RESUMEN
Tumor-derived G-CSF is a well-known factor to aggravate disease progression in various types of cancers. In this study, we investigated a role of G-CSF in squamous cell carcinoma (SCC). High expression of G-CSF in the tumor tissues of esophageal SCC (ESCC) patients correlated with poor prognosis. Murine SCC NR-S1M cells produce considerable amount of G-CSF, which expression is correlated with its metastatic potentials. Deletion of G-CSF in NR-S1M cells mitigated tumor growth and metastasis to lymph node and lung of subcutaneous NR-S1M tumors in the mice. Mechanistically, G-CSF enhanced cell proliferation in autocrine manner in vitro, whereas in NR-S1M tumor-bearing mice, accumulation of plasma G-CSF was associated with expansion of peripheral neutrophils, which led to a decreased proportion of CD8+ T cells. Antibody depletion of neutrophils restored the number of CD8+ T cells and modestly suppressed tumor outgrowth, albeit no changes in distant metastasis. We propose that G-CSF produced by NR-S1M cells facilitates tumor progression in mice through bi-functional effects to promote neutrophil recruitment and tumor cell proliferation, which may render poor prognosis to the ESCC patients with high G-CSF expression.
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Carcinoma de Células Escamosas , Neoplasias Esofágicas , Animales , Ratones , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Infiltración Neutrófila , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas/genética , Proliferación Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
Esophageal squamous cell carcinoma (ESCC) is one of the most fatal types of malignant tumors worldwide. Epitranscriptome, such as N6 -methyladenosine (m6 A) of mRNA, is an abundant post-transcriptional mRNA modification and has been recently implicated to play roles in several cancers, whereas the significance of m6 A modifications is virtually unknown in ESCC. Analysis of tissue microarray of the tumors in 177 ESCC patients showed that higher expression of m6 A demethylase ALKBH5 correlated with poor prognosis and that ALKBH5 was an independent prognostic factor of the survival of patients. There was no correlation between the other demethylase FTO and prognosis. siRNA knockdown of ALKBH5 but not FTO significantly suppressed proliferation and migration of human ESCC cells. ALKBH5 knockdown delayed progression of cell cycle and accumulated the cells to G0/G1 phase. Mechanistically, expression of CDKN1A (p21) was significantly up-regulated in ALKBH5-depleted cells, and m6 A modification and stability of CDKN1A mRNA were increased by ALKBH5 knockdown. Furthermore, depletion of ALKBH5 substantially suppressed tumor growth of ESCC cells subcutaneously transplanted in BALB/c nude mice. Collectively, we identify ALKBH5 as the first m6 A demethylase that accelerates cell cycle progression and promotes cell proliferation of ESCC cells, which is associated with poor prognosis of ESCC patients.
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Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Carcinoma de Células Escamosas de Esófago/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Adulto , Anciano , Enzimas AlkB/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Progresión de la Enfermedad , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Femenino , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Pronóstico , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Shortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [Cnot7/8-double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression (Cnot6/6l-dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in Cnot7/8-dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in Cnot6/6l-dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in Cnot7/8-dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Importantly, Cnot6/6l-dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not.
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Exorribonucleasas/metabolismo , ARN Mensajero/metabolismo , Receptores CCR4/metabolismo , Proteínas Represoras/metabolismo , Animales , Supervivencia Celular/genética , Exorribonucleasas/genética , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Masculino , Ratones Noqueados , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Poli A/genética , Poli A/metabolismo , Subunidades de Proteína , Estabilidad del ARN , ARN Mensajero/genética , Receptores CCR4/genética , Proteínas Represoras/genéticaRESUMEN
Malforminâ A1 (MA1) is a fungus-produced cyclic pentapeptide. MA1 exhibits teratogenicity to plants, fibrinolysis-enhancing activity, and cytotoxicity to mammalian cells. To clarify the cytotoxic mechanism of MA1, we screened for the genes involved in the cytotoxicity of MA1 in monocytoid U937 cells by using a CRISPR/Cas9-based genome-wide knockout library. Screening was performed by positive selection for cells that were resistant to MA1 treatment, and single guide RNAs (sgRNAs) integrated into MA1-resistant cells were analyzed by high-throughput sequencing. As a result of the evaluation of sgRNAs that were enriched in MA1-resistant cells, SQLE, which encodes squalene epoxidase, was identified as a candidate gene. SQLE-depleted U937 cells were viable in the presence of MA1, and squalene epoxidase inhibitor conferred MA1 resistance to wild-type cells. These results indicate that squalene epoxidase is implicated in the cytotoxicity of MA1. This finding represents a new insight into applications of MA1 for treating ischemic diseases.
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Péptidos Cíclicos/farmacología , Escualeno-Monooxigenasa/genética , Aspergillus niger/metabolismo , Sistemas CRISPR-Cas , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Humanos , ARN Guía de Kinetoplastida/genética , Escualeno-Monooxigenasa/metabolismo , Células U937RESUMEN
Shortening of poly(A) tails triggers mRNA degradation; hence, mRNA deadenylation regulates many biological events. In the present study, we generated mice lacking the Cnot1 gene, which encodes an essential scaffold subunit of the CCR4-NOT deadenylase complex in adipose tissues (Cnot1-AKO mice) and we examined the role of CCR4-NOT in adipocyte function. Cnot1-AKO mice showed reduced masses of white adipose tissue (WAT) and brown adipose tissue (BAT), indicating abnormal organization and function of those tissues. Indeed, Cnot1-AKO mice showed hyperinsulinemia, hyperglycemia, insulin resistance, and glucose intolerance and they could not maintain a normal body temperature during cold exposure. Muscle-like fibrous material appeared in both WAT and BAT of Cnot1-AKO mice, suggesting the acquisition of non-adipose tissue characteristics. Gene expression analysis using RNA-sequencing (RNA-seq) showed that the levels of adipose tissue-related mRNAs, including those of metabolic genes, decreased, whereas the levels of inflammatory response-related mRNAs increased. These data suggest that the CCR4-NOT complex ensures proper adipose tissue function by maintaining adipocyte-specific mRNAs at appropriate levels and by simultaneously suppressing mRNAs that would impair adipocyte function if overexpressed.
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Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Factores de Transcripción/genética , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo Pardo/patología , Tejido Adiposo Blanco/patología , Animales , Temperatura Corporal , Células Cultivadas , Regulación de la Expresión Génica , Hiperglucemia/etiología , Hiperglucemia/metabolismo , Hiperinsulinismo/etiología , Hiperinsulinismo/metabolismo , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/metabolismo , Factores de Transcripción/deficienciaRESUMEN
The initial defense against invading pathogenic microbes is the activation of innate immunity by binding of pattern recognition receptors (PRRs) to pathogen associated molecular patterns (PAMPs). To explain the action of PRRs from hagfish, one of the extant jawless vertebrates, we purified the GlcNAc recognition complex (GRC) from serum using GlcNAc-agarose. The GRC comprises four proteins of varying molecular masses: 19 kDa, 26 kDa, 27 kDa, and 31 kDa. Exposure of Escherichia coli to the GRC led to the phagocytic activation of macrophages, revealing the opsonic function of the GRC. The GRC in serum formed a large complex with a molecular mass of approximately 1200 kDa. The GRC bound to Escherichia coli but not to rabbit red blood cells, despite both having GlcNAc on their surface. These structural and binding properties are similar to those of mannose-binding lectin (MBL). The amino acid sequence of a portion of the 31 kDa protein in the GRC matched the amino acid sequence of variable lymphocyte receptor (VLR)-B in some place. According to the Western blot analysis, the 31 kDa protein was recognized by the anti-hagfish VLR-B antiserum. Based on the results, it appears that the GRC functions as a PRR like MBL and that its 31 kDa protein has a structure similar to that of VLR-B.
Asunto(s)
Anguila Babosa , Animales , Conejos , Secuencia de Aminoácidos , Receptores de Reconocimiento de Patrones , Linfocitos , Anticuerpos , Escherichia coliRESUMEN
OBJECTIVES: Residual dissection is a concern in endovascular treatment with a DCB, and there is limited knowledge of hemodynamics at a dissection lesion. Therefore, the objective of this study is to evaluate the mean pressure gradient (MPG) and fractional flow reserve (FFR) at a residual dissection after DCB angioplasty for the superficial femoral artery (SFA). METHODS: A total of 59 cases with residual SFA dissection treated with DCB angioplasty at a single center were analyzed retrospectively. The dissection was classified into 6 types (A-F). The primary endpoints were MPG and FFR at a residual dissection lesion after DCB angioplasty, using evaluation with a pressure wire. RESULTS: The median lesion length was 70 (40-130) mm with 24% popliteal involvement, and 11 cases (18%) had chronic total occlusion. A completion angiogram revealed dissection of types A (n = 33, 56%), B (n = 18, 31%), C (n = 7, 12%), and D (n = 1, 2%). The median MPGs in type A, B, and C cases were 0 (0-2), 0 (0-4), and 3 (0-6) mmHg, with a significant lower in type C cases than in type A cases (A vs C, P = .021). The median FFRs in type A, B, and C cases of 1.0 (.98-1.00), 1.0 (.96-1.00), and .98 (.95-1.00) did not differ significantly among dissection types (A vs B, P = .86; A vs C, P = .055; B vs C, P = .15). CONCLUSIONS: This is the first report of hemodynamics at a SFA dissection. The results suggest that low-grade dissection (types A or B) does not affect MPG and FFR at a SFA lesion. This indicates that a bailout stent may be unnecessary for patients with dissection of types A or B. A further investigation is needed to determine whether a scaffold is required for a SFA lesion with type C dissection.
RESUMEN
Background: Acute lung injury (ALI) is caused by a variety of illnesses, including aspiration pneumonia and sepsis. The CCR4-NOT complex is a large multimeric protein complex that degrades mRNA through poly(A) tail shortening, whereas it also contributes to regulation of transcription and translation. Cnot3 is a scaffold component of the CCR4-NOT complex and is essential for the integrity of the complex; loss of Cnot3 leads to depletion of whole complex. While the significance of cytokine mRNA degradation in limiting inflammation has been established, the roles of CCR4-NOT complex-mediated in ALI remain elusive. Methods: The effects of Cnot3 haploinsufficiency in the pathology and cytokine expression were analyzed in the mouse lungs of acid aspiration-induced acute lung injury. The decay rate and transcription activity of cytokine mRNAs under Cnot3 heterozygous deletion were analyzed in lipopolysaccharide (LPS) -stimulated mouse embryonic fibroblasts (MEFs). Results: Tamoxifen-induced heterozygous deletion of Cnot3 in adult mice (Cnot3 Hetz) did not show body weight loss or any apparent abnormality. Under acid aspiration-induced acute lung injury, Cnot3 Hetz mice exhibited increased pulmonary edema, worse lung pathologies and more severe inflammation compared with wild type mice. mRNA expression of pro-inflammatory genes Il1b and Nos2 were significantly upregulated in the lungs of Cnot3 Hetz mice. Consistently, mRNA expression of Il1b and Nos2 was upregulated in LPS-stimulated Cnot3 Hetz MEFs. Mechanistically, while heterozygous depletion of Cnot3 stabilized both Il1b and Nos2 mRNAs, the nascent pre-mRNA level of Il1b was upregulated in Cnot3 Hetz MEFs, implicating Cnot3-mediated transcriptional repression of Il1b expression in addition to destabilization of Il1b and Nos2 mRNAs. PU.1 (Spi1) was identified as a causative transcription factor to promote Il1b expression under Cnot3 haploinsufficient conditions. Conclusion: CNOT3 plays a protective role in ALI by suppressing expression of pro-inflammatory genes Il1b and Nos2 through both post-transcriptional and transcriptional mechanisms, including mRNA stability control of Spi1.
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Eukaryotic translation initiation factor (eIF)4A-a DEAD-box RNA-binding protein-plays an essential role in translation initiation. Recent reports have suggested helicase-dependent and helicase-independent functions for eIF4A, but the multifaceted roles of eIF4A have not been fully explored. Here we show that eIF4A1 enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA pulldown followed by sequencing revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs, whose translation is rapidly repressed upon mTORC1 inhibition. This selective interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that deletion of EIF4A1 attenuated the translational repression of TOP mRNAs upon mTORC1 inactivation. Moreover, eIF4A1 increases the interaction between TOP mRNAs and LARP1 and, thus, ensures stronger translational repression upon mTORC1 inhibition. Our data show the multimodality of eIF4A1 in modulating protein synthesis through an inhibitory binding partner and provide a unique example of the repressive role of a universal translational activator.
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BACKGROUND: The mechanism underlying cancer cell metastasis from the tumor to regional lymph nodes is not yet fully understood. We hypothesized that peritumoral neutrophil accumulation promotes regional lymph node metastasis in thoracic esophageal squamous cell cancer. METHODS: Between 2010 and 2019, 126 thoracic esophageal squamous cell cancer patients received curative (R0) esophagectomy without preoperative treatment in our hospital. Using paraffin-embedded resected tumors, we performed immunohistochemical analysis of CD16b-positive neutrophil accumulation in the peritumoral area, which was defined as a 1-mm region centered on the border separating the malignant cell nests from the host tissue. The relationship between the density of peritumoral CD16b staining and pathological lymph node metastasis or 5-year overall survival was evaluated. RESULTS: Although the clinicopathological characteristics of CD16b-high and CD16b-low patients did not differ, greater pathological lymph node metastasis (P < .001) and lymphatic invasion by the tumor (P = .024) and a poorer 5-year survival (P = .010) were seen in CD16b-high patients. Moreover, CD16b-positive neutrophil density was generally higher in the peritumoral area than within the tumor itself. Univariate and multivariate analyses showed that CD16b-positive neutrophil accumulation was an independent factor for lymph node metastasis with an odds ratio >25 (P < .001). On the other hand, blood neutrophil counts did not correlate with lymph node metastasis. CONCLUSION: Peritumoral accumulation of CD16b-positive neutrophils is an independent factor strongly correlated with lymph node metastasis in thoracic esophageal squamous cell cancer.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Células Epiteliales/patología , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/cirugía , Esofagectomía , Humanos , Escisión del Ganglio Linfático , Ganglios Linfáticos/patología , Metástasis Linfática/patología , Estadificación de Neoplasias , Neutrófilos , Estudios RetrospectivosRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is the carboxypeptidase to degrade angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) and improves the pathologies of cardiovascular disease and acute respiratory distress syndrome (ARDS)/acute lung injury. B38-CAP is a bacteria-derived ACE2-like carboxypeptidase as potent as human ACE2 and ameliorates hypertension, heart failure and SARS-CoV-2-induced lung injury in mice. Recombinant B38-CAP is prepared with E. coli protein expression system more efficiently than recombinant soluble human ACE2. Here we show therapeutic effects of B38-CAP on abdominal sepsis- or acid aspiration-induced acute lung injury. ACE2 expression was downregulated in the lungs of mice with cecal ligation puncture (CLP)-induced sepsis or acid-induced lung injury thereby leading to upregulation of Ang II levels. Intraperitoneal injection of B38-CAP significantly decreased Ang II levels while upregulated angiotensin 1-7 levels. B38-CAP improved survival rate of the mice under sepsis. B38-CAP suppressed the pathologies of lung inflammation, improved lung dysfunction and downregulated elevated cytokine mRNA levels in the mice with acute lung injury. Thus, systemic treatment with an ACE2-like enzyme might be a potential therapeutic strategy for the patients with severe sepsis or ARDS.
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Lesión Pulmonar Aguda , COVID-19 , Síndrome de Dificultad Respiratoria , Sepsis , Lesión Pulmonar Aguda/patología , Angiotensina II/metabolismo , Enzima Convertidora de Angiotensina 2 , Animales , Carboxipeptidasas/metabolismo , Escherichia coli/metabolismo , Humanos , Pulmón/patología , Ratones , Peptidil-Dipeptidasa A/metabolismo , Sistema Renina-Angiotensina , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , SARS-CoV-2 , Sepsis/complicaciones , Sepsis/tratamiento farmacológico , Sepsis/metabolismoRESUMEN
Metastasis predicts poor prognosis in cancer patients. It has been recognized that specific tumor microenvironment defines cancer cell metastasis, whereas the underlying mechanisms remain elusive. Here we show that Galectin-7 is a crucial mediator of metastasis associated with immunosuppression. In a syngeneic mouse squamous cell carcinoma (SCC) model of NR-S1M cells, we isolated metastasized NR-S1M cells from lymph nodes in tumor-bearing mice and established metastatic NR-S1M cells in in vitro culture. RNA-seq analysis revealed that interferon gene signature was markedly downregulated in metastatic NR-S1M cells compared with parental cells, and in vivo NR-S1M tumors heterogeneously developed focal immunosuppressive areas featured by deficiency of anti-tumor immune cells. Spatial transcriptome analysis (Visium) for the NR-S1M tumors revealed that various pro-metastatic genes were significantly upregulated in immunosuppressive areas when compared to immunocompetent areas. Notably, Galectin-7 was identified as a novel metastasis-driving factor. Galectin-7 expression was induced during tumorigenesis particularly in the microenvironment of immunosuppression, and extracellularly released at later stage of tumor progression. Deletion of Galectin-7 in NR-S1M cells significantly suppressed lymph node and lung metastasis without affecting primary tumor growth. Therefore, Galectin-7 is a crucial mediator of tumor metastasis of SCC, which is educated in the immune-suppressed tumor areas, and may be a potential target of cancer immunotherapy.
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Carcinoma de Células Escamosas , Galectinas , Neoplasias Pulmonares , Microambiente Tumoral , Animales , Ratones , Carcinoma de Células Escamosas/patología , Galectinas/genética , Galectinas/metabolismo , Tolerancia Inmunológica/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ganglios Linfáticos/patología , Microambiente Tumoral/genéticaRESUMEN
Seventeen years after the epidemic of SARS coronavirus, a novel coronavirus SARS-CoV-2-emerged resulting in an unprecedented pandemic. Angiotensin-converting enzyme 2 (ACE2) is an essential receptor for cell entry of SARS-CoV-2 as well as the SARS coronavirus. Despite many similarities to SARS coronavirus, SARS-CoV-2 exhibits a higher affinity to ACE2 and shows higher infectivity and transmissibility, resulting in explosive increase of infected people and COVID-19 patients. Emergence of the variants harboring mutations in the receptor-binding domain of the Spike protein has drawn critical attention to the interaction between ACE2 and Spike and the efficacies of vaccines and neutralizing antibodies. ACE2 is a carboxypeptidase which degrades angiotensin II, B1-bradykinin, or apelin, and thereby is a critical regulator of cardiovascular physiology and pathology. In addition, the enzymatic activity of ACE2 is protective against acute respiratory distress syndrome (ARDS) caused by viral and non-viral pneumonias, aspiration, or sepsis. Upon infection, both SARS-CoV-2 and SARS coronaviruses downregulates ACE2 expression, likely associated with the pathogenesis of ARDS. Thus, ACE2 is not only the SARS-CoV-2 receptor but might also play an important role in multiple aspects of COVID-19 pathogenesis and possibly post-COVID-19 syndromes. Soluble forms of recombinant ACE2 are currently utilized as a pan-variant decoy to neutralize SARS-CoV-2 and a supplementation of ACE2 carboxypeptidase activity. Here, we review the role of ACE2 in the pathology of ARDS in COVID-19 and the potential application of recombinant ACE2 protein for treating COVID-19.
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Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/patología , Síndrome de Dificultad Respiratoria/patología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Sistema Cardiovascular/metabolismo , Regulación hacia Abajo , Humanos , Lesión Pulmonar/patología , Lesión Pulmonar/virología , Dominios Proteicos/genética , Receptores Virales/metabolismo , Sistema Renina-Angiotensina/fisiología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del VirusRESUMEN
COVID-19, caused by SARS-CoV-2, has spread worldwide with dire consequences. To urgently investigate the pathogenicity of COVID-19 and develop vaccines and therapeutics, animal models that are highly susceptible to SARS-CoV-2 infection are needed. In the present study, we established an animal model highly susceptible to SARS-CoV-2 via the intratracheal tract infection in CAG promoter-driven human angiotensin-converting enzyme 2-transgenic (CAG-hACE2) mice. The CAG-hACE2 mice showed several severe symptoms of SARS-CoV-2 infection, with definitive weight loss and subsequent death. Acute lung injury with elevated cytokine and chemokine levels was observed at an early stage of infection in CAG-hACE2 mice infected with SARS-CoV-2. Analysis of the hACE2 gene in CAG-hACE2 mice revealed that more than 15 copies of hACE2 genes were integrated in tandem into the mouse genome, supporting the high susceptibility to SARS-CoV-2. In the developed model, immunization with viral antigen or injection of plasma from immunized mice prevented body weight loss and lethality due to infection with SARS-CoV-2. These results indicate that a highly susceptible model of SARS-CoV-2 infection in CAG-hACE2 mice via the intratracheal tract is suitable for evaluating vaccines and therapeutic medicines.
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Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Animales , COVID-19/patología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas , SARS-CoV-2/aislamiento & purificaciónRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II, thereby improving the pathologies of cardiovascular disease or acute lung injury. Here we show that B38-CAP, an ACE2-like enzyme, is protective against SARS-CoV-2-induced lung injury. Endogenous ACE2 expression is downregulated in the lungs of SARS-CoV-2-infected hamsters, leading to elevation of angiotensin II levels. Recombinant Spike also downregulates ACE2 expression and worsens the symptoms of acid-induced lung injury. B38-CAP does not neutralize cell entry of SARS-CoV-2. However, B38-CAP treatment improves the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, B38-CAP significantly improves lung edema and pathologies of lung injury. These results provide the first in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients.
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Enzima Convertidora de Angiotensina 2/metabolismo , Tratamiento Farmacológico de COVID-19 , COVID-19/prevención & control , Lesión Pulmonar/prevención & control , SARS-CoV-2/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Lesión Pulmonar Aguda , Angiotensina II , Animales , COVID-19/patología , Carboxipeptidasas , Chlorocebus aethiops , Cricetinae , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/patología , Masculino , Ratones , Ratones Transgénicos , Edema Pulmonar/patología , Edema Pulmonar/prevención & control , Glicoproteína de la Espiga del Coronavirus/efectos de los fármacos , Células VeroRESUMEN
The biological significance of deadenylation in global gene expression is not fully understood. Here, we show that the CCR4-NOT deadenylase complex maintains expression of mRNAs, such as those encoding transcription factors, cell cycle regulators, DNA damage response-related proteins, and metabolic enzymes, at appropriate levels in the liver. Liver-specific disruption of Cnot1, encoding a scaffold subunit of the CCR4-NOT complex, leads to increased levels of mRNAs for transcription factors, cell cycle regulators, and DNA damage response-related proteins because of reduced deadenylation and stabilization of these mRNAs. CNOT1 suppression also results in an increase of immature, unspliced mRNAs (pre-mRNAs) for apoptosis-related and inflammation-related genes and promotes RNA polymerase II loading on their promoter regions. In contrast, mRNAs encoding metabolic enzymes become less abundant, concomitant with decreased levels of these pre-mRNAs. Lethal hepatitis develops concomitantly with abnormal mRNA expression. Mechanistically, the CCR4-NOT complex targets and destabilizes mRNAs mainly through its association with Argonaute 2 (AGO2) and butyrate response factor 1 (BRF1) in the liver. Therefore, the CCR4-NOT complex contributes to liver homeostasis by modulating the liver transcriptome through mRNA deadenylation.
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Proteínas de Homeodominio/metabolismo , Hígado/metabolismo , Receptores CCR4/metabolismo , Animales , Citoplasma/metabolismo , Femenino , Proteínas de Homeodominio/genética , Homeostasis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Poli A/genética , Estabilidad del ARN , ARN Mensajero/genética , Receptores CCR4/genética , Ribonucleasas/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores de Transcripción/genéticaRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.
Asunto(s)
Carboxipeptidasas/farmacología , Cardiomegalia/tratamiento farmacológico , Fibrosis/tratamiento farmacológico , Hipertensión/tratamiento farmacológico , Paenibacillus/enzimología , Peptidil-Dipeptidasa A/genética , Angiotensina II/metabolismo , Enzima Convertidora de Angiotensina 2 , Animales , Cardiomegalia/patología , Modelos Animales de Enfermedad , Fibrosis/patología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/prevención & control , Hipertensión/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Peptidil-Dipeptidasa A/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Apelin is an endogenous peptide ligand for the G protein-coupled receptor APJ/AGTRL1/APLNR and is widely expressed throughout human body. In adult hearts Apelin-APJ/Apelin receptor axis is potently inotropic, vasodilatory, and pro-angiogenic and thereby contributes to maintaining homeostasis in normal and pathological hearts. Apelin-APJ/Apelin receptor is also involved in heart development including endoderm differentiation, heart morphogenesis, and coronary vascular formation. APJ/Apelin receptor had been originally identified as an orphan receptor for its sequence similarity to Angiotensin II type 1 receptor, and it was later deorphanized by identification of Apelin in 1998. Both Apelin and Angiotensin II are substrates for Angiotensin converting enzyme 2 (ACE2), which degrades the peptides and thus negatively regulates their agonistic activities. Elabela/Toddler, which shares little sequence homology with Apelin, has been recently identified as a second endogenous APJ ligand. Elabela plays crucial roles in heart development and disease conditions presumably at time points or at areas of the heart different from Apelin. Apelin and Elabela seem to constitute a spatiotemporal double ligand system to control APJ/Apelin receptor signaling in the heart. These expanding knowledges of Apelin systems would further encourage therapeutic applications of Apelin, Elabela, or their synthetic derivatives for cardiovascular diseases.
Asunto(s)
Receptores de Apelina/metabolismo , Apelina/metabolismo , Corazón/fisiología , Angiotensina II/metabolismo , Enzima Convertidora de Angiotensina 2 , Animales , Humanos , Miocardio/metabolismo , Peptidil-Dipeptidasa A/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Pharmacological interventions to enhance fibrinolysis are effective for treating thrombotic disorders. Utilizing the in vitro U937 cell line-based fibrin degradation assay, we had previously found a cyclic pentapeptide malformin A1 (MA1) as a novel activating compound for cellular fibrinolytic activity. The mechanism by which MA1 enhances cellular fibrinolytic activity remains unknown. In the present study, we show that RSK1 is a crucial mediator of MA1-induced cellular fibrinolysis. Treatment with rhodamine-conjugated MA1 showed that MA1 localizes mainly in the cytoplasm of U937 cells. Screening with an antibody macroarray revealed that MA1 induces the phosphorylation of RSK1 at Ser380 in U937 cells. SL0101, an inhibitor of RSK, inhibited MA1-induced fibrinolytic activity, and CRISPR/Cas9-mediated knockout of RSK1 but not RSK2 suppressed MA1-enhanced fibrinolysis in U937 cells. Synthetic active MA1 derivatives also induced the phosphorylation of RSK1. Furthermore, MA1 treatment stimulated phosphorylation of ERK1/2 and MEK1/2. PD98059, an inhibitor of MEK1/2, inhibited MA1-induced phosphorylation of RSK1 and ERK1/2, indicating that MA1 induces the activation of the MEK-ERK-RSK pathway. Moreover, MA1 upregulated the expression of urokinase-type plasminogen activator (uPA) and increased uPA secretion. These inductions were abrogated in RSK1 knockout cells. These results indicate that RSK1 is a key regulator of MA1-induced extracellular fibrinolytic activity.
Asunto(s)
Fibrinólisis/efectos de los fármacos , Péptidos Cíclicos/farmacología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Células U937 , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.