RESUMO
The Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-standing questions about this pathway include how the dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells and how the resistance-nodulation-division transporter Dispatched 1 (Disp, also known as Disp1) regulates this process. Here, we show that inactivation of Disp in Shh-expressing human cells impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. We also show that cholesterol export from Disp-deficient cells is reduced, that these cells contain increased cholesterol amounts in the plasma membrane, and that Shh shedding from Disp-deficient cells is restored by pharmacological membrane cholesterol extraction and by overexpression of transgenic Disp or the structurally related protein Patched 1 (Ptc, also known as Ptch1; a putative cholesterol transporter). These data suggest that Disp can regulate Shh function via controlled cell surface shedding and that membrane cholesterol-related molecular mechanisms shared by Disp and Ptc exercise such sheddase control.
Assuntos
Membrana Celular , Colesterol , Proteínas Hedgehog , Proteínas de Membrana Transportadoras/genética , Células Cultivadas , Proteínas Hedgehog/genética , Humanos , Ligantes , Transdução de SinaisRESUMO
SIGNIFICANCE STATEMENT: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies. BACKGROUND: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. METHODS: To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7. RESULTS: Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. CONCLUSIONS: Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.
Assuntos
Glomérulos Renais , Podócitos , Animais , Camundongos , Humanos , Glomérulos Renais/patologia , Podócitos/metabolismo , Endossomos , Drosophila , Rim , MamíferosRESUMO
Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air-liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Fenamatos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Células A549 , Adulto , Animais , COVID-19/metabolismo , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Citocinas/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Routine manipulation of the mouse genome has become a landmark in biomedical research. Traits that are only associated with advanced developmental stages can now be investigated within a living organism, and the in vivo analysis of corresponding phenotypes and functions advances the translation into the clinical setting. The annexins, a family of closely related calcium (Ca2+)- and lipid-binding proteins, are found at various intra- and extracellular locations, and interact with a broad range of membrane lipids and proteins. Their impacts on cellular functions has been extensively assessed in vitro, yet annexin-deficient mouse models generally develop normally and do not display obvious phenotypes. Only in recent years, studies examining genetically modified annexin mouse models which were exposed to stress conditions mimicking human disease often revealed striking phenotypes. This review is the first comprehensive overview of annexin-related research using animal models and their exciting future use for relevant issues in biology and experimental medicine.
Assuntos
Anexina A1/metabolismo , Lipídeos/química , Pesquisa Translacional Biomédica , Animais , Anexina A2/metabolismo , Anexina A5/metabolismo , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Cálcio/química , Membrana Celular/metabolismo , Diabetes Mellitus/metabolismo , Progressão da Doença , Homeostase , Camundongos , Camundongos Knockout , Nanotecnologia , Neoplasias/metabolismo , Neovascularização Patológica , Peptídeos/química , Fenótipo , Ligação Proteica , Transporte ProteicoRESUMO
Staphylococcus aureus internalization by non-professional phagocytes is considered a main pathogenicity mechanism leading to chronic infections. The well-established mechanism of Staphylococcus aureus internalization is mediated by fibronectin (Fn)-binding proteins (FnBPs), Fn as a bridging molecule and the host cell α5ß1 integrin. We previously identified a novel alternative internalization mechanism in Staphylococcus aureus, which involves the major autolysin Atl and the host cell heat shock cognate protein 70 (Hsc70). Atl-dependent internalization is also employed by the coagulase-negative Staphylococcus epidermidis, where it might represent the major or even sole internalization mechanism, because of the lack of FnBP-homologous proteins. In this study, we aimed to further characterize the Atl-dependent staphylococcal internalization mechanism. We performed biomolecular interaction analysis (BIA) to quantify the adhesive properties of Atl and found multivalent and high affinity interactions of Atl with Fn and Hsc70. Confocal laser scanning microscopy (CLSM) and a flow-cytometric internalization assay in combination with different pharmacological inhibitors suggested an involvement of the α5ß1 integrin, Fn and Hsc70 and subsequent signaling events mediated by Src and phosphoinositide 3 (PI3) kinases in the Atl-dependent staphylococcal uptake by EA.hy 926 cells. Further characterization of the endocytic machinery implicated a role for clathrin-dependent receptor-mediated endocytosis involving actin cytoskeletal rearrangements and microtubules. In conclusion, Atl ubiquitous among staphylococcal species may substitute for the FnBPs ensuring low-level internalization via a mechanism that seems to share important features with the FnBP-mediated staphylococcal uptake potentially being the prerequisite for the development of therapy-resistant chronic infections by staphylococcal strains that lack FnBPs.
Assuntos
Adesinas Bacterianas , Proteínas de Bactérias , Fagócitos/microbiologia , Staphylococcus aureus/patogenicidade , Proteínas de Choque Térmico HSC70 , Humanos , N-Acetil-Muramil-L-Alanina AmidaseRESUMO
Pattern recognition receptors (PRRs) are key elements in the innate immune response. Formyl peptide receptor (FPR) 2 is a PRR that, in addition to proinflammatory, pathogen-derived compounds, also recognizes the anti-inflammatory endogenous ligand annexin A1 (AnxA1). Because the contribution of this signaling axis in viral infections is undefined, we investigated AnxA1-mediated FPR2 activation on influenza A virus (IAV) infection in the murine model. AnxA1-treated mice displayed significantly attenuated pathology upon a subsequent IAV infection with significantly improved survival, impaired viral replication in the respiratory tract, and less severe lung damage. The AnxA1-mediated protection against IAV infection was not caused by priming of the type I IFN response but was associated with an increase in the number of alveolar macrophages (AMs) and enhanced pulmonary expression of the AM-regulating cytokine granulocyte-M-CSF (GM-CSF). Both AnxA1-mediated increase in AM levels and GM-CSF production were abrogated when mouse (m)FPR2 signaling was antagonized but remained up-regulated in mice genetically deleted for mFPR1, an mFPR2 isoform also serving as AnxA1 receptor. Our results indicate a novel protective function of the AnxA1-FPR2 signaling axis in IAV pathology via GM-CSF-associated maintenance of AMs, expanding knowledge on the potential use of proresolving mediators in host defense against pathogens.-Schloer, S., Hübel, N., Masemann, D., Pajonczyk, D., Brunotte, L., Ehrhardt, C., Brandenburg, L.-O., Ludwig, S., Gerke, V., Rescher, U. The annexin A1/FPR2 signaling axis expands alveolar macrophages, limits viral replication, and attenuates pathogenesis in the murine influenza A virus infection model.
Assuntos
Anexina A1/fisiologia , Vírus da Influenza A/fisiologia , Macrófagos Alveolares/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Receptores de Formil Peptídeo/fisiologia , Replicação Viral , Animais , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Vírus da Influenza A/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
Phosphoinositides (PI) and converting enzymes are crucial determinants of organelle identity and morphology. One important endolysosomal specific PI is PI(3,5)P2, generated by the PIKfyve kinase, which orchestrates in combination with Vac14 and Fig4. Dysfunction of this complex leads to large intracellular vacuoles in various cell types and is linked to neurological diseases. Here, we characterize the vacuolization phenotype caused by overexpression of the PIKfyve binding deficient mutant Vac14L156R in podocytes, which represent specialized cells of the kidney. Vacuolization of podocytes, which was associated with strong maturation defects in the endolysosomal system, could be completely rescued by starvation or treatment of cells with the v-ATPase inhibitor Bafilomycin A1. Moreover, we elucidated a strong and reversible de-vacuolization effect of the cholesterol export inhibitor U18666A, which was accompanied by increased basification of the lysosomal pH values. Taken together, our data give new hints to potential therapeutic targets in the treatment of disease linked to intracellular vacuolization.
Assuntos
Meios de Cultura Livres de Soro/farmacologia , Inibidores Enzimáticos/farmacologia , Macrolídeos/farmacologia , Proteínas de Membrana/genética , Podócitos/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Vacúolos/genética , Substituição de Aminoácidos/genética , Células Cultivadas , Alimentos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Podócitos/metabolismo , Podócitos/ultraestrutura , Regulação para Cima/genética , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidoresRESUMO
The vertebrate annexin superfamily (AnxA) consists of 12 members of a calcium (Ca2+) and phospholipid binding protein family which share a high structural homology. In keeping with this hallmark feature, annexins have been implicated in the Ca2+-controlled regulation of a broad range of membrane events. In this review, we identify and discuss several themes of annexin actions that hold a potential therapeutic value, namely, the regulation of the immune response and the control of tissue homeostasis, and that repeatedly surface in the annexin activity profile. Our aim is to identify and discuss those annexin properties which might be exploited from a translational science and specifically, a clinical point of view.
Assuntos
Anexinas/metabolismo , Carcinogênese/metabolismo , Doenças Transmissíveis/metabolismo , Pesquisa Translacional Biomédica , Animais , Biomarcadores/metabolismo , Doenças Transmissíveis/diagnóstico , Interações Hospedeiro-Patógeno , HumanosRESUMO
Emerging infectious diseases and drug-resistant infectious agents call for the development of innovative antimicrobial strategies. With pathogenicity now considered to arise from the complex and bi-directional interplay between a microbe and the host, host cell factor targeting has emerged as a promising approach that might overcome the limitations of classical antimicrobial drug development and could open up novel and efficient therapeutic strategies. Interaction with and modulation of host cell membranes is a recurrent theme in the host-microbe relationship. In this review, we provide an overview of what is currently known about the role of the Ca2+ dependent, membrane-binding annexin protein family in pathogen-host interactions, and discuss their emerging functions as host cell derived auxiliary proteins in microbe-host interactions and host cell targets.
Assuntos
Anexinas/metabolismo , Interações Hospedeiro-Patógeno , Animais , Humanos , Microbiologia , Terapia de Alvo MolecularRESUMO
Annexin A10 is the latest identified member of the annexin family of Ca(2+)- and phospholipid-binding proteins. In previous studies, downregulation of annexin A10 was correlated with dedifferentiation, invasion, and tumor progression, pointing to a possible tumor suppressor role. However, the biochemical characteristics and functions of annexin A10 remain unknown. We show that annexin A10 displays biochemical characteristics atypical for an annexin, indicating a Ca(2+)- and membrane-binding-independent function. Annexin A10 co-localizes with the mRNA-binding proteins SFPQ and PSPC1 at paraspeckles, an only recently discovered nuclear body, and decreases paraspeckle numbers when overexpressed in HeLa cells. In addition, annexin A10 relocates to dark perinucleolar caps upon transcriptional inhibition of RNA polymerase II. We mapped the cap-binding function of annexin A10 to the proximal part of the core domain, which is missing in the short isoform of annexin A10, and show its independence from the remaining functional type II Ca(2+)-binding site. In contrast to this, paraspeckle recruitment required additional core regions and was negatively affected by the mutation of the last type II Ca(2+)-binding site. Additionally, we show that overexpression of annexin A10 in HeLa cells increases their sensitivity to apoptosis and reduces colony formation. The identification of unique nuclear and biochemical characteristics of annexin A10 points towards its membrane-independent role in paraspeckle-associated mRNA regulation or processing.
Assuntos
Anexinas/metabolismo , Núcleo Celular/metabolismo , Animais , Anexinas/análise , Anexinas/genética , Antibióticos Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Núcleo Celular/ultraestrutura , Cães , Doxorrubicina/toxicidade , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Proteínas Nucleares/metabolismo , Fator de Processamento Associado a PTB , Isoformas de Proteínas/metabolismo , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Endocytosis of activated growth factor receptors regulates spatio-temporal cellular signaling. In the case of the EGF receptor, sorting into multivesicular bodies (MVBs) controls signal termination and subsequently leads to receptor degradation in lysosomes. Annexin A1, a Ca(2+)-regulated membrane binding protein often deregulated in human cancers, interacts with the EGF receptor and is phosphorylated by internalized EGF receptor on endosomes. Most relevant for EGF receptor signal termination, annexin A1 is required for the formation of internal vesicles in MVBs that sequester ligand-bound EGF receptor away from the limiting membrane. To elucidate the mechanism underlying annexin A1-dependent EGF receptor trafficking we employed an N-terminally truncated annexin A1 mutant that lacks the EGF receptor phosphorylation site and the site for interaction with its protein ligand S100A11. Overexpression of this dominant-negative mutant induces a delay in EGF-induced EGF receptor transport to the LAMP1-positive late endosomal/lysosomal compartment and impairs ligand-induced EGF receptor degradation. Consistent with these findings, EGF-stimulated EGF receptor and MAP kinase pathway signaling is prolonged. Importantly, depletion of S100A11 also results in a delayed EGF receptor transport and prolonged MAP kinase signaling comparable to the trafficking defect observed in cells expressing the N-terminally truncated annexin A1 mutant. These results strongly suggest that the function of annexin A1 as a regulator of EGF receptor trafficking, degradation and signaling is critically mediated through an N-terminal interaction with S100A11 in the endosomal compartment. This interaction appears to be essential for lysosomal targeting of the EGF receptor, possibly by providing a physical scaffold supporting inward vesiculation in MVBs. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.
Assuntos
Anexina A1/metabolismo , Movimento Celular , Proliferação de Células , Receptores ErbB/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas S100/metabolismo , Anexina A1/antagonistas & inibidores , Anexina A1/genética , Compartimento Celular , Ensaio de Unidades Formadoras de Colônias , Endocitose/fisiologia , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Células HeLa , Humanos , Técnicas Imunoenzimáticas , Proteínas de Membrana Lisossomal/genética , Lisossomos/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Transporte Proteico , Proteínas S100/antagonistas & inibidores , Proteínas S100/genética , Ressonância de Plasmônio de SuperfícieRESUMO
Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3-O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of ß-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Sulfotransferases/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Caderinas/genética , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/genética , Invasividade Neoplásica , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Sulfotransferases/genética , Fator de Transcrição 4 , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Fatores de Transcrição/genética , Células Tumorais Cultivadas , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca2+ levels. Through this they act as Ca2+-regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca2+-regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.
Assuntos
Anexinas , Paladar , Animais , Anexinas/química , Membrana Celular/metabolismo , Transdução de Sinais , Transporte Biológico , Cálcio/metabolismo , Mamíferos/metabolismoAssuntos
Anexinas/metabolismo , Colesterol/metabolismo , Bicamadas Lipídicas/metabolismo , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , LDL-Colesterol/metabolismo , Endossomos/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Membranas Artificiais , Proteína C1 de Niemann-Pick , Fosfatidilserinas/metabolismo , Ligação ProteicaRESUMO
In late 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of coronavirus disease 2019 (COVID-19) emerged in China and spread rapidly around the world, causing an ongoing pandemic of global concern. COVID-19 proceeds with moderate symptoms in most patients, whereas others experience serious respiratory illness that requires intensive care treatment and may end in death. The severity of COVID-19 is linked to several risk factors including male sex, comorbidities, and advanced age. Apart from respiratory complications, further impairments by COVID-19 affecting other tissues of the human body are observed. In this respect, the human kidney is one of the most frequently affected extrapulmonary organs and acute kidney injury (AKI) is known as a direct or indirect complication of SARS-CoV-2 infection. The aim of this work was to investigate the importance of the protein angiotensin-converting enzyme 2 (ACE2) for a possible cell entry of SARS-CoV-2 into human kidney cells. First, the expression of the cellular receptor ACE2 was demonstrated to be decisive for viral SARS-CoV-2 cell entry in human AB8 podocytes, whereas the presence of the transmembrane protease serine 2 (TMPRSS2) was dispensable. Moreover, the ACE2 protein amount was well detectable by mass spectrometry analysis in human kidneys, while TMPRSS2 could be detected only in a few samples. Additionally, a negative correlation of the ACE2 protein abundance to male sex and elderly aged females in human kidney tissues was demonstrated in this work. Last, the possibility of a direct infection of kidney tubular renal structures by SARS-CoV-2 was demonstrated.
Assuntos
COVID-19 , Idoso , Feminino , Humanos , Masculino , Enzima de Conversão de Angiotensina 2 , Rim/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2/metabolismoRESUMO
Upon proinflammatory challenges, endothelial cell surface presentation of the leukocyte receptor P-selectin, together with the stabilizing co-factor CD63, is needed for leukocyte capture and is mediated via demand-driven exocytosis from the Weibel-Palade bodies that fuse with the plasma membrane. We report that neutrophil recruitment to activated endothelium is significantly reduced in mice deficient for the endolysosomal cation channel TPC2 and in human primary endothelial cells with pharmacological TPC2 block. We observe less CD63 signal in whole-mount stainings of proinflammatory-activated cremaster muscles from TPC2 knockout mice. We find that TPC2 is activated and needed to ensure the transfer of CD63 from endolysosomes via Weibel-Palade bodies to the plasma membrane to retain P-selectin on the cell surface of human primary endothelial cells. Our findings establish TPC2 as a key element to leukocyte interaction with the endothelium and a potential pharmacological target in the control of inflammatory leukocyte recruitment.
Assuntos
Selectina-P , Canais de Dois Poros , Camundongos , Humanos , Animais , Selectina-P/metabolismo , Células Endoteliais/metabolismo , Corpos de Weibel-Palade/metabolismo , Adesão Celular , Leucócitos/metabolismo , Endotélio Vascular/metabolismoRESUMO
SARS-CoV-2 is the causative agent of the immune response-driven disease COVID-19 for which new antiviral and anti-inflammatory treatments are urgently needed to reduce recovery time, risk of death and long COVID development. Here, we demonstrate that the immunoregulatory kinase p38 MAPK is activated during viral entry, mediated by the viral spike protein, and drives the harmful virus-induced inflammatory responses. Using primary human lung explants and lung epithelial organoids, we demonstrate that targeting p38 signal transduction with the selective and clinically pre-evaluated inhibitors PH-797804 and VX-702 markedly reduced the expression of the pro-inflammatory cytokines IL6, CXCL8, CXCL10 and TNF-α during infection, while viral replication and the interferon-mediated antiviral response of the lung epithelial barrier were largely maintained. Furthermore, our results reveal a high level of drug synergism of both p38 inhibitors in co-treatments with the nucleoside analogs Remdesivir and Molnupiravir to suppress viral replication of the SARS-CoV-2 variants of concern, revealing an exciting and novel mode of synergistic action of p38 inhibition. These results open new avenues for the improvement of the current treatment strategies for COVID-19.
Assuntos
Antivirais , COVID-19 , Inflamação , Síndrome de COVID-19 Pós-Aguda , SARS-CoV-2 , Proteínas Quinases p38 Ativadas por Mitógeno , Humanos , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/complicações , Inflamação/tratamento farmacológico , Inflamação/virologia , Pulmão , Transdução de SinaisRESUMO
Viral myocarditis is pathologically associated with RNA viruses such as coxsackievirus B3 (CVB3), or more recently, with SARS-CoV-2, but despite intensive research, clinically proven treatment is limited. Here, by use of a transgenic mouse strain (TG) containing a CVB3ΔVP0 genome we unravel virus-mediated cardiac pathophysiological processes in vivo and in vitro. Cardiac function, pathologic ECG alterations, calcium homeostasis, intracellular organization and gene expression were significantly altered in transgenic mice. A marked alteration of mitochondrial structure and gene expression indicates mitochondrial impairment potentially contributing to cardiac contractile dysfunction. An extended picture on viral myocarditis emerges that may help to develop new treatment strategies and to counter cardiac failure.
Assuntos
COVID-19 , Infecções por Coxsackievirus , Miocardite , Viroses , Camundongos , Animais , Camundongos Transgênicos , Enterovirus Humano B , SARS-CoV-2RESUMO
microRNAs are small endogenous noncoding RNAs, which post-transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR-10b and syndecan-1, MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. miR-10b overexpression induced post-transcriptional downregulation of syndecan-1, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and 3'UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan-1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1-deficient cells revealed upregulation of ATF-2, COX-2, cadherin-11, vinculin, actin γ 2, MYL9, transgelin-1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, cyclin-dependent kinase 6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase-dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.
Assuntos
Neoplasias da Mama/patologia , Caderinas/fisiologia , Movimento Celular , MicroRNAs/fisiologia , Sindecana-1/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/fisiologia , Fator 2 Ativador da Transcrição/genética , Comunicação Celular , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Progressão da Doença , Feminino , Humanos , Invasividade Neoplásica , Fosforilação , RNA Mensageiro/análise , RNA Interferente Pequeno/genética , Sindecana-1/fisiologiaRESUMO
Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake.