RESUMO
Thrombospondin-1 (TSP1) inhibits angiogenesis, in part, by interacting with the ubiquitous cell-surface receptor CD47. In endothelial cells, CD47 interacts directly with vascular endothelial growth factor receptor (VEGFR)-2, and TSP1 inhibits VEGFR2 phosphorylation and signaling by disrupting this association. We show that CD47 similarly associates with and regulates VEGFR2 in T cells. TSP1 inhibits phosphorylation of VEGFR2 and its downstream target Src in wild type but not in CD47-deficient human Jurkat and primary murine T cells. VEGFR2 signaling inhibits proliferation and TCR signaling in wild type T cells. However, ligation of CD47 by TSP1 or loss of CD47 expression reverses some inhibitory effects of VEGF on proliferation and T cell activation. We further found that VEGF and VEGFR2 expression are upregulated in CD47-deficient murine CD4(+) and human Jurkat T cells, and the resulting autocrine VEGFR2 signaling enhances proliferation and some TCR responses in the absence of CD47. Thus, CD47 signaling modulates the ability of VEGF to regulate proliferation and TCR signaling, and autocrine production of VEGF by T cells contributes to this regulation. This provides a mechanism to understand the context-dependent effects of TSP1 and VEGF on T cell activation, and reveals an important role for CD47 signaling in regulating T cell production of the major angiogenic factor VEGF.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígeno CD47/imunologia , Tolerância Imunológica , Trombospondina 1/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antígeno CD47/biossíntese , Antígeno CD47/genética , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Neovascularização Patológica/imunologia , Fosforilação , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Quinases da Família src/metabolismoRESUMO
Ribosome assembly begins with conversion of a polycistronic precursor into 18S, 5.8S, and 25S rRNAs. In the ascomycete fungus Candida albicans, rRNA transcription starts 604 nt upstream of the 18S rRNA junction (site A1). One major internal processing site in the 5' external transcribed spacer (A0) occurs 108 nt from site A1. The A0-A1 fragment persists as a stable species during log phase growth and can be used to assess proliferation rates. Separation of the small and large subunit pre-rRNAs occurs at sites A2 and A3 in internal transcribed spacer-1 Saccharomyces cerevisiae pre-rRNA. However, the 5' end of the 5.8S rRNA is represented by only a 5.8S (S) form, and a 7S rRNA precursor of the 5.8S rRNA extends into internal transcribed spacer 1 to site A2, which differs from S. cerevisiae. External transcribed spacer 1 and internal transcribed spacers 1 and 2 show remarkable structural similarity with S. cerevisiae despite low sequence identity. Maturation of C. albicans rRNA resembles other eukaryotes in that processing can occur cotranscriptionally or post-transcriptionally. During rapid proliferation, U3 snoRNA-dependent processing occurs before large and small subunit rRNA separation, consistent with cotranscriptional processing. As cells pass the diauxic transition, the 18S pre-rRNA accumulates into stationary phase as a 23S species, possessing an intact 5' external transcribed spacer extending to site A3. Nutrient addition to starved cells results in the disappearance of the 23S rRNA, indicating a potential role in normal physiology. Therefore, C. albicans reveals new mechanisms that regulate post- versus cotranscriptional rRNA processing.
Assuntos
Candida albicans/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Sequência de Bases , Candida albicans/metabolismo , DNA Polimerase I/metabolismo , DNA Espaçador Ribossômico/genética , Regulação Fúngica da Expressão Gênica , Ordem dos Genes , Dados de Sequência Molecular , Peso Molecular , Conformação de Ácido Nucleico , Precursores de RNA/genética , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , Transcrição GênicaRESUMO
HBR1 (hemoglobin response gene 1) is an essential gene in Candida albicans that positively regulates mating type locus MTLα gene expression and thereby regulates cell type-specific developmental genes. Hbr1p contains a phosphate-binding loop (P-loop), a highly conserved motif characteristic of ATP- and GTP-binding proteins. Recombinant Hbr1p was isolated in an oligomeric state that specifically bound ATP with K(d) â¼2 µM. ATP but not ADP, AMP, GTP, or dATP specifically protected Hbr1p from proteolysis by trypsin. Site-directed mutagenesis of the highly conserved P-loop lysine (K22Q) and the less conserved glycine (G19S) decreased the binding affinity for soluble ATP and ATP immobilized through its γ-phosphate. ATP bound somewhat more avidly than ATPγS to wild type and mutant Hbr1p. Although Hbr1p exhibits sequence motifs characteristic of adenylate kinases, and adenylate kinase and ATPase activities have been reported for the apparent human ortholog of Hbr1p, assays for adenylate kinase activity, autophosphorylation, and ATPase activity proved negative. Overexpression of wild type but not the mutant forms of Hbr1p restored MTlα2 expression in an HBR1/hbr1 mutant, indicating that ATP binding to the P-loop is necessary for this function of Hbr1p.
Assuntos
Trifosfato de Adenosina/química , Candida albicans/metabolismo , Proteínas de Transporte/fisiologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Adenilato Quinase/química , Sequência de Aminoácidos , Sítios de Ligação , Candida/metabolismo , Proteínas de Transporte/biossíntese , Cromatografia em Gel/métodos , Dicroísmo Circular/métodos , Proteínas Fúngicas/biossíntese , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Nucleotídeos/química , Homologia de Sequência de AminoácidosRESUMO
Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent M(r) > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent M(r) 230,000) and CD47 (apparent M(r) > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser(64) and Ser(79). Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser(64).
Assuntos
Antígeno CD47/metabolismo , Heparitina Sulfato/metabolismo , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Transdução de Sinais , Trombospondina 1/fisiologia , Precursor de Proteína beta-Amiloide , Células Endoteliais , Humanos , Células Jurkat , Proteínas do Tecido Nervoso , Serina/metabolismoRESUMO
Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors. We now describe a more potent mechanism by which TSP1 inhibits VEGF receptor-2 (VEGFR2) activation through engaging its receptor CD47. CD47 ligation is known to inhibit downstream signaling targets of VEGFR2, including endothelial nitric-oxide synthase and soluble guanylate cyclase, but direct effects on VEGFR2 have not been examined. Based on FRET and co-immunoprecipitation, CD47 constitutively associated with VEGFR2. Ligation of CD47 by TSP1 abolished resonance energy transfer with VEGFR2 and inhibited phosphorylation of VEGFR2 and its downstream target Akt without inhibiting VEGF binding to VEGFR2. The inhibitory activity of TSP1 in large vessel and microvascular endothelial cells was replicated by a recombinant domain of the protein containing its CD47-binding site and by a CD47-binding peptide derived from this domain but not by the CD36-binding domain of TSP1. Inhibition of VEGFR2 phosphorylation was lost when CD47 expression was suppressed in human endothelial cells and in murine CD47-null cells. These results reveal that anti-angiogenic signaling through CD47 is highly redundant and extends beyond inhibition of nitric oxide signaling to global inhibition of VEGFR2 signaling.
Assuntos
Antígeno CD47/metabolismo , Trombospondina 1/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Células Endoteliais/citologia , Humanos , Camundongos , Microscopia Confocal/métodos , Neovascularização Patológica , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Transdução de Sinais , Trombospondinas/metabolismoRESUMO
Tumor cell migration, invasion, and angiogenesis are important determinants of tumor aggressiveness, and these traits have been associated with the motility stimulating protein autotaxin (ATX). This protein is a member of the ectonucleotide pyrophosphatase and phosphodiesterase family of enzymes, but unlike other members of this group, ATX possesses lysophospholipase D activity. This enzymatic activity hydrolyzes lysophosphatidylcholine to generate the potent tumor growth factor and motogen lysophosphatidic acid (LPA). In the current study, we show a link between ATX expression, LPA, and vascular endothelial growth factor (VEGF) signaling in ovarian cancer cell lines. Exogenous addition of VEGF-A to cultured cells induces ATX expression and secretion, resulting in increased extracellular LPA production. This elevated LPA, acting through LPA(4), modulates VEGF responsiveness by inducing VEGF receptor (VEGFR)-2 expression. Down-regulation of ATX secretion in SKOV3 cells using antisense morpholino oligomers significantly attenuates cell motility responses to VEGF, ATX, LPA, and lysophosphatidylcholine. These effects are accompanied by decreased LPA(4) and VEGFR2 expression as well as by increased release of soluble VEGFR1. Because LPA was previously shown to increase VEGF expression in ovarian cancer, our data suggest a positive feedback loop involving VEGF, ATX, and its product LPA that could affect tumor progression in ovarian cancer cells.
Assuntos
Complexos Multienzimáticos/genética , Neoplasias Ovarianas/genética , Fosfodiesterase I/genética , Pirofosfatases/genética , Fator A de Crescimento do Endotélio Vascular/genética , Anticorpos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , AMP Cíclico/fisiologia , Primers do DNA , Feminino , Deleção de Genes , Humanos , Lisofosfolipídeos/fisiologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Diester Fosfórico Hidrolases , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Transdução de Sinais , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
Radiation, a primary mode of cancer therapy, acutely damages cellular macromolecules and DNA and elicits stress responses that lead to cell death. The known cytoprotective activity of nitric oxide (NO) is blocked by thrombospondin-1, a potent antagonist of NO/cGMP signaling in ischemic soft tissues, suggesting that thrombospondin-1 signaling via its receptor CD47 could correspondingly increase radiosensitivity. We show here that soft tissues in thrombospondin-1-null mice are remarkably resistant to radiation injury. Twelve hours after 25-Gy hindlimb irradiation, thrombospondin-1-null mice showed significantly less cell death in both muscle and bone marrow. Two months after irradiation, skin and muscle units in null mice showed minimal histological evidence of radiation injury and near full retention of mitochondrial function. Additionally, both tissue perfusion and acute vascular responses to NO were preserved in irradiated thrombospondin-1-null hindlimbs. The role of thrombospondin-1 in radiosensitization is specific because thrombospondin-2-null mice were not protected. However, mice lacking CD47 showed radioresistance similar to thrombospondin-1-null mice. Both thrombospondin-1- and CD47-dependent radiosensitization is cell autonomous because vascular cells isolated from the respective null mice showed dramatically increased survival and improved proliferative capacity after irradiation in vitro. Therefore, thrombospondin-1/CD47 antagonists may have selective radioprotective activity for normal tissues.
Assuntos
Antígeno CD47/metabolismo , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Trombospondina 1/metabolismo , Sobrevivência de Tecidos/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Vasos Sanguíneos/patologia , Vasos Sanguíneos/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Membro Posterior/patologia , Membro Posterior/efeitos da radiação , Hipertrofia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias/patologia , Tolerância a Radiação/efeitos da radiação , Trombospondinas/metabolismo , Raios XRESUMO
BACKGROUND: Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4), the predominant Robo in endothelial cells using small interfering RNA technology in vitro. RESULTS: Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells. CONCLUSION: This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.
Assuntos
Células Endoteliais/metabolismo , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Movimento Celular , Células Cultivadas , Quimiotaxia , Células Endoteliais/citologia , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Soro/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas RoundaboutRESUMO
Transitions between yeast and hyphae are essential for Candida albicans pathogenesis. The genetic programs that regulate its hyphal development can be distinguished by embedded versus aerobic surface agar invasion. Hbr1, a regulator of white-opaque switching, is also a positive and negative regulator of hyphal invasion. During embedded growth at 24°C, an HBR1/hbr1 strain formed constitutively filamentous colonies throughout the matrix, resembling EFG1 null colonies, and a subset of long unbranched hyphal aggregates enclosed in a spindle-shaped capsule. Inhibition of adenylate cyclase with farnesol perturbed the filamentation of HBR1/hbr1 cells producing cytokinesis-defective hyphae whereas farnesol treated EFG1 null cells produced abundant opaque-like cells. Point mutations in the Hbr1 ATP-binding domain caused distinct filamentation phenotypes including uniform radial hyphae, hyphal sprouts, and massive yeast cell production. Conversely, aerobic surface colonies of the HBR1 heterozygote on Spider and GlcNAc media lacked filamentation that could be rescued by growth under low (5%) O2. Consistent with these morphogenesis defects, the HBR1 heterozygote exhibited attenuated virulence in a mouse candidemia model. These data define Hbr1 as an ATP-dependent positive and negative regulator of hyphal development that is sensitive to hypoxia.
Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Hifas/metabolismo , Animais , Candida albicans/genética , Candida albicans/patogenicidade , Candidíase , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Hifas/genética , Camundongos , Estrutura Terciária de ProteínaRESUMO
Signaling through the thrombospondin-1 receptor CD47 broadly limits cell and tissue survival of stress, but the molecular mechanisms are incompletely understood. We now show that loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. c-Myc, Klf4, Oct4, and Sox2 expression is elevated in CD47-null endothelial cells, in several tissues of CD47- and thrombospondin-1-null mice, and in a human T cell line lacking CD47. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells and in vivo, whereas CD47 ligation by thrombospondin-1 suppresses c-Myc expression. The inhibitory effects of increasing CD47 levels can be overcome by maintaining c-Myc expression and are absent in cells with dysregulated c-Myc. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.
Assuntos
Antígeno CD47/metabolismo , Células Endoteliais/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Trombospondina 1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Células Endoteliais/citologia , Regulação da Expressão Gênica/fisiologia , Fator 4 Semelhante a Kruppel , Camundongos , Modelos Biológicos , Células-Tronco/citologiaRESUMO
Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to studying multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1-45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models.
Assuntos
Integrinas/química , Ligantes , Nanopartículas/química , Ácidos Nucleicos Peptídicos/química , Animais , Adesão Celular , Linhagem Celular Tumoral , DNA/química , Matriz Extracelular/metabolismo , Biblioteca Gênica , Humanos , Concentração Inibidora 50 , Cinética , Melanoma Experimental , Camundongos , Modelos Químicos , Modelos Moleculares , Metástase Neoplásica , Ligação Proteica , Conformação ProteicaRESUMO
Accidental or therapeutic exposure to ionizing radiation has severe physiological consequences and can result in cell death. We previously demonstrated that deficiency or blockade of the ubiquitously expressed receptor CD47 results in remarkable cell and tissue protection against ischemic and radiation stress. Antagonists of CD47 or its ligand THBS1/thrombospondin 1 enhance cell survival and preserve their proliferative capacity. However the signaling pathways that mediate this cell-autonomous radioprotection are unclear. We now report a marked increase in autophagy in irradiated T-cells and endothelial cells lacking CD47. Irradiated T cells lacking CD47 exhibit significant increases in formation of autophagosomes comprising double-membrane vesicles visualized by electron microscopy and numbers of MAP1LC3A/B(+) puncta. Moreover, we observed significant increases in BECN1, ATG5, ATG7 and a reduction in SQSTM1/p62 expression relative to irradiated wild-type T cells. We observed similar increases in autophagy gene expression in mice resulting from blockade of CD47 in combination with total body radiation. Pharmacological or siRNA-mediated inhibition of autophagy selectively sensitized CD47-deficient cells to radiation, indicating that enhanced autophagy is necessary for the prosurvival response to CD47 blockade. Moreover, re-expression of CD47 in CD47-deficient T cells sensitized these cells to death by ionizing radiation and reversed the increase in autophagic flux associated with survival. This study indicates that CD47 deficiency confers cell survival through the activation of autophagic flux and identifies CD47 blockade as a pharmacological route to modulate autophagy for protecting tissue from radiation injury.
Assuntos
Autofagia , Antígeno CD47/genética , Antígeno CD47/metabolismo , Especificidade de Órgãos/efeitos da radiação , Proteção Radiológica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/genética , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Regulação da Expressão Gênica/efeitos da radiação , Inativação Gênica/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Células Jurkat , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Radiação Ionizante , Proteína Sequestossoma-1 , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Regulação para Cima/efeitos da radiação , Irradiação Corporal TotalRESUMO
Important roles for vascular endothelial growth factor (VEGF) and autotaxin (ATX) have been established for embryonic vasculogenesis and cancer progression. We examined whether these two angiogenic factors cooperate in regulation of endothelial cell migratory responses. VEGF stimulated expression of ATX and LPA1, a receptor for the ATX enzymatic product lysophosphatidic acid (LPA), in human umbilical vein endothelial cells. Knockdown of ATX expression significantly decreased mRNA levels for the receptors LPA1, LPA2, S1P1, S1P2, S1P3, and VEGFR2 and abolished cell migration to lysophosphatidylcholine, LPA, recombinant ATX, and VEGF. Migration to sphingosylphosphorylcholine and sphinogosine-1-phosphate was also reduced in ATX knockdown cells, whereas migration to serum remained unchanged. Furthermore, ATX knockdown decreased Akt2 mRNA levels, whereas LPA treatment strongly stimulated Akt2 expression. We propose that VEGF stimulates LPA production by inducing ATX expression. VEGF also increases LPA1 signaling, which in turn increases Akt2 expression. Akt2 is strongly associated with cancer progression, cellular migration, and promotion of epithelial-mesenchymal transition. These data show a role for ATX in maintaining expression of receptors required for VEGF and lysophospholipids to accelerate angiogenesis. Because VEGF and ATX are upregulated in many cancers, the regulatory mechanism proposed in these studies could apply to cancer-related angiogenesis and cancer progression. These data further suggest that ATX could be a prognostic factor or a target for therapeutic intervention in several cancers.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Complexos Multienzimáticos/metabolismo , Fosfodiesterase I/metabolismo , Pirofosfatases/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/fisiopatologia , Fosfodiesterase I/efeitos dos fármacos , Fosfodiesterase I/genética , Diester Fosfórico Hidrolases , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirofosfatases/efeitos dos fármacos , Pirofosfatases/genética , Interferência de RNA/fisiologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de Ácidos Lisofosfatídicos/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPalpha (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.
Assuntos
Antígeno CD47/metabolismo , GMP Cíclico/metabolismo , Isquemia/metabolismo , Transdução de Sinais , Trombospondinas/metabolismo , Animais , Células Cultivadas , Humanos , Isquemia/genética , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Ligação Proteica , Receptores Imunológicos/metabolismo , Trombospondinas/deficiência , Trombospondinas/genéticaRESUMO
Hemoglobin is an abundant protein in the host vascular compartment and a source of iron, heme, and amino acids for many pathogens. The human fungal pathogen Candida albicans uses hemoglobin as an iron source as well as a signaling molecule to alter gene expression and induce adhesion to several extracellular matrix proteins. We now report that hemoglobin can promote true hyphal morphogenesis. Hemoglobin added to yeast cells at 37 degrees C rapidly induced expression of the hypha-specific genes HWP1 and ECE1 coincident with the pattern of hyphal development. A synthetic medium buffered with phosphate at pH 7.2 and containing physiological glucose (5 mM) and low ammonium ion (0.1 mM) was optimal for the response to hemoglobin. High glucose (110 mM), high ammonium ion (20 mM), and 0.1 mM glutamine were all inhibitory. Heme, free globin, or immobilized hemoglobin could not replicate the activity of hemoglobin to induce germ tubes or hypha-specific gene expression at 37 degrees C under optimized conditions. This implicates the previously described Hb-signaling receptor in hyphal formation. This response was also dependent upon the presence of the morphogenesis regulator Efg1p, but the MAP-kinase specific transcription factor Cph1p was not required. These data define a role for the host-factor hemoglobin in Efg1p-dependent hyphal development.
Assuntos
Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Hemoglobinas/farmacologia , Hifas/crescimento & desenvolvimento , Candida albicans/genética , Candida albicans/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Hifas/efeitos dos fármacos , Hifas/genética , Hifas/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Morfogênese , Nitrogênio/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Candida albicans expresses at least two biochemically distinct fibronectin receptors. Hemoglobin induces expression of a low affinity receptor recognizing the fibronectin cell-binding domain, whereas growth in complex media induces a high affinity receptor recognizing the collagen-binding domain. We now show that sub-inhibitory concentrations of caspofungin and nikkomycin Z, but not fluconazole, induce the high affinity fibronectin receptor in a dose-dependent manner. Macromolecular complexes mechanically sheared from caspofungin-treated cells retained high affinity fibronectin binding that was sensitive to protease, disulfide reduction, and beta (1,3) glucanase digestion. The high affinity fibronectin receptor was not inducible in a Kre9 mutant strain of C. albicans deficient in beta (1,6) glucans. Conversely, a mutant strain lacking the fibronectin binding protein Als5p showed no defects in induction of high or low affinity fibronectin receptors. Heterozygous mutants of a regulator of white-opaque phenotypic switching, HBR1, lacked any detectable high affinity fibronectin receptor expression in response to caspofungin, and re-introduction of the gene restored activity. Therefore, sub-inhibitory dosages of caspofungin induce a high affinity fibronectin receptor that is distinct from the known receptor Als5p and is dependent on beta (1,6) glucans and HBR1.
Assuntos
Aminoglicosídeos/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Integrina alfa5beta1/biossíntese , Peptídeos Cíclicos/farmacologia , beta-Glucanas/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Caspofungina , Equinocandinas , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Teste de Complementação Genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Lipopeptídeos , Substâncias Macromoleculares/metabolismo , Mutação , Peptídeo Hidrolases/metabolismoRESUMO
Adhesion to host cells and tissues is important for several steps in the pathogenesis of disseminated Candida albicans infections. Although such adhesion is evident in vivo and for C. albicans grown in vitro in complex medium, some adhesive activities are absent when cultures are grown in defined media. However, addition of hemoglobin to defined media restores binding and adhesion to several host proteins. This activity of hemoglobin is independent of iron acquisition and is mediated by a cell surface hemoglobin receptor. In addition to regulating expression of adhesion receptors, hemoglobin rapidly induces expression of several genes. One of these, a heme oxygenase, allows the pathogen to utilize exogenous heme or hemoglobin to acquire iron and to produce the cytoprotective molecules alpha-biliverdin and carbon monoxide. The specific recognition of and responses to hemoglobin demonstrate a unique adaptation of C. albicans to be both a commensal and an opportunistic pathogen in humans.
Assuntos
Candida albicans/efeitos dos fármacos , Candida albicans/fisiologia , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Adesões Focais/fisiologia , Heme Oxigenase (Desciclizante)/metabolismo , Hemoglobinas/metabolismo , Hemoglobinas/farmacologia , Sítios de Ligação , Matriz Extracelular/efeitos dos fármacos , Adesões Focais/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ligação ProteicaRESUMO
Phenotypic switching from the white to the opaque phase is a necessary step for mating in the pathogenic fungus Candida albicans. Suppressing switching during vascular dissemination of the organism may be advantageous, because opaque cells are more susceptible to host defenses. A repressor of white-opaque switching, HBR1 (hemoglobin response gene 1), was identified based on its specific induction following growth in the presence of exogenous hemoglobin. Deletion of a single HBR1 allele allowed opaque phase switching and mating competence, accompanied by a lack of detectable MTL alpha1 and alpha2 gene expression and enhanced MTLa1 gene expression. Conversely, overexpression of Hbr1p or exposure to hemoglobin increased MTLalpha gene expression. The a1/alpha2 repressed target gene CAG1 was derepressed in the same mutant in a hemoglobin-sensitive manner. Regulation of CAG1 by hemoglobin required an intact MTLa1 gene. Several additional Mtlp targets were perturbed in HBR1 mutants in a manner consistent with commitment to an a mating phenotype, including YEL007w, MFalpha, HST6, and RAM2. Therefore, Hbr1 is part of a host factor-regulated signaling pathway that controls white-opaque switching and mating in the absence of allelic deletion at the MTL locus.
Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Genes Fúngicos Tipo Acasalamento , Hemoglobinas/farmacologia , Alelos , Sequência de Aminoácidos , Candida albicans/genética , Primers do DNA , Proteínas Fúngicas/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Hemoglobinas/metabolismo , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Candida albicans is an opportunistic pathogen that has adapted uniquely to life in mammalian hosts. One of the host factors recognized by this yeast is hemoglobin, which binds to a specific cell surface receptor. In addition to its regulating the expression of adhesion receptors on the yeast, we have found that hemoglobin induces the expression of a C. albicans heme oxygenase (CaHmx1p). Hemoglobin transcriptionally induces the CaHMX1 gene independent of the presence of inorganic iron in the medium. A Renilla luciferase reporter driven by the CaHMX1 promoter demonstrated rapid activation of transcription by hemoglobin and (cobalt protoporphyrin IX) globin but not by apoglobin or other proteins. In contrast, iron deficiency or exogenous hemin did not activate the reporter until after 3 h, suggesting that induction of the promoter by hemoglobin is mediated by receptor signaling rather than heme or iron flux into the cell. As observed following disruption of the Saccharomyces cerevisiae ortholog, HMX1, a CaHMX1 null mutant was unable to grow under iron restriction. This suggests a role for CaHmx1p in inorganic iron acquisition. CaHMX1 encodes a functional heme oxygenase. Exogenous heme or hemoglobin is exclusively metabolized to alpha-biliverdin. CaHMX1 is required for utilization of these exogenous substrates, indicating that C. albicans heme oxygenase confers a nutritional advantage for growth in mammalian hosts.