RESUMO
Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging activity could be an effective strategy for breast cancer treatment.
Assuntos
Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Metilação de DNA , Células Epiteliais/metabolismo , Superóxido Dismutase/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Epigênese Genética , Células Epiteliais/citologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/metabolismo , Transplante HeterólogoRESUMO
Adolescent experiences of social deprivation result in profound and enduring perturbations in adult behavior, including impaired sensorimotor gating. The behavioral deficits induced by adolescent social isolation in rats can be ameliorated by antipsychotic drugs blocking dopamine D2 receptors in the prefrontal cortex (PFC) or by chronic administration of a cannabinoid CB1 receptor antagonist. The patterning and abundance of D2 receptors in the PFC evolves concurrently with CB1 receptors through the period of adolescence. This evidence suggests that mature expression and/or surface distribution of D2 and CB1 receptors may be influenced by the adolescent social environment. We tested this hypothesis using electron microscopic immunolabeling to compare the distribution of CB1 and D2 receptors in the PFC of adult male Sprague-Dawley rats that were isolated or socially reared throughout the adolescent transition period. Prepulse inhibition (PPI) of acoustic startle was assessed as a measure of sensorimotor gating. Social isolation reduced PPI and selectively decreased dendritic D2 immunogold labeling in the PFC. However, the decrease was only evident in dendrites that were not contacted by axon terminals containing CB1. There was no apparent change in the expression of CB1 or D2 receptors in presynaptic terminals. The D2 deficit therefore may be tempered by local CB1-mediated retrograde signaling. This suggests a biological mechanism whereby the adolescent social environment can persistently influence cortical dopaminergic activity and resultant behavior.
Assuntos
Córtex Pré-Frontal/metabolismo , Receptor CB1 de Canabinoide/fisiologia , Receptores de Dopamina D2/fisiologia , Isolamento Social , Estimulação Acústica , Animais , Interpretação Estatística de Dados , Dendritos/metabolismo , Dendritos/fisiologia , Dendritos/ultraestrutura , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Neurônios/metabolismo , Neurônios/ultraestrutura , Córtex Pré-Frontal/fisiologia , Córtex Pré-Frontal/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Receptores Pré-Sinápticos/fisiologia , Receptores Pré-Sinápticos/ultraestrutura , Reflexo de Sobressalto , Filtro Sensorial/efeitos dos fármacos , Filtro Sensorial/fisiologiaRESUMO
Apolipoproteins, such as apolipoprotein A-I (apoA-I), can stimulate cholesterol efflux from cells expressing the ATP binding cassette transporter A1 (ABCA1). The nature of the molecular interaction between these cholesterol acceptors and ABCA1 is controversial, and models suggesting a direct protein-protein interaction or indirect association have been proposed. To explore this issue, we performed competition binding and chemical cross-linking assays using six amphipathic plasma proteins and an 18 amino acid amphipathic helical peptide. All seven proteins stimulated lipid efflux and inhibited the cross-linking of apoA-I to ABCA1. Cross-linking of apoA-I to ABCA1 was saturable and occurred at high affinity (Kd of 7.0 +/- 1.9 nM), as was cross-linking of apoA-II. After binding to ABCA1, apoA-I rapidly dissociated (half-life of 25 min) from the complex and was released back into the medium. A mutant form of ABCA1 (W590S) that avidly binds apoA-I but fails to promote cholesterol efflux released apoA-I with similar kinetics but without transfer of cholesterol to apoA-I. Thus, a high-affinity, saturable, protein-protein interaction occurs between ABCA1 and all of its amphipathic protein ligands. Dissociation of the complex leads to the cellular release of cholesterol and the apolipoprotein. However, dissociation is not dependent on cholesterol transfer, which is a clearly separable event, distinguishable by ABCA1 mutants.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteínas/metabolismo , Colesterol/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adenoviridae , Transporte Biológico/fisiologia , Linhagem Celular Transformada , Membrana Celular/metabolismo , Humanos , Metabolismo dos Lipídeos , Substâncias Macromoleculares , Mutação , Peptídeos/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução GenéticaRESUMO
Peroxisome proliferator-activated receptor (PPAR)-gamma is a nuclear hormone receptor, with a well-established role in adipogenesis and glucose metabolism. Over the past 3 years several laboratories have reported that this protein can influence macrophage responses to a variety of inflammatory stimuli. The effect of PPAR-gamma activation on macrophage lipid uptake, cholesterol efflux, and cytokine production have all recently been examined in several in-vitro culture systems. In addition, PPAR-gamma ligands have been shown to influence atherosclerotic lesion formation in murine models of that disease. This review attempts to summarize and critically evaluate that work and its implications for the use of PPAR-gamma activators in understanding and treating the pathogenetic processes that contribute to atherosclerotic plaque formation.
Assuntos
Inflamação/fisiopatologia , Metabolismo dos Lipídeos , Macrófagos/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Arteriosclerose/tratamento farmacológico , Arteriosclerose/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Humanos , Macrófagos/citologia , Fatores de Transcrição/farmacologiaRESUMO
Mutations in the ATP-binding cassette transporter A1 (ABCA1) transporter are associated with Tangier disease and a defect in cellular cholesterol efflux. The amino terminus of the ABCA1 transporter has two putative in-frame translation initiation sites, 60 amino acids apart. A cluster of hydrophobic amino acids form a potentially cleavable signal sequence in this 60-residue extension. We investigated the functional role of this extension and found that it was required for stable protein expression of transporter constructs containing any downstream transmembrane domains. The extension directed transporter translocation across the ER membrane with an orientation that resulted in glycosylation of amino acids immediately distal to the signal sequence. Neither the native signal sequence nor a green fluorescent protein tag, fused at the amino terminus, was cleaved from ABCA1. The green fluorescent protein fusion protein had efflux activity comparable with wild type ABCA1 and demonstrated a predominantly plasma membrane distribution in transfected cells. These data establish a requirement for the upstream 60 amino acids of ABCA1. This region contains an uncleaved signal anchor sequence that positions the amino terminus in a type II orientation leading to the extracellular presentation of an approximately 600-amino acid loop in which loss-of-function mutations cluster in Tangier disease patients.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Especificidade de Anticorpos , Colesterol/metabolismo , DNA Complementar , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), the transcription factor target of the anti-diabetic thiazolidinedione (TZD) drugs, is reported to mediate macrophage differentiation and inflammatory responses. Using PPAR-gamma-deficient stem cells, we demonstrate that PPAR-gamma is neither essential for myeloid development, nor for such mature macrophage functions as phagocytosis and inflammatory cytokine production. PPAR-gamma is required for basal expression of CD36, but not for expression of the other major scavenger receptor responsible for uptake of modified lipoproteins, SR-A. In wild-type macrophages, TZD treatment divergently regulated CD36 and class A macrophage-scavenger receptor expression and failed to induce significant cellular cholesterol accumulation, indicating that TZDs may not exacerbate macrophage foam-cell formation.
Assuntos
Diferenciação Celular/fisiologia , Colesterol/metabolismo , Macrófagos/citologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Antígenos CD36/imunologia , Sondas de DNA , Hipoglicemiantes/farmacologia , Lipoproteínas LDL/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tiazóis/farmacologiaRESUMO
Previous research has shown that homeless youth have high rates of suicidal ideation, sexual abuse, and abuse of alcohol and other drugs. However, little is known about how these rates differ by gender and ethnicity. Our objective was to describe patterns of sexual abuse, alcohol and other drug use, and indicators of suicidal behaviors in homeless adolescents and to determine gender and ethnic differences in these factors. We used secondary data analysis of data from surveys completed by 96 homeless youth whose average age was 17.9 years. Over 60% of the sample reported a history of sexual abuse; the majority were under the age of 12 years when they first tried alcohol, marijuana, and cocaine; 56.3% had injected drugs, and 46.9% had tried inhalants. During the past 12 months, 35.1% had seriously considered suicide and 12.3% had actually attempted suicide at least once. Significantly more Hispanics than Whites had considered suicide (chi 2 = 4.31, p = .038). A disproportionate number of Hispanics (95% of the sample) reported a history of sexual abuse. Participants with a history of sexual abuse were significantly more likely than those who did not have a history of sexual abuse to have used alcohol and/or marijuana (chi 2 = 9.93, p < .01) and to have considered suicide in the past 12 months (F = 14.93, p < .001). We found that sexual abuse history is greater in this sample than in the general population and is particularly prevalent among Hispanic/Latino subjects. As in other studies, sexual abuse was more common among females than among males. High prevalence of sexual abuse, alcohol and other drug use, and suicidal behaviors in this sample of homeless youth underscores the need to develop and test community-based interventions to improve their health status.
Assuntos
Comportamento do Adolescente , Alcoolismo/epidemiologia , Abuso Sexual na Infância/estatística & dados numéricos , Jovens em Situação de Rua/estatística & dados numéricos , Psicologia do Adolescente , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Tentativa de Suicídio/estatística & dados numéricos , Adolescente , Comportamento do Adolescente/psicologia , Distribuição por Idade , Alcoolismo/complicações , Alcoolismo/psicologia , Abuso Sexual na Infância/psicologia , Comorbidade , Feminino , Inquéritos Epidemiológicos , Hispânico ou Latino/psicologia , Hispânico ou Latino/estatística & dados numéricos , Jovens em Situação de Rua/psicologia , Humanos , Masculino , Avaliação das Necessidades , Prevalência , Psicologia do Adolescente/estatística & dados numéricos , Fatores de Risco , Distribuição por Sexo , Transtornos Relacionados ao Uso de Substâncias/complicações , Transtornos Relacionados ao Uso de Substâncias/psicologia , Tentativa de Suicídio/psicologia , Inquéritos e Questionários , Estados Unidos/epidemiologia , População Branca/psicologia , População Branca/estatística & dados numéricosRESUMO
There is an increasing prevalence in the United States of intergenerational surrogate parenting. In fact, grandmother-headed households that include grandchildren without their parents are the fastest growing intergenerational surrogate family combination. This article reviews the intergenerational surrogate family phenomenon relative to its impact on the grandmothers' health and offers resources and implications for nursing.
Assuntos
Educação Infantil/psicologia , Família/psicologia , Relação entre Gerações , Poder Familiar/psicologia , Criança , Humanos , Prevalência , Estados Unidos/epidemiologiaRESUMO
TNA and the Texas Department of Insurance collaborated on an integrated, education program that has provided benefits for all--graduate nursing students get an opportunity to learn about managed care issues and policies; TDI has gained the clinical expertise of the nursing profession in order to best evaluate quality of care.
Assuntos
Educação Continuada em Enfermagem/organização & administração , Programas de Assistência Gerenciada/organização & administração , Papel do Profissional de Enfermagem , Humanos , Autonomia Profissional , Garantia da Qualidade dos Cuidados de Saúde/organização & administração , Sociedades de Enfermagem/organização & administração , Texas , Apoio ao Desenvolvimento de Recursos Humanos/organização & administraçãoRESUMO
Microbial pathogens frequently take advantage of host systems for their pathogenesis. Shedding of cell surface molecules as soluble extracellular domains (ectodomains) is one of the host responses activated during tissue injury. In this study, we examined whether pathogenic bacteria can modulate shedding of syndecan-1, the predominant syndecan of host epithelia. Our studies found that overnight culture supernatants of Pseudomonas aeruginosa and Staphylococcus aureus enhanced the shedding of syndecan-1 ectodomains, whereas culture supernatants of several other Gram-negative and Gram-positive bacteria had only low levels of activity. Because supernatants from all tested strains of P. aeruginosa (n = 9) enhanced syndecan-1 shedding by more than 4-fold above control levels, we focused our attention on this Gram-negative bacterium. Culture supernatants of P. aeruginosa increased shedding of syndecan-1 in both a concentration- and time-dependent manner, and augmented shedding by various host cells. A 20-kDa shedding enhancer was partially purified from the supernatant through ammonium sulfate precipitation and gel chromatography, and identified by N-terminal sequencing as LasA, a known P. aeruginosa virulence factor. LasA was subsequently determined to be a syndecan-1 shedding enhancer from the findings that (i) immunodepletion of LasA from the partially purified sample resulted in abrogation of its activity to enhance shedding and (ii) purified LasA increased shedding in a concentration-dependent manner. Our results also indicated that LasA enhances syndecan-1 shedding by activation of the host cell's shedding mechanism and not by direct interaction with syndecan-1 ectodomains. Enhanced syndecan-1 shedding may be a means by which pathogenic bacteria take advantage of a host mechanism to promote their pathogenesis.
Assuntos
Proteínas de Bactérias , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Proteoglicanas/metabolismo , Pseudomonas aeruginosa/fisiologia , Meios de Cultivo Condicionados , Pseudomonas aeruginosa/patogenicidade , Sindecanas , VirulênciaRESUMO
The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated. To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.
Assuntos
Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Proteoglicanas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-3/farmacologia , Animais , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Cinética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glicoproteínas de Membrana/química , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Concentração Osmolar , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteoglicanas/química , Receptores de Trombina/agonistas , Receptores de Trombina/antagonistas & inibidores , Receptores de Trombina/fisiologia , Sindecana-1 , Sindecana-4 , Sindecanas , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Gene deletion studies indicate that the macrophage scavenger receptor A (SR-A) protects mice from LPS-induced endotoxemia. Paradoxically, cultured human monocyte-derived macrophages down-regulate SR-A expression when exposed to LPS. We found that human THP-1 monocyte/macrophages decrease SR-A expression in response to LPS independent of their differentiation status. In contrast, primary and elicited mouse peritoneal macrophages as well as the J774A.1 and RAW264.7 mouse macrophage lines increase SR-A expression in response to LPS. Exposure to LPS caused J774A.1 and RAW264.7 cells to increase SR-A transcripts by 3- and 5-fold, respectively. LPS caused a concomitant 3-fold increase in SR-A protein levels and increased cell membrane expression of the receptor. RAW264.7 cells increased SR-A transcript levels in response to LPS at concentrations as low as 1 ng/ml, and the response was saturated at 10 ng/ml. The LPS induction of SR-A transcripts required continual protein synthesis and began at 8 h, peaked by 16 h, and persisted for at least 48 h. LPS induction did not increase SR-A gene transcription or affect alternative transcript splicing, but mildly increased mature transcript stability and proceeded in the presence of actinomycin D. Finally, treatment of RAW264.7 cells with TNF-alpha did not induce SR-A transcript levels, indicating that a TNF-alpha autocrine/paracrine signaling mechanism alone is not sufficient to recapitulate the LPS induction of SR-A transcripts. The induction of SR-A expression by LPS-stimulated mouse macrophages is the opposite of the down-regulation of SR-A reported in human monocyte-derived macrophages and may have implications for the observed resistance mice show toward endotoxemia.
Assuntos
Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana , Monócitos/metabolismo , Receptores Imunológicos/biossíntese , Receptores de Lipoproteínas , Processamento Alternativo/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular , Dactinomicina/farmacologia , Relação Dose-Resposta Imunológica , Feminino , Meia-Vida , Humanos , Imunofenotipagem , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Receptores Imunológicos/genética , Receptores Depuradores , Receptores Depuradores Classe A , Receptores Depuradores Classe B , Fatores de Tempo , Transcrição Gênica/imunologia , Ativação Transcricional/imunologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
In platelets and other secretory cells, protein kinase C (PKC) plays a role in exocytosis stimulated by physiological extracellular signals, although its linkage to the secretory machinery is poorly understood. We investigated whether Rab6, a GTP-binding protein that fractionates with platelet alpha-granules, may be involved in linking these processes. We found that Rab6 contains two PKC consensus phosphorylation sites that are evolutionarily conserved. In platelets metabolically labelled with [(32)P]P(i), Rab6 phosphorylation was induced by phorbol esters or by thrombin. This phosphorylation was blocked by a specific PKC inhibitor (Ro-31-8220), but not by a p38 mitogen-activated protein kinase inhibitor (PD-169316). Physiological stimulation of platelets caused a PKC-dependent translocation of Rab6 from platelet particulate fractions, nearly doubling the fraction of Rab6 in the cytosol. A human Rab6 isoform (Rab6C) that is preferentially expressed in human platelet RNA was cloned and its phosphorylation by PKC was characterized. Rab6C incorporated up to 2 mol of [(32)P]P(i) per mol of active protein. Rab6C bound GDP and GTP with K(d) values of 113+/-12 and 119+/-27 nM respectively, and hydrolysed GTP at a rate of 100+/-15 micromol of GTP/mol of Rab6C per min. PKC phosphorylation of Rab6C increased the affinity for GTP by 3-fold, although it had lesser effects on GDP (1.6-fold). Phosphorylation did not alter the GTPase activity. In summary, thrombin activation of platelets leads to PKC-dependent phosphorylation of Rab6 and a translocation of Rab6 to the cytosol. We suggest that PKC phosphorylation may be an important mechanism through which Rab functional interactions in vesicle trafficking and secretion can be altered in response to an external stimulus.
Assuntos
Plaquetas/metabolismo , Guanosina Difosfato/sangue , Guanosina Trifosfato/sangue , Proteína Quinase C/sangue , Proteínas rab de Ligação ao GTP/sangue , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Plaquetas/efeitos dos fármacos , Bovinos , Sequência Consenso , Citosol/metabolismo , Primers do DNA/genética , Inibidores de Dissociação do Nucleotídeo Guanina/sangue , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Fosforilação , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/fisiologia , Isoformas de Proteínas/sangue , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Homologia de Sequência de Aminoácidos , Trombina/farmacologia , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genéticaRESUMO
In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins-VAMP, SNAP-25, and syntaxin 4-that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 +/- 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.
Assuntos
Plaquetas/fisiologia , Proteínas de Membrana/sangue , Proteínas do Tecido Nervoso/sangue , Trombina/fisiologia , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Sequência de Bases , Plaquetas/efeitos dos fármacos , Fusão Celular/fisiologia , Membrana Celular/fisiologia , Clonagem Molecular , Citosol/metabolismo , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Munc18 , Fosforilação , Ativação Plaquetária , Reação em Cadeia da Polimerase , Proteína Quinase C/sangue , Proteínas Qa-SNARE , Proteínas Recombinantes/biossíntese , Proteínas SNARE , Proteína 25 Associada a Sinaptossoma , Trombina/farmacologiaRESUMO
The heparan sulfate on the surface of all adherent cells modulates the actions of a large number of extracellular ligands. Members of both cell surface heparan sulfate proteoglycan families, the transmembrane syndecans and the glycosylphosphoinositide-linked glypicans, bind these ligands and enhance formation of their receptor-signaling complexes. These heparan sulfate proteoglycans also immobilize and regulate the turnover of ligands that act at the cell surface. The extracellular domains of these proteoglycans can be shed from the cell surface, generating soluble heparan sulfate proteoglycans that can inhibit interactions at the cell surface. Recent analyses of genetic defects in Drosophila melanogaster, mice, and humans confirm most of these activities in vivo and identify additional processes that involve cell surface heparan sulfate proteoglycans. This chapter focuses on the mechanisms underlying these activities and on the cellular functions that they regulate.
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Animais , Membrana Celular/metabolismo , Proteoglicanas de Heparan Sulfato/biossíntese , HumanosRESUMO
The activity of fibroblast growth factor 2 (FGF-2) is stringently controlled. Inactive in undisturbed tissues, it is activated during injury and is critical for tissue repair. We find that this control can be imposed by the soluble syndecan-1 ectodomain, a heparan sulfate proteoglycan shed from cell surfaces into wound fluids. The ectodomain potently inhibits heparin-mediated FGF-2 mitogenicity because of the poorly sulfated domains in its heparin sulfate chains. Degradation of these regions by platelet heparanase produces heparin-like heparin sulfate fragments that markedly activate FGF-2 mitogenicity and are found in wound fluids. These results establish a novel physiological control for FGF-2 and suggest new ways to modulate FGF activity.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Glucuronidase , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Receptores Proteína Tirosina Quinases , Sítios de Ligação/fisiologia , Exsudatos e Transudatos/química , Exsudatos e Transudatos/metabolismo , Fibrinolíticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Glicosídeo Hidrolases/farmacologia , Heparina/farmacologia , Heparitina Sulfato/metabolismo , Humanos , Glicoproteínas de Membrana/efeitos dos fármacos , Mitógenos/metabolismo , Oligossacarídeos/farmacologia , Ligação Proteica , Proteoglicanas/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Solubilidade , Sindecana-1 , Sindecanas , Ferimentos e Lesões/metabolismoRESUMO
The syndecan family of transmembrane heparan sulfate proteoglycans is abundant on the surface of all adherent mammalian cells. Syndecans bind and modify the action of various growth factors/cytokines, proteases/antiproteases, cell adhesion molecules, and extracellular matrix components. Syndecan expression is highly regulated during wound repair, a process orchestrated by many of these effectors. Each syndecan ectodomain is shed constitutively by cultured cells, but the mechanism and significance of this shedding are not understood. Therefore, we examined (i) whether physiological agents active during wound repair influence syndecan shedding, and (ii) whether wound fluids contain shed syndecan ectodomains. Using SVEC4-10 endothelial cells we find that certain proteases and growth factors accelerate shedding of the syndecan-1 and -4 ectodomains. Protease-accelerated shedding is completely inhibited by serum-containing media. Thrombin activity is duplicated by the 14-amino acid thrombin receptor agonist peptide that directly activates the thrombin receptor and is not inhibited by serum. Epidermal growth factor family members accelerate shedding but FGF-2, platelet-derived growth factor-AB, transforming growth factor-beta, tumor necrosis factor-alpha, and vascular endothelial cell growth factor 165 do not. Shed ectodomains are soluble, stable in the conditioned medium, have the same size core proteins regardless whether shed at a basal rate, or accelerated by thrombin or epidermal growth factor-family members and are found in acute human dermal wound fluids. Thus, shedding is accelerated by activation of at least two distinct receptor classes, G protein-coupled (thrombin) and protein tyrosine kinase (epidermal growth factor). Proteases and growth factors active during wound repair can accelerate syndecan shedding from cell surfaces. Regulated shedding of syndecans suggests physiological roles for the soluble proteoglycan ectodomains.
Assuntos
Endotélio Vascular/fisiologia , Substâncias de Crescimento/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento/fisiologia , Receptores de Trombina/fisiologia , Sequência de Aminoácidos , Anfirregulina , Anticorpos , Membrana Celular/metabolismo , Células Cultivadas , Família de Proteínas EGF , Fatores de Crescimento Endotelial/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Epitopos , Receptores ErbB/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Glicoproteínas/farmacologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Linfocinas/farmacologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteoglicanas/análise , Proteoglicanas/química , Proteínas Recombinantes/farmacologia , Pele/lesões , Sindecana-1 , Sindecana-4 , Sindecanas , Acetato de Tetradecanoilforbol/farmacologia , Trombina/farmacologia , Fator de Crescimento Transformador alfa/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Ferimentos e Lesões/fisiopatologiaRESUMO
Cells invade extracellular matrices in a regulated manner at specific times and places during normal development. A dramatic example is trophoblast invasion of the uterine wall. Previous studies have shown that differentiation of trophoblasts to an invasive phenotype is accompanied by temporally and spatially regulated switching of their integrin repertoire. In the first trimester human placenta, alpha 6 integrins are restricted to cytotrophoblast (CTB) stem cells and downregulated in invasive CTBs, whereas alpha 5 beta 1 and alpha 1 beta 1 integrins are upregulated in differentiating and invasive CTBs. The goal of the present study was to determine whether these changes have functional consequences for CTB invasiveness. Using an in vitro invasion model, we determined first that aggregates of invading first trimester CTBs in vitro undergo the same pattern of integrin switching as was observed in situ, thereby validating the utility of the model. We then showed that antibody perturbation of interactions involving laminin or collagen type IV and their integrin alpha 1/beta 1 receptor inhibited invasion by CTBs, whereas perturbing interactions between fibronectin and the alpha 5/beta 1 fibronectin receptor accelerated invasion. Finally, we report that later gestation CTBs, which display greatly decreased invasive capacity, are also unable to upregulate alpha 1 beta 1 complexes, providing further evidence that this integrin is critical for CTB invasion. This gestational regulation is transcriptional. These data indicate that integrin switching observed during differentiation in situ has significant functional consequences for CTB invasion. The data suggest further that differentiating CTBs upregulate counterbalancing invasion-accelerating and invasion-restraining adhesion mechanisms. We propose that this contributes to regulating the depth of CTB invasion during normal implantation.
Assuntos
Implantação do Embrião , Integrinas/metabolismo , Trofoblastos/citologia , Northern Blotting , Diferenciação Celular , Movimento Celular/fisiologia , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fibronectinas/metabolismo , Humanos , Imuno-Histoquímica , Laminina/metabolismo , Modelos Biológicos , Receptores de Colágeno , Receptores de Fibronectina , Trofoblastos/metabolismoRESUMO
We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.