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Here, we identify that Caveolin-2 (Cav-2), an integral membrane protein, controls adipocyte hypertrophy in association with nuclear lamina. In the hypertrophy stage of adipogenesis, pY19-Cav-2 association with lamin A/C facilitated the disengagement of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) from lamin A/C and repressed Cav-2 promoter at the nuclear periphery for epigenetic activation of Cav-2, and thereby promoted C/EBPα and PPARγ-induced adipocyte hypertrophy. Stable expression of Cav-2 was required and retained by phosphorylation, deubiquitination, and association with lamin A/C for the adipocyte hypertrophy. However, obese adipocytes exhibited augmented Cav-2 stability resulting from the up-regulation of lamin A/C over lamin B1, protein tyrosine phosphatase 1B (PTP1B), and nuclear deubiquitinating enzyme (DUB), Uchl5. Our findings show a novel epigenetic regulatory mechanism of adipocyte hypertrophy by Cav-2 at the nuclear periphery.
Assuntos
Lamina Tipo A , PPAR gama , Humanos , Camundongos , Animais , PPAR gama/genética , PPAR gama/metabolismo , Lamina Tipo A/metabolismo , Lâmina Nuclear/metabolismo , Caveolina 2/genética , Caveolina 2/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Adipócitos/metabolismo , Hipertrofia/metabolismo , Diferenciação Celular , Adipogenia/genética , Células 3T3-L1RESUMO
Caveolin-2 is a protein suitable for the study of interactions of caveolins with other proteins and lipids present in caveolar lipid rafts. Caveolin-2 has a lower tendency to associate with high molecular weight oligomers than caveolin-1, facilitating the study of its structural modulation upon association with other proteins or lipids. In this paper, we have successfully expressed and purified recombinant human caveolin-2 using E. coli. The structural changes of caveolin-2 upon interaction with a lipid bilayer of liposomes were characterized using bioinformatic prediction models, circular dichroism, differential scanning calorimetry, and fluorescence techniques. Our data support that caveolin-2 binds and alters cholesterol-rich domains in the membranes through a CARC domain, a type of cholesterol-interacting domain in its sequence. The far UV-CD spectra support that the purified protein keeps its folding properties but undergoes a change in its secondary structure in the presence of lipids that correlates with the acquisition of a more stable conformation, as shown by differential scanning calorimetry experiments. Fluorescence experiments using egg yolk lecithin large unilamellar vesicles loaded with 1,6-diphenylhexatriene confirmed that caveolin-2 adsorbs to the membrane but only penetrates the core of the phospholipid bilayer if vesicles are supplemented with 30% of cholesterol. Our study sheds light on the caveolin-2 interaction with lipids. In addition, we propose that purified recombinant caveolin-2 can provide a new tool to study protein-lipid interactions within caveolae.
Assuntos
Caveolina 1 , Escherichia coli , Humanos , Escherichia coli/metabolismo , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Cavéolas/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Bicamadas Lipídicas/metabolismoRESUMO
OBJECTIVES: Caveolin family proteins, including caveolin-1 (Cav-1), caveolin-2 (Cav-2), and caveolin-3 (Cav-3), are identified as the principal protein components of caveolae in mammalian cells. Circulating form of caveolin family proteins can be used as a good potential biomarker for predicting disease. METHODS: To investigate the clinical significance of the serological levels of caveolin family proteins in patients with systemic lupus erythematosus (SLE), we evaluated the soluble serum levels of caveolin family proteins in patients with SLE by enzyme-linked immunosorbent assay (ELISA) and assessed their associations with various known clinical variables. RESULTS: The major findings of our study are as follows: Cav-2 was not detected in serum of SLE patients and normal controls (NCs). Serum Cav-1 and Cav-3 levels were higher in SLE patients compared with NCs. There were no significant correlations between serum Cav-1 and Cav-3 levels and SLE disease activity. Further analysis showed that serum Cav-3 may be more valuable as a marker than serum Cav-1 in SLE patients. CONCLUSION: Serum levels of Cav-1 and Cav-3 might have a diagnostic role in patients with SLE. However, their predictive and prognostic value was not determined. Further studies are necessary to determine the potential clinical significance of these assays in SLE.
Assuntos
Biomarcadores , Caveolinas , Lúpus Eritematoso Sistêmico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Caveolina 1/biossíntese , Caveolina 1/sangue , Caveolina 3/biossíntese , Caveolina 3/sangue , Caveolinas/biossíntese , Caveolinas/sangue , Criança , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Adulto JovemRESUMO
N-myristoylation is a ubiquitous protein lipidation in eukaryotes, but regulatory roles for myristoylation on proteins still remain to be explored. Here, we show that N-myristoylation of Caveolin-2 (Cav-2) controls insulin signaling. Alternative translation initiation (ATI)-yielded truncated form of non-N-myristoylable Cav-2ß and various conditional Cav-2 mutants were compared to full-length form of N-myristoylable Cav-2α. Insulin induced insulin receptor (IR) tyrosine kinase-catalyzed Tyr-19 phosphorylation of N-myristoylable M14A Cav-2 and triggered activation of IR signaling cascade. In contrast, insulin induced ubiquitination of non-N-myristoylable M1A and G2A Cav-2 to facilitate protein-tyrosine phosphatase 1B interaction with IR which desensitized IR signaling through internalization. Metabolic labeling and click chemistry showed palmitoylation of M14A but not M1A and G2A Cav-2. Insulin did not induce phosphorylation of M1A and G2A Cav-2 and Cav-2ß. Like Cav-2α, G2A Cav-2 and Cav-2ß formed large homo-oligomers localized in lipid rafts. These findings show Cav-2 N-myristoylation plays a crucial role to coordinate its phosphorylation, palmitoylation, and ubiquitination to control insulin signaling.
Assuntos
Caveolina 2/metabolismo , Insulina/fisiologia , Transdução de Sinais , Animais , Caveolina 2/química , Linhagem Celular , Humanos , Lipoilação , Microdomínios da Membrana/metabolismo , Ácido Mirístico/metabolismo , Fosforilação , Ratos , Receptor de Insulina/metabolismo , Tirosina/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: The bromodomain and extra-terminal domain (BET) family of proteins, especially BRD4 play an important role in epigenetic regulation, and are essential for cell survival and also are promising anticancer targets. This study aims to analyze the effect of BRD4 on the cell growth and progression of pancreatic cancer and novel mechanisms involved. METHODS: Expression of BRD4 in pancreatic cancer and paired adjacent noncancerous tissues from 76 patients was analyzed by western blotting, immunohistochemistry, and real time PCR. Its correlation with the clinicopathological characteristics and prognosis of pancreatic cancer patients was analyzed. The effects of BRD4 on the cell proliferation were detected by colony formation assay and sulforhodamine B assay. Migration and invasion were determined by Transwell assays, and the effect of BRD4 on subcutaneous tumor formation was verified in nude mice. Cell cycle analysis was detected by flow cytometry. The potential downstream targets of BRD4 and related molecular mechanisms were clarified by RNA sequencing, chromatin immunoprecipitation and dual luciferase reporter assay. RESULTS: BRD4 was overexpressed in pancreatic cancer. Biological results showed that BRD4 functioned as tumor promoter, facilitated cell proliferation, migration and invasion in vitro and in vivo. Further, caveolin-2 was selected as the downstream gene of BRD4 by RNA sequencing. Caveolin-2 overexpression can partially reverse the decreased cell growth ability caused by BRD4 knockdown, but did not affect cell migration and invasion. Chromatin immunoprecipitation assay and dual luciferase reporter assay revealed BRD4 could bind to the promoter region of caveolin-2 and upregulate caveolin-2 expression. Clinical data further indicated a positive correlation between BRD4 and caveolin-2 expression. BRD4 (high)/caveolin-2 (high) correlated with shorter overall survival of patients with pancreatic cancer. Multivariate analysis revealed that both BRD4 and caveolin-2 were independent factors. CONCLUSIONS: Our findings reveal the oncogenic effects of BRD4 in pancreatic cancer and elucidate a possible mechanism by which BRD4 and caveolin-2 act to enhance cell growth. Targeting the BRD4-caveolin-2 interaction by development of BET inhibitors will be a therapeutic strategy for pancreatic cancer.
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Caveolin-2 (Cav-2) is expressed in a variety of cell tissue, and it has also been found in renal tissue. The expression of Cav-2 in proximal tubules is still unclear. The aim of this study was to carry out a complete evaluation of the expression pattern of Cav-2 in rat renal cortex to clarify and deepen the knowledge about the localization of Cav-2 in the proximal tubules and also to evaluate its presence in urine. Male Wistar rats were used to assess Cav-2 expression by Western blot analysis in homogenates, apical, and basolateral membranes from kidney cortex, in lysates and total plasma membranes from renal cortical cell suspensions, in urine, and in urinary exosomes. Cav-2 was clearly expressed in renal cortex homogenates and in both apical and basolateral membranes isolated from kidney cortex, with a greater expression on the former membranes. It was also observed in lysates and in plasma membranes from cortical cell suspensions. Moreover, Cav-2 was found in urine and in its exosomal fraction. These results confirmed the presence of Cav-2 in proximal tubule cells in the kidney of healthy rats, and showed for the first time its expression at the apical membrane of these cells and in urine. Besides, urinary exosomal pathway could be involved in Cav-2 urinary excretion under normal conditions. We observed an increase in the urinary abundance of Cav-2 in two models of acute kidney injury, and thus we proposed the urinary excretion of Cav-2 as a potential biomarker of kidney injury.
Assuntos
Injúria Renal Aguda/urina , Caveolina 2/urina , Membrana Celular/genética , Exossomos/metabolismo , Túbulos Renais Proximais/metabolismo , Injúria Renal Aguda/patologia , Animais , Biomarcadores/urina , Membrana Celular/patologia , Exossomos/patologia , Túbulos Renais Proximais/patologia , Masculino , Ratos , Ratos WistarRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive gastrointestinal tumors, with an overall 5-year survival rate less than 8%. The dismal prognosis is mainly due to aggressive potential for metastasis. Hence, there is an urgent need for a better understanding of the molecular mechanisms underlying pancreatic cancer invasion and metastasis to improve the unfavorable overall survival (OS) of PDAC patients. In this study, we identified microRNA-29a (miR-29a) as an important tumor suppressor, which was downregulated in PDAC tissues. Moreover, miR-29a counteracted MUC16-mediated migration and invasion. In the pancreatic cancer cells, MUC16 upregulated c-Myc expression, which enhanced c-Myc binding to E-box in the miR-29a promoter and inhibited miR-29a transcription. Thus, miR-29a was negatively correlated with both MUC16 expression and serum CA125 levels. Furthermore, caveolin 2 (CAV2) was demonstrated to be the target of miR-29a by bioinformatics and luciferase reporter assays, and high CAV2 expression was responsible for a poor prognosis, especially in the subgroup with normal CA125 levels. Thus, the present study explains why high levels of serum CA125 are correlated with PDAC metastasis, highlighting the predictive value of this marker in PDAC patients.
Assuntos
Antígeno Ca-125/sangue , Caveolina 2/genética , Proteínas de Membrana/sangue , MicroRNAs/genética , Metástase Neoplásica/genética , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Humanos , Metástase Neoplásica/patologia , Neoplasias Pancreáticas/patologia , PrognósticoRESUMO
Insulin resistance, defined as attenuated sensitivity responding to insulin, impairs insulin action. Direct causes and molecular mechanisms of insulin resistance have thus far remained elusive. Here we show that alternative translation initiation (ATI) of Caveolin-2 (Cav-2) regulates insulin sensitivity. Cav-2ß isoform yielded by ATI desensitizes insulin receptor (IR) via dephosphorylation by protein-tyrosine phosphatase 1B (PTP1B), and subsequent endocytosis and lysosomal degradation of IR, causing insulin resistance. Blockage of Cav-2 ATI protects against insulin resistance by preventing Cav-2ß-PTP1B-directed IR desensitization, thereby normalizing insulin sensitivity and glucose uptake. Our findings show that Cav-2ß is a negative regulator of IR signaling, and identify a mechanism causing insulin resistance through control of insulin sensitivity via Cav-2 ATI.
Assuntos
Antígenos CD/metabolismo , Caveolina 2/metabolismo , Resistência à Insulina/genética , Iniciação Traducional da Cadeia Peptídica/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Células 3T3 , Animais , Antígenos CD/genética , Caveolina 2/genética , Códon de Iniciação/genética , Endocitose , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteólise , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor de Insulina/genéticaRESUMO
This study aimed to investigate the effects of transient receptor potential vanilloid 4 (TRPV4) inhibition on blood-brain barrier (BBB) integrity and the expressions of caveolae structural proteins caveolin-1 and caveolin-2 in rats with focal cerebral ischemia and reperfusion. BBB permeability was assessed by Evans blue extravasation. The mRNA and protein expressions of caveolin-1 and caveolin-2 were determined by RT-PCR, Western blot and immunohistochemistry assays. We found that BBB permeability significantly increased and reaches its peak at 72 h of reperfusion in cerebral ischemia-reperfusion rats and is able to be ameliorated by administration of HC-067047, an antagonist of TRPV4. Additionally, it shows a significant upregulation of caveolin-1 and caveolin-2 expression in cerebral microvessels of ischemic tissue. However, treatment with HC-067047 was shown to downregulate caveolin-1 and caveolin-2 expression during cerebral ischemia-reperfusion. This study demonstrates that inhibition of TRPV4 ameliorates BBB leakage induced by ischemia-reperfusion injury through the downregulation of caveolin-1 and caveolin-2.
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BACKGROUND: We recently demonstrated that the heart of late pregnant (LP) rodents is more prone to ischemia/reperfusion (I/R) injury compared to non-pregnant rodents. Lipids, particularly polyunsaturated fatty acids, have received special attention in the field of cardiovascular research. Here, we explored whether Intralipid (ITLD) protects the heart against I/R injury in LP rodents and investigated the mechanisms underlying this protection. METHODS AND RESULTS: In-vivo female LP rat hearts or ex-vivo isolated Langendorff-perfused LP mouse hearts were subjected to ischemia followed by reperfusion with PBS or ITLD (one bolus of 5mg/kg of 20% in in-vivo and 1% in ex-vivo). Myocardial infarct size, mitochondrial calcium retention capacity, genome-wide expression profiling, pharmacological inhibition and co-immunoprecipitation were performed. One bolus of ITLD at reperfusion significantly reduced the in-vivo myocardial infarct size in LP rats (23.3±2% vs. 55.5±3.4% in CTRL, p<0.01). Postischemic administration of ITLD also protected the LP hearts against I/R injury ex-vivo. ITLD significantly increased the threshold for the opening of the mitochondrial permeability transition pore in response to calcium overload (nmol-calcium/mg-mitochondrial protein: 290±17 vs. 167±10 in CTRL, p<0.01) and significantly increased phosphorylation of STAT3 (1.8±0.08 vs. 1±0.16 in CTRL, p<0.05) and GSK-3ß (2.63±0.55 vs. 1±0.0.34 in CTRL, p<0.05). The ITLD-induced cardioprotection was fully abolished by Stattic, a specific inhibitor of STAT3. Transcriptome analysis revealed caveolin 2 (Cav2) was significantly upregulated by ITLD in hearts of LP rats under I/R injury. Co-immunoprecipitation experiments showed that Cav2 interacts with STAT3. CONCLUSIONS: ITLD protects the heart in late pregnancy against I/R injury by inhibiting the mPTP opening through Cav2/STAT3/GSK-3ß pathway.
Assuntos
Caveolina 2/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Fosfolipídeos/farmacologia , Substâncias Protetoras/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Óleo de Soja/farmacologia , Animais , Cálcio/metabolismo , Análise por Conglomerados , Modelos Animais de Doenças , Emulsões/administração & dosagem , Emulsões/farmacologia , Feminino , Perfilação da Expressão Gênica , Camundongos , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Poro de Transição de Permeabilidade Mitocondrial , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Permeabilidade , Fosfolipídeos/administração & dosagem , Fosforilação , Gravidez , Substâncias Protetoras/administração & dosagem , Ligação Proteica , Ratos , Óleo de Soja/administração & dosagem , Fatores de Tempo , TranscriptomaRESUMO
Association of Caveolin-2 in the inner nuclear membrane specifically with A-type lamin is crucial for the maintenance of its Tyr-19 phosphorylation to promote insulin-response epigenetic activation at the nuclear periphery. Here, we identify that pY19-Caveolin-2 in the inner nuclear membrane exists as homo-oligomeric forms and the A-type lamin is required for sustenance of its oligomeric status. Oligomerization-defective and hence pY19-dephosphorylated monomeric Caveolin-2 in the inner nuclear membrane is unable to carry out Caveolin-2-mediated epigenetic activation of Egr-1 and JunB genes and transactivation of Elk-1 and STAT3 in response to insulin. The homo-oligomeric pY19-Caveolin-2 localizes in and recruits epigenetic modifiers to the A-type lamin-enriched inner nuclear membrane microdomain for the epigenetic activation. Our data show that A-type lamin-dependent Caveolin-2 homo-oligomerization in the inner nuclear membrane microdomain is a precondition for pY19-Caveolin-2-mediated insulin-response epigenetic activation at the nuclear periphery.
Assuntos
Caveolina 2/metabolismo , Epigênese Genética/efeitos dos fármacos , Insulina/farmacologia , Lamina Tipo A/metabolismo , Membrana Nuclear/efeitos dos fármacos , Caveolina 2/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Lamina Tipo A/genética , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Membrana Nuclear/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Tirosina , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Here, we demonstrate that insulin receptor (IR) tyrosine kinase catalyzes Tyr-19 and Tyr-27 phosphorylation of caveolin-2 (cav-2), leading to stimulation of signaling proteins downstream of IR, and that the catalysis is dependent on fatty acylation status of cav-2, promoting its interaction with IR. Cav-2 is myristoylated at Gly-2 and palmitoylated at Cys-109, Cys-122, and Cys-145. The fatty acylation deficient mutants are unable to localize in the plasma membrane and not phosphorylated by IR tyrosine kinase. IR interacts with the C-terminal domain of cav-2 containing the cysteines for palmitoylation. IR mutants, Y999F and K1057A, but not W1220S, fail interaction with cav-2. Insulin receptor substrate-1 (IRS-1) is recruited to interact with the IR-catalyzed phospho-tyrosine cav-2, which facilitates IRS-1 association with and activation by IR to initiate IRS-1-mediated downstream signaling. Cav-2 fatty acylation and tyrosine phosphorylation are necessary for the IRS-1-dependent PI3K-Akt and ERK activations responsible for glucose uptake and cell survival and proliferation. In conclusion, fatty acylated cav-2 is a new substrate of IR tyrosine kinase, and the fatty acylation and phosphorylation of cav-2 present novel mechanisms by which insulin signaling is activated.
Assuntos
Caveolina 2/metabolismo , Ácidos Graxos/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Acilação/efeitos dos fármacos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Biocatálise/efeitos dos fármacos , Caveolina 2/química , Linhagem Celular , Cisteína/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Insulina/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lipoilação/efeitos dos fármacos , Camundongos , Mitógenos/farmacologia , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacosRESUMO
The role of caveolin-2 (cav-2), independently of caveolin-1 (cav-1) and caveolae, has remained elusive. Our data show that cav-2 exists in the plasma membrane (PM) in cells lacking cav-1 and forms homo-oligomeric complexes. Cav-2 did not interact with cavin-1 and cavin-2 in the PM. Rab6-GTP was required for the microtubule-dependent exocytic transport of cav-2 from the Golgi to the PM independently of cav-1. The cav-2-oligomerized noncaveolar microdomain was unaffected by cholesterol depletion and protected from shearing of silica-coated PM. Activation of insulin receptor (IR) was processed in the microdomain. Actin depolymerization affected the formation and sustenance of cav-2-oligomerized noncaveolar microdomain and attenuated IR recruitment to the microdomain thereby inhibiting IR signaling activation. Cav-2 shRNA stable cells and the cells ectopically expressing an oligomerization domain truncation mutant, cav-2∆47-86 exhibited retardation of IR signaling activation via the noncaveolar microdomain. Elevation in status of cav-2 expression rendered the noncaveolar activation of IR signaling in cav-1 down-regulated or/and cholesterol-depleted cells. Our findings reveal a novel homo-oligomeric cav-2 microdomain responsible for regulating activation of IR signaling in the PM.
Assuntos
Citoesqueleto de Actina/metabolismo , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Insulina/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Transporte Biológico , Western Blotting , Cavéolas/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Células Cultivadas , Fibroblastos/citologia , Guanosina Trifosfato/metabolismo , Imunoprecipitação , Insulina/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Insulina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Frações SubcelularesRESUMO
The aim of the present study was to demonstrate the location of the different members of the caveolin (cav) family in human muscle spindles. Twenty spindles of three human muscles (vastus medialis, ischiocavernosus, bulbospongiosus) from 12 cadavers were immunohistochemically stained for cav-1, cav-2, and cav-3, and the equatorial and polar regions evaluated. All layers of the outer and inner spindle capsule and all blood vessels within the spindle stained for cav-1 and cav-2. In the muscle spindle, intrafusal muscle fibres stained selectively for cav-3, but with a patchy appearance. Caveolinopathies may therefore also include changes in muscle spindle function.
Assuntos
Caveolinas/biossíntese , Fusos Musculares/metabolismo , Músculo Esquelético/metabolismo , Idoso , Idoso de 80 Anos ou mais , Cadáver , Feminino , Humanos , Imuno-Histoquímica , MasculinoRESUMO
Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are primary cicatricial alopecia that cause a major impact on quality of life due to irreversible hair loss and symptoms as itching, burning and pain. They are characterized by permanent loss of hair follicle stem cells (HFSCs) by pathomechanisms still poorly understood, resulting in poor efficacy of currently available treatments. Caveolae are flask-shaped lipid rafts invaginated within the plasma membrane of multiple cell types. Although their role in the HF physiology and pathophysiology is relatively unknown, we have previously demonstrated that the primary structural component of caveolae (caveolin-1 or Cav1) is upregulated in FFA. Thus, we propose to investigate the expression and localization of caveolae-associated structural proteins (Cav1, Cav2, and Cavin-1) and HFSCs (identified by K15) in both LPP and FFA. We analyzed 4 patients with LPP biopsied in affected and non-affected (NA) scalp, 4 patients with FFA biopsied in affected scalp and 4 healthy controls. Affected scalp of LPP and FFA demonstrated increased levels of Cav1 and Cavin-1 compared with HC and LPP-NA. Moreover, Cav1, Cav2 and Cavin1 all exhibit high colocalization with K15 and their expression appears to be negatively correlated, supporting the hypothesis that these proteins are important players in LPP/FFA and may serve as therapeutic targets in future treatments.
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Alopecia , Cavéolas , Caveolina 1 , Folículo Piloso , Líquen Plano , Regulação para Cima , Humanos , Alopecia/patologia , Alopecia/metabolismo , Folículo Piloso/patologia , Folículo Piloso/metabolismo , Líquen Plano/metabolismo , Líquen Plano/patologia , Pessoa de Meia-Idade , Feminino , Caveolina 1/metabolismo , Masculino , Cavéolas/metabolismo , Couro Cabeludo/patologia , Adulto , Queratina-15/metabolismo , Idoso , Biópsia , Fibrose , Células-Tronco/metabolismo , Células-Tronco/patologia , Proteínas de Ligação a RNA/metabolismoRESUMO
Here, we report that Caveolin-2 (Cav-2) is a cell cycle regulator in the mitotic clonal expansion (MCE) for adipogenesis. For the G2/M phase transition and re-entry into the G1 phase, dephosphorylated Cav-2 by protein tyrosine phosphatase 1B (PTP1B) controlled epigenetic activation of Ccnb1, Cdk1, and p21 in a lamin A/C-dependent manner, thereby ensuring the survival of preadipocytes. Cav-2, associated with lamin A/C, recruited the repressed promoters of Ccnb1 and Cdk1 for activation, and disengaged the active promoter of p21 from lamin A/C for inactivation through histone H3 modifications at the nuclear periphery. Cav-2 deficiency abrogated the histone H3 modifications and impeded the transactivation of Ccnb1, Cdk1, and p21, leading to a delay in mitotic entry, retardation of re-entry into G1 phase, and the apoptotic cell death of preadipocytes. Re-expression of Cav-2 restored the G2/M phase transition and G1 phase re-entry, preadipocyte survival, and adipogenesis in Cav-2-deficient preadipocytes. Our study uncovers a novel mechanism by which cell cycle transition and apoptotic cell death are controlled for adipocyte hyperplasia.
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Adipócitos , Adipogenia , Proteína Quinase CDC2 , Caveolina 2 , Inibidor de Quinase Dependente de Ciclina p21 , Mitose , Adipogenia/genética , Animais , Mitose/genética , Adipócitos/metabolismo , Adipócitos/citologia , Camundongos , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Caveolina 2/genética , Caveolina 2/metabolismo , Sobrevivência Celular/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Células 3T3-L1 , Apoptose/genéticaRESUMO
Here, we show that insulin induces palmitoylation turnover of Caveolin-2 (Cav-2) in adipocytes. Acyl protein thioesterases-1 (APT1) catalyzes Cav-2 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase 21 (ZDHHC21) repalmitoylation of the depalmitoylated Cav-2 for the turnover, thereby controlling insulin receptor (IR)-Cav-2-insulin receptor substrate-1 (IRS-1)-Akt-driven signaling. Insulin-induced palmitoylation turnover of Cav-2 facilitated glucose uptake and fat storage through induction of lipogenic genes. Cav-2-, APT1-, and ZDHHC21-deficient adipocytes, however, showed increased induction of lipolytic genes and glycerol release. In addition, white adipose tissues from insulin sensitive and resistant obese patients exhibited augmented expression of LYPLA1 (APT1) and ZDHHC20 (ZDHHC20). Our study identifies the specific enzymes regulating Cav-2 palmitoylation turnover, and reveals a new mechanism by which insulin-mediated lipid metabolism is controlled in adipocytes.
Assuntos
Adipócitos , Caveolina 2 , Proteínas Substratos do Receptor de Insulina , Insulina , Metabolismo dos Lipídeos , Lipoilação , Receptor de Insulina , Humanos , Adipócitos/metabolismo , Animais , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Camundongos , Caveolina 2/metabolismo , Caveolina 2/genética , Receptor de Insulina/metabolismo , Receptor de Insulina/genética , Insulina/metabolismo , Obesidade/metabolismo , Obesidade/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Aciltransferases/metabolismo , Aciltransferases/genética , Transdução de Sinais , Resistência à Insulina , Células 3T3-L1 , MasculinoRESUMO
AIMS: To explore the accumulation of lipid droplets (LDs) and its relationship with lipid metabolism, and epithelial-mesenchymal transition (EMT) in the carcinogenesis processes in the oral cavity. METHODS: LDs were stained by oil red O. Forty-eight oral squamous cell carcinomas (OSCC), 78 oral potentially malignant disorders (OPMDs) and 25 normal tissue sections were included to explore the LDs surface protein caveolin-2 and perilipin-3, lipid metabolism-related molecule FABP5 and EMT biomarker E-cadherin expression by immunohistochemical staining. RESULTS: The accumulation of LDs was observed in OPMDs and OSCCs compared with normal tissues (p<0.05). In general, an increasing trend of caveolin-2, perilipin-3 and FABP5 expression was detected from the normal to OPMDs to OSCC groups (p<0.05). Additionally, caveolin-2, perilipin-3 and FABP5 expression were positively correlated with epithelial dysplasia in OPMDs, whereas E-cadherin positivity was negatively correlated with histopathological grade in both OPMDs and OSCC, respectively. A negative correlation of caveolin-2 (p<0.01, r =-0.1739), and FABP5 (p<0.01, r =-0.1880) with E-cadherin expression was detected. The caveolin-2 (p<0.0001, r=0.2641) and perilipin-3 (p<0.05, r=0.1408) staining was positively correlated with FABP5. Increased caveolin-2 expression was related to local recurrence and worse disease-free survival (p<0.05). CONCLUSION: In the oral epithelial carcinogenesis process, LDs begin to accumulate early in the precancerous stage. LDs may be the regulator of FABP5-associated lipid metabolism and may closely related to the process of EMT; caveolin-2 could be the main functional protein.
RESUMO
PURPOSE: Our microRNA expression signature of renal cell carcinoma revealed that miR-218 expression was significantly decreased in cancer tissues, suggesting that miR-218 is a candidate tumor suppressor. We investigated the functional significance of miR-218 in cancer cells and identified what are to our knowledge novel miR-218 mediated cancer pathways in renal cell carcinoma. MATERIALS AND METHODS: Gain of function studies using mature miR-218 were performed to investigate cell proliferation, migration and invasion in the A498 and 786-O renal cell carcinoma cell lines. To identify miR-218 mediated molecular pathways and responsible genes in renal cell carcinoma, we used gene expression and in silico database analyses. Loss of function assays were performed to investigate the functional significance of miR-218 target genes. RESULTS: Restoration of mature miR-218 significantly inhibited RCC cell proliferation, migration and invasion. Gene expression studies and luciferase reporter assays showed that CAV2 involved in the focal adhesion pathway was directly regulated by miR-218. A silencing study of CAV2 revealed significant inhibition of cell proliferation, migration and invasion. CAV2 mRNA and protein expression was significantly up-regulated in renal cell carcinoma clinical specimens. CONCLUSIONS: Loss of tumor suppressive miR-218 enhances cancer cell migration and invasion through dysregulation of the focal adhesion pathway, especially CAV2 as an oncogenic function in renal cell carcinoma. Tumor suppressive microRNA mediated cancer pathways and responsible genes provide new insights into the potential mechanisms of renal cell carcinoma oncogenesis and metastasis.
Assuntos
Carcinoma de Células Renais/genética , Caveolina 2/metabolismo , Movimento Celular/genética , Neoplasias Renais/genética , MicroRNAs/genética , Western Blotting , Carcinoma de Células Renais/patologia , Adesão Celular/genética , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Adesões Focais/genética , Adesões Focais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Renais/patologia , MicroRNAs/metabolismo , Invasividade Neoplásica/patologia , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Transdução de Sinais/genética , Estatísticas não Paramétricas , Células Tumorais CultivadasRESUMO
Background: Oral squamous cell carcinoma (OSCC) accounts for 90% of oral malignancies, which may be preceded by oral potentially malignant disorders (OPMDs). Cancer progression involves the downregulation of epithelial markers (E-cadherin) and the upregulation of mesenchymal markers (N-cadherin), which together characterise the epithelial-mesenchymal transition (EMT). Furthermore, caveolin can act on cell adhesion and migration events that regulate the expression of the E-cadherin/α-ß-catenin complex, thus favouring aggressive biological behaviour. This study aimed to analyse the immunoexpression of E-cadherin, N-cadherin and caveolin-2 at different stages of oral carcinogenesis to identify reliable biomarkers to predict malignant potential. Methods: Expressions of E-cadherin and N-cadherin in 14 normal oral mucosae (NOM), 14 OPMD and 33 OSCC specimens were evaluated using immunohistochemistry. Clinicopathological parameters were also assessed. Results: E-cadherin immunoexpression was significantly reduced during the progression of oral carcinogenesis (P = 0.0018). N-cadherin immunoexpression did not show any statistical differences between these groups. However, a representative number of N-cadherin-positive OSCC cases did not express E-cadherin. The expression of caveolin-2 increased significantly with the progression of the disease, from NOM to OSCC (P value: 0.0028). Conclusion: These findings indicate that cadherin switch and caveolin-2 immunoexpression may be regulatory events in oral carcinogenesis.