RESUMO
The two paralogous Arabidopsis genes MAINTENANCE OF MERISTEMS (MAIN) and MAINTENANCE OF MERISTEMS LIKE1 (MAIL1) encode a conserved retrotransposon-related plant mobile domain and are known to be required for silencing of transposable elements (TE) and for primary root development. Loss of function of either MAIN or MAIL1 leads to release of heterochromatic TEs, reduced condensation of pericentromeric heterochromatin, cell death of meristem cells and growth arrest of the primary root soon after germination. Here, we show that they act in one protein complex that also contains the inactive isoform of PROTEIN PHOSPHATASE 7 (PP7), which is named PROTEIN PHOSPHATASE 7-LIKE (PP7L). PP7L was previously shown to be important for chloroplast biogenesis and efficient chloroplast protein synthesis. We show that loss of PP7L function leads to the same root growth phenotype as loss of MAIL1 or MAIN. In addition, pp7l mutants show similar silencing defects. Double mutant analyses confirmed that the three proteins act in the same molecular pathway. The primary root growth arrest, which is associated with cell death of stem cells and their daughter cells, is a consequence of genome instability. Our data demonstrate so far unrecognized functions of an inactive phosphatase isoform in a protein complex that is essential for silencing of heterochromatic elements and for maintenance of genome stability in dividing cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Elementos de DNA Transponíveis/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Inativação Gênica , Germinação , Heterocromatina/genética , Isoenzimas , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/fisiologia , Mutação , Proteínas Nucleares/genética , Fenótipo , Fosfoproteínas Fosfatases/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Retroelementos/genéticaRESUMO
Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain. The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the ß1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether ß1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1ß (IL-1ß)-induced inflammatory in vitro BBB model. We demonstrate that IL-1ß increases cell-matrix adhesion and induces a redistribution of active ß1 integrins to the basal surface. In particular, binding of α5ß1 integrin to its ligand fibronectin is enhanced and α5ß1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5ß1 integrin reduces the IL-1ß-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs). These data imply that IL-1ß-induced inflammation not only destabilizes tight junctions but also increases α5ß1 integrin-dependent cell-matrix adhesion to fibronectin.
Assuntos
Encéfalo/irrigação sanguínea , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Integrina alfa5beta1/metabolismo , Interleucina-1beta/farmacologia , Leucócitos Mononucleares/fisiologia , Migração Transendotelial e Transepitelial , Barreira Hematoencefálica , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Fibronectinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina alfa5/metabolismo , Integrina alfa5beta1/antagonistas & inibidores , Integrina beta1/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
Integrin α1ß1 is an adhesion receptor that binds to collagen and laminin. It regulates cell adhesion, cytoskeletal organization, and migration. The cytoplasmic tail of the α1 subunit consists of 15 amino acids and contains six positively charged lysine residues. In this study, we present evidence that the α1 integrin cytoplasmic tail (α1CT) directly associates with phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Since the association was disrupted by calcium, magnesium and phosphate ions, this interaction appears to be in ionic nature. Here, the peptide-lipid interaction was driven by the conserved KIGFFKR motif. The exchange of both two potential phospholipid-binding lysines for glycines in the KIGFFKR motif increased α1ß1 integrin-specific adhesion and F-actin cytoskeleton formation compared to cells expressing the unmodified α1 subunit, whereas only mutation of the second lysine at position 1171 increased levels of constitutively active α1ß1 integrins on the cell surface. In addition, enhanced focal adhesion formation and increased phosphorylation of focal adhesion kinase, but decreased phosphorylation of AKT was observed in these cells. We conclude that the KIGFFKR motif, and in particular lysine1171 is involved in the dynamic regulation of α1ß1 integrin activity and that the interaction of α1CT with phosphoinositides may contribute to this process.
Assuntos
Integrina alfa1 , Proteínas Proto-Oncogênicas c-akt , Integrina alfa1/química , Fosfatidilinositóis , Lisina , Adesão Celular/genéticaRESUMO
Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Aterosclerose/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Mutação com Ganho de Função , Técnicas de Inativação de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Mutação com Perda de Função , Camundongos , Transdução de SinaisRESUMO
The transcriptional repressors Hey1 and Hey2 are primary target genes of Notch signaling in the cardiovascular system and induction of Hey gene expression is often interpreted as activation of Notch signaling. Here we report that treatment of primary human endothelial cells with serum or fresh growth medium led to a strong wave of Hey1 and Hey2 transcription lasting for approximately three hours. Transcription of other Notch target genes (Hes1, Hes5, ephrinB2, Dll4) was however not induced by serum in endothelial cells. Gamma secretase inhibition or expression of dominant-negative MAML1 did not prevent the induction of Hey genes indicating that canonical Notch signaling is dispensable. Pretreatment with soluble BMP receptor Alk1, but not Alk3, abolished Hey gene induction by serum. Consequently, the Alk1 ligand BMP9 stimulated Hey gene induction in endothelial cells. Several other cell types however did not show such a strong BMP signaling and consequently only a very mild induction of Hey genes. Taken together, the experiments revealed that bone morphogenic proteins within the serum of cell culture medium are potent inducers of endothelial Hey1 and Hey2 gene expression within the first few hours after medium change.
Assuntos
Receptores de Activinas Tipo II/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas Repressoras/genética , Soro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Linhagem Celular , Meios de Cultura , Células Endoteliais/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/farmacologia , Humanos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/metabolismo , Proteína Smad5/metabolismoRESUMO
Wound repair is a quiescent mechanism to restore barriers in multicellular organisms upon injury. In chronic wounds, however, this program prematurely stalls. It is known that patterns of extracellular signals within the wound fluid are crucial to healing. Extracellular pH (pHe) is precisely regulated and potentially important in signaling within wounds due to its diverse cellular effects. Additionally, sufficient oxygenation is a prerequisite for cell proliferation and protein synthesis during tissue repair. It was, however, impossible to study these parameters in vivo due to the lack of imaging tools. Here, we present luminescent biocompatible sensor foils for dual imaging of pHe and oxygenation in vivo. To visualize pHe and oxygen, we used time-domain dual lifetime referencing (tdDLR) and luminescence lifetime imaging (LLI), respectively. With these dual sensors, we discovered centripetally increasing pHe-gradients on human chronic wound surfaces. In a therapeutic approach, we identify pHe-gradients as pivotal governors of cell proliferation and migration, and show that these pHe-gradients disrupt epidermal barrier repair, thus wound closure. Parallel oxygen imaging also revealed marked hypoxia, albeit with no correlating oxygen partial pressure (pO2)-gradient. This highlights the distinct role of pHe-gradients in perturbed healing. We also found that pHe-gradients on chronic wounds of humans are predominantly generated via centrifugally increasing pHe-regulatory Na+/H+-exchanger-1 (NHE1)-expression. We show that the modification of pHe on chronic wound surfaces poses a promising strategy to improve healing. The study has broad implications for cell science where spatial pHe-variations play key roles, e.g. in tumor growth. Furthermore, the novel dual sensors presented herein can be used to visualize pHe and oxygenation in various biomedical fields.
Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes , Reepitelização , Úlcera Varicosa/metabolismo , Idoso , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Hipóxia Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Feminino , Humanos , Concentração de Íons de Hidrogênio , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Masculino , Microscopia de Fluorescência/métodos , Pessoa de Meia-Idade , Imagem Óptica/métodos , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Úlcera Varicosa/patologiaRESUMO
SIGNIFICANCE: Several redox compounds, including respiratory burst oxidase homologs (Rboh) and iron chelate reductases have been identified in animal and plant plasma membrane (PM). Studies using molecular biological, biochemical, and proteomic approaches suggest that PM redox systems of plants are involved in signal transduction, nutrient uptake, transport, and cell wall-related processes. Function of PM-bound redox systems in oxidative stress will be discussed. RECENT ADVANCES: Present knowledge about the properties, structures, and functions of these systems are summarized. Judging from the currently available data, it is likely that electrons are transferred from cytosolic NAD(P)H to the apoplast via quinone reductases, vitamin K, and a cytochrome b561. In tandem with these electrons, protons might be transported to the apoplastic space. CRITICAL ISSUES: Recent studies suggest localization of PM-bound redox systems in microdomains (so-called lipid or membrane rafts), but also organization of these compounds in putative and high molecular mass protein complexes. Although the plant flavocytochrome b family is well characterized with respect to its function, the molecular mechanism of an electron transfer reaction by these compounds has to be verified. Localization of Rboh in other compartments needs elucidation. FUTURE DIRECTIONS: Plant members of the flavodoxin and flavodoxin-like protein family and the cytochrome b561 protein family have been characterized on the biochemical level, postulated localization, and functions of these redox compounds need verification. Compositions of single microdomains and interaction partners of PM redox systems have to be elucidated. Finally, the hypothesis of an electron transfer chain in the PM needs further proof.