RESUMEN
A central question in Hedgehog (Hh) signaling is how evolutionarily conserved components of the pathway might use the primary cilium in mammals but not fly. We focus on Suppressor of fused (Sufu), a major Hh regulator in mammals, and reveal that Sufu controls protein levels of full-length Gli transcription factors, thus affecting the production of Gli activators and repressors essential for graded Hh responses. Surprisingly, despite ciliary localization of most Hh pathway components, regulation of Gli protein levels by Sufu is cilium-independent. We propose that Sufu-dependent processes in Hh signaling are evolutionarily conserved. Consistent with this, Sufu regulates Gli protein levels by antagonizing the activity of Spop, a conserved Gli-degrading factor. Furthermore, addition of zebrafish or fly Sufu restores Gli protein function in Sufu-deficient mammalian cells. In contrast, fly Smo is unable to translocate to the primary cilium and activate the mammalian Hh pathway. We also uncover a novel positive role of Sufu in regulating Hh signaling, resulting from its control of both Gli activator and repressor function. Taken together, these studies delineate important aspects of cilium-dependent and cilium-independent Hh signal transduction and provide significant mechanistic insight into Hh signaling in diverse species.
Asunto(s)
Cilios/metabolismo , Evolución Molecular , Proteínas Hedgehog/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Animales , Proteína Axina , Línea Celular Transformada , Drosophila , Proteínas de Drosophila/metabolismo , Humanos , Ratones , Proteínas Nucleares/metabolismo , Receptores Patched , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Represoras/genética , Receptor Smoothened , Complejos de Ubiquitina-Proteína Ligasa , Regulación hacia Arriba , Pez Cebra , Proteínas de Pez Cebra/metabolismo , Proteína Gli2 con Dedos de Zinc , Proteína Gli3 con Dedos de ZincRESUMEN
The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Zinc/química , Ácido Anhídrido Hidrolasas , Secuencias de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/química , Separación Celular , Cromosomas/ultraestructura , Roturas del ADN de Doble Cadena , Daño del ADN , Citometría de Flujo , Silenciador del Gen , Genoma , Genómica , Células HEK293 , Histonas/química , Humanos , Proteína Homóloga de MRE11 , Microscopía Fluorescente/métodos , Mutación , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Recombinación Genética , Proteínas Supresoras de Tumor/químicaRESUMEN
Ubiquitination plays an important role in the DNA damage response. We identified a novel interaction of the E3 ubiquitin ligase RNF8 with Nbs1, a key regulator of DNA double-strand break (DSB) repair. We found that Nbs1 is ubiquitinated both before and after DNA damage and is a direct ubiquitination substrate of RNF8. We also identified key residues on Nbs1 that are ubiquitinated by RNF8. By using laser microirradiation and live-cell imaging, we observed that RNF8 and its ubiquitination activity are important for promoting optimal binding of Nbs1 to DSB-containing chromatin. We also demonstrated that RNF8-mediated ubiquitination of Nbs1 contributes to the efficient and stable binding of Nbs1 to DSBs and is important for HR-mediated DSB repair. Taken together, these studies suggest that Nbs1 is one important target of RNF8 to regulate DNA DSB repair.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga/fisiología , Proteínas Nucleares/metabolismo , Ubiquitinación/fisiología , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Recombinación Homóloga/efectos de la radiación , Humanos , Rayos Láser/efectos adversos , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligasas , Ubiquitinación/efectos de la radiaciónRESUMEN
Desmogleins (Dsg2) are the major components of desmosomes. Dsg2 has five extracellular tandem cadherin domains (EC1-EC5) for cell-cell interaction. We had previously confirmed the Dsg2 antibody and its epitope (named KC21) derived from EC2 domain suppressing epithelial-mesenchymal transition and invasion in human cancer cell lines. Here, we screened six peptide fragments derived from EC2 domain and found that KR20, the parental peptide of KC21, was the most potent one on suppressing endothelial colony-forming cell (ECFC) tube-like structure formation. KC21 peptide also attenuated migration but did not disrupt viability and proliferation of ECFCs, consistent with the function to inhibit VEGF-mediated activation of p38 MAPK but not AKT and ERK. Animal studies showed that KC21 peptides suppressed capillary growth in Matrigel implant assay and inhibited oxygen-induced retinal neovascularization. The effects were comparable to bevacizumab (Bev). In conclusion, KC21 peptide is an angiogenic inhibitor potentially useful for treating angiogenesis-related diseases.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Desmogleína 2/farmacología , Células Progenitoras Endoteliales/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Neovascularización Retiniana/prevención & control , Animales , Bevacizumab/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Células Progenitoras Endoteliales/metabolismo , Humanos , Hipoxia/complicaciones , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Neovascularización Retiniana/etiología , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/fisiopatología , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: We sought to clarify the response of endothelial connexins to hyperlipidemia and lipid-lowering therapy. METHODS AND RESULTS: Aortic endothelial gap junctions were analyzed by en face immunoconfocal microscopy and electron microscopy in C57BL/6 mice subjected to the following regimens: (1) normal chow (NC) for 3 months (3 mo), (2) NC for 9 mo, (3) NC for 3 mo, followed by a cholesterol-enriched diet (CED) for 6 mo, (4) NC for 3 mo and CED for 6 mo, with simvastatin in the final week, and (5) (in apoprotein E [apoE]-deficient mice) NC and examined at 3 mo and 7 to 9 mo. In wild-type mice, connexin37 (Cx37) and Cx40 were markedly downregulated in the CED-fed animals compared with those fed NC (CED vs 9-mo NC, 77% reduction in Cx37 and 65% reduction in Cx40; both P<0.01). After simvastatin treatment, Cx40 remained depressed, but Cx37 recovered to 94% of the level found in non-cholesterol-fed animals (P<0.01). Electron microscopy demonstrated that gap junctions were smaller in animals fed the CED compared with those given simvastatin and with controls fed NC (P<0.01). Endothelial connexins were rare in the atherosclerotic plaques of apoE-deficient mice. CONCLUSIONS: Mouse aortic endothelial gap junctions and connexins are downregulated during long-term hyperlipidemia. Short-term treatment with simvastatin leads to recovery of Cx37 expression but not Cx40 expression.
Asunto(s)
Conexinas/metabolismo , Endotelio Vascular/metabolismo , Hiperlipidemias/tratamiento farmacológico , Hiperlipidemias/metabolismo , Simvastatina/administración & dosificación , Animales , Aorta/patología , Conexinas/efectos de los fármacos , Regulación hacia Abajo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/ultraestructura , Femenino , Uniones Comunicantes/metabolismo , Uniones Comunicantes/ultraestructura , Hiperlipidemias/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Distribución Tisular , Proteína alfa-5 de Unión Comunicante , Proteína alfa-4 de Unión ComunicanteRESUMEN
We investigated the effect of nicotine on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). We also evaluated whether the effect requires activation of acetyl choline receptors sensitive to nicotine (nAChRs) and is altered by statins. The results showed that expression of Cx43 protein is reduced by nicotine in a dose-dependent manner (6 x 10(-4) M nicotine vs control, 33% reduction, p < 0.01), though Cx43 mRNA is up-regulated (6 x 10(-4) M nicotine vs control, 36% increase, p < 0.01). Concomitantly, the communication function, determined by fluorescence recovery after photobleaching, is decreased (6 x 10(-4) M nicotine vs control, 38% reduction, p < 0.05). Such a down-regulation of Cx43 gap junctions by nicotine disappears in the presence of the nAChRs antagonist, dihydro-beta-erythroidine, and protease inhibitors leupeptin plus N-acetyl-Leu-Leu-Norleu-al (ALLN). Similarly, the effect of nicotine is attenuated by statins, including fluvastatin, lovastatin, pravastatin, and simvastatin, even at the presence of mevalonate. We concluded that i) nicotine down-regulates Cx43 expression and gap-junctional communication in HUVEC via post-transcriptional modification, which involves enhancement of Cx43 proteolysis; ii) the effect of nicotine is mediated via activation of nAChRs; and iii) the effect of nicotine is attenuated by statins through mechanisms outside the hypolipidemic pathway.
Asunto(s)
Conexina 43/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Endotelio Vascular/metabolismo , Uniones Comunicantes/efectos de los fármacos , Nicotina/farmacología , Venas Umbilicales/metabolismo , Células Cultivadas , Conexina 43/genética , Endotelio Vascular/ultraestructura , Uniones Comunicantes/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Ácido Mevalónico/farmacología , Antagonistas Nicotínicos/farmacología , Fotoblanqueo , Inhibidores de Proteasas/farmacología , ARN Mensajero , Receptores Nicotínicos/metabolismo , Venas Umbilicales/citologíaRESUMEN
Carbohydrate antigen 125 (CA-125), traditionally a tumor marker for screening, diagnosis, and monitoring in ovarian malignancy, had recently been shown increasing evidence and more extensively recognized/explored as a novel surrogate of heart failure (HF). The exact mechanisms underlying the pathophysiologic link between elevated serum CA-125 concentration and HF may be multi-factorial, with both mechanical and inflammatory process including numerous potential cytokines involved. Accumulating data had consistently indicated its diagnostic and prognostic role in HF patients in various clinical settings, however, there is limited clinical information regarding the incremental value or head-to-head comparison of such marker to other well-established HF markers. In this brief review, we aimed to discuss the biosynthesis, and potential insights of underlying pathophysiologies associated with CA-125 secretion in the scenarios of cardiac structural/functional alterations and HF, and further explored its current usage and roles in several recent reports.