RESUMO
Epilepsy is a heterogeneous and chronic neurological condition of undefined etiology in the majority of cases. Similarly, the pathogenesis of the unprovoked seizures that lead to epilepsy is not known. We are interested in the factors that modify inherent seizure susceptibility, with a particular focus on those occurring during the prenatal and early postnatal periods. Female Sprague-Dawley rats were bred in-house or transported during pregnancy at one of two gestational days (G9 or G16). The effects of transport stress, maternal behavior, and offspring sex were then examined in terms of how they were related to provoked seizure susceptibility to kainic acid (KA) or a model of febrile convulsions (FCs) on postnatal day 14 (P14). We also examined the pattern of neuronal activation in the hippocampus and amygdala as indicated by the density of FosB protein immunoreactivity (FosB-ir). Results demonstrated only a small and inconsistent effect of transport alone, suggesting that the groups differed slightly prior to experimental manipulations. However, the influence of maternal behaviors such as licking and grooming (LG), arched back nursing (ABN), and dam-off time (DO) exerted a much stronger effect on the offspring. Dams designated as high LG gave birth to smaller litters, had pups that weighed less, had greater seizure susceptibility and severity, and had more FosB-ir neurons predominantly in the ventral hippocampus and the medial subnucleus of the amygdala (MeA). We also found a sex-dependent effect such that P14 males were smaller than their female littermates and had a greater seizure susceptibility and severity. Taken together, these results suggest an impact of prenatal and postnatal factors, as well as sex, on seizure susceptibility in young animals.
Assuntos
Epilepsia/etiologia , Comportamento Materno/fisiologia , Efeitos Tardios da Exposição Pré-Natal , Caracteres Sexuais , Estresse Psicológico/complicações , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Encéfalo/patologia , Corticosterona/sangue , Modelos Animais de Doenças , Suscetibilidade a Doenças , Epilepsia/patologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Feminino , Ácido Caínico/toxicidade , Lipopolissacarídeos/toxicidade , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Estresse Psicológico/sangue , Fatores de TempoRESUMO
OBJECTIVE: Using the stable HSPA1A (HSP70-1) promoter-driven luciferase reporter HepG2 cells (HepG2/HSPA1A cells) to assess the overall toxicity of coke oven emissions. METHODS: The stable HepG2/HSPA1A cells were treated with different concentrations of coke oven emissions (COEs) collected from the top, side, and bottom of a coke oven battery for 24 h. After the treatments, luciferase activity, cell viability, malondialdehyde (MDA) concentration, Olive tail moment, and micronuclei frequency were determined, respectively. RESULTS: The bottom COEs induced significant increases (P < 0.01) in relative luciferase activity up to 1.4 times the control level at 0.15 µg/L. The low dose of side COEs (0.02 µg/L) led to a significant increase (P < 0.01) in relative luciferase activity that progressively increased to 2.1 times the control level at 65.4 µg/L. The top COEs produced a strong dose-dependent induction of relative luciferase activity up to over 5 times the control level at the highest concentration tested (202 µg/L). In HepG2/HSPA1A cells treated with the bottom COEs, relative luciferase activity was positively correlated with MDA concentration (r = 0.404, P < 0.05). For the three COEs samples, positive correlations were observed between relative luciferase activity and Olive tail moment and micronuclei frequency. CONCLUSION: The relative luciferase activity in HepG2/HSPA1A cells can sensitively reflect the overall toxicity of COEs. The stable HepG2/HSPA1A cells can be used for rapid screening of the overall toxicity of complex air pollutants in the workplace.
Assuntos
Coque/toxicidade , Proteínas de Choque Térmico HSP70/genética , Genes Reporter , Células Hep G2 , Humanos , Luciferases/genética , Malondialdeído/análise , Micronúcleos com Defeito Cromossômico , Exposição Ocupacional , Regiões Promotoras Genéticas , Testes de ToxicidadeRESUMO
OBJECTIVE: We examined the molecular mediators of postoperative choke-vessel growth. Our focus was the possible overlap between choke-vessel growth and arteriogenesis. METHODS: A rat perforator flap model, encompassing four vascular territories, was used. Flaps were surgically elevated, re-inset, and allowed to survive for one, three, five, or seven days. Tissue samples for Western and histological analyses were collected from the choke zone along the dorsal midline. Tissue from territories linked by the choke zone was analyzed to distinguish between global and local effects. The proteins examined included CD11b, ICAM-1, and MMP-2, three markers associated with arteriogenesis, as well as Hsp70 and vascular endothelial growth factor, markers of physiological stress and hypoxia/ischemia. RESULTS: Arteriogenesis markers, as shown by Western analysis, increased at three and five days after flap elevation, and the increase was localized by immunohistochemistry to the growing arteries and veins. The marker of physiological stress increased at Days 5 and 7. The hypoxia-ischemia marker did not increase in the choke zone. CONCLUSIONS: The growth of choke arteries and veins proceeds in an inflammatory environment that resembles arteriogenesis. Ischemia did not appear to play a role in choke-vessel changes.
Assuntos
Artérias/crescimento & desenvolvimento , Neovascularização Fisiológica , Procedimentos de Cirurgia Plástica/métodos , Retalhos Cirúrgicos/irrigação sanguínea , Animais , Biomarcadores/análise , Hipóxia , Inflamação , Isquemia , Ratos , Cirurgia PlásticaRESUMO
OBJECTIVE AND DESIGN: Angiotensin II (Ang II) induces the proinflammatory nuclear factor kappaB (NF-kappaB) in the vasculature. Heat shock and elevated levels of heat shock proteins (Hsps) decrease Ang II-induced NF-kappaB transcriptional activity and inflammation, but little is known about the role of specific Hsps. Here we used small interfering RNA (siRNA) technology to examine the role of Hsp27 in the Ang II-induced NF-kappaB signaling pathway. METHODS: Hsp27 siRNA was transfected into rat aortic vascular smooth muscle (A10) and 48 hrs later, the cells were stressed with 100 nM of Ang II for up to 24 hrs. Hsp27 levels were determined by immunofluorescence microscopy and Western analysis and inhibitor kappaB-alpha (I kappaB-alpha), the p65 subunit of NF-kappaB, and I kappaB kinase (IKK) levels were determined by Western analysis. RESULTS: When Hsp27 was specifically knocked down with Hsp27 siRNA in A10 cells there was a trend toward an increase in Ang II-induced phosphorylated p65. I kappaB-alpha and IKK-beta levels were not changed by the knockdown of Hsp27. CONCLUSION: Hsp27 may regulate the phosphorylation of the p65 subunit of NF-kappaB in the Ang II-induced signaling pathway of NF-kappaB in A10 cells. The proinflammatory effects of Ang II on NF-kappaB in vascular smooth muscle cells may be through a non-canonical pathway and be dependent on p65 phosphorylation.
Assuntos
Angiotensina II/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição RelA/metabolismo , Animais , Aorta/citologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP27/genética , Fosforilação , Ratos , Transdução de SinaisRESUMO
OBJECTIVES: Heat shock and elevated expression of heat shock proteins suppress activation of the pro-inflammatory transcription factor NF-kappaB. We hypothesized that knocking down the expression of heat shock factor-1 (HSF-1) with RNAi technology would exacerbate angiotensin (Ang) II-induced inflammatory injury in vascular smooth muscle cells (VSMC). METHODS: Rat aorta A10 cells and human intestinal smooth muscle cells were grown without transfection or with transfection with HSF-1 small interfering RNA (siRNA), or negative control siRNA. Cells were stimulated with Ang II (100 nM) to activate the NF-kappaB signaling pathway. RESULTS: HSF-1 siRNA significantly knocked down HSF-1 expression, and one of the downstream heat shock proteins (Hsp), Hsp27, in both cells lines. HSF-1 siRNA also affected cells stressed with heat shock or Ang II treatment. Ang II induced activation of NF-kappaB and AP-1 in untransfected VSMCs, however, Ang II induced significantly higher activities of these pro-inflammatory transcription factors in HSF-1 siRNA transfected cells. Control siRNA had no apparent effect on HSF-1 and Hsp27 expression and Ang II-induced NF-kappaB and AP-1 activation. CONCLUSIONS: These data indicate that the knock down of HSF-1 exacerbates Ang II-induced inflammation in VSMCs, and suggests that heat shock proteins protect against inflammatory injury by suppression of pro-inflammatory transcription factors such as NF-kappaB and AP-1.
Assuntos
Proteínas de Ligação a DNA/genética , Músculo Liso Vascular/imunologia , Miócitos de Músculo Liso/imunologia , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/genética , Angiotensina II/farmacologia , Animais , Aorta , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Proteínas de Choque Térmico HSP27 , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/metabolismo , Humanos , Intestinos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Estimulação Química , Fatores de Transcrição/metabolismo , Transfecção/métodosRESUMO
Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.
Assuntos
Doença de Huntington/metabolismo , Corpos de Inclusão/metabolismo , Ferro/metabolismo , Acetilcisteína/farmacologia , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Desferroxamina/metabolismo , Éxons , Proteínas de Choque Térmico/metabolismo , Corpos de Inclusão/ultraestrutura , Microscopia Confocal , Oxirredução , Fenantridinas/metabolismo , Ratos , Proteínas Recombinantes de FusãoRESUMO
The purpose of the present experiment was to characterize changes in TrkB signaling in the rat visual system resulting from exposure to enriched environment. Female Sprague-Dawley rats were placed in enriched or impoverished conditions for 1, 7 or 28 days. Levels of BDNF protein and its predominant receptor TrkB were examined in the retina, superior colliculus and visual cortex. In the retina, 1 day of enrichment increased full-length TrkB and after 28 days increased BDNF. In the superior colliculus, enrichment for 7 days reduced full-length TrkB and after 28 days increased BDNF and full-length TrkB. One day of enrichment significantly increased BDNF, reduced full-length TrkB and increased truncated TrkB in the visual cortex. Consequently, we further investigated whether exposure to enriched environment and the subsequent changes in BDNF and TrkB translates into a neuroprotective effect on retinal ganglion cells (RGCs) following transection of the optic nerve. Although exogenous intraocular application of BDNF provides neuroprotection to RGCs after axotomy, the endogenous increase in BDNF in the retina after 28 days of enrichment had no effect on RGC survival. While enriched housing conditions offer a model of non-invasive rehabilitation treatment for injury and modulates changes in BDNF and TrkB levels, these molecular changes did not translate into a neuroprotective effect on RGCs following transection of the optic nerve.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Meio Ambiente , Receptor trkB/metabolismo , Células Ganglionares da Retina/fisiologia , Transdução de Sinais/fisiologia , Vias Visuais , Análise de Variância , Animais , Animais Recém-Nascidos , Axotomia/métodos , Western Blotting/métodos , Contagem de Células/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imunoprecipitação/métodos , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Vias Visuais/citologia , Vias Visuais/efeitos dos fármacos , Vias Visuais/metabolismoRESUMO
Androgen deprivation in males has detrimental effects on various tissues and bodily functions, some of which can be restored by estradiol (E2) administration. We investigated how the duration of androgen deprivation affects the autoregulation of estrogen receptors (ERs) levels in core brain areas associated with sexual behavior and cognition, as well as in pelvic floor muscles (PFM). We also measured c-Fos levels in brain areas associated with sexual behavior shortly after the rats mated. Prolonged castration increases ERα levels in the preoptic area (POA) and E2 treatment reverses these effects. In the POA, c-Fos levels after mating are not affected by the duration of androgen deprivation and/or E2 treatment. ERß levels in the POA as well as c-Fos levels in the POA and the core area of nucleus accumbens correlate with the mounting frequency for E2-treated Short-Term castrates. Additionally, ERß levels in the medial amygdala are positively correlated with the mounting frequency of Long-Term castrates that received E2 treatment. In the hippocampus, ERs are downregulated only when E2 is administered early after castration, whereas downregulation of ERα in the prefrontal cortex only occurs with delayed E2 treatment. Early, but not delayed, E2 treatment after castration increases ERß levels in the bulbocavernosus and ERα levels in the levator ani of male rats. Our data suggest that the duration of androgen deprivation may influence the autoregulation of ERs by E2 treatment in select brain areas and pelvic floor muscles of male rats.
Assuntos
Androgênios/deficiência , Encéfalo/metabolismo , Músculo Esquelético/metabolismo , Diafragma da Pelve/fisiologia , Receptores de Estrogênio/metabolismo , Animais , Western Blotting , Encéfalo/efeitos dos fármacos , Castração , Estradiol/administração & dosagem , Estrogênios/administração & dosagem , Homeostase , Masculino , Músculo Esquelético/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Distribuição Aleatória , Ratos Long-Evans , Comportamento Sexual Animal/efeitos dos fármacos , Comportamento Sexual Animal/fisiologia , Fatores de TempoRESUMO
Cell death from cerebral ischemia is a dynamic process. In the minutes to days after an ischemic insult, progressive changes in cellular morphology occur. Associated with these events is the regulation of competing programs of gene expression; some are protective against ischemic insult, and others contribute to delayed cell death. Many genes involved in these processes have been identified, but individually, these findings have provided only limited insight into the systems biology of cerebral ischemia. Attempts to characterize the coordinated expression of large numbers of genes in cerebral ischemia has only recently become possible. Today, DNA microarray technology provides a powerful tool for investigating parallel expression changes for thousands of genes at one time. In this study, adult mice were subjected to 30 minutes of hypoxia-ischemia (HI), and the hippocampus was examined 12 hours later for differential gene expression using a 15K high-density mouse EST array. The genomic response to HI is complex, affecting approximately 7% of the total number of ESTs examined. Assigning differentially expressed ESTs to molecular functional groups revealed that HI affects many pathways including the molecular chaperones, transcription factors, kinases, and calcium ion binding genes. A comprehensive list of regulated genes should prove valuable in advancing our understanding of the pathogenesis of cerebral ischemia.
Assuntos
Química Encefálica/genética , Hipocampo/fisiologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Regulação da Expressão Gênica/fisiologia , Hipóxia-Isquemia Encefálica/patologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análiseRESUMO
The small heat shock protein Hsp27 has been shown to protect neurons from apoptosis. We have recently shown the expression of Hsp27 in a subset of injured adult retinal ganglion cells (RGCs), a response that is muted by the administration of brain-derived neurotrophic factor. This work has suggested a role for Hsp27 in the long-term survival of RGCs following injury. The purpose of this study was to investigate the expression of Hsp27 during postnatal retinal development, based on Hsp27's role as a neuronal survival factor and on its up-regulation in the adult injured retina. Expression of Hsp27 in the developing retina was examined at various times postnatally (between P0 and P24) by using immunohistochemical techniques. We report that Hsp27 expression peaks in the ganglion cell layer between P6 and P12 and is not detected at earlier (P0-P3) or later (P15-P24) times. Double labeling of the Hsp27-positive cells with Fluorogold applied to the superior colliculus confirmed that Hsp27-positive cells in the ganglion cell layer are RGCs. We have shown developmentally regulated expression of Hsp27 in RGCs of the postnatal rat. The retinal expression of Hsp27 correlates temporally with innervation of the tectum by late-born RGCs and with onset of spontaneous retinotectal activity. We propose that the expression of Hsp27 may play an important role in retinal development during a critical period of RGC functional connectivity with the superior colliculus.
Assuntos
Proteínas de Choque Térmico/biossíntese , Retina/crescimento & desenvolvimento , Células Ganglionares da Retina/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Colículos Superiores/metabolismo , Vias Visuais/metabolismoRESUMO
Angiotensin II (Ang II) is a potent vasoconstrictor and induces inflammation and end-organ injury through its activation of the proinflammatory transcription factor, nuclear factor-kappaB (NF-kappaB). Heat shock (HS) treatment with subsequent expression of heat shock proteins (Hsps) is an effective strategy for tissue protection against oxidative injuries. Recently, HS and Hsps have been shown to interact with NF-kappaB in tissue injury. In this study, we investigated whether HS could protect against Ang II-induced hypertension and inflammation by inhibiting NF-kappaB. Sprague-Dawley rats were divided into control and HS groups. Control and 24-hour post-heat shocked rats were treated with Ang II. At days 1, 3, 5, 7, 11, and 14 after Ang II administration, systolic blood pressures were measured by tail-cuff plethysmography, and aorta tissues were collected. Aorta NF-kappaB deoxyribonucleic acid-binding activity was measured by electrophoretic mobility shift assay, and NF-kappaB p65 subunit, Hsp70, Hsp27, and interleukin-6 (IL-6) expressions were measured by Western analysis. HS treatment significantly decreased Ang II-induced hypertension. The activation of NF-kappaB in aorta by Ang II was suppressed by HS treatment. The elevated expression of IL-6 induced by Ang II treatment was also decreased by HS treatment. Although Ang II treatment induced an increase in Hsp70 and Hsp27, HS treatment induced a greater elevation of Hsp70 and Hsp27 expression. HS treatment protects against Ang II-induced hypertension and inflammation. This protection may relate to the interaction of Hsps and the NF-kappaB pathway.
Assuntos
Angiotensina II/farmacologia , Aorta/metabolismo , Resposta ao Choque Térmico/fisiologia , Hipertensão/prevenção & controle , Inflamação/prevenção & controle , Acrilatos/farmacologia , Angiotensina II/administração & dosagem , Angiotensina II/toxicidade , Animais , Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Choque Térmico HSP70/metabolismo , Temperatura Alta/uso terapêutico , Hidralazina/farmacologia , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Imidazóis/farmacologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B , Norepinefrina/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Tiofenos/farmacologia , Fator de Transcrição RelARESUMO
Animals exposed to an enriched environment display features of neural plasticity such as an increased brain volume, enhanced number of dendritic spines, as well as enlarged synapses. Here we report the first description of molecular plasticity in the mammalian retina, as revealed by gene expression. A marked upregulation of both NGFI-A and Arc, two candidate-plasticity genes, was observed in adult rats that had been exposed to an enriched environment for 3 weeks. This increase was paralleled by an increase in the expression of the late genes GAP-43 and Synapsin I, which also indicated changes in retinal connectivity. Our results suggest that both NGFI-A and Arc may regulate mechanisms of plasticity that had been invoked by heightened complexity of the visual environment.
Assuntos
Proteínas do Olho/genética , Regulação da Expressão Gênica , Plasticidade Neuronal/genética , Retina/metabolismo , Animais , Contagem de Células , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Meio Ambiente , Comportamento Exploratório , Proteínas do Olho/biossíntese , Proteína GAP-43/biossíntese , Proteína GAP-43/genética , Genes Precoces , Manobra Psicológica , Abrigo para Animais , Proteínas Imediatamente Precoces/biossíntese , Masculino , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-Dawley , Retina/citologia , Sinapsinas/biossíntese , Sinapsinas/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: Since insulin stimulates nitric oxide (NO) production and an increase in NO following heat shock is required for myocardial heat shock protein 70 (Hsp70) synthesis, we hypothesized that insulin would enhance myocardial Hsp70 synthesis by augmenting NO signaling. We examined whether a physiologic dose of insulin increased myocardial Hsp70 in unstressed and heat shock treated rats. METHODS: Adult male Sprague-Dawley rats were assigned to groups: (1) control, (2) insulin injected (200 microU/gm body weight), (3) heat shock treated (core body temperature 42 degrees C for 15 min), (4) heat shock and insulin treated, (5) L-nitroarginine methyl ester (L-NAME) and heat shock and insulin treated, (6) sodium nitroprusside (SNP) and heat shock and insulin treated. Six hours later, myocardial Hsp70 content and localization was analyzed. RESULTS: Hsp70 was increased in heat shock treated hearts (120.6+/-16.8 ng/mg protein, P < 0.001) vs. control (12.9+/-2.0 ng/mg protein), or insulin treated hearts (15.5+/-0.83 ng/mg protein). In addition, Hsp70 was increased in the heat shock and insulin treated hearts (164.4+/-7.53 ng/mg protein) compared to control, insulin only (P = 0.001) or heat shock only treated hearts (P = 0.01). L-NAME did not abolish the insulin induced increase in Hsp70 in heat shocked hearts (195.2+/-13.4 ng/mg protein, P = 0.21) and SNP did not further enhance Hsp70 in the insulin and heat shocked group (188.9+/-8.2 ng/mg protein, P = 0.71). Western analysis and confocal microscopy revealed a lowlevel expression of myocardial Hsp70 in response to insulin. Hsp70 was localized primarily in blood vessels after insulin or heat shock treatments. CONCLUSIONS: Insulin caused a low-level expression of myocardial Hsp70 and potentiated Hsp70 synthesis in response to heat shock. The ability of insulin to potentiate Hsp70 after heat shock is independent of NO signaling as it was not altered by either LNAME or SNP pretreatment. Blood vessels appear to be the primary site of Hsp70 after insulin or heat shock treatment.
Assuntos
Proteínas de Choque Térmico HSP70/biossíntese , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Miocárdio/metabolismo , Animais , Western Blotting/métodos , Inibidores Enzimáticos/farmacologia , Fator VIII/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Coração/efeitos dos fármacos , Resposta ao Choque Térmico/efeitos dos fármacos , Temperatura Alta , Técnicas Imunoenzimáticas/métodos , Masculino , Microscopia de Fluorescência/métodos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Faloidina/imunologia , Ratos , Ratos Sprague-Dawley , Vasodilatadores/farmacologiaRESUMO
Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant that induces deoxyribonucleic acid (DNA) damage. The inducible heat shock protein (HspA1A) can function as a molecular chaperone; however, its role in DNA repair remains largely unknown. In the present study, human bronchial epithelial cells (16HBE) stably transfected with plasmids carrying HspA1A gene or shRNAs against HspA1A were treated with BaP. DNA damage levels of the cells were evaluated by comet assay. Results suggest that HspA1A could protect cells against DNA damage and facilitate the decrease of DNA damage levels during the first 2 h of DNA repair. DNA repair capacity (DRC) of Benzo(a)pyrene diol epoxide (BPDE)-DNA adducts was evaluated by host cell reactivation assay in the stable 16HBE cells transfected with luciferase reporter vector PCMVluc pretreated with BPDE. Compared with control cells, cells overexpressing HspA1A showed higher DRC (p < 0.01 at 10 µM BPDE and p < 0.05 at 20 µM BPDE, respectively), while knockdown of HspA1A inhibited DNA repair (p < 0.05 at 10 µM BPDE). Moreover, casein kinase 2 (CK2) was shown to interact with HspA1A by mass spectrometry and co-immunoprecipitation assays. The two proteins were co-localized in the cell nucleus and perinuclear region during DNA repair, and were identified by confocal laser scanning microscope. In addition, cells overexpressing HspA1A showed an increased CK2 activity after BaP treatment compared with control cells (p < 0.01). Our results suggest that HspA1A facilitates DNA repair after BaP treatment. HspA1A also interacts with CK2 and enhances the kinase activities of CK2 during DNA repair.
Assuntos
Benzo(a)pireno/toxicidade , Brônquios/patologia , Caseína Quinase II/metabolismo , Reparo do DNA/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas de Choque Térmico HSP70/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoproteção/efeitos dos fármacos , Adutos de DNA/metabolismo , Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , TransfecçãoRESUMO
HSPA1A (HSP70-1) is a highly inducible heat shock gene up-regulated in response to environmental stresses and pollutants. The aim of our study was to evaluate the sensitivity of the stable metabolically competent HepG2 cells containing a human HSPA1A promoter-driven luciferase reporter (HepG2-luciferase cells) for assessing the toxicity of organic pollutants present in air. The HepG2-luciferase cells were validated by heat shock treatment and testing three organic compounds (pyrene, benzo[a]pyrene, and formaldehyde) that are ubiquitous in the air. The maximal level of HSPA1A (HSP70-1) and relative luciferase activity induced by heat shock were over three and nine times the control level, respectively. Pyrene, benzo[a]pyrene, and formaldehyde all induced significantly elevated levels of relative luciferase activity in a dose-dependent manner. Extractable organic matter (EOM) from urban traffic and coke oven emissions in ambient air were tested on the HepG2-luciferase cells. The traffic EOM induced significant increase in relative luciferase activity at concentrations of picogram per liter. The coke oven EOM produced a strong dose-dependent induction of relative luciferase activity up to six times the control value. Significant increases in relative luciferase activity were observed at concentrations that were as low, or lower than the concentrations that the tested organic pollutants decreased cell viability, and increased malondialdehyde concentration, Olive tail moment, and micronuclei frequency. Therefore, we conclude that the HepG2-luciferase cells are a valuable tool for rapid screening of the overall toxicity of organic pollutants present in air.
Assuntos
Poluentes Atmosféricos/toxicidade , Proteínas de Choque Térmico HSP70/genética , Luciferases/metabolismo , Benzo(a)pireno/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Formaldeído/toxicidade , Genes Reporter , Proteínas de Choque Térmico HSP70/metabolismo , Células Hep G2 , Humanos , Luciferases/genética , Malondialdeído/metabolismo , Regiões Promotoras Genéticas , Pirenos/toxicidadeRESUMO
After heat shock or other metabolic stress, heat shock proteins (Hsps) are expressed at high levels in all tissues and cells. The highly inducible 70 kDa heat shock protein (Hsp70) is associated with improved post-ischemic myocardial contractile recovery. Similarly, the small 27 kDa heat shock protein (Hsp27), that is abundant in muscle, is also linked with improved myocardial function after ischemic injury. Various Hsps have pro-survival functions that include chaperone, anti-apoptotic and/or anti-inflammatory activity. In this review we will summarize our understanding of myocardial protection and present evidence for protection having time dependent aspects that appear to be stimulus dependent.
Assuntos
Proteínas de Choque Térmico HSP70/fisiologia , Proteínas de Choque Térmico/fisiologia , Resposta ao Choque Térmico/fisiologia , Inflamação/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Inflamação/complicações , Inflamação/metabolismo , Insulina/fisiologia , Isquemia Miocárdica/complicações , Isquemia Miocárdica/metabolismo , Substâncias Protetoras/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Heat shock proteins (Hsps) are highly conserved proteins that are induced in response to various physiological and environmental stressors. HspB1 (Hsp27) is a prominent member of the small Hsps family and is strongly induced during the stress response. Notably, HspB1 has powerful neuroprotective effects, increasing the survival of cells subjected to cytotoxic stimuli. This is especially relevant to the study of the retina, where cells are subject to death due to retinal disease and injury. While HspB1 shows constitutive expression in some areas of the mammalian retina, of particular interest is the upregulation of the protein in response to ischemia and oxidative stress, traumatic nerve injury, and elevated intraocular pressure and glaucoma. Several mechanisms have been proposed to account for the cytoprotective actions of HspB1, including its role as a molecular chaperone, a stabilizer of the cytoskeleton, and a regulator of apoptosis. This review will focus on the role of HspB1 in the retina, emphasizing effects on retinal ganglion cells, by analyzing the expression, induction by stressors, and mechanisms of its neuroprotective function. Finally, the potential of HspB1 as a clinical therapeutic will be examined.