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1.
Immunity ; 47(2): 251-267.e7, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28813658

RESUMO

Despite considerable research connecting cellular metabolism with differentiation decisions, the underlying mechanisms that translate metabolite-sensitive activities into unique gene programs are still unclear. We found that aspects of the interleukin-2 (IL-2)-sensitive effector gene program in CD4+ and CD8+ T cells in type 1 conditions (Th1) were regulated by glutamine and alpha-ketoglutarate (αKG)-induced events, in part through changes in DNA and histone methylation states. We further identified a mechanism by which IL-2- and αKG-sensitive metabolic changes regulated the association of CCCTC-binding factor (CTCF) with select genomic sites. αKG-sensitive CTCF sites were often associated with loci containing IL-2- and αKG-sensitive genome organization patterns and gene expression in T cells. IL-2- and αKG-sensitive CTCF sites in T cells were also associated with genes from developmental pathways that had αKG-sensitive expression in embryonic stem cells. The data collectively support a mechanism wherein CTCF serves to translate αKG-sensitive metabolic changes into context-dependent differentiation gene programs.


Assuntos
Diferenciação Celular , Interleucina-2/metabolismo , Ácidos Cetoglutáricos/metabolismo , Proteínas Repressoras/metabolismo , Células Th1/imunologia , Animais , Fator de Ligação a CCCTC , Diferenciação Celular/genética , Células Cultivadas , Microambiente Celular , Metilação de DNA , Feminino , Regulação da Expressão Gênica , Glutamina/metabolismo , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Repressoras/genética
2.
Proc Natl Acad Sci U S A ; 120(49): e2315096120, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38011564

RESUMO

Hidradenitis suppurativa (HS) is a complex inflammatory skin disease with undefined mechanistic underpinnings. Here, we investigated HS epithelial cells and demonstrated that HS basal progenitors modulate their lineage restriction and give rise to pathogenic keratinocyte clones, resulting in epidermal hyperproliferation and dysregulated inflammation in HS. When comparing to healthy epithelial stem/progenitor cells, in HS, we identified changes in gene signatures that revolve around the mitotic cell cycle, DNA damage response and repair, as well as cell-cell adhesion and chromatin remodeling. By reconstructing cell differentiation trajectory and CellChat modeling, we identified a keratinocyte population specific to HS. This population is marked by S100A7/8/9 and KRT6 family members, triggering IL1, IL10, and complement inflammatory cascades. These signals, along with HS-specific proinflammatory cytokines and chemokines, contribute to the recruitment of certain immune cells during the disease progression. Furthermore, we revealed a previously uncharacterized role of S100A8 in regulating the local chromatin environment of target loci in HS keratinocytes. Through the integration of genomic and epigenomic datasets, we identified genome-wide chromatin rewiring alongside the switch of transcription factors (TFs), which mediated HS transcriptional profiles. Importantly, we identified numerous clinically relevant inflammatory enhancers and their coordinated TFs in HS basal CD49fhigh cells. The disruption of the S100A enhancer using the CRISPR/Cas9-mediated approach or the pharmacological inhibition of the interferon regulatory transcription factor 3 (IRF3) efficiently reduced the production of HS-associated inflammatory regulators. Our study not only uncovers the plasticity of epidermal progenitor cells in HS but also elucidates the epigenetic mechanisms underlying HS pathogenesis.


Assuntos
Hidradenite Supurativa , Humanos , Hidradenite Supurativa/genética , Pele/metabolismo , Epigenômica , Epigênese Genética , Células-Tronco/metabolismo , Cromatina/metabolismo
3.
Blood ; 142(6): 574-588, 2023 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-37192295

RESUMO

Tyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML-committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the HIF-1-mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Proteínas Tirosina Quinases , Camundongos , Humanos , Animais , Proteínas Tirosina Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco Neoplásicas/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Resistencia a Medicamentos Antineoplásicos
4.
J Cell Sci ; 135(4)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35099001

RESUMO

Mitochondrial dysfunction causes severe congenital cardiac abnormalities and prenatal/neonatal lethality. The lack of sufficient knowledge regarding how mitochondrial abnormalities affect cardiogenesis poses a major barrier for the development of clinical applications that target mitochondrial deficiency-induced inborn cardiomyopathies. Mitochondrial morphology, which is regulated by fission and fusion, plays a key role in determining mitochondrial activity. Dnm1l encodes a dynamin-related GTPase, Drp1, which is required for mitochondrial fission. To investigate the role of Drp1 in cardiogenesis during the embryonic metabolic shift period, we specifically inactivated Dnm1l in second heart field-derived structures. Mutant cardiomyocytes in the right ventricle (RV) displayed severe defects in mitochondrial morphology, ultrastructure and activity. These defects caused increased cell death, decreased cell survival, disorganized cardiomyocytes and embryonic lethality. By characterizing this model, we reveal an AMPK-SIRT7-GABPB axis that relays the reduced cellular energy level to decrease transcription of ribosomal protein genes in cardiomyocytes. We therefore provide the first genetic evidence in mouse that Drp1 is essential for RV development. Our research provides further mechanistic insight into how mitochondrial dysfunction causes pathological molecular and cellular alterations during cardiogenesis.


Assuntos
Dinaminas , Proteínas Ribossômicas , Animais , Dinaminas/genética , Dinaminas/metabolismo , Coração/embriologia , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
5.
J Cell Sci ; 135(17)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35946425

RESUMO

Mitral and tricuspid valves are essential for unidirectional blood flow in the heart. They are derived from similar cell sources, and yet congenital dysplasia affecting both valves is clinically rare, suggesting the presence of differential regulatory mechanisms underlying their development. Here, we specifically inactivated Dicer1 in the endocardium during cardiogenesis and found that Dicer1 deletion caused congenital mitral valve stenosis and regurgitation, whereas it had no impact on other valves. We showed that hyperplastic mitral valves were caused by abnormal condensation and extracellular matrix (ECM) remodeling. Our single-cell RNA sequencing analysis revealed impaired maturation of mesenchymal cells and abnormal expression of ECM genes in mutant mitral valves. Furthermore, expression of a set of miRNAs that target ECM genes was significantly lower in tricuspid valves compared to mitral valves, consistent with the idea that the miRNAs are differentially required for mitral and tricuspid valve development. We thus reveal miRNA-mediated gene regulation as a novel molecular mechanism that differentially regulates mitral and tricuspid valve development, thereby enhancing our understanding of the non-association of inborn mitral and tricuspid dysplasia observed clinically.


Assuntos
MicroRNAs , Valva Tricúspide , Matriz Extracelular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Valva Mitral , Valva Tricúspide/anormalidades
6.
J Pharmacol Exp Ther ; 388(2): 546-559, 2024 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-37914412

RESUMO

Vesicants such as arsenicals and mustards produce highly painful cutaneous inflammatory and blistering responses, hence developed as chemical weapons during World War I/II. Here, using lewisite and sulfur mustard surrogates, namely phenylarsine oxide (PAO) and 2-chloroethyl ethyl sulfide (CEES), respectively, we defined a common underlying mechanism of toxic action by these two distinct classes of vesicants. Murine skin exposure to these chemicals causes tissue destruction characterized by increase in skin bifold thickness, Draize score, infiltration of inflammatory cells, and apoptosis of epidermal and dermal cells. RNA sequencing analysis identified ∼346 inflammatory genes that were commonly altered by both PAO and CEES, along with the identification of cytokine signaling activation as the top canonical pathway. Activation of several proinflammatory genes and pathways is associated with phosphorylation-dependent activation of heat shock protein 90α (p-HSP90α). Topical treatment with known HSP90 inhibitors SNX-5422 and IPI-504 post PAO or CEES skin challenge significantly attenuated skin damage including reduction in overall skin injury and clinical scores. In addition, highly upregulated inflammatory genes Saa3, Cxcl1, Ccl7, IL-6, Nlrp3, Csf3, Chil3, etc. by both PAO and CEES were significantly diminished by treatment with HSP90 inhibitors. These drugs not only reduced PAO- or CEES-induced p-HSP90α expression but also its client proteins NLRP3 and pP38 and the expression of their target inflammatory genes. Our data confirm a critical role of HSP90 as a shared underlying molecular target of toxicity by these two distinct vesicants and provide an effective and novel medical countermeasure to suppress vesicant-induced skin injury. SIGNIFICANCE STATEMENT: Development of effective and novel mechanism-based antidotes that can simultaneously block cutaneous toxic manifestations of distinct vesicants is important and urgently needed. Due to difficulties in determining the exact nature of onsite chemical exposure, a potent drug that can suppress widespread cutaneous damage may find great utility. Thus, this study identified HSP90 as a common molecular regulator of cutaneous inflammation and injury by two distinct warfare vesicants, arsenicals and mustards, and HSP90 inhibitors afford significant protection against skin damage.


Assuntos
Arsenicais , Substâncias para a Guerra Química , Gás de Mostarda , Humanos , Animais , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Substâncias para a Guerra Química/toxicidade , Irritantes , Pele , Gás de Mostarda/toxicidade , Arsenicais/metabolismo , Arsenicais/farmacologia
7.
J Neurosci ; 42(25): 5085-5101, 2022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-35589390

RESUMO

Endosomal sorting plays a fundamental role in directing neural development. By altering the temporal and spatial distribution of membrane receptors, endosomes regulate signaling pathways that control the differentiation and function of neural cells. Several genes linked to inherited demyelinating peripheral neuropathies, known as Charcot-Marie-Tooth (CMT) disease, encode proteins that directly interact with components of the endosomal sorting complex required for transport (ESCRT). Our previous studies demonstrated that a point mutation in the ESCRT component hepatocyte growth-factor-regulated tyrosine kinase substrate (HGS), an endosomal scaffolding protein that identifies internalized cargo to be sorted by the endosome, causes a peripheral neuropathy in the neurodevelopmentally impaired teetering mice. Here, we constructed a Schwann cell-specific deletion of Hgs to determine the role of endosomal sorting during myelination. Inactivation of HGS in Schwann cells resulted in motor and sensory deficits, slowed nerve conduction velocities, delayed myelination and hypomyelinated axons, all of which occur in demyelinating forms of CMT. Consistent with a delay in Schwann cell maturation, HGS-deficient sciatic nerves displayed increased mRNA levels for several promyelinating genes and decreased mRNA levels for genes that serve as markers of myelinating Schwann cells. Loss of HGS also altered the abundance and activation of the ERBB2/3 receptors, which are essential for Schwann cell development. We therefore hypothesize that HGS plays a critical role in endosomal sorting of the ERBB2/3 receptors during Schwann cell maturation, which further implicates endosomal dysfunction in inherited peripheral neuropathies.SIGNIFICANCE STATEMENT Schwann cells myelinate peripheral axons, and defects in Schwann cell function cause inherited demyelinating peripheral neuropathies known as CMT. Although many CMT-linked mutations are in genes that encode putative endosomal proteins, little is known about the requirements of endosomal sorting during myelination. In this study, we demonstrate that loss of HGS disrupts the endosomal sorting pathway in Schwann cells, resulting in hypomyelination, aberrant myelin sheaths, and impairment of the ERBB2/3 receptor pathway. These findings suggest that defective endosomal trafficking of internalized cell surface receptors may be a common mechanism contributing to demyelinating CMT.


Assuntos
Doença de Charcot-Marie-Tooth , Animais , Doença de Charcot-Marie-Tooth/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Camundongos , Doenças do Sistema Nervoso Periférico , RNA Mensageiro , Células de Schwann/metabolismo
8.
FASEB J ; 36(7): e22412, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35713587

RESUMO

The cellular adaptive response to hypoxia relies on the expression of hypoxia-inducible factors (HIFs), HIF-1 and HIF-2. HIFs regulate global gene expression changes during hypoxia that are necessary for restoring oxygen homeostasis and promoting cell survival. In the early stages of hypoxia, HIF-1 is elevated, whereas at the later stages, HIF-2 becomes the predominant form. What governs the transition between the two HIFs (the HIF switch) and the role of miRNAs in this regulation are not completely clear. Genome-wide expression studies on the miRNA content of RNA-induced silencing complexes (RISC) in HUVECs exposed to hypoxia compared to the global miRNA-Seq analysis revealed very specific differences between these two populations. We analyzed the miRNA and mRNA composition of RISC at 2 h (mainly HIF-1 driven), 8 h (HIF-1 and HIF-2 elevated), and 16 h (mainly HIF-2 driven) in a gene ontology context. This allowed for determining the direct impact of the miRNAs in modulating the cellular signaling pathways involved in the hypoxic adaptive response. Our results indicate that the miRNA-mRNA RISC components control the adaptive responses, and this does not always rely on the miRNA transcriptional elevations during hypoxia. Furthermore, we demonstrate that the hypoxic levels of the vast majority of HIF-1-dependent miRNAs (including miR-210-3p) are also HIF-2 dependent and that HIF-2 governs the expression of 11 specific miRNAs. In summary, the switch from HIF-1 to HIF-2 during hypoxia provides an important level of miRNA-driven control in the adaptive pathways in endothelial cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , MicroRNAs , Complexo de Inativação Induzido por RNA , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular/genética , Células Endoteliais/metabolismo , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(46): 28847-28858, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33127760

RESUMO

CHD7 encodes an ATP-dependent chromatin remodeling factor. Mutation of this gene causes multiple developmental disorders, including CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth/development, Genital abnormalities, and Ear anomalies) syndrome, in which conotruncal anomalies are the most prevalent form of heart defects. How CHD7 regulates conotruncal development remains unclear. In this study, we establish that deletion of Chd7 in neural crest cells (NCCs) causes severe conotruncal defects and perinatal lethality, thus providing mouse genetic evidence demonstrating that CHD7 cell-autonomously regulates cardiac NCC development, thereby clarifying a long-standing controversy in the literature. Using transcriptomic analyses, we show that CHD7 fine-tunes the expression of a gene network that is critical for cardiac NCC development. To gain further molecular insights into gene regulation by CHD7, we performed a protein-protein interaction screen by incubating recombinant CHD7 on a protein array. We find that CHD7 directly interacts with several developmental disorder-mutated proteins including WDR5, a core component of H3K4 methyltransferase complexes. This direct interaction suggested that CHD7 may recruit histone-modifying enzymes to target loci independently of its remodeling functions. We therefore generated a mouse model that harbors an ATPase-deficient allele and demonstrates that mutant CHD7 retains the ability to recruit H3K4 methyltransferase activity to its targets. Thus, our data uncover that CHD7 regulates cardiovascular development through ATP-dependent and -independent activities, shedding light on the etiology of CHD7-related congenital disorders. Importantly, our data also imply that patients carrying a premature stop codon versus missense mutations will likely display different molecular alterations; these patients might therefore require personalized therapeutic interventions.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Coração/embriologia , Trifosfato de Adenosina/metabolismo , Alelos , Animais , Síndrome CHARGE/genética , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Cardiopatias Congênitas/genética , Camundongos , Camundongos Knockout , Mutação , Crista Neural/embriologia , Crista Neural/metabolismo , Organogênese/fisiologia
10.
J Am Soc Nephrol ; 33(4): 747-768, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35110364

RESUMO

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45+ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.


Assuntos
Cistos , Doenças Renais Policísticas , Animais , Cílios/metabolismo , Cistos/genética , Rim/metabolismo , Camundongos , Mutação , Doenças Renais Policísticas/metabolismo
11.
Hum Mutat ; 43(1): 74-84, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34747535

RESUMO

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.


Assuntos
Neurilemoma , Neurofibromatoses , Cromossomos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neurilemoma/genética , Neurilemoma/patologia , Neurofibromatoses/genética , Proteína SMARCB1/genética , Fatores de Transcrição/genética
12.
Physiol Genomics ; 54(2): 45-57, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34890513

RESUMO

Recent studies have identified at least 20 different kidney cell types based upon chromatin structure and gene expression. Histone deacetylases (HDACs) are epigenetic transcriptional repressors via deacetylation of histone lysines resulting in inaccessible chromatin. We reported that kidney epithelial HDAC1 and HDAC2 activity is critical for maintaining a healthy kidney and preventing fluid-electrolyte abnormalities. However, to what extent does Hdac1/Hdac2 knockdown affect chromatin structure and subsequent transcript expression in the kidney? To answer this question, we used single nucleus assay for transposase-accessible chromatin-sequencing (snATAC-seq) and snRNA-seq to profile kidney nuclei from male and female, control, and littermate kidney epithelial Hdac1/Hdac2 knockdown mice. Hdac1/Hdac2 knockdown resulted in significant changes in the chromatin structure predominantly within the promoter region of gene loci involved in fluid-electrolyte balance such as the aquaporins, with both increased and decreased accessibility captured. Moreover, Hdac1/Hdac2 knockdown resulted different gene loci being accessible with a corresponding increased transcript number in the kidney, but among all mice only 24%-30% of chromatin accessibility agreed with transcript expression (e.g., open chromatin and increased transcript). To conclude, although chromatin structure does affect transcription, ∼70% of the differentially expressed genes cannot be explained by changes in chromatin accessibility and HDAC1/HDAC2 had a minimal effect on these global patterns. Yet, the genes that are targets of HDAC1 and HDAC2 are critically important for maintaining kidney function.


Assuntos
Cromatina/genética , Células Epiteliais/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Rim/metabolismo , Transcriptoma/genética , Animais , Aquaporina 1/genética , Aquaporina 1/metabolismo , Aquaporina 2/genética , Aquaporina 2/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Rim/citologia , Masculino , Camundongos Knockout , RNA-Seq/métodos
13.
J Biol Chem ; 296: 100034, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33148698

RESUMO

ST6Gal-I, an enzyme upregulated in numerous malignancies, adds α2-6-linked sialic acids to select membrane receptors, thereby modulating receptor signaling and cell phenotype. In this study, we investigated ST6Gal-I's role in epithelial to mesenchymal transition (EMT) using the Suit2 pancreatic cancer cell line, which has low endogenous ST6Gal-I and limited metastatic potential, along with two metastatic Suit2-derived subclones, S2-013 and S2-LM7AA, which have upregulated ST6Gal-I. RNA-Seq results suggested that the metastatic subclones had greater activation of EMT-related gene networks than parental Suit2 cells, and forced overexpression of ST6Gal-I in the Suit2 line was sufficient to activate EMT pathways. Accordingly, we evaluated expression of EMT markers and cell invasiveness (a key phenotypic feature of EMT) in Suit2 cells with or without ST6Gal-I overexpression, as well as S2-013 and S2-LM7AA cells with or without ST6Gal-I knockdown. Cells with high ST6Gal-I expression displayed enrichment in mesenchymal markers (N-cadherin, slug, snail, fibronectin) and cell invasiveness, relative to ST6Gal-I-low cells. Contrarily, epithelial markers (E-cadherin, occludin) were suppressed in ST6Gal-I-high cells. To gain mechanistic insight into ST6Gal-I's role in EMT, we examined the activity of epidermal growth factor receptor (EGFR), a known EMT driver. ST6Gal-I-high cells had greater α2-6 sialylation and activation of EGFR than ST6Gal-I-low cells. The EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR activation, mesenchymal marker expression, and invasiveness in Suit2 and S2-LM7AA, but not S2-013, lines. Collectively, these results advance our understanding of ST6Gal-I's tumor-promoting function by highlighting a role for ST6Gal-I in EMT, which may be mediated, at least in part, by α2-6-sialylated EGFR.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Neoplasias Pancreáticas/patologia , Sialiltransferases/fisiologia , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Pancreáticas/enzimologia , beta-D-Galactosídeo alfa 2-6-Sialiltransferase
14.
Hum Mol Genet ; 29(17): 2855-2871, 2020 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-32766788

RESUMO

DOCK3 is a member of the DOCK family of guanine nucleotide exchange factors that regulate cell migration, fusion and viability. Previously, we identified a dysregulated miR-486/DOCK3 signaling cascade in dystrophin-deficient muscle, which resulted in the overexpression of DOCK3; however, little is known about the role of DOCK3 in muscle. Here, we characterize the functional role of DOCK3 in normal and dystrophic skeletal muscle. Utilizing Dock3 global knockout (Dock3 KO) mice, we found that the haploinsufficiency of Dock3 in Duchenne muscular dystrophy mice improved dystrophic muscle pathologies; however, complete loss of Dock3 worsened muscle function. Adult Dock3 KO mice have impaired muscle function and Dock3 KO myoblasts are defective for myogenic differentiation. Transcriptomic analyses of Dock3 KO muscles reveal a decrease in myogenic factors and pathways involved in muscle differentiation. These studies identify DOCK3 as a novel modulator of muscle health and may yield therapeutic targets for treating dystrophic muscle symptoms.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , Distrofia Muscular de Duchenne/genética , Proteínas do Tecido Nervoso/genética , Animais , Diferenciação Celular/genética , Movimento Celular/genética , Sobrevivência Celular/genética , Humanos , Camundongos , Camundongos Knockout , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Mioblastos/metabolismo , Transcriptoma/genética
15.
J Virol ; 95(21): e0135721, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34406867

RESUMO

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.


Assuntos
Evolução Molecular , Heparitina Sulfato/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Adaptação Biológica , Animais , Sítios de Ligação , Chlorocebus aethiops , Efeito Citopatogênico Viral , DNA Complementar , Furina/metabolismo , Heparina/metabolismo , Interações Hospedeiro-Patógeno , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Ensaio de Placa Viral , Ligação Viral
16.
J Autoimmun ; 132: 102883, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35987175

RESUMO

IgA nephropathy (IgAN) is an autoimmune disease characterized by the deposition of galactose-deficient IgA1 (Gd-IgA1)-containing immune complexes in the kidneys. Elevated serum levels of Gd-IgA1, the main autoantigen in IgAN, are associated with mucosal infections and poor renal outcome in IgAN patients, but little is known about the activation of IgA1-secreting cells overproducing this autoantigen. We found that in peripheral blood mononuclear cells (PBMCs), cytokine stimulation elevated Gd-IgA1 production in B cells from IgAN patients but not in those from healthy controls (p < 0.01). These results were replicated in immortalized B cells derived from PBMCs of IgAN patients and healthy controls. Using single-cell transcriptomics, we identified subsets of IgA1-secreting cells from IgAN patients, but not from healthy controls, with decreased expression of C1GALT1 in response to cytokine stimulation. The C1GALT1-encoded glycosyltransferase is responsible for addition of galactose to IgA1 O-glycans, and its reduced activity is associated with elevated serum levels of Gd-IgA1. These newly identified subsets of IgA1-secreting cells with reduced C1GALT1 expression exhibited reduced expression of several genes related to cytokine-mediated signaling, including those encoding phosphatases, such as SOCS1. siRNA knock-down of SOCS1, and the related SOCS3, increased Gd-IgA1 production in cells derived from PBMCs of healthy controls, indicating a role of these regulators in abnormal cytokine signaling and Gd-IgA1 overproduction. These results revealed that specific subsets of IgA1-secreting cells may be responsible for autoantigen production in IgAN due to abnormal regulation of cytokine-mediated signaling, a process that may occur in inflammatory responses in IgAN patients.


Assuntos
Glomerulonefrite por IGA , Humanos , Autoantígenos , Galactose , Citocinas , Monoéster Fosfórico Hidrolases , Leucócitos Mononucleares , Imunoglobulina A
17.
Cell Mol Life Sci ; 78(21-22): 7061-7080, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34636989

RESUMO

Accumulation of misfolded proteins in ER activates the unfolded protein response (UPR), a multifunctional signaling pathway that is important for cell survival. The UPR is regulated by three ER transmembrane sensors, one of which is inositol-requiring protein 1 (IRE1). IRE1 activates a transcription factor, X-box-binding protein 1 (XBP1), by removing a 26-base intron from XBP1 mRNA that generates spliced XBP1 mRNA (XBP1s). To search for XBP1 transcriptional targets, we utilized an XBP1s-inducible human cell line to limit XBP1 expression in a controlled manner. We also verified the identified XBP1-dependent genes with specific silencing of this transcription factor during pharmacological ER stress induction with both an N-linked glycosylation inhibitor (tunicamycin) and a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) (thapsigargin). We then compared those results to the XBP1s-induced cell line without pharmacological ER stress induction. Using next-generation sequencing followed by bioinformatic analysis of XBP1-binding motifs, we defined an XBP1 regulatory network and identified XBP1 as a repressor of PUMA (a proapoptotic gene) and IRE1 mRNA expression during the UPR. Our results indicate impairing IRE1 activity during ER stress conditions accelerates cell death in ER-stressed cells, whereas elevating XBP1 expression during ER stress using an inducible cell line correlated with a clear prosurvival effect and reduced PUMA protein expression. Although further studies will be required to test the underlying molecular mechanisms involved in the relationship between these genes with XBP1, these studies identify a novel repressive role of XBP1 during the UPR.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Endorribonucleases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Proteína 1 de Ligação a X-Box/genética , Linhagem Celular , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/genética , Células HaCaT , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Transdução de Sinais/genética , Resposta a Proteínas não Dobradas/genética
18.
Lab Invest ; 101(10): 1382-1393, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34158590

RESUMO

Hepatorenal fibrocystic disease (HRFCD) is a genetically inherited disorder related to primary cilia dysfunction in which patients display varying levels of fibrosis, bile duct expansion, and inflammation. In mouse models of HRFCD, the phenotype is greatly impacted by the genetic background in which the mutation is placed. Macrophages are a common factor associated with progression of HRFCD and are also strongly influenced by the genetic background. These data led us to hypothesize that macrophage subtypes that change in relation to the genetic background are responsible for the variable phenotypic outcomes in HRFCD. To test this hypothesis, we utilized a mouse model of HRFCD (Ift88Orpk mice) on the C57BL/6 and BALB/c inbred backgrounds that have well-documented differences in macrophage subtypes. Our analyses of infiltrating macrophage subtypes confirm that genetic strain influences the subtype of infiltrating macrophage present during normal postnatal liver development and in Ift88Orpk livers (Ly6clo in C57BL/6 vs Ly6chi in BALB/c). Each infiltrating macrophage subtype was similarly associated with a unique phenotypic outcome as analysis of liver tissue shows that C57BL/6 Ift88Orpk mice have increased bile duct expansion, but reduced levels of fibrosis compared to BALB/c Ift88Orpk livers. RNA sequencing data suggest that the ability to infiltrate macrophage subtypes to influence the phenotypic outcome may be due to unique ligand-receptor signaling between infiltrating macrophages and cilia dysfunctional biliary epithelium. To evaluate whether specific macrophage subtypes cause the observed phenotypic divergence, we analyzed the liver phenotype in BALB/c Ift88Orpk mice on a CCR2-/- background. Unexpectedly, the loss of Ly6chi macrophages, which were strongly enriched in BALB/c Ift88Orpk mice, did not significantly alter liver fibrosis. These data indicate that macrophage subtypes may correlate with HRFCD phenotypic outcome, but do not directly cause the pathology.


Assuntos
Cirrose Hepática , Macrófagos , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Fígado/metabolismo , Macrófagos/classificação , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenótipo
19.
J Immunol ; 203(9): 2472-2484, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31562212

RESUMO

Innate lymphoid cells (ILCs) are strategically positioned at mucosal barrier surfaces where they respond quickly to infection or injury. Therefore, we hypothesized that ILCs are key contributors to the early immune response in the intestine against Listeria monocytogenes Using a modified strain of L. monocytogenes that mimics human gastrointestinal listeriosis in mice, we find ILCs to be essential for control of early replication of L. monocytogenes in the intestine as well as for restricted dissemination of bacteria to peripheral tissues. Specifically, group 1 ILCs (ILC1s) and group 3 ILCs (ILC3s) respond to infection with proliferation and IFN-γ and IL-22 production. Mechanistically, we show that the transcription factor STAT4 is required for the proliferative and IFN-γ effector response by ILC1s and ILC3s, and loss of STAT4 signaling in the innate immune compartment results in an inability to control bacterial growth and dissemination. Interestingly, STAT4 acts acutely as a transcription factor to promote IFN-γ production. Together, these data illustrate a critical role for ILCs in the early responses to gastrointestinal infection with L. monocytogenes and identify STAT4 as a central modulator of ILC-mediated protection.


Assuntos
Gastroenteropatias/imunologia , Listeriose/imunologia , Linfócitos/imunologia , Fator de Transcrição STAT4/fisiologia , Animais , Proteínas de Homeodomínio/fisiologia , Imunidade Inata , Interferon gama/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia
20.
Cell Mol Biol Lett ; 26(1): 11, 2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33730996

RESUMO

Inositol requiring enzyme 1 alpha (IRE1α) is one of three signaling sensors in the unfolding protein response (UPR) that alleviates endoplasmic reticulum (ER) stress in cells and functions to promote cell survival. During conditions of irrevocable stress, proapoptotic gene expression is induced to promote cell death. One of the three signaling stressors, IRE1α is an serine/threonine-protein kinase/endoribonuclease (RNase) that promotes nonconventional splicing of XBP1 mRNA that is translated to spliced XBP1 (XBP1s), an active prosurvival transcription factor. Interestingly, elevated IRE1α and XBP1s are both associated with poor cancer survival and drug resistance. In this study, we used next-generation sequencing analyses to demonstrate that triazoloacridone C-1305, a microtubule stabilizing agent that also has topoisomerase II inhibitory activity, dramatically decreases XBP1s mRNA levels and protein production during ER stress conditions, suggesting that C-1305 does this by decreasing IRE1α's endonuclease activity.


Assuntos
Acridinas/farmacologia , Endorribonucleases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Splicing de RNA/genética , Triazóis/farmacologia , Proteína 1 de Ligação a X-Box/genética , Acridinas/química , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Himecromona/análogos & derivados , Himecromona/química , Himecromona/farmacologia , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Triazóis/química
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