RESUMO
Early life environmental exposure, particularly during perinatal period, can have a life-long impact on organismal development and physiology. The biological rationale for this phenomenon is to promote physiological adaptations to the anticipated environment based on early life experience. However, perinatal exposure to adverse environments can also be associated with adult-onset disorders. Multiple environmental stressors induce glucocorticoids, which prompted us to investigate their role in developmental programming. Here, we report that perinatal glucocorticoid exposure had long-term consequences and resulted in diminished CD8 T cell response in adulthood and impaired control of tumor growth and bacterial infection. We found that perinatal glucocorticoid exposure resulted in persistent alteration of the hypothalamic-pituitary-adrenal (HPA) axis. Consequently, the level of the hormone in adults was significantly reduced, resulting in decreased CD8 T cell function. Our study thus demonstrates that perinatal stress can have long-term consequences on CD8 T cell immunity by altering HPA axis activity.
Assuntos
Infecções Bacterianas/imunologia , Desenvolvimento Embrionário/imunologia , Glucocorticoides/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/genética , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Dexametasona/farmacologia , Desenvolvimento Embrionário/genética , Feminino , Glucocorticoides/imunologia , Glucocorticoides/metabolismo , Humanos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Interleucina-4/farmacologia , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Neoplasias/induzido quimicamente , Neoplasias/genética , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/imunologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Receptores de Glucocorticoides/genética , Transdução de Sinais/genéticaRESUMO
Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas , Antivirais/farmacologia , Imunidade Inata , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Proteínas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , S-Adenosilmetionina , Replicação ViralRESUMO
Uridylation occurs pervasively on mRNAs, yet its mechanism and significance remain unknown. By applying TAIL-seq, we identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A-tails (less than â¼ 25 nt) in vitro. PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A-tails. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U-tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , Células HeLa , Humanos , MicroRNAs/metabolismo , Poli A/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , Uridina Monofosfato/metabolismoRESUMO
RNase III Drosha initiates microRNA (miRNA) maturation by cleaving a primary miRNA transcript and releasing a pre-miRNA with a 2 nt 3' overhang. Dicer recognizes the 2 nt 3' overhang structure to selectively process pre-miRNAs. Here, we find that, unlike prototypic pre-miRNAs (group I), group II pre-miRNAs acquire a shorter (1 nt) 3' overhang from Drosha processing and therefore require a 3'-end mono-uridylation for Dicer processing. The majority of let-7 and miR-105 belong to group II. We identify TUT7/ZCCHC6, TUT4/ZCCHC11, and TUT2/PAPD4/GLD2 as the terminal uridylyl transferases responsible for pre-miRNA mono-uridylation. The TUTs act specifically on dsRNAs with a 1 nt 3' overhang, thereby creating a 2 nt 3' overhang. Depletion of TUTs reduces let-7 levels and disrupts let-7 function. Although the let-7 suppressor, Lin28, induces inhibitory oligo-uridylation in embryonic stem cells, mono-uridylation occurs in somatic cells lacking Lin28 to promote let-7 biogenesis. Our study reveals functional duality of uridylation and introduces TUT7/4/2 as components of the miRNA biogenesis pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , MicroRNAs/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA Nucleotidiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , Uridina Monofosfato/metabolismo , Sequência de Bases , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas de Ligação a RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNARESUMO
Multiple deadenylases are known in vertebrates, the PAN2-PAN3 (PAN2/3) and CCR4-NOT (CNOT) complexes, and PARN, yet their differential functions remain ambiguous. Moreover, the role of poly(A) binding protein (PABP) is obscure, limiting our understanding of the deadenylation mechanism. Here, we show that CNOT serves as a predominant nonspecific deadenylase for cytoplasmic poly(A)+ RNAs, and PABP promotes deadenylation while preventing premature uridylation and decay. PAN2/3 selectively trims long tails (>â¼150 nt) with minimal effect on transcriptome, whereas PARN does not affect mRNA deadenylation. CAF1 and CCR4, catalytic subunits of CNOT, display distinct activities: CAF1 trims naked poly(A) segments and is blocked by PABPC, whereas CCR4 is activated by PABPC to shorten PABPC-protected sequences. Concerted actions of CAF1 and CCR4 delineate the â¼27 nt periodic PABPC footprints along shortening tail. Our study unveils distinct functions of deadenylases and PABPC, re-drawing the view on mRNA deadenylation and regulation.
Assuntos
Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Receptores CCR4/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular/metabolismo , Citoplasma/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Células HEK293 , Células HeLa , Humanos , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Poli A/metabolismo , Proteínas de Ligação a Poli(A)/genética , Poliadenilação , RNA Mensageiro/genética , Receptores CCR4/genética , Fatores de Transcrição/genética , TranscriptomaRESUMO
During the maternal-to-zygotic transition (MZT), maternal RNAs are actively degraded and replaced by newly synthesized zygotic transcripts in a highly coordinated manner. However, it remains largely unknown how maternal mRNA decay is triggered in early vertebrate embryos. Here, through genome-wide profiling of RNA abundance and 3' modification, we show that uridylation is induced at the onset of maternal mRNA clearance. The temporal control of uridylation is conserved in vertebrates. When the homologs of terminal uridylyltransferases TUT4 and TUT7 (TUT4/7) are depleted in zebrafish and Xenopus, maternal mRNA clearance is significantly delayed, leading to developmental defects during gastrulation. Short-tailed mRNAs are selectively uridylated by TUT4/7, with the highly uridylated transcripts degraded faster during the MZT than those with unmodified poly(A) tails. Our study demonstrates that uridylation plays a crucial role in timely mRNA degradation, thereby allowing the progression of early development.
Assuntos
Embrião de Mamíferos/enzimologia , Embrião não Mamífero/enzimologia , Nucleotidiltransferases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transcriptoma , Xenopus laevis/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Camundongos Endogâmicos ICR , Nucleotidiltransferases/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Cancer cells can develop an immunosuppressive tumor microenvironment to control tumor-infiltrating lymphocytes. The underlying mechanisms still remain unclear. Here, we report that mouse and human colon cancer cells acquire lymphocyte membrane proteins including cellular markers such as CD4 and CD45. We observed cell populations harboring both a tumor-specific marker and CD4 in the tumor microenvironment. Sorted cells from these populations were capable of forming organoids, identifying them as cancer cells. Live imaging analysis revealed that lymphocyte membrane proteins were transferred to cancer cells via trogocytosis. As a result of the transfer in vivo, cancer cells also acquired immune regulatory surface proteins such as CTLA4 and Tim3, which suppress activation of immune cells [T. L. Walunas et al, Immunity 1, 405-413 (1994) and L. Monney et al., Nature 415, 536-541 (2002)]. RNA sequencing analysis of ex vivo-cocultured splenocytes with trogocytic cancer cells showed reductions in Th1 activation and natural killer cell signaling pathways compared with the nontrogocytic control. Cancer cell trogocytosis was confirmed in the patient-derived xenograft models of colorectal cancer and head and neck cancer. These findings suggest that cancer cells utilize membrane proteins expressed in lymphocytes, which in turn contribute to the development of the immunosuppressive tumor microenvironment.
Assuntos
Linfócitos T CD4-Positivos/citologia , Antígeno CTLA-4/metabolismo , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Linfócitos do Interstício Tumoral/citologia , Animais , Células CACO-2 , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Hematopoéticas/citologia , Humanos , Sistema Imunitário , Imunossupressores , Células Jurkat , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Organoides/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Trogocitose , Microambiente TumoralRESUMO
Eukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAIL-seq (mRNA TAIL-seq [mTAIL-seq]) with enhanced sequencing depth for mRNAs (by â¼1000-fold compared with the previous version). The improved method allows us to investigate the regulation of poly(A) tails in Drosophila oocytes and embryos. We found that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs, with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAIL-seq data with ribosome profiling data, we found a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tails in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems.
Assuntos
Oócitos/metabolismo , Poli A/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Animais , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Células HeLa , Humanos , Poli A/química , Poli A/genética , Poliadenilação , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/química , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Adiponectin (Acrp30) is an adipokine associated with protection from cardiovascular disease, insulin resistance, and inflammation. Although its effects are conventionally attributed to binding Adipor1/2 and T-cadherin, its abundance in circulation, role in ceramide metabolism, and homology to C1q suggest an overlooked role as a lipid-binding protein, possibly generalizable to other C1q/TNF-related proteins (CTRPs) and C1q family members. To investigate this, adiponectin, representative family members, and variants were expressed in Expi293 cells and tested for binding to lipids in liposomes using density centrifugation. Binding to physiological lipids were also analyzed using gradient ultracentrifugation, liquid chromatography-mass spectrometry, and shotgun lipidomics. Interestingly, adiponectin selectively bound several anionic phospholipids and sphingolipids, including phosphatidylserine, ceramide-1-phosphate, glucosylceramide, and sulfatide, via the C1q domain in an oligomerization-dependent fashion. Binding to lipids was observed in liposomes, low-density lipoproteins, cell membranes, and plasma. Other CTRPs and C1q family members (Cbln1, CTRP1, CTRP5, and CTRP13) also bound similar lipids. These findings suggest that adiponectin and CTRPs function not only as hormones, but also as lipid opsonins, as may other C1q family proteins.
Assuntos
Adiponectina/metabolismo , Complemento C1q/metabolismo , Fosfolipídeos/metabolismo , Esfingolipídeos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adipocinas/metabolismo , Adiponectina/genética , Animais , Ânions , Membrana Celular , LDL-Colesterol , Humanos , Metabolismo dos Lipídeos , Lipidômica , Lipoproteínas/metabolismo , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Opsonizantes/metabolismo , PlasmaRESUMO
Global investigation of the 3' extremity of mRNA (3'-terminome), despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequences and relative paucity of mRNA. We here develop a method, TAIL-seq, to sequence the very end of mRNA molecules. TAIL-seq allows us to measure poly(A) tail length at the genomic scale. Median poly(A) length is 50-100 nt in HeLa and NIH 3T3 cells. Poly(A) length correlates with mRNA half-life, but not with translational efficiency. Surprisingly, we discover widespread uridylation and guanylation at the downstream of poly(A) tail. The U tails are generally attached to short poly(A) tails (<25 nt), while the G tails are found mainly on longer poly(A) tails (>40 nt), implicating their generic roles in mRNA stability control. TAIL-seq is a potent tool to dissect dynamic control of mRNA turnover and translational control, and to discover unforeseen features of RNA cleavage and tailing.
Assuntos
Regiões 3' não Traduzidas , Genoma , MicroRNAs/genética , Estabilidade de RNA , Análise de Sequência de RNA/métodos , Animais , Sequência de Bases , Regulação da Expressão Gênica , Guanina/metabolismo , Meia-Vida , Células HeLa , Humanos , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Células NIH 3T3 , Poliadenilação , Transdução de Sinais , Uridina/metabolismoRESUMO
Early development depends heavily on accurate control of maternally inherited mRNAs, and yet it remains unknown how maternal microRNAs are regulated during maternal-to-zygotic transition (MZT). We here find that maternal microRNAs are highly adenylated at their 3' ends in mature oocytes and early embryos. Maternal microRNA adenylation is widely conserved in fly, sea urchin, and mouse. We identify Wispy, a noncanonical poly(A) polymerase, as the enzyme responsible for microRNA adenylation in flies. Knockout of wispy abrogates adenylation and results in microRNA accumulation in eggs, whereas overexpression of Wispy increases adenylation and reduces microRNA levels in S2 cells. Wispy interacts with Ago1 through protein-protein interaction, which may allow the effective and selective adenylation of microRNAs. Thus, adenylation may contribute to the clearance of maternally deposited microRNAs during MZT. Our work provides mechanistic insights into the regulation of maternal microRNAs and illustrates the importance of RNA tailing in development.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , MicroRNAs/metabolismo , Poli A/genética , Polinucleotídeo Adenililtransferase/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Dados de Sequência Molecular , Oócitos/crescimento & desenvolvimento , Polinucleotídeo Adenililtransferase/genéticaRESUMO
Prader-Willi Syndrome (PWS) is caused by loss of expression of paternally expressed genes in the human 15q11.2-q13 imprinting domain. A set of imprinted genes that are active on the paternal but silenced on the maternal chromosome are intricately regulated by a bipartite imprinting center (PWS-IC) located in the PWS imprinting domain. In past work, we discovered that euchromatic histone lysine N-methyltransferase-2 (EHMT2/G9a) inhibitors were capable of un-silencing PWS-associated genes by restoring their expression from the maternal chromosome. Here, in mice lacking the Ehmt2 gene, we document un-silencing of the imprinted Snrpn/Snhg14 gene on the maternal chromosome in the late embryonic and postnatal brain. Using PWS and Angelman syndrome patient derived cells with either paternal or maternal deletion of 15q11-q13, we have found that chromatin of maternal PWS-IC is closed and has compact 3D folding confirmation. We further show that a new and distinct noncoding RNA preferentially transcribed from upstream of the PWS-IC interacts with EHMT2 and forms a heterochromatin complex to silence gene expression of SNRPN in CIS on maternal chromosome. Taken together, these findings demonstrate that allele-specific recruitment of EHMT2 is required to maintain the maternal imprints. Our findings provide novel mechanistic insights and support a new model for imprinting maintenance of the PWS imprinted domain.
RESUMO
Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2-knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.
RESUMO
Deadenylation generally constitutes the first and pivotal step in eukaryotic messenger RNA decay. Despite its importance in posttranscriptional regulations, the kinetics of deadenylation and its regulation remain largely unexplored. Here we identify La ribonucleoprotein 1, translational regulator (LARP1) as a general decelerator of deadenylation, which acts mainly in the 30-60-nucleotide (nt) poly(A) length window. We measured the steady-state and pulse-chased distribution of poly(A)-tail length, and found that deadenylation slows down in the 30-60-nt range. LARP1 associates preferentially with short tails and its depletion results in accelerated deadenylation specifically in the 30-60-nt range. Consistently, LARP1 knockdown leads to a global reduction of messenger RNA abundance. LARP1 interferes with the CCR4-NOT-mediated deadenylation in vitro by forming a ternary complex with poly(A)-binding protein (PABP) and poly(A). Together, our work reveals a dynamic nature of deadenylation kinetics and a role of LARP1 as a poly(A) length-specific barricade that creates a threshold for deadenylation.
Assuntos
Exorribonucleases , Proteínas de Ligação a RNA , Exorribonucleases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a Poli(A)/genética , Regulação da Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Poli A/metabolismoRESUMO
Antiviral immune mediators, including interferons and their downstream effectors, are critical for host defense yet can become detrimental when uncontrolled. Here, we identify a macrophage-mediated anti-inflammatory mechanism that limits type I interferon (IFN-I) responses. Specifically, we found that cellular stress and pathogen recognition induce Oncostatin M (OSM) production by macrophages. OSM-deficient mice succumbed to challenge with influenza or a viral mimic due to heightened IFN-I activation. Macrophage-derived OSM restricted excessive IFN-I production by lung epithelial cells following viral stimulation. Furthermore, reconstitution of OSM in the respiratory tract was sufficient to protect mice lacking macrophage-derived OSM against morbidity, indicating the importance of local OSM production. This work reveals a host strategy to dampen inflammation in the lung through the negative regulation of IFN-I by macrophages.
RESUMO
IgE mediates allergic responses by coating mast cell or basophil surfaces and inducing degranulation upon binding a specific allergen. IgE can also be spontaneously produced in the absence of foreign allergens; yet the origin, regulation, and functions of such "natural" IgE still remain largely unknown. Here, we find that glucocorticoids enhance the production of IgE in B cells both in vivo and ex vivo without antigenic challenge. Such IgE production is promoted by B cell-intrinsic glucocorticoid receptor signaling that reinforces CD40 signaling and synergizes with the IL-4/STAT6 pathway. In addition, we found that rare B cells in the mesenteric lymph nodes are responsible for the production of glucocorticoid-inducible IgE. Furthermore, locally produced glucocorticoids in the gut may induce natural IgE during perturbations of gut homeostasis, such as dysbiosis. Notably, mice preemptively treated with glucocorticoids were protected from subsequent pathogenic anaphylaxis. Together, our results suggest that glucocorticoids, classically considered to be broadly immunosuppressive, have a selective immunostimulatory role in B cells.
Assuntos
Anafilaxia , Glucocorticoides , Alérgenos , Anafilaxia/metabolismo , Animais , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Imunoglobulina E/metabolismo , Mastócitos , CamundongosRESUMO
The lipid raft-resident protein, MAL2, has been implicated as contributing to the pathogenesis of several malignancies, including breast cancer, but the underlying mechanism for its effects on tumorigenesis is unknown. Here, we show that MAL2-mediated lipid raft formation leads to HER2 plasma membrane retention and enhanced HER2 signaling in breast cancer cells. We demonstrate physical interactions between HER2 and MAL2 in lipid rafts using proximity ligation assays. Super-resolution structured illumination microscopy imaging displays the structural organization of the HER2/Ezrin/NHERF1/PMCA2 protein complex. Formation of this protein complex maintains low intracellular calcium concentrations in the vicinity of the plasma membrane. HER2/MAL2 protein interactions in lipid rafts are enhanced in trastuzumab-resistant breast cancer cells. Our findings suggest that MAL2 is crucial for lipid raft formation, HER2 signaling, and HER2 membrane stability in breast cancer cells, suggesting MAL2 as a potential therapeutic target.
Assuntos
Neoplasias da Mama/patologia , Proteínas do Citoesqueleto/metabolismo , Resistencia a Medicamentos Antineoplásicos , Microdomínios da Membrana/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Fosfoproteínas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Receptor ErbB-2/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Proteínas do Citoesqueleto/genética , Endocitose , Feminino , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Fosfoproteínas/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptor ErbB-2/genética , Trocadores de Sódio-Hidrogênio/genética , Trastuzumab/farmacologia , Células Tumorais CultivadasRESUMO
The intestine is a site of direct encounter with the external environment and must consequently balance barrier defense with nutrient uptake. To investigate how nutrient uptake is regulated in the small intestine, we tested the effect of diets with different macronutrient compositions on epithelial gene expression. We found that enzymes and transporters required for carbohydrate digestion and absorption were regulated by carbohydrate availability. The "on-demand" induction of this machinery required γδ T cells, which regulated this program through the suppression of interleukin-22 production by type 3 innate lymphoid cells. Nutrient availability altered the tissue localization and transcriptome of γδ T cells. Additionally, transcriptional responses to diet involved cellular remodeling of the epithelial compartment. Thus, this work identifies a role for γδ T cells in nutrient sensing.
Assuntos
Carboidratos da Dieta/administração & dosagem , Carboidratos da Dieta/metabolismo , Enterócitos/fisiologia , Interleucinas/metabolismo , Mucosa Intestinal/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T/fisiologia , Adaptação Fisiológica , Animais , Comunicação Celular , Proteínas Alimentares/administração & dosagem , Digestão , Regulação da Expressão Gênica , Interleucinas/genética , Absorção Intestinal , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Camundongos Endogâmicos C57BL , Nutrientes/administração & dosagem , Nutrientes/metabolismo , Subpopulações de Linfócitos T/imunologia , Transcrição Gênica , Transcriptoma , Interleucina 22RESUMO
Enhanced stemness in colorectal cancer has been reported and it contributes to aggressive progression, but the underlying mechanisms remain unclear. Here we report a Wnt ligand, Dickkopf-2 (DKK2) is essential for developing colorectal cancer stemness. Genetic depletion of DKK2 in intestinal epithelial or stem cells reduced tumorigenesis and expression of the stem cell marker genes including LGR5 in a model of colitis-associated cancer. Sequential mutations in APC, KRAS, TP53, and SMAD4 genes in colonic organoids revealed a significant increase of DKK2 expression by APC knockout and further increased by additional KRAS and TP53 mutations. Moreover, DKK2 activates proto-oncogene tyrosine-protein kinse Src followed by increased LGR5 expressing cells in colorectal cancer through degradation of HNF4α1 protein. These findings suggest that DKK2 is required for colonic epithelial cells to enhance LGR5 expression during the progression of colorectal cancer.
RESUMO
T cells maintain a quiescent state prior to activation. As inappropriate T cell activation can cause disease, T cell quiescence must be preserved. Despite its importance, the mechanisms underlying the "quiescent state" remain elusive. Here, we identify BTG1 and BTG2 (BTG1/2) as factors responsible for T cell quiescence. BTG1/2-deficient T cells show an increased proliferation and spontaneous activation due to a global increase in messenger RNA (mRNA) abundance, which reduces the threshold to activation. BTG1/2 deficiency leads to an increase in polyadenylate tail length, resulting in a greater mRNA half-life. Thus, BTG1/2 promote the deadenylation and degradation of mRNA to secure T cell quiescence. Our study reveals a key mechanism underlying T cell quiescence and suggests that low mRNA abundance is a crucial feature for maintaining quiescence.