RESUMO
Sphingolipids are intrinsic components of membrane lipid rafts. The abnormal accumulation of these molecules may introduce architectural and functional changes in these domains, leading to cellular dysfunction. Galactosylsphingosine (psychosine) is a pathogenic lipid raft-associated molecule whose accumulation leads to brain deterioration and irreversible neurological handicap in the incurable leukodystrophy Krabbe disease (KD). The relevance of clearing excessive levels of pathogenic psychosine from lipid rafts in therapy for KD has not been investigated. The work presented here demonstrates that psychosine inhibits raft-mediated endocytosis in neural cells. In addition, although in vitro enzyme reconstitution is sufficient for the reversal of related endocytic defects in affected neural cells, traditional in vivo enzyme therapies in the mouse model of KD appear to be insufficient for complete removal of pathogenic levels of raft-associated psychosine. This work describes a mechanism that may contribute to limiting the in vivo efficacy of traditional therapies for KD.
Assuntos
Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/terapia , Microdomínios da Membrana/efeitos dos fármacos , Neurônios/patologia , Psicosina/farmacologia , Animais , Animais Recém-Nascidos , Transplante de Medula Óssea/métodos , Encéfalo/patologia , Células Cultivadas , Toxina da Cólera/metabolismo , Clatrina , Modelos Animais de Doenças , Endocitose/genética , Endocitose/fisiologia , Terapia de Reposição de Enzimas/métodos , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Humanos , Espectrometria de Massas , Camundongos , Proteína Quinase C/metabolismo , Psicosina/metabolismo , Fatores de TempoRESUMO
This study characterized the therapeutic benefits of combining hematogenous cell replacement with lentiviral-mediated gene transfer of galactosylceramidase (GALC) in Twitcher mice, a bona fide model for Krabbe disease. Bone marrow cells and GALC-lentiviral vectors were administered intravenously without any preconditioning to newborn Twitcher pups before postnatal day 2. Treated Twitchers survived up to 4 months of age. GALC activity remained less than 5% of normal values in the nervous system for the first 2 months after treatment and reached approximately 30% in long-term-surviving mice. Long-term reconstitution of GALC activity in the nervous system was provided primarily by infiltrating macrophages and to a lesser extent by direct lentiviral transduction of neural cells. Treated Twitchers had significant preservation of myelin, with a G-ratio (ratio of the axon diameter to the diameter of the myelinated fiber) in sciatic nerve myelin of 0.75 +/- 0.08 compared with 0.85 +/- 0.10 in untreated mutants. Although treated mutants had improved locomotor activities during their long-term survival, they died with symptoms of progressive neurological degeneration, indistinguishable from those seen in untreated Twitchers. Examination of long-lived Twitchers showed that treated mutants were not protected from developing degeneration of axons throughout the neuroaxis. These results suggest that GALC deficiency not only affects myelinating glia but also leads to neuronal dysfunction. The contemporaneous neuropathology might help to explain the limited efficacy of current gene and cell therapies.
Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/terapia , Transtornos Heredodegenerativos do Sistema Nervoso/terapia , Lentivirus/genética , Leucodistrofia de Células Globoides/terapia , Animais , Animais Recém-Nascidos , Transplante de Medula Óssea/métodos , Modelos Animais de Doenças , Galactosilceramidase/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/fisiopatologia , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/fisiopatologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Regeneração Nervosa/genética , Taxa de Sobrevida , Resultado do TratamentoRESUMO
AIM: To evaluate the status of activation of the intestinal dendritic cells (DCs) and T lymphocytes (T cells) from surgical specimens of human colon and adenocarcinoma, and the potential effect of administration of interleukin 2 (IL-2). METHODS: Patients undergoing colectomy for cancer were randomized to receive subcutaneous IL-2 (12million UI/day) (treated group; n=10) for 3days before operation or no treatment (control group, n=10). DCs and T cells were isolated and purified from the lamina propria (LP) of segments of normal colon and adenocarcinoma of both groups. Cell phenotype was determined by expression of membrane receptors. Interaction between DC and T cells was assesses by a mixed leukocyte reaction using naïve T cells co-cultured with DCs. CD4+ T-cell polarization was studied by intracellular staining with monoclonal antibodies for interleukin-4 and interferon-gamma. RESULTS: CD4+ T cells were significantly less in tumour than in LP (p<0.05) in both treated and control groups. IL-2 did not modify the number of any of the T-cell subsets analysed. In contrast, T cells isolated from LP and neoplasm of treated patients produced more interferon-gamma and less interleukin-4 (p<0.05 vs. controls). IL-2 administration significantly increased (p<0.05) the number of mature, myeloid and plasmocytoid DCs compared to controls. Allogeneic naïve T cells were polarized toward a Th1 type of response which appeared to be mediated by IL-2 activated DCs. CONCLUSIONS: systemic IL-2 treatment may have immunomodulatory properties on intestinal DC maturation and drive a Th1 mediated anti-neoplastic response.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/administração & dosagem , Linfócitos T CD4-Positivos/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Células Dendríticas/efeitos dos fármacos , Interleucina-2/administração & dosagem , Mucosa Intestinal/efeitos dos fármacos , Adenocarcinoma/cirurgia , Idoso , Linfócitos T CD4-Positivos/metabolismo , Colectomia , Neoplasias do Colo/cirurgia , Células Dendríticas/metabolismo , Feminino , Humanos , Mucosa Intestinal/citologia , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
It has been shown that each manipulation of the mammary region, including breast surgery, may stimulate prolactin secretion. However, it has also been observed that in more than 50% of breast cancer patients surgical removal of the tumor is not followed by enhanced prolactin secretion. This might be indicative of an altered psychoneuroendocrine control of the mammary gland, which could lead to the onset of more biologically aggressive breast cancer. In fact, surgery-induced hyperprolactinemia has been proven to be associated with a better prognosis in terms of survival in node-negative breast cancer patients. The present study was performed to investigate the impact of postoperative hyperprolactinemia on the disease-free survival (DFS) of breast cancer patients with axillary node involvement. The study included 100 consecutive node-positive breast cancer patients who were followed for at least 10 years. Surgery-induced hyperprolactinemia occurred in 45/100 (45%) patients without any significant correlation with the main prognostic variables including number of involved nodes and ER status. The two groups of patients received the same adjuvant therapies. After a median follow-up of 151 months, the recurrence rate in patients with surgery-induced hyperprolactinemia was significantly lower than in patients with no postoperative hyperprolactinemia (23/45 vs 43/55, p<0.01). Moreover, DFS was significantly longer in hyperprolactinemic patients than in patients who had no enhanced secretion of prolactin postoperatively. In agreement with the results described previously in node-negative breast cancer, our study demonstrates the favorable prognostic significance of surgery-induced hyperprolactinemia in terms of DFS duration also in breast cancer patients with axillary node involvement, independent of the other well-known prognostic variables, thereby confirming that the psychoneuroendocrine status of cancer patients may influence the prognosis of their disease.
Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Hiperprolactinemia/diagnóstico , Hiperprolactinemia/etiologia , Adulto , Idoso , Neoplasias da Mama/complicações , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Hiperprolactinemia/complicações , Hiperprolactinemia/patologia , Metástase Linfática/patologia , Pessoa de Meia-Idade , Prognóstico , Fatores de TempoRESUMO
Felty's syndrome (FS) is a rare complication (less than 1%) of rheumatoid arthritis (RA), with the clinical feature of splenomegaly and neutropenia. Approximately 10-40% of FS patients have an expansion of peripheral blood large granular lymphocytes (LGL). This cell population mainly consists of two subsets: cytotoxic T cells (CD8+, CD57+) and natural killer cells (CD3-,CD8-,CD56+). It has been hypothesised that LGL expansion could be responsible for neutropenia by suppressing neutrophil precursors in the bone marrow, but various mechanisms have been proposed to explain this association. We report a case of a 60-year-old woman with rheumatoid factor positive RA who developed LGL expansion responsible for splenomegaly, but without neutropenia. In conclusion, LGL expansion is an uncommon complication of RA and may be responsible for both FS and clinical pictures resembling FS.
Assuntos
Síndrome de Felty/complicações , Leucemia Linfoide/complicações , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Caveolae are vesicular invaginations of the plasma membrane, and function as 'message centers' for regulating signal transduction events. Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolar membrane domains in skeletal muscle and in the heart. Several mutations within the coding sequence of the human caveolin-3 gene (located at 3p25) have been identified. Mutations that lead to a loss of approximately 95% of caveolin-3 protein expression are responsible for a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans. By contrast, upregulation of the caveolin-3 protein is associated with Duchenne muscular dystrophy (DMD). Thus, tight regulation of caveolin-3 appears essential for maintaining normal muscle health and homeostasis.
Assuntos
Cavéolas/metabolismo , Caveolinas/genética , Caveolinas/metabolismo , Distrofias Musculares/genética , Animais , Cavéolas/química , Caveolina 3 , Modelos Animais de Doenças , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismoRESUMO
Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.
Assuntos
Caveolinas/metabolismo , Ciclo Celular/fisiologia , Ciclinas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Caspase 3 , Caspases/metabolismo , Caveolina 1 , Separação Celular , Células Cultivadas , Meios de Cultura Livres de Soro , Inibidor de Quinase Dependente de Ciclina p21 , Inibidores Enzimáticos/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Citometria de Fluxo , Genes Reporter , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estaurosporina/farmacologiaRESUMO
Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma virus-transformed cells. Previous studies have shown that caveolin-1 is phosphorylated on tyrosine 14 by c-Src and that lipid modification of c-Src is required for this phosphorylation event to occur in vivo. Phosphocaveolin-1 (Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain largely unknown. Here, we show that palmitoylation of caveolin-1 at a single site (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kinase. Furthermore, upon evaluating a battery of nonreceptor and receptor tyrosine kinases, we demonstrate that the tyrosine phosphorylation of caveolin-1 by c-Src is a highly selective event. We show that Src-induced tyrosine phosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dually acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH(2)-terminal domain of Galpha(i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, when these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptide derived from the extreme N terminus of c-Src, the tyrosine phosphorylation of caveolin-1 is abrogated; the same peptide lacking myristoylation has no inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-14) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactions as important regulatory factors for coupling caveolin-1 to the c-Src tyrosine kinase in vivo.
Assuntos
Caveolinas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Ácido Palmítico/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Antígenos CD36/metabolismo , Células COS , Proteína Tirosina Quinase CSK , Caveolina 1 , Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Ácido Mirístico/metabolismo , Fosforilação , Tirosina/metabolismo , Domínios de Homologia de src , Quinases da Família srcRESUMO
Caveolins 1, 2, and 3 are the principal proteins of caveolae, the vesicular invaginations of the plasma membrane. Several reports have suggested that caveolin-1 may have a role in cellular transformation and tumorigenesis. We studied the expression of caveolin-1 and caveolin-2 in normal epithelium, adenoma, and adenocarcinoma of the colon and their possible role in tumorigenesis. Formalin-fixed, paraffin-embedded sections of 41 cases of adenocarcinoma and 13 cases of adenoma of the colon were stained immunohistochemically with anti-caveolin-1 and anti-caveolin-2 antibodies. The expression of caveolin-1 was elevated in the overwhelming majority of the adenocarcinomas, while most normal colonic epithelium and adenomas showed little or no staining. There was significant statistical correlation of the expression of caveolin-1 with adenocarcinoma but not with tumor stage. Expression of caveolin-2 was undetectable in all of the normal colonic glands, adenomas, and carcinomas. We discuss the possible clinical implications of our findings within the context of caveolins and signal transduction.
Assuntos
Adenocarcinoma/metabolismo , Caveolinas/biossíntese , Neoplasias do Colo/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adenoma/metabolismo , Adenoma/patologia , Adenoma/cirurgia , Caveolina 1 , Caveolina 2 , Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/cirurgia , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Transdução de SinaisRESUMO
Caveolin-1 is a principal structural component of caveolae membranes. These membrane microdomains participate in the regulation of signaling, transcytosis, and cholesterol homeostasis at the plasma membrane. In the present study, we determined the effect of caveolin-1 expression on cellular cholesterol efflux mediated by high-density lipoprotein (HDL). We evaluated this effect in parental NIH/3T3 cells as well as in two transformed NIH/3T3 cell lines in which caveolin-1 protein levels are dramatically downregulated. Compared with parental NIH/3T3 cells, these two transformed cell lines effluxed cholesterol more rapidly to HDL. In addition, NIH/3T3 cells harboring caveolin-1 antisense also effluxed cholesterol more rapidly to HDL. However, this effect was not due to changes in total cellular cholesterol content. We further showed that chronic HDL exposure reduced caveolin-1 protein expression in NIH/3T3 cells. HDL exposure also inhibited caveolin-1 promoter activity, suggesting a direct negative effect of HDL on caveolin-1 gene transcription. Moreover, we showed that HDL-induced downregulation of caveolin-1 prevents the uptake of oxidized low-density lipoprotein in human endothelial cells. These data suggest a novel proatherogenic role for caveolin-1, i.e., regarding the uptake and/or transcytosis of modified lipoproteins.
Assuntos
Caveolinas/fisiologia , Colesterol/metabolismo , Lipoproteínas HDL/farmacologia , Fatores de Transcrição , Células 3T3 , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Caveolina 1 , Caveolinas/genética , Linhagem Celular Transformada , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Genes abl , Genes ras , Cinética , Luciferases/genética , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas , Proteína de Ligação a Elemento Regulador de Esterol 1 , TransfecçãoRESUMO
The potential role of caveolin-1 in apoptosis remains controversial. Here, we investigate whether caveolin-1 expression is proapoptotic or antiapoptotic using a well-defined antisense approach. We show that NIH/3T3 cells harboring antisense caveolin-1 are resistant to staurosporine-induced apoptosis, as assessed using cell morphology, DNA content, caspase 3 activation, and focal adhesion kinase cleavage. Importantly, sensitivity to apoptosis is recovered when caveolin-1 levels are restored. Conversely, recombinant stable expression of caveolin-1 in T24 bladder carcinoma cells sensitizes these cells to caspase 3 activation. Consistent with the observations using NIH/3T3 cells, downregulation of caveolin-1 in T24 cells substantially diminishes caspase 3-like activity. Loss of sensitivity to apoptotic stimulation is recovered by inhibition of the phosphatidylinositol 3-kinase pathway using LY-294002, suggesting a possible mechanism for the sensitizing effect of caveolin-1. Thus our results suggest that caveolin-1 may act as a coupling or sensitizing factor in signaling apoptotic cell death in both fibroblastic (NIH/3T3) and epithelial (T24) cells.
Assuntos
Apoptose/fisiologia , Caveolinas/genética , Células Epiteliais/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Caveolina 1 , Cromonas/farmacologia , DNA Antissenso , Regulação para Baixo/fisiologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/enzimologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Expressão Gênica/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Estaurosporina/farmacologia , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/enzimologia , Neoplasias da Bexiga UrináriaRESUMO
Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.
Assuntos
Caveolinas/metabolismo , Substâncias de Crescimento/metabolismo , Proteínas/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Células 3T3 , Adipócitos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Cavéolas/metabolismo , Caveolina 1 , Caveolinas/genética , Caveolinas/imunologia , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Feminino , Proteína Adaptadora GRB7 , Humanos , Insulina/metabolismo , Insulina/farmacologia , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Transdução de Sinais , Vanadatos/farmacologiaRESUMO
Caveolin-3 is the principal structural protein of caveolae in striated muscle. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (DeltaTFT and Pro --> Leu) within the CAV3 gene. We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly and are rapidly degraded. The mechanism by which LGMD-1C mutants of caveolin-3 are degraded remains unknown. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination-proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. In the presence of MG-132, LGMD-1C caveolin-3 mutants accumulated within the endoplasmic reticulum and did not reach the plasma membrane. LGMD-1C mutants of caveolin-3 behave in a dominant negative fashion, causing intracellular retention and degradation of wild-type caveolin-3. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; wild-type caveolin-3 was not degraded and reached the plasma membrane. These results may have clinical implications for treatment of patients with LGMD-1C.
Assuntos
Caveolinas/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Complexos Multienzimáticos/metabolismo , Distrofias Musculares/genética , Mutação , Ubiquitinas/metabolismo , Células 3T3 , Animais , Caveolina 3 , Caveolinas/genética , Inibidores de Cisteína Proteinase/farmacologia , Proteínas do Citoesqueleto/química , Hidrólise , Glicoproteínas de Membrana/química , Camundongos , Complexos Multienzimáticos/antagonistas & inibidores , Complexo de Endopeptidases do ProteassomaRESUMO
Caveolin-1 is a principal component of caveolae membranes. In NIH 3T3 cells, caveolin-1 expression is dramatically up-regulated in confluent cells and localizes at areas of cell-cell contact. However, it remains unknown whether caveolin-1 is involved in cell-cell signaling. Here, we examine the potential role of caveolin-1 in regulating beta-catenin signaling. beta-Catenin plays a dual role in the cell, linking E-cadherin to the actin cytoskeleton and in Wnt signaling by forming a complex with members of the lymphoid enhancing factor (Lef-1) family of transcription factors. We show that E-cadherin, beta-catenin, and gamma-catenin (plakoglobin) are all concentrated in caveolae membranes. Moreover, we demonstrate that activation of beta-catenin/Lef-1 signaling by Wnt-1 or by overexpression of beta-catenin itself is inhibited by caveolin-1 expression. We also show that recombinant expression of caveolin-1 in caveolin-1 negative cells is sufficient to recruit beta-catenin to caveolae membranes, thereby blocking beta-catenin-mediated transactivation. These results suggest that caveolin-1 expression can modulate Wnt/beta-catenin/Lef-1 signaling by regulating the intracellular localization of beta-catenin.
Assuntos
Caveolinas , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transativadores , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra , Células 3T3 , Animais , Caveolina 1 , Membrana Celular/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Ativação Transcricional , Proteínas Wnt , Proteína Wnt1 , beta CateninaRESUMO
The cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma pRB protein. Cyclin D1 protein levels are elevated by mitogenic and oncogenic signaling pathways, and antisense mRNA to cyclin D1 inhibits transformation by the ras, neu, and src oncogenes, thus linking cyclin D1 regulation to cellular transformation. Caveolins are the principal protein components of caveolae, vesicular plasma membrane invaginations that also function in signal transduction. We show here that caveolin-1 expression levels inversely correlate with cyclin D1 abundance levels in transformed cells. Expression of antisense caveolin-1 increased cyclin D1 levels, whereas caveolin-1 overexpression inhibited expression of the cyclin D1 gene. Cyclin D1 promoter activity was selectively repressed by caveolin-1, but not by caveolin-3, and this repression required the caveolin-1 N terminus. Maximal inhibition of the cyclin D1 gene promoter by caveolin-1 was dependent on the cyclin D1 promoter T-cell factor/lymphoid enhancer factor-1-binding site between -81 to -73. The T-cell factor/lymphoid enhancer factor sequence was sufficient for repression by caveolin-1. We suggest that transcriptional repression of the cyclin D1 gene may contribute to the inhibition of transformation by caveolin-1.
Assuntos
Caveolinas , Ciclina D1/genética , Regulação da Expressão Gênica , Proteínas de Membrana/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Aminoácidos , Animais , Células CHO , Caveolina 1 , Membrana Celular/fisiologia , Cricetinae , Meios de Cultura Livres de Soro , Proteínas de Ligação a DNA/metabolismo , Humanos , Fator 1 de Ligação ao Facilitador Linfoide , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Successful beta-cell replacement therapy in insulin-dependent (type I) diabetes is hindered by the scarcity of human donor tissue and by the recurrence of autoimmune destruction of transplanted beta cells. Availability of non-beta cells, capable of releasing insulin and escaping autoimmune recognition, would therefore be important for diabetes cell therapy. We developed rat pituitary GH3 cells stably transfected with a furin-cleavable human proinsulin cDNA linked to the rat PRL promoter. Two clones (InsGH3/clone 1 and 7) were characterized in vitro with regard to basal and stimulated insulin release and proinsulin transgene expression. Mature insulin secretion was obtained in both clones, accounting for about 40% of total released (pro)insulin-like products. Immunocytochemistry of InsGH3 cells showed a cytoplasmic granular insulin staining that colocalized with secretogranin II (SGII) immunoreactivity. InsGH3 cells/clone 7 contained and released in vitro significantly more insulin than clone 1. Secretagogue-stimulated insulin secretion was observed in both InsGH3 clones either under static or dynamic conditions, indicating that insulin was targeted also to the regulated secretory pathway. Proinsulin mRNA levels were elevated in InsGH3 cells, being significantly higher than in betaTC3 cells. Moreover, proinsulin gene expression increased in response to various stimuli, thereby showing the regulation of the transfected gene at the transcriptional level. In conclusion, these data point to InsGH3 cells as a potential beta-cell surrogate even though additional engineering is required to instruct them to release insulin in response to physiologic stimulations.
Assuntos
Transplante de Células/métodos , Células Clonais/transplante , Hipófise/citologia , Proinsulina/genética , Transfecção , Animais , Cálcio/metabolismo , Cromograninas , Células Clonais/química , Células Clonais/metabolismo , Colforsina/farmacologia , DNA Complementar/genética , Diabetes Mellitus Tipo 1/terapia , Eletroforese Capilar , Regulação da Expressão Gênica , Humanos , Insulina/análise , Insulina/metabolismo , Secreção de Insulina , Camundongos , Mitógenos/farmacologia , Neuropeptídeos/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Proinsulina/análise , Regiões Promotoras Genéticas/genética , Proteínas/análise , RNA Mensageiro/análise , Radioimunoensaio , Ratos , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Transgenes/fisiologiaRESUMO
In a companion article, we describe the engineering and characterization of pituitary GH3 cell clones stably transfected with a furin-cleavable human insulin cDNA (InsGH3 cells). This article describes the performance of InsGH3 (clones 1 and 7) cell grafts into streptozotocin (STZ)-induced diabetic nude mice. Subcutaneous implantation of 2 x 10(6) InsGH3 cells resulted in the progressive reversal of hyperglycemia and diabetic symptoms, even though the progressive growth of the transplanted cells (clone 7) eventually led to glycemic levels below the normal mouse range. Proinsulin transgene expression was maintained in harvested InsGH3 grafts that, conversely, lose the expression of the prolactin (PRL) gene. Elevated concentrations of circulating mature human insulin were detected in graft recipients, demonstrating that proinsulin processing by InsGH3 cells did occur in vivo. Histologic analysis showed that transplanted InsGH3 grew in forms of encapsulated tumors composed of cells with small cytoplasms weakly stained for the presence of insulin. Conversely, intense insulin immunoreactivity was detected in graft-draining venules. Compared to pancreatic betaTC3 cells, InsGH3 cells showed in vitro a higher rate of replication, an elevate resistance to apoptosis induced by serum deprivation and proinflammatory cytokines, and significantly higher antiapoptotic Bcl-2 protein levels. Moreover, InsGH3 cells were resistant to the streptozotocin toxicity that, in contrast, reduced betaTC3 cell viability to 50-60% of controls. In conclusion, proinsulin gene expression and mature insulin secretion persisted in transplanted InsGH3 cells that reversed hyperglycemia in vivo. InsGH3 cells might represent a potential beta-cell surrogate because they are more resistant than pancreatic beta cells to different apoptotic insults and might therefore be particularly suitable for encapsulation.
Assuntos
Transplante de Células/métodos , Células Clonais/transplante , Diabetes Mellitus Tipo 1/terapia , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Animais , Apoptose , Divisão Celular , Sobrevivência Celular , Células Clonais/metabolismo , Diabetes Mellitus Experimental/terapia , Formazans , Sobrevivência de Enxerto , Hibridização In Situ , Secreção de Insulina , Ilhotas Pancreáticas/química , Masculino , Camundongos , Camundongos Nus , Hipófise/citologia , Proinsulina/genética , Proteínas Proto-Oncogênicas c-bcl-2/análise , Ratos , Pele , Sais de TetrazólioRESUMO
Caveolin-3 is the principal structural protein of caveolae membrane domains in striated muscle cells. Caveolin-3 mRNA and protein expression are dramatically induced during the differentiation of C2C12 skeletal myoblasts, coincident with myoblast fusion. In these myotubes, caveolin-3 localizes to the sarcolemma (muscle cell plasma membrane), where it associates with the dystrophin-glycoprotein complex. However, it remains unknown what role caveolin-3 plays in myoblast differentiation and myotube formation. Here, we employ an antisense approach to derive stable C2C12 myoblasts that fail to express the caveolin-3 protein. We show that C2C12 cells harboring caveolin-3 antisense undergo differentiation and express normal amounts of four muscle-specific marker proteins. However, C2C12 cells harboring caveolin-3 antisense fail to undergo myoblast fusion and, therefore, do not form myotubes. Interestingly, treatment with specific p38 mitogen-activated protein kinase inhibitors blocks both myotube formation and caveolin-3 expression, but does not affect the expression of other muscle-specific proteins. In addition, we find that three human rhabdomyosarcoma cell lines do not express caveolin-3 and fail to undergo myoblast fusion. Taken together, these results support the idea that caveolin-3 expression is required for myoblast fusion and myotube formation, and suggest that p38 is an upstream regulator of caveolin-3 expression.
Assuntos
Caveolinas , Diferenciação Celular , Regulação para Baixo , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Esquelético/metabolismo , Caveolina 3 , Fusão Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Oligonucleotídeos Antissenso/farmacologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
T cell-mediated inflammation is considered to play a key role in the pathogenic mechanisms sustaining multiple sclerosis (MS). Caspase-1, formerly designated IL-1beta-converting enzyme, is crucially involved in immune-mediated inflammation because of its pivotal role in regulating the cellular export of IL-1beta and IL-18. We studied the role of caspase-1 in experimental autoimmune encephalomyelitis (EAE), the animal model for MS. Caspase-1 is transcriptionally induced during EAE, and its levels correlate with the clinical course and transcription rate of proinflammatory cytokines such as TNF-alpha, IL-1beta, IFN-gamma, and IL-6. A reduction of EAE incidence and severity is observed in caspase-1-deficient mice, depending on the immunogenicity and on the amount of the encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide used. In caspase-1-deficient mice, reduced EAE incidence correlates with defective development of anti-MOG IFN-gamma-producing Th1 cells. Finally, pharmacological blockade of caspase-1 in Biozzi AB/H mice, immunized with spinal cord homogenate or MOG35-55 peptide, by the caspase-1-inhibitor Z-Val-Ala-dl -Asp-fluoromethylketone, significantly reduces EAE incidence in a preventive but not in a therapeutic protocol. These results indicate that caspase-1 plays an important role in the early stage of the immune-mediated inflammatory process leading to EAE, thus representing a possible therapeutic target in the acute phase of relapsing remitting MS.
Assuntos
Caspase 1/fisiologia , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/imunologia , Bainha de Mielina/fisiologia , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Clorometilcetonas de Aminoácidos/administração & dosagem , Animais , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Doenças Autoimunes/fisiopatologia , Caspase 1/deficiência , Caspase 1/genética , Inibidores de Caspase , Inibidores de Cisteína Proteinase/administração & dosagem , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/prevenção & controle , Feminino , Imunossupressores/administração & dosagem , Bombas de Infusão Implantáveis , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/patologia , Fármacos Neuroprotetores/administração & dosagem , RNA Mensageiro/biossíntese , Medula Espinal/imunologia , Células Th1/enzimologia , Células Th1/imunologia , Regulação para Cima/imunologiaRESUMO
This study was performed to determine if rat articular chondrocytes express caveolin, the structural protein of caveolae, and to determine differences in the distribution of the caveolin subtypes 1, 2 and 3 in knee joints of newborn and adult rats. All three subtypes of caveolin were detected in adult cartilage by immunocytochemical staining. In newborn rats, only caveolin-1 was found in the hyaline cartilage. Caveolin-1, -2 and -3 messenger RNA and protein were also detected in chondrocyte cell cultures. Ultrastructural investigations of cell culture and cartilage tissue revealed the presence of caveolae at the plasma membrane of chondrocytes. These findings represent the first report on the different expression of caveolin isoforms, in particular the expression of the muscle cell-specific caveolin-3 in chondrocytes. There is evidence that caveolin-2 and -3 are upregulated during growth and development of articular cartilage, suggesting a role for caveolins in chondrocyte differentiation.