohort B: MRR, 88.9%; ORR, 100%). Median TTMR was 1.87 mo. PFS and OS were not reached with a median follow-up of 6.5 and 8.3 mo for Cohorts A and B, respectively. The most common adverse events (AEs) were rash (44.4%), neutropenia (25.9%), and leukopenia (22.2%), with most AEs classified as grade 1 or 2. Grade ≥ 3 AEs included neutropenia (11.1%), lymphopenia (11.1%), and leukopenia (7.4%). No grade 5 AEs were noted. All bleeding events were grade 1; none were associated with drug-related atrial fibrillation or hypertension. Although the follow-up duration was relatively short, the study met the primary endpoint. Therefore, tirabrutinib monotherapy is considered to be highly effective for both untreated and relapsed/refractory WM with a manageable safety profile. (JapicCTI-173646).


Assuntos
Imidazóis/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Feminino , Genótipo , Humanos , Imidazóis/administração & dosagem , Imidazóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Gradação de Tumores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Resultado do Tratamento , Macroglobulinemia de Waldenstrom/diagnóstico , Macroglobulinemia de Waldenstrom/etiologia
5.
Nat Commun ; 11(1): 3194, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581241

RESUMO

Ph+ acute lymphoblastic leukemia (ALL) is characterized by the expression of an oncogenic fusion kinase termed BCR-ABL1. Here, we show that interleukin 7 receptor (IL7R) interacts with the chemokine receptor CXCR4 to recruit BCR-ABL1 and JAK kinases in close proximity. Treatment with BCR-ABL1 kinase inhibitors results in elevated expression of IL7R which enables the survival of transformed cells when IL7 was added together with the kinase inhibitors. Importantly, treatment with anti-IL7R antibodies prevents leukemia development in xenotransplantation models using patient-derived Ph+ ALL cells. Our results suggest that the association between IL7R and CXCR4 serves as molecular platform for BCR-ABL1-induced transformation and development of Ph+ ALL. Targeting this platform with anti-IL7R antibody eliminates Ph+ ALL cells including those with resistance to commonly used ABL1 kinase inhibitors. Thus, anti-IL7R antibodies may provide alternative treatment options for ALL in general and may suppress incurable drug-resistant leukemia forms.


Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores CXCR4/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Feminino , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-7/farmacologia , Subunidade alfa de Receptor de Interleucina-7/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-7/genética , Camundongos , Camundongos Mutantes , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores CXCR4/genética , Transdução de Sinais/efeitos dos fármacos
6.
Mol Pharmacol ; 98(2): 72-87, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32474443

RESUMO

G protein-coupled receptors (GPCRs) are biologic switches that transduce extracellular stimuli into intracellular responses in the cell. Temporally resolving GPCR transduction pathways is key to understanding how cell signaling occurs. Here, we investigate the kinetics and dynamics of the activation and early signaling steps of the CXC chemokine receptor (CXCR) 4 in response to its natural ligands CXC chemokine ligand (CXCL) 12 and macrophage migration inhibitory factor (MIF), using Förster resonance energy transfer-based approaches. We show that CXCR4 presents a multifaceted response to CXCL12, with receptor activation (≈0.6 seconds) followed by a rearrangement in the receptor/G protein complex (≈1 seconds), a slower dimer rearrangement (≈1.7 seconds), and prolonged G protein activation (≈4 seconds). In comparison, MIF distinctly modulates every step of the transduction pathway, indicating distinct activation mechanisms and reflecting the different pharmacological properties of these two ligands. Our study also indicates that CXCR4 exhibits some degree of ligand-independent activity, a relevant feature for drug development. SIGNIFICANCE STATEMENT: The CXC chemokine ligand (CXCL) 12/CXC chemokine receptor (CXCR) 4 axis represents a well-established therapeutic target for cancer treatment. We demonstrate that CXCR4 exhibits a multifaceted response that involves dynamic receptor dimer rearrangements and that is kinetically embedded between receptor-G protein complex rearrangements and G protein activation. The alternative endogenous ligand macrophage migration inhibitory factor behaves opposite to CXCL12 in each assay studied and does not lead to G protein activation. This detailed understanding of the receptor activation may aid in the development of more specific drugs against this target.


Assuntos
Quimiocina CXCL12/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Cinética , Ligação Proteica , Multimerização Proteica , Transdução de Sinais
7.
Anticancer Res ; 40(6): 3221-3229, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487616

RESUMO

BACKGROUND/AIM: Chemokines are cytokines involved not only in inflammatory but also in inappropriate response of the immune system in breast cancer (BC) progression. We examined the diagnostic usefulness of CXCL12, CXCR4 and CA 15-3 in BC patients, based on ROC curve analysis. MATERIALS AND METHODS: The study group consisted of 100 patients with BC; the control group consisted of 35 women with benign breast disease and 35 healthy patients. The median concentration of chemokines was measured by ELISA and that of CA 15-3 by chemiluminescent microparticle immunoassay. RESULTS: The concentrations of CXCL12 and CXCR4 in the BC group were significantly higher than those in the control groups. The AUC value of CXCL12 (0.7502) was the highest of all the chemokines measured in the BC patients. CONCLUSION: There may be a link between CXCL12, CXCR4 and BC that can assist in the diagnosis, markedly when combined with CA 15-3.


Assuntos
Neoplasias da Mama/genética , Quimiocina CXCL12/genética , Receptores CXCR4/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Quimiocina CXCL12/metabolismo , Feminino , Humanos , Receptores CXCR4/metabolismo , Transdução de Sinais
8.
J Vis Exp ; (159)2020 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-32538902

RESUMO

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.


Assuntos
Transferência Adotiva , Subpopulações de Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Movimento Celular , Receptores CXCR4/metabolismo , Retroviridae/metabolismo , Animais , Formação de Anticorpos , Linfócitos B/imunologia , Diferenciação Celular , Imunoglobulina M/imunologia , Antígenos Comuns de Leucócito , Camundongos , Receptores CXCR4/genética , Transdução de Sinais
9.
Anticancer Res ; 40(5): 2725-2737, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366418

RESUMO

BACKGROUND/AIM: Glioblastoma (GB) is the most aggressive type of tumor in the central nervous system and is characterized by resistance to therapy and abundant vasculature. Tumor vessels contribute to the growth of GB, and the tumor microenvironment is thought to influence tumor vessels. We evaluated the molecular communication between human GB cells and human brain microvascular endothelial cells (HBMEC) in vitro. MATERIALS AND METHODS: We investigated whether GB-conditioned media (GB-CM) influenced HBMEC proliferation and migration, as well as the levels of MMP-9, CXCL12, CXCR4, CXCR7, VEGFs, VEGFR-2, and WNT5a in HBMEC. RESULTS: Although HBMEC proliferation was not modified, increased HBMEC migration was detected after GB-CM treatment. Furthermore, treatment of HBMEC with GB-CM resulted in increased levels of MMP-9 and CXCR4. The levels of WNT5a, VEGFs and VEGFR-2 were not affected. CONCLUSION: GB-secreted factors lead to increased endothelial cell migration and to increased levels of MMP-9 and CXCR4.


Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Movimento Celular , Células Endoteliais/patologia , Glioblastoma/patologia , Metaloproteinase 9 da Matriz/metabolismo , Receptores CXCR4/metabolismo , Neoplasias Encefálicas/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo
10.
PLoS One ; 15(4): e0232536, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353075

RESUMO

Sperm migration towards an oocyte in the female reproductive tract is an important step for successful fertilization. Although several sperm-chemotactic factors have been identified in mammals, it is unclear whether these chemoattractants contribute to sperm migration towards an oocyte that is the final destination for sperm. Furthermore, chemoattractants for bovine sperm are still undiscovered even though the follicular fluid attracts sperm in cattle. Here, we demonstrated that a single bovine cumulus-oocyte complex (COC) had the ability to attract sperm, suggesting that the COC secreted sperm chemoattractants. We identified stromal cell-derived factor 1 (SDF1), which was expressed in COCs, and its receptor CXCR4 in sperm, as a candidate. Our results showed that bovine sperm preferentially migrated to the area with a high SDF1 concentration and occasionally showed turn movements by asymmetric flagellar bends during the migration. We also demonstrated that increasing the intracellular Ca2+ concentration via Ca2+ channels was related to SDF1-induced sperm chemotaxis. Finally, a CXCR4 inhibitor significantly suppressed the in vitro bovine sperm migration towards a COC. Taken together, we propose that SDF1 is a chemotactic factor for bovine sperm to regulate their migration towards an oocyte via the CXCR4 receptor.


Assuntos
Quimiocina CXCL12/metabolismo , Quimiotaxia/fisiologia , Receptores CXCR4/metabolismo , Motilidade Espermática/fisiologia , Animais , Bovinos , Células do Cúmulo/metabolismo , Feminino , Fertilização In Vitro/veterinária , Microscopia Intravital , Masculino , Oócitos/metabolismo , Espermatozoides/fisiologia
11.
PLoS Biol ; 18(4): e3000656, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271748

RESUMO

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here, we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide cross-linking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 ß1-strand, while also recapitulating earlier findings from nuclear magnetic resonance, modeling and crystallography of homologous receptors. A cross-linking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor targeting small molecules and biologics with novel pharmacology.


Assuntos
Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Animais , Sítios de Ligação , Western Blotting , Quimiocina CXCL12/genética , Cisteína/química , Cisteína/genética , Dissulfetos/química , Citometria de Fluxo , Células HEK293 , Humanos , Insetos/citologia , Modelos Moleculares , Mutação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores CXCR4/genética , beta-Arrestinas/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-32330088

RESUMO

Chlamydia pneumoniae infection could play a role in atherosclerosis. Toll-like receptor 2 (TLR2) and C-X-C motif chemokine receptor 4 (CXCR4) have been both shown to be involved in atherosclerosis. However, whether and how TLR2/CXCR4 cross talk is involved in C. pneumoniae infection-induced atherosclerosis remains to be determined. Our study aims to demonstrate that C. pneumoniae infection induced the cross talk between TLR2 and CXCR4 to mediate C. pneumoniae infection-induced vascular smooth muscle cell (VSMC) migration and even accelerate atherosclerosis. We first found that C. pneumoniae infection increased the aortic lesion size (en face), cross-sectional lesion area, and lipid content in aortic root lesion, which were both significantly reduced in apolipoprotein E-null (ApoE-/-)TLR2-/- or CXCR4-blocked ApoE-/- mice and were almost reversed in CXCR4-blocked ApoE-/-TLR2-/- mice. Subsequently, our data showed that C. pneumoniae infection-induced increases in VSMC contents in the atherosclerotic lesion were remarkably suppressed in ApoE-/-TLR2-/- mice or CXCR4-blocked ApoE-/- mice, and were further decreased in CXCR4-blocked ApoE-/-TLR2-/- mice. We then demonstrated that the increase in VSMC migratory capacity caused by C. pneumoniae infection was inhibited by either TLR2 or CXCR4 depletion, and downregulating both TLR2 and CXCR4 further decreased C. pneumoniae infection-induced VSMC migration by suppressing the infection-stimulated F-actin reorganization through the inhibition of the phosphorylation of focal adhesion kinase. Taken together, our data indicate that TLR2/CXCR4 coassociation facilitates C. pneumoniae infection-induced acceleration of atherosclerosis by inducing VSMC migration via focal adhesion kinase-mediated F-actin reorganization.NEW & NOTEWORTHY Toll-like receptor 2 (TLR2) and C-X-C motif chemokine receptor 4 (CXCR4) have both been shown to be involved in atherosclerosis. We demonstrate for the first time the presence of TLR2/CXCR4 coassociation during Chlamydia pneumoniae infection-induced atherosclerosis. Amazingly, blocking of both TLR2 and CXCR4 significantly retards and even almost reverses this infection-induced atherosclerosis. Our work reveals new mechanisms about C. pneumoniae infection-induced atherosclerosis and identifies potential new therapeutic targets for the prevention and treatment of atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Infecções por Chlamydophila/complicações , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores CXCR4/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Aterosclerose/microbiologia , Movimento Celular , Infecções por Chlamydophila/metabolismo , Infecções por Chlamydophila/microbiologia , Camundongos , Fosforilação
13.
Am J Respir Cell Mol Biol ; 63(2): 244-254, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32275835

RESUMO

Delayed lung repair leads to alveolopleural fistulae, which are a major cause of morbidity after lung resections. We have reported that intrapleural hypercapnia is associated with delayed lung repair after lung resection. Here, we provide new evidence that hypercapnia delays wound closure of both large airway and alveolar epithelial cell monolayers because of inhibition of epithelial cell migration. Cell migration and airway epithelial wound closure were dependent on Rac1-GTPase activation, which was suppressed by hypercapnia directly through the upregulation of AMP kinase and indirectly through inhibition of injury-induced NF-κB-mediated CXCL12 (pleural CXC motif chemokine 12) release, respectively. Both these pathways were independently suppressed, because dominant negative AMP kinase rescued the effects of hypercapnia on Rac1-GTPase in uninjured resting cells, whereas proteasomal inhibition reversed the NF-κB-mediated CXCL12 release during injury. Constitutive overexpression of Rac1-GTPase rescued the effects of hypercapnia on both pathways as well as on wound healing. Similarly, exogenous recombinant CXCL12 reversed the effects of hypercapnia through Rac1-GTPase activation by its receptor, CXCR4. Moreover, CXCL12 transgenic murine recipients of orthotopic tracheal transplantation were protected from hypercapnia-induced inhibition of tracheal epithelial cell migration and wound repair. In patients undergoing lobectomy, we found inverse correlation between intrapleural carbon dioxide and pleural CXCL12 levels as well as between CXCL12 levels and alveolopleural leak. Accordingly, we provide first evidence that high carbon dioxide levels impair lung repair by inhibiting epithelial cell migration through two distinct pathways, which can be restored by recombinant CXCL12.


Assuntos
Dióxido de Carbono/efeitos adversos , Lesão Pulmonar/fisiopatologia , Pulmão/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Feminino , Humanos , Hipercapnia/metabolismo , Pulmão/metabolismo , Lesão Pulmonar/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos
14.
Life Sci ; 249: 117534, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32156548

RESUMO

Tumors are dynamic tissue masses, so requiring continuous exposure to the host cells, nurturing them into pave a path for tumor growth and metastasis. C-X-C chemokine ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) is the key signaling for such aim. Gathering knowledge about the activity within this axis would deepen our insight into the utmost importance this signaling taken to attract and cross-connect multiple cells within the tumor microenvironment (TME) aiming for tumor progression and metastasis. The concept behind this review is to underscore the multi-tasking roles taken by CXCL12/CXCR4 signaling in tumor metastasis, and to also suggest some strategies to target the activities within this axis.


Assuntos
Quimiocina CXCL12/metabolismo , Metástase Neoplásica , Neoplasias/patologia , Receptores CXCR4/metabolismo , Microambiente Tumoral , Humanos , Neoplasias/metabolismo , Transdução de Sinais
15.
Arch Med Res ; 51(4): 297-302, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32169299

RESUMO

BACKGROUND: Potential agents that can effectively treat hepatocellular carcinoma (HCC) are being continuously explored. METHODS: Celastrol extracted from roots of an ancient Chinese herb, Tripterygium wilfordii (Thunder god vine), has been identified as a potential anti-tumor agent. In this study, the molecular mechanisms underlying the action of celastrol on cell proliferation and chemokine CXCR4-related signal pathway associated with tumor growth were investigated. RESULTS: The CXCR4 expression was diminished by celastrol treatment in a dose-dependent manner, and its downstream associated pathways, including PI3K and Akt were also downregulated. Celastrol also significantly attenuated proliferation and migration ability of HCC cells, and induced cell apoptosis in vitro. Additionally, significant inhibition of HCC growth was observed in the celastrol-treated group as compared with the control group in vivo as well. CONCLUSION: Celastrol is capable of attenuating cell proliferation and inducing apoptosis, leading to inhibition of HCC growth through the suppression of CXCR4-related signal pathway.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Receptores CXCR4/metabolismo , Triterpenos/uso terapêutico , Animais , Carcinoma Hepatocelular/patologia , Proliferação de Células , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Transdução de Sinais , Triterpenos/farmacologia
16.
Mol Carcinog ; 59(4): 390-398, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32037613

RESUMO

Brain metastasis is a leading cause of death worldwide, but the mechanism involved remains unclear. Stromal cell-derived factor-1 (SDF-1)/C-X-C motif chemokine receptor 4 (CXCR4) signaling has been reported to induce the directed metastasis of cancers, and adenosine A2A receptor activation suppresses the SDF-1/CXCR4 interaction. However, whether A2A receptor activation implicates the SDF-1/CXCR4 signaling pathway and thus modulates brain metastasis remains unclear. In this study, Western blot was performed to evaluate the protein levels. Cell invasion and migration assays were used to estimate the metastasis ability of PC-9 cells. The viability of cells was demonstrated by lactate dehydrogenase and cell proliferation assays. And the findings in vitro were further identified in nude mice. Notably, adenosine A2A receptor activation inhibited the proliferation and viability of PC-9 cells and thus suppressed the brain metastasis. A2A receptor stimulation protected the function of blood-brain barrier (BBB). The suppression of brain metastasis and the protection of BBB by A2A receptor relied on SDF-1/CXCR4 signaling, and treatment using A2A receptor agonist and CXCR4 antagonist protected the nude mice from malignancy metastasis in vivo. Adenosine A2A receptor activation suppressed the brain metastasis by implicating the SDF-1/CXCR4 axis and protecting the BBB.


Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/prevenção & controle , Quimiocina CXCL12/metabolismo , Neoplasias Pulmonares/prevenção & controle , Receptor A2A de Adenosina/metabolismo , Receptores CXCR4/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/secundário , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Humanos , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenetilaminas/farmacologia , Receptores CXCR4/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
17.
PLoS One ; 15(2): e0228674, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040489

RESUMO

In order to accomplish their physiological functions leukocytes have the capability to migrate. As a prerequisite they need to adopt a polarized cell shape, forming a leading edge at the front and a uropod at rear pole. In this study we explored the capability of chronic lymphocytic leukaemia (CLL) cells to adopt this leukocyte-specific migration phenotype. Furthermore, we studied the impact of the Toll-like receptor 9 (TLR9) agonists CpGs type A, B and C and the antagonist oligodesoxynucleotide (ODN) INH-18 on the cell polarization and migration process of primary human CLL cells. Upon cultivation, a portion of purified CLL cells adopted polarized cell shapes spontaneously (range 10-38%). Stimulation with CpG ODNs type B (ODN 2006) and CpGs type C (ODN 2395) significantly increased the frequency of morphologically polarized CLL cells, while ODN INH-18 was hardly able to act antagonistically. Like in human hematopoietic stem and progenitor cells, in morphologically polarized CLL cells CXCR4 was redistributed to the leading edge and CD50 to the uropod. Coupled to the increased frequencies of morphologically polarized cells, CpGs type B and C stimulated CLL cells showed higher migration activities in vitro and following intravenous injection higher homing frequencies to the bone marrow of immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Thus, presumably independent of TLR-9 signaling, CpGs type B and C promote the cellular polarization process of CLL cells and their ability to migrate in vitro and in vivo.


Assuntos
Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Oligodesoxirribonucleotídeos/farmacologia , Animais , Células da Medula Óssea/citologia , Membrana Celular/metabolismo , Movimento Celular , Proliferação de Células , Forma Celular , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Transplante de Neoplasias , Receptores CXCR4/metabolismo , Receptor Toll-Like 9/metabolismo
18.
J Leukoc Biol ; 107(4): 635-647, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32057138

RESUMO

Neutropenia and impaired functions were common manifestation in antiretroviral therapy (ART) in both naïve and experienced PLWHA. Granulopoiesis can be divided into two phases: lineage determination and committed granulopoiesis. However, stage-specific impairment of granulopoiesis in PLWHA with neutropenia remains unclear. A total of 48 ART-naïve and 49 ART-experienced PLWHA from 2016 to 2018 were recruited and divided into non-, mild-, and moderate-to-severe-neutropenia groups according to their neutrophil counts. The bone marrow aspirates and peripheral blood were collected and analyzed by multicolor flow cytometry for granulocyte subsets, hematopoietic stem/progenitor cells (HSPC), apoptosis, and emigration and retention of different subsets. Compared with healthy donors, the percentages of circulating segmented neutrophils were significantly decreased along with an increase of immature neutrophils in both groups. ART-naïve patients with moderate to severe neutropenia exhibited decreased proportion and accelerated apoptosis of relative mature segmented neutrophils. In contrast, ART-experienced patients with neutropenia displayed decreased proportion of granulocyte macrophage progenitors, indicating a defect at a stage of lineage determination. Meanwhile, ART-experienced patients with neutropenia also the expression of CXCR4 segmented neutrophils, suggesting an increased retention of segmented neutrophils inn the bone marrow. ART-naïve patients with neutropenia is caused by increased apoptosis of relatively differentiated neutrophils at committed granulopoiesis, whereas impaired lineage determination and enhanced retention of segmented neutrophils contribute to in ART-experienced patients.


Assuntos
Síndrome de Imunodeficiência Adquirida/patologia , Granulócitos/patologia , Neutropenia/patologia , Síndrome de Imunodeficiência Adquirida/sangue , Adulto , Medula Óssea/patologia , Feminino , Células-Tronco Hematopoéticas/patologia , Humanos , Masculino , Neutropenia/sangue , Neutrófilos/patologia , Receptores CXCR4/metabolismo , Receptores de Interleucina-8B/metabolismo
19.
Nat Cell Biol ; 22(3): 266-273, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32042179

RESUMO

Chemoattractant gradients frequently guide migrating cells. To achieve the most directional signal, such gradients should be maintained with concentrations around the dissociation constant (Kd)1-6 of the chemoreceptor. Whether this actually occurs in animals is unknown. Here we investigate whether a moving tissue, the zebrafish posterior lateral line primordium, buffers its attractant in this concentration range to achieve robust migration. We find that the Cxcl12 (also known as Sdf1) attractant gradient ranges from 0 to 12 nM, values similar to the 3.4 nM Kd of its receptor Cxcr4. When we increase the Kd of Cxcl12 for Cxcr4, primordium migration is less directional. Furthermore, a negative-feedback loop between Cxcl12 and its clearance receptor Ackr3 (also known as Cxcr7) regulates the Cxcl12 concentrations. Breaking this negative feedback by blocking the phosphorylation of the cytoplasmic tail of Ackr3 also results in less directional primordium migration. Thus, directed migration of the primordium is dependent on a close match between the Cxcl12 concentration and the Kd of Cxcl12 for Cxcr4, which is maintained by buffering of the chemokine levels. Quantitative modelling confirms the plausibility of this mechanism. We anticipate that buffering of attractant concentration is a general mechanism for ensuring robust cell migration.


Assuntos
Movimento Celular , Quimiocinas/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Quimiocina CXCL12/metabolismo , Retroalimentação Fisiológica , Humanos , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
20.
Am J Physiol Renal Physiol ; 318(3): F741-F753, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32068458

RESUMO

Glomerular parietal epithelial cell (PEC) activation, as revealed by de novo expression of CD44 and cell migration toward the injured filtration barrier, is a hallmark of podocyte injury-driven focal segmental glomerulosclerosis (FSGS). However, the signaling pathway that mediates activation of PECs in response to podocyte injury is unknown. The present study focused on CD44 signaling, particularly the roles of two CD44-related chemokines, migration inhibitory factor (MIF) and stromal cell-derived factor 1 (SDF1), and their common receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), in the NEP25/LMB2 mouse podocyte-toxin model of FSGS. In the early phase of the disease, CD44-positive PECs were locally evident on the opposite side of the intact glomerular tuft and subsequently increased in the vicinity of synechiae with podocyte loss. Expression of MIF and SDF1 was first increased in injured podocytes and subsequently transferred to activated PECs expressing CD44 and CXCR4. In an immortalized mouse PEC (mPEC) line, recombinant MIF and SDF1 (rMIF and rSDF1, respectively) individually increased CD44 and CXCR4 mRNA and protein levels. rMIF and rSDF1 stimulated endogenous MIF and SDF1 production. rMIF- and rSDF1-induced mPEC migration was suppressed by CD44 siRNA. However, MIF and SDF1 inhibitors failed to show any impact on proteinuria, podocyte number, and CD44 expression in NEP25/LMB2 mice. Our data suggest that injured podocytes upregulate MIF and SDF1 that stimulate CD44 expression and CD44-mediated migration, which is enhanced by endogenous MIF and SDF1 in PECs. This biphasic expression pattern of the chemokine-CD44 axis in podocytes and PECs may be a novel mechanism of "podocyte-PEC cross-talk" signaling underlying podocyte injury-driven FSGS.


Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Quimiocina CXCL12/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Receptores de Hialuronatos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Podócitos/fisiologia , Receptores CXCR4/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Cápsula Glomerular , Movimento Celular , Células Cultivadas , Quimiocina CXCL12/genética , Antígenos de Histocompatibilidade Classe II/genética , Receptores de Hialuronatos/genética , Camundongos , Camundongos Endogâmicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Interferência de RNA , Receptores CXCR4/genética , Regulação para Cima
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA