RESUMO
BACKGROUND: Cationic lipids are the most widely used nonviral vectors for gene delivery. Upon complexation to DNA, they offer a nonimmunogenic alternative to viral gene transfer. Unfortunately, their in vivo application has been limited due to a serum-associated inhibition of transfection. As a result, significant research effort has focused on overcoming this deleterious effect of serum. METHODS: To better understand this phenomenon, we investigated the influence of lipoplex colloidal stability on gene transfection in the presence of serum. In addition, conditions of the reaction medium were modulated and their effects on collidal stability and subsequent in vitro transfection efficiency were studied. RESULTS: The colloidal stability of the cationic lipid-DNA complexes, which depended on the charge ratio, determined the efficiency of in vitro transfection in the presence of serum. In particular, large-sized, colloidally unstable complexes of over 700 nm mean diameter induced efficient transfection in the presence or absence of serum. Conversely, colloidally stable complexes of less than 250 nm in size resulted in efficient transfection only in the absence of serum. Furthermore, for the same charge ratio, both colloidally stable and unstable lipoplexes could be obtained depending on the degree to which various solution parameters (NaCl concentration, cationic lipid acyl chain length, pH and DNA concentration) were altered. In each case, only those complexes lacking colloidal stability resulted in high levels of in vitro transfection in the presence of serum. This phenomenon was shown to be independent of both the percent DNA internalized and of the lamellar organization of the cationic lipid/DNA lipoplexes. CONCLUSIONS: Through the modulation of various mixture conditions, large-sized lipoplexes can be formed which are resistant to the transfection-inhibiting effect of serum.
Assuntos
Cátions/química , Vetores Genéticos , Lipídeos/química , Transfecção/fisiologia , Células 3T3 , Acilação , Animais , Linhagem Celular , DNA/química , Fluorescência , Genes Reporter , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Luz , Luciferases/genética , Luciferases/metabolismo , Camundongos , Plasmídeos , Espalhamento de Radiação , Cloreto de Sódio/química , Transfecção/métodosRESUMO
Plasmid expression vectors containing angiotensinogen (ATG) cDNA were complexed to cationic liposomes and injected into the renal artery of unilaterally nephrectomized rats to evaluate the effect of intrarenal ATG cDNA on arterial blood pressure and the renin-angiotensin system. Systolic blood pressures measured by tail cuff on days 12, 16, and 18 after transfection were significantly higher in rats that received ATG cDNA than in control rats that received the lac Z reporter gene. Plasma renin activity and plasma ATG concentration were unchanged. These results provide direct evidence that the availability of intrarenal ATG may be instrumental in the development of systemic hypertension.
Assuntos
Angiotensinogênio/biossíntese , Hipertensão Renal/genética , Nefrectomia , Transfecção/fisiologia , Angiotensinogênio/sangue , Angiotensinogênio/genética , Animais , Pressão Sanguínea/fisiologia , DNA Complementar/biossíntese , DNA Complementar/genética , Frequência Cardíaca/fisiologia , Hemodinâmica/fisiologia , Hipertensão Renal/patologia , Hipertensão Renal/fisiopatologia , Rim/patologia , Lipossomos , Masculino , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Renina/metabolismo , Transgenes/genéticaRESUMO
Association of a targeting ligand such as transferrin, or an endosome disrupting peptide such as GALA, with cationic liposome-DNA complexes ('lipoplexes') results in a significant enhancement of transfection of several cell types (Simões S et al, Gene Therapy 1998; 5: 955-964). Although these strategies can overcome some of the barriers to gene delivery by lipoplexes, the mechanisms by which they actually enhance tranfection is not known. In studies designed to establish the targeting specificity of transferrin, we found that apo-transferrin enhances transfection to the same extent as transferrin, indicating that internalization of the lipoplexes is mostly independent of transferrin receptors. These observations were reinforced by results obtained from competitive inhibition studies either by preincubating the cells with an excess of free ligand or with various 'receptor-blocking' lipoplexes. Transfection of cells in the presence of drugs that interfere with the endocytotic pathway provided additional insights into the mechanisms of gene delivery by transferrin- or GALA-lipoplexes. Our results indicate that transferrin-lipoplexes deliver transgenes by endocytosis primarily via a non-receptor-mediated mechanism, and that acidification of the endosomes is partially involved in this process.
Assuntos
DNA/metabolismo , Técnicas de Transferência de Genes , Lipossomos/metabolismo , Peptídeos/metabolismo , Transferrina/metabolismo , Sobrevivência Celular , DNA/química , Endocitose , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Lipossomos/química , Peptídeos/química , Receptores da Transferrina/fisiologia , Transfecção/fisiologia , Transferrina/químicaRESUMO
1. Caveolae are small invaginations of the plasma membrane that have recently been implicated in signal transduction. In the present study, we have investigated whether caveolins, the principal protein of caveolae, also modulate volume-regulated anion channels (VRACs). 2. ICl,swell, the cell swelling-induced chloride current through VRACs, was studied in three caveolin-1-deficient cell lines: Caco-2, MCF-7 and T47D. 3. Electrophysiological measurements showed that ICl, swell was very small in these cells and that transient expression of caveolin-1 restored ICl,swell. The caveolin-1 effect was isoform specific: caveolin-1beta but not caveolin-1alpha upregulated VRACs. This correlated with a different subcellular distribution of caveolin-1alpha (perinuclear location) from caveolin-1beta (perinuclear and peripheral). 4. To explain the modulation of ICl, swell by caveolin-1 we propose that caveolin increases the availability of VRACs in the plasma membrane or, alternatively, that it plays a crucial role in the signal transduction cascade of VRACs.
Assuntos
Caveolinas , Canais de Cloreto/fisiologia , Ativação do Canal Iônico/fisiologia , Proteínas de Membrana/fisiologia , Animais , Western Blotting , Células CACO-2 , Bovinos , Caveolina 1 , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Tamanho Celular/fisiologia , Eletrofisiologia , Técnica Direta de Fluorescência para Anticorpo , Humanos , Isomerismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Microscopia Confocal , Técnicas de Patch-Clamp , Transdução de Sinais/fisiologia , Transfecção/fisiologiaRESUMO
The 5-hydroxytryptamine 5-HT1A receptor was one of the first G protein coupled receptors whose cDNA and gene were isolated by molecular cloning methods. Transfection of the cDNA of this receptor into cells previously bearing no 5-HT receptors has resulted in the acquisition of large amounts of information regarding potential signal transduction pathways linked to the receptor, correlations of receptor structure to its various functions, and pharmacological properties of the receptor. Transfection studies with the 5-HT1A receptor have generated critical new information that might otherwise have been elusive. This information notably includes the discovery of unsuspected novel signalling linkages, the elucidation of the mechanisms of receptor desensitization, the refinement of models of the receptor pharmacophore, and the development of silent receptor antagonists, among others. The current review summarizes the most important studies of the recombinant 5-HT1A receptor in the decade since the identification of its cDNA.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Receptores de Serotonina/fisiologia , Transdução de Sinais/fisiologia , Transfecção/fisiologia , Alelos , Animais , Células HeLa , Humanos , Receptores de Serotonina/química , Receptores de Serotonina/genética , Receptores 5-HT1 de Serotonina , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
Notch plays an important role in cell fate decisions in uncommitted proliferative cells, including neurogenesis, but is believed to not have a role in postmitotic cells. We have shown previously that Notch1 is highly expressed in embryonal mouse and human brain, but surprisingly it continues to be expressed at low levels in the adult brain. The function of Notch1 in postmitotic neurons in mammals is unknown. To better understand the potential role of Notch1 in mature central nervous system neurons we studied the effect of Notch1 transfection on neurite outgrowth in primary neocortex hippocampal neurons. Transfection at two days in vitro with full length Notch1 inhibited neurite outgrowth. Transfection at five to six days in vitro, after neurite outgrowth was established, led to apparent regression of neurites. These effects were enhanced when truncated constitutively active forms of Notch1 were introduced. Co-transfection with Numb, a physiological inhibitor of Notch, blocked Notch's effect on neurite outgrowth. We also examined whether Notch1 could activate C-promoter binding factor (CBF1) transcription factor using C-promoter binding factor-luciferase constructs, and demonstrated that this signal transduction pathway is present and can be activated in postmitotic neurons. Our results show that in postmitotic neurons Notch1 influences neurite morphology, and can activate its native signal transduction pathway. These data strongly suggest that Notch1 may play a physiologically important role in the central nervous system beyond neurogenesis.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Neuritos/fisiologia , Neurônios/fisiologia , Receptores de Superfície Celular , Fatores de Transcrição , Animais , Proteínas de Ligação ao Cálcio , Células Cultivadas , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Mitose/fisiologia , Neuritos/ultraestrutura , Plasticidade Neuronal/fisiologia , Neurônios/ultraestrutura , Plasmídeos/genética , Biossíntese de Proteínas , Proteínas/genética , Receptor Notch1 , Proteínas Serrate-Jagged , Sinapses/fisiologia , Ativação Transcricional/genética , Transfecção/genética , Transfecção/fisiologiaRESUMO
Adenovirus transfers genes to a wide range of cell types, but its application to neurons has been hampered by its reduced efficiency of infection as compared with that for glia. To achieve neuron-targeted gene transfer, we have produced an adenovirus carrying the reporter lacZ gene driven by the SCG10 minimum promoter containing the neural-restrictive silencer element (NRSE), which element selectively represses the transcription of genes in non-neuronal cells. When rat hippocampal slice cultures were infected with NRSE-bearing adenovirus, beta-galactosidase-positive cells were mostly pyramidal and granular neurons, whereas infection with virus carrying a mutated NRSE resulted in beta-galactosidase expression in both neurons and glia. The results suggest that the adenovirus carrying NRSE to be a useful tool for neurontargeted gene transfer.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Neurônios/fisiologia , Proteínas Repressoras/genética , Infecções por Adenoviridae/enzimologia , Animais , Técnicas In Vitro , Mutação/fisiologia , Neuroglia/enzimologia , Neurônios/enzimologia , Células Piramidais/enzimologia , Ratos , Ratos Sprague-Dawley , Transfecção/fisiologia , beta-Galactosidase/metabolismoRESUMO
The high-affinity mammalian brain L-proline transporter (PROT) belongs to the GAT1 gene family, which includes Na- and Cl-dependent plasma membrane carriers for neurotransmitters, osmolites, and metabolites. These transporters couple substrate flux to transmembrane electrochemical gradients, particularly the Na gradient. In the nervous system, transporters clear synapses and help to replenish transmitters in nerve terminals. The localization of PROT to specific excitatory terminals in rat forebrain suggests a role for this carrier in excitatory transmission (). We investigated the voltage regulation and electrogenicity of this novel transporter, using human embryonic kidney (HEK) 293 cells stably transfected with rat PROT cDNA. In physiological solutions between -140 and -40 mV, L-proline (PRO) and its six-member ring congener L-pipecolate (PIP) induced inward current. The current-voltage relationship and the variance of current fluctuations were similar for PRO- and PIP-induced current, and the ratio of induced variance to the mean current ranged from 20 to 60 fA. Des-Tyr-Leu-enkephalin (GGFL), a competitive peptide inhibitor of PROT, reduced the rat PROT-associated current to control levels. GGFL alone did not elicit currents, and the GGFL-sensitive substrate-induced current was absent in nontransfected cells. Finally, GGFL inhibited PROT-mediated transport only when applied to the extracellular face of PROT. These data suggest that (1) PROT uptake is electrogenic, (2) individual transporter currents are voltage-independent, and (3) GGFL is a nonsubstrate inhibitor that interacts either with an extracellular domain of PROT or in an externally accessible pore.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Encéfalo/metabolismo , Encefalinas/farmacologia , Proteínas de Membrana Transportadoras/fisiologia , Ácidos Pipecólicos/farmacologia , Prolina/farmacologia , Animais , Linhagem Celular , Condutividade Elétrica , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacologia , Humanos , Rim/citologia , Rim/embriologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Prolina/antagonistas & inibidores , Prolina/farmacocinética , Ratos , Transfecção/fisiologiaRESUMO
The transcriptional downregulation of the SERCA2 gene is studied using neonatal rat cardiomyocytes stimulated with endothelin-1 to induce hypertrophy. Liposome-based transfection of cells with a 1.9 kb SERCA2 promoter fragment directed expression of a reporter gene identical to the downregulation of genomic SERCA2 expression by endothelin-1. Results of a new gene gun technology for transient transfection of cardiomyocytes with a RSV-beta-galactosidase construct are reported. This new method for propelling DNA-coated gold beads into cardiomyocytes is extremely suitable for directly testing promoter/reporter gene DNA constructs since the transfection efficiency (approximately 10%) appears to be higher than traditional transfection methods.
Assuntos
Biolística/normas , ATPases Transportadoras de Cálcio/genética , Cardiomegalia/genética , Regulação da Expressão Gênica/fisiologia , Retículo Sarcoplasmático/enzimologia , Transfecção/fisiologia , Animais , Cardiomegalia/patologia , Células Cultivadas , Endotelina-1/farmacologia , Expressão Gênica/efeitos dos fármacos , Miocárdio/citologia , Ratos , Transcrição Gênica/fisiologiaRESUMO
The molecular mechanisms by which GH regulates insulin-like growth factor (IGF-I) gene expression remain obscure. One difficulty has been the lack of established GH-responsive cell lines that express the IGF-I gene. To develop such a cell line, we used rat C6 glioma cells which, as determined by RNase protection assay, express the IGF-I gene but not the GH receptor gene. To confer GH responsiveness, C6 cells were cotransfected with vectors that express the GH receptor (pRc/CMV WTrGHR) and Jak2 (pRc/CMV Jak2). GH responsiveness was demonstrated using luciferase reporter genes containing either the Sis-inducible element from the c-fos gene (pTK81-SIE-Luc) or 6 copies of the GH-responsive GAS-like element (GLE) from the rat spi2.1 gene (pSpi-GLE-Luc). The SIE is activated by binding of STAT1 and 3, whereas the GLE binds STAT5. In cells cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and either pTK81-SIE-Luc or pSpi GLE-Luc, treatment with 500 ng/ml GH for 24 h stimulated a 3.1- and 1.7-fold increase in luciferase activity, respectively. These data suggest that in C6 cells cotransfected with pRc/CMV WTrGHR and pRc/CMV Jak2, GH activates STAT1, 3, and 5. To determine whether GH-responsive IGF-I promoter activity could be demonstrated, C6 cells were cotransfected with pRc/CMV WTrGHR, pRc/ CMV Jak2, and an IGF-I-luciferase fusion gene that contained a fragment of the rat IGF-I gene that extended from -412 in the 5'-flanking region of exon 1 to the Met-22 in exon 3. GH stimulated a modest, but reproducible, 1.7-fold increase in luciferase activity in these cells, suggesting that a GH-responsive element is present in this region of the IGF-I gene. To better localize the GH-responsive element, cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2 plus one of several IGF-I-luciferase fusion genes containing either fragments of one of the two promoters in the IGF-I gene or a fragment of intron 2 that includes a GH-responsive DNase I hypersensitivity site. For all constructs, treatment with GH for 24 h did not stimulate a significant increase in luciferase activity, suggesting that GH-responsive sequences are not located in these specific regions of the IGF-I gene or that GH-directed transcription of the IGF-I gene is mediated via several different regions of the IGF-I gene and the effect of any one of these regions in isolation was not sufficiently robust to be detected in this model system. In summary, transient expression of the GH receptor and Jak2 in C6 cells creates a GH-responsive system that activates STAT1, 3, and 5. Moreover, a fragment of the IGF-I gene that contains exons 1 and 2, a fragment of exon 3, and introns 1 and 2 is GH responsive using this model system.
Assuntos
Glioma/genética , Hormônio do Crescimento/fisiologia , Fator de Crescimento Insulin-Like I/genética , Regiões Promotoras Genéticas/fisiologia , Animais , Expressão Gênica/fisiologia , Glioma/patologia , Hormônio do Crescimento/farmacologia , Coelhos , Ratos , Receptores da Somatotropina/genética , Transdução de Sinais/fisiologia , Transfecção/fisiologia , Células Tumorais CultivadasRESUMO
Although androgens have myriad effects on the skeleton, the regulation of androgen action in bone is not well understood. Androgen receptors (ARs) are known to play an important role in mediating androgen action. We have examined the effects of androgens and other sex steroids on AR levels in osteoblastic cells in vitro using two clonal human cell lines, SaOS-2 and U-2 OS. AR protein levels were quantitated both by specific androgen binding studies and Western analyses, and AR messenger RNA was measured with RNase protection assays. Potential changes in AR functionality was assessed by reporter assays. Treatment of osteoblastic cells with the nonaromatizable androgen 5alpha-dihydrotestosterone (DHT) increased specific androgen binding 2-to 4-fold. Similar increases in AR protein levels were documented by Western analysis in both cell lines. The androgen-mediated increase in receptor levels was time and dose dependent as well as androgen specific. Steady-state AR messenger RNA levels were also increased by DHT. When AR concentrations in osteoblastic cells were elevated with exogenous receptor, there was an enhancement of DHT responsiveness, measured by increased trans-activation of an androgen-responsive promoter. Thus, androgen exposure increased androgen receptor protein levels and specific androgen binding in osteoblastic cells. Androgen action as measured by androgen-mediated transcriptional activation is enhanced in the presence of elevated AR levels. Consequently, these studies have revealed an additional means by which androgens may modulate skeletal metabolism.
Assuntos
Androgênios/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Regulação para Cima/fisiologia , Linhagem Celular , DNA Complementar/genética , Di-Hidrotestosterona/farmacologia , Humanos , Concentração Osmolar , Osteoblastos/metabolismo , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Testosterona/farmacologia , Ativação Transcricional/fisiologia , Transfecção/fisiologiaRESUMO
Extrarenal 25-hydroxyvitamin D3-1alpha-hydroxylase is believed to play a major role in the pathogenesis of hypercalcemia associated with various types of granulomatous and lymphoproliferative diseases and certain solid tumors. In this paper, we describe the cloning of the cytochrome P450 component of the extrarenal enzyme from a human nonsmall cell lung carcinoma, SW 900. The cytochrome P450 for the extrarenal 1alpha-hydroxylase has an amino acid sequence identical to that of the cytochrome P450 component of the CYP1alpha, the renal form of the enzyme, and appears to be a product of the same gene. CYP1alpha messenger RNA (mRNA) and 1alpha-hydroxylase enzyme activity were detected in two (SW 900, SK-Luci-6) of a series of five nonsmall cell lung carcinoma cell lines. All five lung cell lines were cultured with the same medium under the same conditions, but only two of the five expressed 1alpha-hydroxylase enzyme; two others (WT-E, Calu-1) expressed high levels of the reciprocally regulated enzyme, 25-hydroxyvitamin D3-24-hydroxylase, with its specific cytochrome P450 component, CYP24. Although under basal conditions the lung cell line SW 900 expressed only CYP1alpha and showed 1alpha-hydroxylase enzyme activity, when treated with small concentrations of 1alpha,25-dihydroxyvitamin D3 or high concentrations of 25-hydroxyvitamin D3, it began to express CYP24 and exhibit 24-hydroxylase enzyme activity. Somewhat surprisingly, SW 900 cells still had detectable CYP1alpha mRNA some 24 h after vitamin D treatment despite the fact that 1alpha-hydroxylase enzyme activity was unmeasurable. These data are consistent with the emerging hypothesis that vitamin D through its active form does not directly turn off CYP1alpha mRNA production but, rather, strongly stimulates CYP24, thereby masking CYP1alpha activity. The factor(s) responsible for the basal expression of CYP1alpha in SW 900 and SK-Luci-6 is currently unknown.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Neoplasias Pulmonares/metabolismo , Esteroide Hidroxilases/metabolismo , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Animais , Células COS , Citocromo P-450 CYP1A1/genética , Sistema Enzimático do Citocromo P-450/genética , Humanos , RNA Mensageiro/metabolismo , Esteroide Hidroxilases/genética , Transfecção/fisiologia , Células Tumorais Cultivadas , Vitamina D/farmacologia , Vitamina D3 24-HidroxilaseRESUMO
BACKGROUND: Restenosis after percutaneous coronary intervention remains a serious clinical problem. Progress in local gene therapy to prevent restenosis has been hindered by concerns over the safety and efficacy of viral vectors and the limited efficiency of nonviral techniques. This study investigates the use of adjunctive ultrasound to enhance nonviral gene delivery. METHODS AND RESULTS: Cultured porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were transfected with naked or liposome-complexed luciferase reporter plasmid for 3 hours. Ultrasound exposure (USE) for 60 seconds at 1 MHz, 0.4 W/cm2, 30 minutes into this transfection period enhanced luciferase activity 48 hours later by 7.5-fold and 2. 4-fold, respectively. Luciferase activity after lipofection of ECs was similarly enhanced 3.3-fold by adjunctive USE. USE had no effect on cell viability, although it inhibited VSMC but not EC proliferation. CONCLUSIONS: Adjunctive USE was associated with enhanced transgene expression in VSMCs and ECs and reduced VSMC but not EC proliferation in vitro, which suggests that ultrasound-assisted local gene therapy has potential as an antirestenotic therapy.
Assuntos
Vasos Sanguíneos/diagnóstico por imagem , Vasos Sanguíneos/fisiologia , Expressão Gênica/efeitos da radiação , Genes Reporter/genética , Transfecção/fisiologia , Animais , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/diagnóstico por imagem , Luciferases/genética , Luciferases/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/diagnóstico por imagem , Suínos , UltrassonografiaRESUMO
Clusterin is a widely expressed, well conserved, secreted glycoprotein, which is highly induced in tissues regressing as a consequence of apoptotic cell death in vivo. It has recently been shown that clusterin expression is only confined to surviving cells following the induction of apoptosis in vitro, suggesting that it is involved in cell survival rather than death. In the hypothesis that clusterin may be implicated in cellular responses to stress, clusterin gene expression was analyzed in the A431 human epidermoid cancer cell line following heat shock and oxidative stress. Our results show that both a transient heat shock (20 min at 42 degrees C) and various oxidative stresses, including hydrogen peroxide, superoxide anion, hyperoxia and UVA exposure, induce a strong increase in clusterin mRNA levels as assessed by northern blot. Nuclear run-on analysis suggests that transcriptional activation is involved in inducing clusterin mRNA in response to heat shock. Using pulse-chase analysis of control and heat shocked cells, it is shown that clusterin mRNA is translated and secreted, thus resulting in increased extracellular levels of the protein following heat shock. To investigate the function of clusterin in response to these stresses, clusterin anti-sense transfectants that stably express virtually no clusterin at the mRNA and protein level were generated in A431 cells. These anti-sense transfectants are shown to be highly sensitive to apoptotic cell death induced by heat shock or oxidative stress compared with wild-type A431 cells or control transfectants. Taken together, our results show that clusterin gene expression is induced in response to heat shock and oxidative stress in human A431 cells, and confers cellular protection against heat shock and oxidative stress.
Assuntos
Apoptose/fisiologia , Expressão Gênica/fisiologia , Glicoproteínas/genética , Temperatura Alta , Chaperonas Moleculares , Estresse Oxidativo/fisiologia , Choque/fisiopatologia , Clusterina , Glicoproteínas/metabolismo , Humanos , Estresse Oxidativo/genética , Choque/patologia , Transcrição Gênica/fisiologia , Transfecção/fisiologia , Células Tumorais CultivadasRESUMO
Phosducin (Phd) regulates the function of G proteins by its ability to tightly bind Gbetagamma subunits. Because the internalization of opioid receptors as well as the activity of adenylyl cyclase (AC) activity depends on G proteins, we tested Phd on these parameters. NG 108-15 hybrid cells stably expressing the phosphoprotein were challenged with [D-penicillamine2,D-penicillamine5]enkephalin to inhibit cAMP generation, demonstrating an increased efficacy of the opioid on AC. Studying the binding of [35S]guanosine-5'-O-(gamma-thio)-triphosphate to membranes from Phd overexpressing cells, we found that [D-penicillamine2, D-penicillamine5 ]enkephalin failed, in the presence of Phd (0.1 nM), to elevate incorporation of the nucleotide. Phd also strongly inhibited opioid-stimulated GTPase activity. NG 108-15 cells were also employed to investigate the effect of Phd on opioid receptor internalization. Control cells and cells overexpressing Phd were transiently transfected to express mu-opioid receptors fused to green fluorescence protein. In controls and in Phd overexpressing cells confocal microscopy identified fluorescence associated with the membrane. Time-lapse series microscopy of living control cells challenged with etorphine (1 microM) revealed receptor internalization within 30 min. In contrast, Phd overexpressing cells largely failed to respond to the opioid. Thus, in Phd overexpressing cells, opioids exhibit an increased efficacy despite the inhibitory action of the phosphoprotein on opioid-stimulated incorporation of [35S]guanosine-5'-O-(gamma-thio)-triphosphate. We suggest that inhibition of GTPase stabilizes the opioid-induced G protein Gi-GTP complex, which is believed to enhance AC inhibition. Finally, scavenging of Gbetagamma by Phd attenuates internalization of opioid receptors, which may contribute to the efficacy of opioids.
Assuntos
Proteínas do Olho/fisiologia , Fosfoproteínas/fisiologia , Receptores Opioides/fisiologia , AMP Cíclico/metabolismo , DNA/biossíntese , D-Penicilina (2,5)-Encefalina , Encefalinas/farmacologia , Proteínas do Olho/biossíntese , Citometria de Fluxo , GTP Fosfo-Hidrolases/metabolismo , Reguladores de Proteínas de Ligação ao GTP , Glioma , Proteínas de Fluorescência Verde , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Células Híbridas , Proteínas Luminescentes/genética , Microscopia Confocal , Proteínas do Tecido Nervoso/biossíntese , Neuroblastoma , Fosfoproteínas/biossíntese , Ensaio Radioligante , Receptores Opioides/genética , Proteínas Recombinantes de Fusão/fisiologia , Transfecção/fisiologia , Células Tumorais CultivadasRESUMO
1. Protein kinase A (PKA) modulation of tetrodotoxin-resistant (TTX-r) voltage-gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned alpha-subunit of the rat sensory neurone-specific TTX-r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intracellular I-II loop had been eliminated by site-directed mutagenesis (serine to alanine). 2. In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I-II loops expressed as glutathione-S-transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I-II loop. 3. SNS and SNS(SA) channels were transiently expressed in COS-7 cells and their electrophysiological properties compared. In wild-type SNS channels, forskolin and 8-bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I-II loop PKA consensus sites caused a marked reduction in PKA modulation of wild-type channels. 4. Under control conditions, the voltage dependence of activation of SNS(SA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(SA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild-type channels under basal conditions.5. We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I-II loop.
Assuntos
AMP Cíclico/fisiologia , Canais de Sódio/fisiologia , Tetrodotoxina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Células COS , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Resistência a Medicamentos , Eletrofisiologia , Glutationa Transferase/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Conformação Molecular , Mutagênese Sítio-Dirigida/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.8 , Mapeamento de Peptídeos , Fosfopeptídeos/metabolismo , Fosforilação , Ratos , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/genética , Transfecção/genética , Transfecção/fisiologiaRESUMO
A soluble protein that specifically bound growth hormone (GH) was characterized in culture medium of a COS-7 cell line transfected with the cDNA of the full-length chicken GH receptor (cGHR). Incubation of culture medium with 125I-labeled human GH resulted in the formation of a single specific complex with high affinity (KD = 0.36 nM) and apparent molecular weight of 75 kDa. The production of large quantities of GH-binding protein (GHBP) amounting to, per hour, 23% of the cell's GHR, points to the importance of partial proteolysis for GHR turnover. Considerable amounts of GHBP were also detected in a cytosolic fraction. These results strongly suggest that in chicken, as in rabbit and monkey, the GHBP is generated, at least partially, by proteolytic cleavage of the membrane-anchored GHR.
Assuntos
Proteínas de Transporte/biossíntese , Hormônio do Crescimento/química , Peptídeo Hidrolases/metabolismo , Animais , Ligação Competitiva/fisiologia , Células COS , Galinhas , Chlorocebus aethiops , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Reagentes de Ligações Cruzadas/química , Meios de Cultura , Citosol , Eletroforese em Gel de Poliacrilamida , Receptores da Somatotropina/análise , Contagem de Cintilação , Transfecção/fisiologiaRESUMO
Several proteins belonging to the ATP-binding cassette superfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1). We measured whole cell swelling-activated Cl- currents (ICl,swell) in parental cells and cells expressing wild-type MDR1 or a phosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671 replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbol ester reduced the rate of increase in ICl,swell only in cells that express MDR1. PKC stimulation had no effect on steady-state ICl,swell. Stimulation of protein kinase A (PKA) with 8-bromoadenosine 3',5'-cyclic monophosphate reduced steady-state ICl, swell only in MDR1-expressing cells. PKA stimulation had no effect on the rate of ICl,swell activation. The effects of stimulation of PKA and PKC on ICl,swell were additive (i.e., decrease in the rate of activation and reduction in steady-state ICl,swell). The effects of PKA and PKC stimulation were absent in cells expressing the phosphorylation-defective mutant. In summary, it is likely that phosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl- channels by independent mechanisms and that Ser-661, Ser-667, and Ser-671 are involved in the responses of ICl,swell to stimulation of PKA and PKC. These results support the notion that MDR1 phosphorylation affects ICl,swell.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Canais de Cloreto/fisiologia , Proteína Quinase C/metabolismo , Células 3T3 , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Animais , Membrana Celular/metabolismo , Canais de Cloreto/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Complementar/genética , Condutividade Elétrica , Ativação Enzimática/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação/fisiologia , Fosforilação , Transfecção/fisiologiaRESUMO
The human glucagon-like peptide-1 (GLP-1) receptor mediates the insulinotropic effects of the incretin hormone GLP-1. It is expressed in a cell- and tissue-specific manner. Recently, we cloned the 5'-region of the GLP-1 receptor gene and found that tissue and cell specificity is lost by 5'-deletion to -574. In this region proximal to the main transcription start point three putative binding sites for Sp1 were localized. Now, in vitro binding of Sp1 was shown by deoxyribonuclease footprint analysis with DNA fragments using either recombinant Sp1 or nuclear extracts from HIT cells. To elucidate the roles of the three Sp1-binding sites, we mutated each of the sites individually as well as in different combinations. The activity of each construct was analyzed in comparison to the wild-type promoter. Mutation of two adjacent Sp1-binding sites showed a clear reduction of activity. Contrasting results were obtained after mutation of the third, more distal Sp1-binding site. Here, a clear increase (approximately 150%) revealed a silencing effect of this cis-regulatory element, possibly resembling a Sp3-binding site. Electrophoretic mobility shift analysis revealed binding of Sp1 and Sp3, which was demonstrated by supershifts using specific antibodies. Cotransfection with Sp1 and Sp3 expression vectors in insect cells lacking endogenous Sp factors clearly demonstrated the involvement of Sp1 and Sp3. Therefore, the basal activity of the GLP-1 receptor gene is mediated by two proximal Sp1-binding sites, whereas a more distal site acts as a repressor.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Receptores de Glucagon/genética , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Sítios de Ligação/fisiologia , Western Blotting , Linhagem Celular , Pegada de DNA , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Eletroforese , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Dados de Sequência Molecular , Mutação , Plasmídeos/fisiologia , Regiões Promotoras Genéticas/fisiologia , Fator de Transcrição Sp3 , Transfecção/fisiologiaRESUMO
Zac1 is a new zinc finger protein that concomitantly controls apoptosis and cell cycle arrest through separate pathways. The mouse Zac1 gene is mainly expressed in the pituitary gland and in different brain areas. In this study regional and cellular expression of Zac1 in the pituitary gland was determined by in situ hybridization. Zac1 messenger RNA was abundantly expressed in the anterior pituitary lobe compared with that in the intermediate and posterior lobes. Zac1 transcripts were found in all hormone-secreting cell types, with the highest levels in GH- and PRL-producing cells. To investigate the impact of Zac1 in pituitary cell proliferation, we ablated the endogenous Zac1 gene by antisense treatment in two murine cell types, AtT-20 and TtT/GF, that are representative of granular and agranular cell lineages, respectively. The decline in Zac1 protein levels under antisense treatment was accompanied by increased DNA synthesis in clonal corticotroph and folliculo-stellate cells, as demonstrated by enhanced [3H]thymidine incorporation (36% and 50%, respectively). Antisense oligonucleotides against Zac1 controlled cell proliferation in a dose-dependent way, and mutagenized antisense oligonucleotides were inert. Conclusively, our data provide the first evidence of a role for Zac1 in pituitary growth control.