RESUMEN
Synaptic abnormalities have been described in individuals with autism spectrum disorders (ASD). The cell-adhesion molecule Neuroligin-3 (Nlgn3) has an essential role in the function and maturation of synapses and NLGN3 ASD-associated mutations disrupt hippocampal and cortical function. Here we show that Wnt/ß-catenin signaling increases Nlgn3 mRNA and protein levels in HT22 mouse hippocampal cells and primary cultures of rat hippocampal neurons. We characterized the activity of mouse and rat Nlgn3 promoter constructs containing conserved putative T-cell factor/lymphoid enhancing factor (TCF/LEF)-binding elements (TBE) and found that their activity is significantly augmented in Wnt/ß-catenin cell reporter assays. Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis experiments revealed that endogenous ß-catenin binds to novel TBE consensus sequences in the Nlgn3 promoter. Moreover, activation of the signaling cascade increased Nlgn3 clustering and co- localization with the scaffold PSD-95 protein in dendritic processes of primary neurons. Our results directly link Wnt/ß-catenin signaling to the transcription of the Nlgn3 gene and support a functional role for the signaling pathway in the dysregulation of excitatory/inhibitory neuronal activity, as is observed in animal models of ASD.
Asunto(s)
Trastorno del Espectro Autista/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Hipocampo/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Trastorno del Espectro Autista/fisiopatología , Células Cultivadas , Embrión de Mamíferos , Femenino , Células HEK293 , Hipocampo/fisiopatología , Humanos , Masculino , Ratones , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-DawleyRESUMEN
Two distantly located promoter regions regulate the dynamic expression of RUNX genes during development: distal P1 and proximal P2 promoters. We have recently described that ß-catenin increases total Runx1 mRNA levels in human CD34(+) hematopoietic progenitors and enhances spatial proximity with its translocation partner ETO. Here, we report that induction of Wnt/ß-catenin signaling in HL60 and Jurkat leukemia-derived cell lines and CD34(+) progenitors selectively activate the production of the longer distal P1-Runx1 mRNA isoform. Gain- and loss-of-function experiments revealed that the differential increase in P1-Runx1 expression is accomplished through a minimal ß-catenin responsive region that includes a highly conserved TCF/LEF-binding element, located -20/-16 bp upstream of the canonical distal P1-Runx1 transcription start site. We conclude that the distal P1-Runx1 promoter is a direct transcriptional target of Wnt/ß-catenin signaling that may be important in normal hematopoiesis or its transition into malignant stem cells during the onset or progression of leukemia.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Leucemia/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Jurkat , Leucemia/patología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteína 1 Compañera de Translocación de RUNX1 , Factores de Transcripción/genética , Vía de Señalización Wnt , beta Catenina/genéticaRESUMEN
In the present study, we investigated whether cellular damage, as demonstrated by lactate dehydrogenase (LDH) release in the human fallopian tube (FT) infected by Neisseria gonorrhoeae (Ngo), correlated with high levels of nitric oxide synthase (NOS) mRNA and enzyme activity. Infection with Ngo induced a significant increase (~35-fold) in mRNA transcripts of the inducible isoform of NOS. Paradoxically, a reduction in NOS enzyme activity was observed in infected cultures, suggesting that gonococcal infection possibly influences translation of iNOS mRNA to the enzyme. In addition, treatment with the NOS inhibitor TRIM did not prevent gonococcal-induced cellular damage. In contrast, the addition of the inhibitor L-NAME induced a 40% reduction in LDH release, which correlated with a ~50% reduction in gonococcal numbers. Moreover, treatment of normal FT explants with an exogenous NO donor, SNAP, did not induce significant cellular damage. Taken together, our data suggest that NO does not contribute to cellular damage during infection of the human FT with Neisseria gonorrhoeae.
Asunto(s)
Trompas Uterinas/microbiología , L-Lactato Deshidrogenasa/metabolismo , Neisseria gonorrhoeae/enzimología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , ARN Mensajero/metabolismo , Células Cultivadas , Trompas Uterinas/patología , Femenino , Humanos , Factores de TiempoRESUMEN
BACKGROUND: Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae (Ngo) can lead to acute salpingitis, an inflammatory condition resulting in damage primarily to the ciliated cells, with loss of ciliary activity and sloughing of the cells from the epithelium. Recently, we have shown that Ngo infection induced apoptosis in FT epithelium cells by a TNF-alpha dependent mechanism that could contribute to the cell and tissue damage observed in gonococcal salpingitis. AIM: To investigate the apoptosis-related genes expressed during apoptosis induction in cultured FT epithelial cells infected in vitro by Ngo. MATERIALS AND METHODS: In the current study, we used cDNA macroarrays and real time PCR to identify and determine the expression levels of apoptosis related genes during the in vitro gonococci infection of FT epithelial cells. RESULTS: Significant apoptosis was induced following infection with Ngo. Macroarray analysis identified the expression of multiple genes of the TNF receptor family (TNFRSF1B, -4, -6, -10A, -10B and -10D) and the Bcl-2 family (BAK1, BAX, BLK, HRK and MCL-1) without differences between controls and infected cells. This lack of difference was confirmed by RT-PCR of BAX, Bcl-2, TNFRS1A (TNFR-I) and TNFRSF1B (TNFR-II). CONCLUSION: Several genes related to apoptosis are expressed in primary cultures of epithelial cells of the human Fallopian tube. Infection with Ngo induces apoptosis without changes in the pattern of gene expression of several apoptosis-related genes. RESULTS strongly suggest that Ngo regulates apoptosis in the FT by post-transcriptional mechanisms that need to be further addressed.
Asunto(s)
Apoptosis/genética , Células Epiteliales/microbiología , Trompas Uterinas/microbiología , Neisseria gonorrhoeae/fisiología , Salpingitis/microbiología , Células Cultivadas , Células Epiteliales/patología , Trompas Uterinas/patología , Femenino , Regulación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salpingitis/patologíaRESUMEN
Background: Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae (Ngo) can lead to acute salpingitis, an inflammatory condition resulting in damage primarily to the ciliated cells, with loss of ciliary activity and sloughing of the cells from the epithelium. Recently, we have shown that Ngo infection induced apoptosis in FT epithelium cells by a TNF-alpha dependent mechanism that could contribute to the cell and tissue damage observed in gonococcal salpingitis. Aim: To investigate the apoptosis-related genes expressed during apoptosis induction in cultured FT epithelial cells infected in vitro by Ngo. Materials and Methods: In the current study, we used cDNA macroarrays and real time PCR to identify and determine the expression levels of apoptosis related genes during the in vitro gonococci infection of FT epithelial cells. Results: Significant apoptosis was induced following infection with Ngo. Macroarray analysis identified the expression of multiple genes of the TNF receptor family (TNFRSF1B, -4, -6, -10A, -10B and -10D) and the Bcl-2 family (BAK1, BAX, BLK, HRK and MCL-1) without differences between controls and infected cells. This lack of difference was confirmed by RT-PCR of BAX, Bcl-2, TNFRS1A (TNFR-I) and TNFRSF1B (TNFR-II). Conclusion: Several genes related to apoptosis are expressed in primary cultures of epithelial cells of the human Fallopian tube. Infection with Ngo induces apoptosis without changes in the pattern of gene expression of several apoptosis-related genes. Results strongly suggest that Ngo regulates apoptosis in the FT by post-transcriptional mechanisms that need to be further addressed.