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1.
Nat Commun ; 15(1): 588, 2024 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-38238288

RESUMO

Despite significant research, mechanisms underlying the failure of islet beta cells that result in type 2 diabetes (T2D) are still under investigation. Here, we report that Sox9, a transcriptional regulator of pancreas development, also functions in mature beta cells. Our results show that Sox9-depleted rodent beta cells have defective insulin secretion, and aging animals develop glucose intolerance, mimicking the progressive degeneration observed in T2D. Using genome editing in human stem cells, we show that beta cells lacking SOX9 have stunted first-phase insulin secretion. In human and rodent cells, loss of Sox9 disrupts alternative splicing and triggers accumulation of non-functional isoforms of genes with key roles in beta cell function. Sox9 depletion reduces expression of protein-coding splice variants of the serine-rich splicing factor arginine SRSF5, a major splicing enhancer that regulates alternative splicing. Our data highlight the role of SOX9 as a regulator of alternative splicing in mature beta cell function.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Humanos , Processamento Alternativo/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Splicing de RNA
2.
Mol Cell ; 74(1): 32-44.e8, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30846318

RESUMO

Excessive levels of saturated fatty acids are toxic to cells, although the basis for this lipotoxicity remains incompletely understood. Here, we analyzed the transcriptome, lipidome, and genetic interactions of human leukemia cells exposed to palmitate. Palmitate treatment increased saturated glycerolipids, accompanied by a transcriptional stress response, including upregulation of the endoplasmic reticulum (ER) stress response. A comprehensive genome-wide short hairpin RNA (shRNA) screen identified >350 genes modulating lipotoxicity. Among previously unknown genetic modifiers of lipotoxicity, depletion of RNF213, a putative ubiquitin ligase mutated in Moyamoya vascular disease, protected cells from lipotoxicity. On a broader level, integration of our comprehensive datasets revealed that changes in di-saturated glycerolipids, but not other lipid classes, are central to lipotoxicity in this model. Consistent with this, inhibition of ER-localized glycerol-3-phosphate acyltransferase activity protected from all aspects of lipotoxicity. Identification of genes modulating the response to saturated fatty acids may reveal novel therapeutic strategies for treating metabolic diseases linked to lipotoxicity.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Glicerídeos/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Palmítico/toxicidade , Aciltransferases/genética , Aciltransferases/metabolismo , Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático/genética , Regulação Enzimológica da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Células K562 , Metabolismo dos Lipídeos/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismo
3.
Mol Metab ; 4(11): 834-45, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26629407

RESUMO

OBJECTIVE: Circadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion. METHODS: We established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock. RESULTS: Bioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption. CONCLUSIONS: Our study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes.

4.
Nat Cell Biol ; 17(9): 1145-57, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26302406

RESUMO

E-cadherin and p120 catenin (p120) are essential for epithelial homeostasis, but can also exert pro-tumorigenic activities. Here, we resolve this apparent paradox by identifying two spatially and functionally distinct junctional complexes in non-transformed polarized epithelial cells: one growth suppressing at the apical zonula adherens (ZA), defined by the p120 partner PLEKHA7 and a non-nuclear subset of the core microprocessor components DROSHA and DGCR8, and one growth promoting at basolateral areas of cell-cell contact containing tyrosine-phosphorylated p120 and active Src. Recruitment of DROSHA and DGCR8 to the ZA is PLEKHA7 dependent. The PLEKHA7-microprocessor complex co-precipitates with primary microRNAs (pri-miRNAs) and possesses pri-miRNA processing activity. PLEKHA7 regulates the levels of select miRNAs, in particular processing of miR-30b, to suppress expression of cell transforming markers promoted by the basolateral complex, including SNAI1, MYC and CCND1. Our work identifies a mechanism through which adhesion complexes regulate cellular behaviour and reveals their surprising association with the microprocessor.


Assuntos
Caderinas/fisiologia , Cateninas/metabolismo , MicroRNAs/metabolismo , Quinases da Família src/metabolismo , Junções Aderentes/metabolismo , Animais , Antígenos CD , Células CACO-2 , Proteínas de Transporte/metabolismo , Cães , Humanos , Células Madin Darby de Rim Canino , MicroRNAs/genética , Transporte Proteico , Interferência de RNA , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , delta Catenina
5.
Essays Biochem ; 53: 83-93, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22928510

RESUMO

The AJC (apical junctional complex) of vertebrate epithelial cells orchestrates cell-cell adhesion and tissue barrier function. In addition, it plays a pivotal role in signalling. Several protein components of the AJC, e.g. the cytoplasmic proteins ß-catenin, p120-catenin and ZO (Zonula Occludens)-2, can shuttle to the nucleus, where they interact with transcription factors to regulate gene expression and cell proliferation. Other junctional proteins, e.g. angiomotin, α-catenin and cingulin, are believed to act by sequestering either transcription factors, such as YAP (Yes-associated protein), or regulators of small GTPases, such as GEF (guanine-nucleotide-exchange factor)-H1, at junctions. The signalling activities of AJC proteins are triggered by different extracellular and intracellular cues, including cell density, and physiological or pathological activation of developmentally regulated pathways, such as the Wnt pathway. The interplay between junctional protein complexes, the actin cytoskeleton and signalling pathways is of crucial importance in the regulation of gene expression and cell proliferation.


Assuntos
Proliferação de Células , Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Proteínas de Junções Íntimas/genética , Animais , Células Epiteliais/metabolismo , Proteínas de Junções Íntimas/metabolismo , Transcrição Gênica , Via de Sinalização Wnt
6.
Ann N Y Acad Sci ; 1257: 125-32, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22671598

RESUMO

Cingulin, paracingulin, and PLEKHA7 are proteins localized in the cytoplasmic region of the apical junctional complex of vertebrate epithelial cells. Cingulin has been detected at tight junctions (TJs), whereas paracingulin has been detected at both TJs and adherens junctions (AJs) and PLEKHA7 has been detected at AJs. One function of cingulin and paracingulin is to regulate the activity of Rho family GTPases at junctions through their direct interaction with guanidine exchange factors of RhoA and Rac1. Cingulin also contributes to the regulation of transcription of several genes in different types of cultured cells, in part through its ability to modulate RhoA activity. PLEKHA7, together with paracingulin, is part of a protein complex that links E-cadherin to the microtubule cytoskeleton at AJs. In this paper, we review the current knowledge about these proteins, including their discovery, the characterization of their expression, localization, structure, molecular interactions, and their roles in different developmental and disease model systems.


Assuntos
Junções Aderentes/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Junções Íntimas/metabolismo , Células Epiteliais/citologia , Humanos , Transdução de Sinais , Proteína da Zônula de Oclusão-1/metabolismo
7.
Mol Membr Biol ; 28(7-8): 427-44, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21781017

RESUMO

Small GTPases of the Rho family (RhoA, Rac1, and Cdc42) and the Ras family GTPase Rap1 are essential for the assembly and function of epithelial cell-cell junctions. Through their downstream effectors, small GTPases modulate junction formation and stability, primarily by orchestrating the polymerization and contractility of the actomyosin cytoskeleton. The major upstream regulators of small GTPases are guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Several GEFs and a few GAPs have been localized at epithelial junctions, and bind to specific junctional proteins. Thus, junctional proteins can regulate small GTPases at junctions, through their interactions with GEFs and GAPs. Here we review the current knowledge about the mechanisms of regulation of small GTPases by junctional proteins. Understanding these mechanisms will help to clarify at the molecular level how small GTPases control the morphogenesis and physiology of epithelial tissues, and how they are disregulated in disease.


Assuntos
Células Epiteliais/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Junções Íntimas/enzimologia , Actomiosina/genética , Actomiosina/metabolismo , Junções Aderentes/enzimologia , Junções Aderentes/genética , Animais , Citoesqueleto/genética , Citoesqueleto/metabolismo , Desmossomos/enzimologia , Desmossomos/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Morfogênese/genética , Junções Íntimas/genética , Vertebrados , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
8.
J Biol Chem ; 286(19): 16743-50, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21454477

RESUMO

Paracingulin is a 160-kDa protein localized in the cytoplasmic region of epithelial tight and adherens junctions, where it regulates RhoA and Rac1 activities by interacting with guanine nucleotide exchange factors. Here, we investigate the molecular mechanisms that control the recruitment of paracingulin to cell-cell junctions. We show that paracingulin forms a complex with the tight junction protein ZO-1, and the globular head domain of paracingulin interacts directly with ZO-1 through an N-terminal region containing a conserved ZIM (ZO-1-Interaction-Motif) sequence. Recruitment of paracingulin to cadherin-based cell-cell junctions in Rat1 fibroblasts requires the ZIM-containing region, whereas in epithelial cells removal of this region decreases the junctional localization of paracingulin at tight junctions but not at adherens junctions. Depletion of ZO-1, but not ZO-2, reduces paracingulin accumulation at tight junctions. A yeast two-hybrid screen identifies both ZO-1 and the adherens junction protein PLEKHA7 as paracingulin-binding proteins. Paracingulin forms a complex with PLEKHA7 and its interacting partner p120ctn, and the globular head domain of paracingulin interacts directly with a central region of PLEKHA7. Depletion of PLEKHA7 from Madin-Darby canine kidney cells results in the loss of junctional localization of paracingulin and a decrease in its expression. In summary, we characterize ZO-1 and PLEKHA7 as paracingulin-interacting proteins that are involved in its recruitment to epithelial tight and adherens junctions, respectively.


Assuntos
Junções Aderentes/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/química , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Animais , Células CACO-2 , Caderinas/química , Cateninas/química , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Cães , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Técnicas do Sistema de Duplo-Híbrido , Proteína da Zônula de Oclusão-1 , delta Catenina
9.
Mol Membr Biol ; 28(2): 123-35, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21166484

RESUMO

Cingulin (CGN) and paracingulin (CGNL1) are structurally related proteins that regulate Rho family GTPases by recruiting guanine nucleotide exchange factors to epithelial junctions. Although the subcellular localization of cingulin and paracingulin is likely to be essential for their role as adaptor proteins, nothing is known on their in vivo localization, and their dynamics of exchange with the junctional membrane. To address these questions, we generated stable clones of MDCK cells expressing fluorescently tagged cingulin and paracingulin. By FRAP analysis, cingulin and paracingulin show a very similar dynamic behaviour, with recovery curves and mobile fractions that are distinct from ZO-1, and indicate a rapid exchange with a cytosolic pool. Interestingly, only paracingulin, but not cingulin, is peripherally localized in isolated cells, requires the integrity of the microtubule cytoskeleton to be stably anchored to junctions, and associates with E-cadherin. In contrast, both proteins require the integrity of the actin cytoskeleton to maintain their junctional localization. Although cingulin and paracingulin form a complex and can interact in vitro, the junctional recruitment and the dynamics of membrane exchange of paracingulin is independent of cingulin, and vice-versa. In summary, cingulin and paracingulin show a similar dynamic behaviour, but partially distinct localizations and functional interactions with the cytoskeleton, and are recruited independently to junctions.


Assuntos
Junções Aderentes/metabolismo , Membrana Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Junções Aderentes/fisiologia , Junções Aderentes/ultraestrutura , Animais , Caderinas/metabolismo , Linhagem Celular , Citoesqueleto/metabolismo , Cães , Imunofluorescência , Rim/citologia , Proteínas dos Microtúbulos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/fisiologia , Junções Íntimas/ultraestrutura
10.
PLoS One ; 5(8): e12207, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20808826

RESUMO

The pleckstrin-homology-domain-containing protein PLEKHA7 was recently identified as a protein linking the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Here we characterize the expression, tissue distribution and subcellular localization of PLEKHA7 by immunoblotting, immunofluorescence microscopy, immunoelectron microscopy, and northern blotting in mammalian tissues. Anti-PLEKHA7 antibodies label the junctional regions of cultured kidney epithelial cells by immunofluorescence microscopy, and major polypeptides of M(r) approximately 135 kDa and approximately 145 kDa by immunoblotting of lysates of cells and tissues. Two PLEKHA7 transcripts ( approximately 5.5 kb and approximately 6.5 kb) are detected in epithelial tissues. PLEKHA7 is detected at epithelial junctions in sections of kidney, liver, pancreas, intestine, retina, and cornea, and its tissue distribution and subcellular localization are distinct from ZO-1. For example, PLEKHA7 is not detected within kidney glomeruli. Similarly to E-cadherin, p120 ctn, beta-catenin and alpha-catenin, PLEKHA7 is concentrated in the apical junctional belt, but unlike these adherens junction markers, and similarly to afadin, PLEKHA7 is not localized along the lateral region of polarized epithelial cells. Immunoelectron microscopy definitively establishes that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of 28 nm from the plasma membrane. In summary, we show that PLEKHA7 is a cytoplasmic component of the epithelial adherens junction belt, with a subcellular localization and tissue distribution that is distinct from that of ZO-1 and most AJ proteins, and we provide the first description of its distribution and localization in several tissues.


Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Células Epiteliais/citologia , Espaço Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Animais , Especificidade de Anticorpos , Biomarcadores/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Linhagem Celular , Cães , Células Epiteliais/metabolismo , Humanos , Rim/citologia , Camundongos , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteína da Zônula de Oclusão-1
11.
Ann N Y Acad Sci ; 1165: 88-98, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19538293

RESUMO

Tight junctions (TJ) regulate the passage of solutes across epithelial sheets, contribute to the establishment and maintenance of epithelial apico-basal polarity and are involved in the regulation of gene expression and cell proliferation. Cingulin, a Mr 140 kDa protein localized in the cytoplasmic region of TJ, is not directly required for TJ formation and epithelial polarity but regulates RhoA signaling, through its interaction with the RhoA activator GEF-H1, and gene expression. Here we describe in more detail the effect of cingulin mutation in embryoid bodies (EB) on gene expression, by identifying the genes that show the highest degree of up- or downregulation, and the putative canonical pathways that might be affected by cingulin. Furthermore, we show that full-length canine GEF-H1, produced in baculovirus-infected insect cells, interacts with regions both in the cingulin globular head, and in the coiled-coil rod domain. These results extend our previous studies and provide new perspectives for the mechanistic analysis of cingulin function.


Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/fisiologia , Transdução de Sinais/genética , Junções Íntimas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Comunicação Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Mutação , Proteína rhoA de Ligação ao GTP/genética
12.
Biochim Biophys Acta ; 1778(3): 601-13, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18339298

RESUMO

The region of cytoplasm underlying the tight junction (TJ) contains several multimolecular protein complexes, which are involved in scaffolding of membrane proteins, regulation of cytoskeletal organization, establishment of polarity, and signalling to and from the nucleus. In this review, we summarize some of the most recent advances in understanding the identity of these proteins, their domain organization, their protein interactions, and their functions in vertebrate organisms. Analysis of knockdown and knockout model systems shows that several TJ proteins are essential for the formation of epithelial tissues and early embryonic development, whereas others appear to have redundant functions.


Assuntos
Junções Íntimas/fisiologia , Animais , Citoplasma/química , Citoplasma/genética , Citoplasma/fisiologia , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Modelos Biológicos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Complexos Multiproteicos , Domínios PDZ , Transdução de Sinais , Junções Íntimas/química , Junções Íntimas/genética
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