RESUMEN
The serotonin transporter (SERT) readily takes up serotonin (5-HT), thereby regulating the availability of 5-HT within the intestine. In the absence of SERT, 5-HT remains in the interstitial space and has the potential to aberrantly activate the many 5-HT receptors distributed on the epithelium, immune cells and enteric neurons. Perturbation of SERT is common in many gastrointestinal disorders as well as mouse models of colitis. Select commensal microbes regulate intestinal SERT levels, but the mechanism of this regulation is poorly understood. Additionally, ethanol upregulates SERT in the brain and dendritic cells, but its effects in the intestine have never been examined. We report that the intestinal commensal microbe Limosilactobacillus (previously classified as Lactobacillus) reuteri ATCC PTA 6475 secretes 83.4 mM ethanol. Consistent with the activity of L. reuteri alcohol dehydrogenases, we found that L. reuteri tolerated various levels of ethanol. Application of L. reuteri conditioned media or exogenous ethanol to human colonic T84 cells was found to upregulate SERT at the level of mRNA. A 4-(4-(dimethylamino) phenyl)-1-methylpyridinium (APP+) uptake assay confirmed the functional activity of SERT. These findings were mirrored in mouse colonic organoids, where L. reuteri metabolites and ethanol were found to upregulate SERT at the apical membrane. Finally, in a trinitrobenzene sulphonic acid model of acute colitis, we observed that mice treated with L. reuteri maintained SERT at the colon membrane compared with mice receiving phosphate buffered saline vehicle control. These data suggest that L. reuteri metabolites, including ethanol, can upregulate SERT and may be beneficial for maintaining intestinal homeostasis with respect to serotonin signalling.
Asunto(s)
Colitis , Mucosa Intestinal/metabolismo , Limosilactobacillus reuteri , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Animales , Colitis/terapia , Etanol , Limosilactobacillus reuteri/química , Ratones , Serotonina , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismoRESUMEN
Necrotizing enterocolitis (NEC), the most common neonatal gastrointestinal emergency, results in significant mortality and morbidity, yet its pathogenesis remains unclear. Argininosuccinate lyase (ASL) is the only enzyme in mammals that is capable of synthesizing arginine. Arginine has several homeostatic roles in the gut and its deficiency has been associated with NEC. Because enterocytes are the primary sites of arginine synthesis in neonatal mammals, we evaluated the consequences of disruption of arginine synthesis in the enterocytes on the pathogenesis of NEC. We devised a novel approach to study the role of enterocyte-derived ASL in NEC by generating and characterizing a mouse model with enterocyte-specific deletion of Asl (Asl(flox/flox); VillinCre(tg/+), or CKO). We hypothesized that the presence of ASL in a cell-specific manner in the enterocytes is protective in the pathogenesis of NEC. Loss of ASL in enterocytes resulted in an increased incidence of NEC that was associated with a proinflammatory state and increased enterocyte apoptosis. Knockdown of ASL in intestinal epithelial cell lines resulted in decreased migration in response to lipopolysaccharide. Our results show that enterocyte-derived ASL has a protective role in NEC.
Asunto(s)
Argininosuccinatoliasa/metabolismo , Enterocolitis Necrotizante/prevención & control , Enterocitos/enzimología , Animales , Animales Recién Nacidos , Apoptosis , Argininosuccinatoliasa/genética , Aciduria Argininosuccínica/enzimología , Aciduria Argininosuccínica/genética , Línea Celular , Movimiento Celular , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/inducido químicamente , Enterocolitis Necrotizante/enzimología , Enterocolitis Necrotizante/genética , Enterocolitis Necrotizante/inmunología , Enterocolitis Necrotizante/patología , Enterocitos/inmunología , Enterocitos/patología , Humanos , Fórmulas Infantiles , Recién Nacido , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Noqueados , Infiltración Neutrófila , Interferencia de ARN , Ratas , Factores de Tiempo , TransfecciónRESUMEN
This study was aimed at identifying the molecular mechanisms by which ceramide inhibits telomerase activity in the A549 human lung adenocarcinoma cell line. C(6)-ceramide (20 microm) caused a significant reduction of telomerase activity at 24 h as detected using the telomeric repeat amplification protocol, and this inhibition correlated with decreased telomerase reverse transcriptase (hTERT) protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses showed that C(6)-ceramide significantly decreased hTERT mRNA in a time-dependent manner. Electrophoretic mobility shift and supershift assays demonstrated that the binding activity of c-Myc transcription factor to the E-box sequence on the hTERT promoter was inhibited in response to C(6)-ceramide at 24 h. These results were also confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box deleted sequences cloned upstream of a luciferase reporter gene. Further analysis using RT-PCR and Western blotting showed that c-Myc protein but not its mRNA levels were decreased in response to C(6)-ceramide at 24 h. The effects of ceramide on the c-Myc protein were shown to be due to a reduction in half-life via increased ubiquitination. Similar results were obtained by increased endogenous ceramide levels in response to nontoxic concentrations of daunorubicin, resulting in the inhibition of telomerase and c-Myc activities. Furthermore, the elevation of endogenous ceramide by overexpression of bacterial sphingomyelinase after transient transfections also induced the inhibition of telomerase activity with concomitant decreased hTERT and c-Myc protein levels. Taken together, these results show for the first time that both exogenous and endogenous ceramides mediate the modulation of telomerase activity via decreased hTERT promoter activity caused by rapid proteolysis of the ubiquitin-conjugated c-Myc transcription factor.
Asunto(s)
Ceramidas/farmacología , Regiones Promotoras Genéticas , Telomerasa/genética , Telomerasa/metabolismo , Transcripción Genética/efectos de los fármacos , Adenocarcinoma , Cicloheximida/farmacología , Proteínas de Unión al ADN , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Genes myc/efectos de los fármacos , Semivida , Humanos , Luciferasas/genética , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/antagonistas & inhibidores , Transfección , Células Tumorales Cultivadas , Ubiquitinas/metabolismoRESUMEN
This study was designed to analyze whether ceramide, a bioeffector of growth suppression, plays a role in the regulation of telomerase activity in A549 cells. Telomerase activity was inhibited significantly by exogenous C(6)-ceramide, but not by the biologically inactive analog dihydro-C(6)-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 microm C(6)-ceramide at 24 h. Moreover, analysis of phosphatidylserine translocation from the inner to the outer plasma membrane by flow cytometry and of poly(ADP-ribose) polymerase degradation by Western blotting showed that ceramide treatment (20 microm for 24 h) had no apoptotic effects. Trypan blue exclusion, [(3)H]thymidine incorporation, and cell cycle analyses, coupled with clonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 microm concentration at 24 h resulted in the cell cycle arrest of the majority of the cell population at G(0)/G(1) with no detectable cell death. These results suggest that the inhibition of telomerase by ceramide is not a consequence of cell death but is correlated with growth arrest. Next, to determine the role of endogenous ceramide in telomerase modulation, A549 cells were transiently transfected with an expression vector containing the full-length bacterial sphingomyelinase cDNA (b-SMase). The overexpression of b-SMase, but not exogenously applied purified b-SMase enzyme, resulted in significantly decreased telomerase activity compared with controls, showing that the increased endogenous ceramide is sufficient for telomerase inhibition. Moreover, treatment of A549 cells with daunorubicin at 1 microm for 6 h resulted in the inhibition of telomerase, which correlated with the elevation of endogenous ceramide levels and growth arrest. Finally, stable overexpression of human glucosylceramide synthase, which attenuates ceramide levels by converting ceramide to glucosylceramide, prevented the inhibitory effects of C(6)-ceramide and daunorubicin on telomerase. Therefore, these results provide novel data showing for the first time that ceramide is a candidate upstream regulator of telomerase.