RESUMEN
Background: The activity, localization, and substrate specificity of the protein phosphatase 2A (PP2A) heterotrimer are controlled by various regulatory B subunits. PR72 belongs to the B'' gene family and has been shown to be upregulated in human heart failure. However, little is known about the functions of PR72 in the myocardium. Methods: To address this issue, we generated a transgenic mouse model with heart-specific overexpression of PP2A-PR72. Biochemical and physiological methods were used to determine contractility, Ca2+ cycling parameters, and protein phosphorylation. Results: A 2.5-fold increase in PR72 expression resulted in moderate cardiac hypertrophy. Maximal ventricular pressure was increased in catheterized transgenic mice (TG) compared to wild-type (WT) littermates. This was accompanied by an increased shortening of sarcomere length and faster relaxation at the single-cell level in TG. In parallel with these findings, the peak amplitude of Ca2+ transients was increased, and the decay in intracellular Ca2+ levels was shortened in TG compared to WT. The changes in Ca2+ cycling in TG were also evident from an increase in the full duration and width at half maximum of Ca2+ sparks. Consistent with the contractile data, phosphorylation of phospholamban at threonine-17 was higher in TG hearts. The lower expression of the Na+/Ca2+ exchanger may also contribute to the hypercontractile state in transgenic myocardium. Conclusion: Our results suggest that PP2A-PR72 plays an important role in regulating cardiac contractile function and Ca2+ cycling, indicating that the upregulation of PR72 in heart failure is an attempt to compensate functionally.
RESUMEN
The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to ß-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (ß and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show ß-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to ß-adrenergic stimulation in the myocardium.
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Adrenérgicos , Proteína Fosfatasa 2 , Adrenérgicos/metabolismo , Adrenérgicos/farmacología , Animales , Calcio/metabolismo , Isoproterenol/farmacología , Ratones , Ratones Noqueados , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Fosforilación , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Protein phosphatase 2A (PP2A) represents a heterotrimer that is responsible for the dephosphorylation of important regulatory myocardial proteins. This study was aimed to test whether the phosphorylation of PP2A-B56α at Ser41 by PKC is involved in the regulation of myocyte Ca2+ cycling and contraction. For this purpose, heart preparations of wild-type (WT) and transgenic mice overexpressing the nonphosphorylatable S41A mutant form (TG) were stimulated by administration of the direct PKC activator phorbol 12-myristate 13-acetate (PMA), and functional effects were studied. PKC activation was accompanied by the inhibition of PP2A activity in WT cardiomyocytes, whereas this effect was absent in TG. Consistently, the increase in the sarcomere length shortening and the peak amplitude of Ca2+ transients after PMA administration in WT cardiomyocytes was attenuated in TG. However, the costimulation with 1 µM isoprenaline was able to offset these functional deficits. Moreover, TG hearts did not show an increase in the phosphorylation of the myosin-binding protein C after administration of PMA but was detected in corresponding WT. PMA modulated voltage-dependent activation of the L-type Ca2+ channel (LTCC) differently in the two genotypes, shifting V1/2a by +1.5 mV in TG and by -2.4 mV in WT. In the presence of PMA, ICaL inactivation remained unchanged in TG, whereas it was slower in corresponding WT. Our data suggest that PKC-activated enhancement of myocyte contraction and intracellular Ca2+ signaling is mediated by phosphorylation of B56α at Ser41, leading to a decrease in PP2A activity.NEW & NOTEWORTHY The importance of the serine-41 phosphorylation site on B56α in reducing PP2A activity was demonstrated for the first time using a transgenic mutation model. Direct activation of PKC inhibits PP2A, leading to increased phosphorylation of MyBP-C in cardiomyocytes. The increased phosphorylation of contractile proteins is influenced by the PKC-phosphoB56α-PP2A signaling cascade resulting in improved intracellular Ca2+ handling and enhanced contractility and relaxation. PKC-mediated inhibition of PP2A also leads to modulation of the LTCC activation and inactivation kinetics.
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Miocitos Cardíacos , Proteína Fosfatasa 2 , Animales , Isoproterenol/farmacología , Ratones , Contracción Muscular , Miocitos Cardíacos/metabolismo , Fosforilación , Proteína Fosfatasa 2/metabolismoRESUMEN
ICER (inducible cAMP early repressor) isoforms are transcriptional repressors encoded by the Crem (cAMP responsive element modulator) gene. They were linked to the regulation of a multitude of cellular processes and pathophysiological mechanisms. Here, we show for the first time that two independent induction patterns for CREM repressor isoforms exist in the heart, namely for ICER and smICER (small ICER), which are induced in response to ß-adrenergic stimulation in a transient- and saturation-like manner, respectively. This time-shifted induction pattern, driven by two internal promoters in the Crem gene, leads to the predominant transcription of smIcer after prolonged ß-adrenergic stimulation. Using an ICER knockout mouse model with preserved smICER induction, we show that the transient-like induction of Icer itself has minor effects on gene regulation, cardiac hypertrophy or contractile function in the heart. We conclude that the functions previously linked to ICER may be rather attributed to smICER, also beyond the cardiac background.
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Agonistas Adrenérgicos beta/farmacología , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Receptores Adrenérgicos beta/genética , Animales , Cardiomegalia/tratamiento farmacológico , Línea Celular , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células HEK293 , Corazón/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
Adenylyl cyclases (AC) are essential for the normal and pathophysiological response of many cells. In cardiomyocytes, the predominant AC isoforms are AC5 and AC6. Specific AC5 inhibition was suggested as an option for the treatment of heart failure potentially advantageous over ß-blockers. We previously reported an interaction between the calcium-binding protein annexin A4 (ANXA4) and AC5 in human embryonic kidney 293 (HEK293) cells and an inhibition of cyclic adenosine monophosphate (cAMP) production in cardiomyocytes. Here, we investigated whether ANXA4 is able to differentiate between AC5 and AC6. In transfected HEK293 cells, ANXA4 specifically co-immunoprecipitated with AC5 and not with AC6, via its N-terminal domain. Both ANXA4 and a peptide comprising the ANXA4 N-terminal sequence (A4N1-22 ) decreased the cAMP production in AC5 and not in AC6 expressing cells. In line with ACs inhibition, in myocytes from ANXA4-deficient mice, ß-adrenoceptor (ßAR) stimulation led to a higher increase of the L-type calcium current (ICaL ) and to an excessive action potential duration (APD) prolongation as compared to wild-type cardiomyocytes. This enhanced response was reversed in the presence of A4N1-22 peptide likely via specific AC5 inhibition. We conclude that via the N-terminal domain ANXA4 inhibits AC5 not AC6, and that A4N1-22 as a specific AC5 inhibitor could serve as a novel therapeutic tool for the treatment of AC5-linked diseases.
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Potenciales de Acción/fisiología , Adenilil Ciclasas/metabolismo , Anexina A4/metabolismo , Corazón/fisiología , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Células Musculares/metabolismoRESUMEN
RATIONALE: A higher expression/activity of type 1 serine/threonine protein phosphatase 1 (PP1) may contribute to dephosphorylation of cardiac regulatory proteins triggering the development of heart failure. OBJECTIVE: Here, we tested the putatively protective effects of PP1 inhibitor-2 (I2) overexpression using a heart failure model induced by chronic ß-adrenergic stimulation. METHODS AND RESULTS: Transgenic (TG) and wild-type (WT) mice were subjected to isoprenaline (ISO) or isotonic NaCl solution supplied via osmotic minipumps for 7â¯days. I2 overexpression was associated with a depressed PP1 activity. Basal contractility was unchanged in catheterized mice and isolated cardiomyocytes between TGNaCl and WTNaCl. TGISO mice exhibited more fibrosis and a higher expression of hypertrophy marker proteins as compared to WTISO. After acute administration of ISO, the contractile response was accompanied by a higher sensitivity in TGISO as compared to WTISO. In contrast to basal contractility, the peak amplitude of [Ca]i and SR Ca load were reduced in TGNaCl as compared to WTNaCl. These effects were normalized to WT levels after chronic ISO stimulation. Cardiomyocyte relaxation and [Ca]i decay kinetics were hastened in TGISO as compared to WTISO, which can be explained by a higher phospholamban phosphorylation at Ser16. Chronic catecholamine stimulation was followed by an enhanced expression of GSK3ß, whereas the phosphorylation at Ser9 was lower in TG as compared to the corresponding WT group. This resulted in a higher I2 phosphorylation that may reactivate PP1. CONCLUSION: Our findings suggest that the basal desensitization of ß-adrenergic signaling and the depressed Ca handling in TG by inhibition of PP1 is restored by a GSK3ß-dependent phosphorylation of I2.
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Calcio/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Oncogénicas/metabolismo , Proteína Fosfatasa 1/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN , Insuficiencia Cardíaca/metabolismo , Chaperonas de Histonas , Humanos , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Miocitos Cardíacos/efectos de los fármacos , Sarcómeros/efectos de los fármacos , Sarcómeros/genética , Cloruro de Sodio/farmacologíaRESUMEN
Annexin A4 (AnxA4), a Ca(2+)- and phospholipid-binding protein, is up-regulated in the human failing heart. In this study, we examined the impact of AnxA4 on ß-adrenoceptor (ß-AR)/cAMP-dependent signal transduction. Expression of murine AnxA4 in human embryonic kidney (HEK)293 cells dose-dependently inhibited cAMP levels after direct stimulation of adenylyl cyclases (ACs) with forskolin (FSK), as determined with an exchange protein activated by cAMP-Förster resonance energy transfer (EPAC-FRET) sensor and an ELISA (control vs. +AnxA4: 1956 ± 162 vs. 1304 ± 185 fmol/µg protein; n = 8). Disruption of the anxA4 gene led to a consistent increase in intracellular cAMP levels in isolated adult mouse cardiomyocytes, with heart-directed expression of the EPAC-FRET sensor, stimulated with FSK, and as determined by ELISA, also in mouse cardiomyocytes stimulated with the ß-AR agonist isoproterenol (ISO) (anxA4a(+/+) vs. anxA4a(-/-): 5.1 ± 0.3 vs. 6.7 ± 0.6 fmol/µg protein) or FSK (anxA4a(+/+) vs. anxA4a(-/-): 1891 ± 238 vs. 2796 ± 343 fmol/µg protein; n = 9-10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membrane-bound AC type 5 (AC5). As a functional consequence of AnxA4-mediated AC inhibition, AnxA4 inhibited the FSK-induced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10-fold vs. control; n = 4 transfections) and reduced the FSK-induced phosphorylation of the CRE-binding protein (CREB) measured on Western blots (control vs. +AnxA4: 150 ± 17% vs. 105 ± 10%; n = 6) and by the use of the indicator of CREB activation caused by phosphorylation (ICAP)-FRET sensor, indicating CREB phosphorylation. Inactivation of AnxA4 in anxA4a(-/-) mice was associated with an increased cardiac response to ß-AR stimulation. Together, these results suggest that AnxA4 is a novel direct negative regulator of AC5, adding a new facet to the functions of annexins.
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Adenilil Ciclasas/metabolismo , Anexina A4/metabolismo , Membrana Celular/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Adenilil Ciclasas/genética , Animales , Anexina A4/genética , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Membrana Celular/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Fosforilación/fisiologíaRESUMEN
Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large-scale production of cardiomyocytes (CMs) for translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high-efficiency procedure for generating CMs both in two-dimensional (2D) and three-dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost-efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin-free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time-course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture-induced maturation of the resulting CMs. This suggested that hPSC-CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC-CMs to functional readouts and thus define the cornerstones of culture-induced maturation.
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Diferenciación Celular , Corazón/fisiología , Células Madre Pluripotentes/citología , Humanos , Mesodermo/citología , Miocitos Cardíacos/citologíaRESUMEN
Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser(41) of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca(2+) release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser(41). This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca(2+) homeostasis.
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Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis/fisiología , Proteína Quinasa C-alfa/metabolismo , Proteína Fosfatasa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Sustitución de Aminoácidos , Animales , Retículo Endoplásmico/genética , Células HEK293 , Homeostasis/efectos de los fármacos , Humanos , Mutación Missense , Fenilefrina/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteína Quinasa C-alfa/genética , Proteína Fosfatasa 2/genética , Serina/genética , Serina/metabolismo , Células Sf9 , SpodopteraRESUMEN
OBJECTIVE: There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT(3/4) receptor-modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo. METHODS: Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca(2+) measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunohistochemical analysis. RESULTS: Tropisetron suppressed collagen synthesis induced by transforming growth factor ß1 (TGFß1). This effect was independent of 5-HT(3/4) receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFß1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin. CONCLUSION: Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma.
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Colágeno/biosíntesis , Indoles/farmacología , Receptores Nicotínicos/metabolismo , Esclerodermia Sistémica/tratamiento farmacológico , Células 3T3 , Adulto , Anciano , Animales , Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Dermis/efectos de los fármacos , Dermis/metabolismo , Dermis/patología , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/tratamiento farmacológico , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Ratones , Persona de Mediana Edad , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Antagonistas de la Serotonina/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Tropisetrón , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Calsequestrin (CSQ) is a Ca(2+) storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca(2+) signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TG(CSQ)) was associated with heart failure, attenuation of SR Ca(2+) release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TG(CxT)) could be beneficial for impaired intracellular Ca(2+) signaling and contractile function. Indeed, the depressed intracellular Ca(2+) concentration ([Ca](i)) peak amplitude in TG(CSQ) was normalized by co-overexpression in TG(CxT) myocytes. This effect was associated with changes in the expression of cardiac Ca(2+) regulatory proteins. For example, the protein level of the L-type Ca(2+) channel Ca(v)1.2 was higher in TG(CxT) compared with TG(CSQ). Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression was reduced in TG(CxT) compared with TG(CSQ), whereas JUN expression and [(3)H]ryanodine binding were lower in both TG(CxT) and TG(CSQ) compared with wild-type hearts. As a result of these expressional changes, the SR Ca(2+) load was higher in both TG(CxT) and TG(CSQ) myocytes. In contrast to the improved cellular Ca(2+), transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TG(CxT) compared with TG(CSQ). Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca(2+) release compared with TG(CSQ) mice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to ß-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca(2+) signaling.
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Calcio/metabolismo , Calsecuestrina/metabolismo , Proteínas Portadoras/metabolismo , Ventrículos Cardíacos/metabolismo , Proteínas Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Señalización del Calcio/fisiología , Calsecuestrina/genética , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Fibrosis , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/patología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Transgénicos , Proteínas Musculares/genética , Contracción Miocárdica/fisiología , Miocitos Cardíacos/citología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Laboratory breeding conditions of the model organism C. elegans do not correspond with the conditions in its natural soil habitat. To assess the consequences of the differences in environmental conditions, the effects of air composition, medium and bacterial food on reproductive fitness and/or dietary-choice behavior of C. elegans were investigated. The reproductive fitness of C. elegans was maximal under oxygen deficiency and not influenced by a high fractional share of carbon dioxide. In media approximating natural soil structure, reproductive fitness was much lower than in standard laboratory media. In seminatural media, the reproductive fitness of C. elegans was low with the standard laboratory food bacterium E. coli (γ-Proteobacteria), but significantly higher with C. arvensicola (Bacteroidetes) and B. tropica (ß-Proteobacteria) as food. Dietary-choice experiments in semi-natural media revealed a low preference of C. elegans for E. coli but significantly higher preferences for C. arvensicola and B. tropica (among other bacteria). Dietary-choice experiments under quasi-natural conditions, which were feasible by fluorescence in situ hybridization (FISH) of bacteria, showed a high preference of C. elegans for Cytophaga-Flexibacter-Bacteroides, Firmicutes, and ß-Proteobacteria, but a low preference for γ-Proteobacteria. The results show that data on C. elegans under standard laboratory conditions have to be carefully interpreted with respect to their biological significance.
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Caenorhabditis elegans/fisiología , Aptitud Genética , Presión del Aire , Animales , Bacteroides , Conducta Animal/fisiología , Betaproteobacteria , Caenorhabditis elegans/microbiología , Cytophaga , Ecosistema , Flexibacter , Alimentos , Gammaproteobacteria , Aptitud Genética/fisiología , Hibridación Fluorescente in Situ , Oxígeno/análisis , Suelo , Microbiología del SueloRESUMEN
Physiological polyamines are required in various biological processes. In the current study, we used norspermidine, a structural analog of the natural polyamine spermidine, to investigate polyamine uptake in the model organism Caenorhabditis elegans. Norspermidine was found to have two remarkable effects: it is toxic for the nematode, without affecting its food, Escherichia coli; and it hampers RNA interference. By characterizing a norspermidine-resistant C. elegans mutant strain that has been isolated in a genetic screen, we demonstrate that both effects, as well as the uptake of a fluorescent polyamine-conjugate, depend on the transporter protein CATP-5, a novel P(5B)-type ATPase. To our knowledge, CATP-5 represents the first P(5)-type ATPase that is associated with the plasma membrane, being expressed in the apical membrane of intestinal cells and the excretory cell. Moreover, genetic interaction studies using C. elegans polyamine synthesis mutants indicate that CATP-5 has a function redundant to polyamine synthesis and link reduced polyamine levels to retarded postembryonic development, reduced brood size, shortened life span, and small body size. We suggest that CATP-5 represents a crucial component of the pharmacologically important polyamine transport system, the molecular nature of which has not been identified so far in metazoa.
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Adenosina Trifosfatasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de la Membrana/metabolismo , Poliaminas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/clasificación , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Transporte Biológico Activo , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Expresión Génica , Genes de Helminto , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Interferencia de ARN/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Espermidina/análogos & derivados , Espermidina/metabolismo , Espermidina/toxicidadRESUMEN
To elucidate the function of Omega class glutathione transferases (GSTs) (EC 2.5.1.18) in multicellular organisms, the GSTO-1 from Caenorhabditis elegans (GSTO-1; C29E4.7) was investigated. Disc diffusion assays using Escherichia coli overexpressing GSTO-1 provided a test of resistance to long-term exposure under oxidative stress. After affinity purification, the recombinant GSTO-1 had minimal catalytic activity toward classic GST substrates but displayed significant thiol oxidoreductase and dehydroascorbate reductase activity. Microinjection of the GSTO-1-promoter green fluorescent protein construct and immunolocalization by electron microscopy localized the protein exclusively in the intestine of all postembryonic stages of C. elegans. Deletion analysis identified an approximately 300-nucleotide sequence upstream of the ATG start site necessary for GSTO-1 expression. Site-specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression. Similarly, RNA interference (RNAi) of Elt-2 indicated the involvement of this gut-specific transcription factor in GSTO-1 expression. Transcriptional up-regulation under stress conditions of GSTO-1 was confirmed by analyzing promoter-reporter constructs in transgenic C. elegans strains. To investigate the function of GSTO-1 in vivo, transgenic animals overexpressing GSTO-1 were generated exhibiting an increased resistance to juglone-, paraquat-, and cumene hydroperoxide-induced oxidative stress. Specific silencing of the GSTO-1 by RNAi created worms with an increased sensitivity to several prooxidants, arsenite, and heat shock. We conclude that the stress-responsive GSTO-1 plays a key role in counteracting environmental stress.
Asunto(s)
Proteínas de Caenorhabditis elegans/clasificación , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Glutatión Transferasa/clasificación , Glutatión Transferasa/metabolismo , Estrés Oxidativo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Factores de Transcripción GATA/genética , Factores de Transcripción GATA/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Genes Reporteros/genética , Glutatión Transferasa/química , Glutatión Transferasa/genética , Humanos , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The mitogen-activated protein kinase (MAPK) pathways and insulin-like signaling play pivotal roles in cellular stress response. Using an anti-phospho-SAPK/JNK antibody and a daf-16::GFP-based reporter assay, the present study shows in Caenorhabditis elegans that ambient temperature (1-37 degrees C) specifically influences the activation (phosphorylation) of the MAP kinase JNK-1 as well as the nuclear translocation of DAF-16, the main downstream target of insulin-like signaling. Activated JNK-1 was detected only in neuronal cells, and JNK-1 was found to be controlled by the MAPK JKK-1 under heat stress. Comparative analyses on the wildtype and a jnk-1 deletion mutant revealed a promoting influence of JNK-1 on both nuclear DAF-16 translocations and DAF-16 target gene (superoxide dismutase 3, sod-3) expressions within peripheral, non-neuronal tissue. Consequently, the mutant exhibited a reduced thermal tolerance and reproductive fitness at higher temperatures. These results provide evidence of indirect interactions between neuronal MAPK and peripheral insulin-like signaling in response to environmental stimuli (temperature, H2O2).