RESUMEN
Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, renal cysts, obesity and polydactyly. BBS genes have been implicated in ciliogenesis, hedgehog signaling and retinal pigment epithelium maturation. BBS1 and BBS5 are members of the BBSome, implicated in cilia transport of proteins, and BBS10 is a member of the chaperonin-complex, mediating BBSome assembly. In this study, involvement of BBS1, BBS5 and BBS10 in ciliogenesis and hedgehog signaling were investigated in BBS-defective patient fibroblasts as well as in RPE-hTERT cells following siRNA-mediated knockdown of the BBS genes. Furthermore, the ability of BBS1-defective induced pluripotent stem-cells (iPSCs) to differentiate into RPE cells was assessed. We report that cells lacking functional BBS5 or BBS10 have a reduced number of primary cilia, whereas cells lacking functional BBS1 display shorter primary cilia compared to wild-type cells. Hedgehog signaling was substantially impaired and Smoothened, a component of hedgehog signaling, was trapped inside the cilia of the BBS-defective cells, even in the absence of Smoothened agonist. Preliminary results demonstrated the ability of BBS1-defective iPSC to differentiate into RPE-65 expressing RPE-like cells. The BBS1-/--defective RPE-like cells were less pigmented, compared to RPE-like cells differentiated from control iPSCs, indicating an impact of BBS1 on RPE maturation.
Asunto(s)
Síndrome de Bardet-Biedl/metabolismo , Chaperoninas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Síndrome de Bardet-Biedl/patología , Línea Celular , Cilios/metabolismo , Cilios/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de SeñalRESUMEN
Primary cilia are sensory organelles that coordinate multiple cellular signaling pathways, including Hedgehog (HH), Wingless/Int (WNT) and Transforming Growth Factor-ß (TGF-ß) signaling. Similarly, primary cilia have been implicated in regulation of mTOR signaling, in which Tuberous Sclerosis Complex proteins 1 and 2 (TSC1/2) negatively regulate protein synthesis by inactivating the mTOR complex 1 (mTORC1) at energy limiting states. Here we report that TSC1 and TSC2 regulate Smoothened (SMO)-dependent HH signaling in mouse embryonic fibroblasts (MEFs). Reduced SMO-dependent expression of Gli1 was demonstrated in both Tsc1-/- and Tsc2-/- cells, and we found that Tsc1 is required for TGF-ß induced phosphorylation of SMAD2/3 and subsequent expression of the HH signaling effector and transcription factor GLI2. Hedgehog signaling was restored in Tsc1-/- cells after exogenous expression of Gli2, whereas rapamycin restored HH signaling in Tsc2-/- cells. Furthermore, we observed that Tsc1-/- MEFs display significantly elongated cilia, whereas cilia in Tsc2-/- MEFs were shorter than normal. The elongated cilium phenotype of Tsc1-/- MEFs is likely due to increased mTORC1-dependent autophagic flux observed in these cells, as both the autophagic flux and the cilia length phenotype was restored by rapamycin. In addition, ciliary length control in Tsc1-/- MEFs was also influenced by reduced expression of Gli2, which compromised expression of Wnt5a that normally promotes cilia disassembly. In summary, our results support distinct functions of Tsc1 and Tsc2 in cellular signaling as the two genes affect ciliary length control and HH signaling via different mechanisms.
Asunto(s)
Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Proteínas Hedgehog/genética , Ratones Noqueados , Interferencia de ARN , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteína Gli2 con Dedos de Zinc/genética , Proteína Gli2 con Dedos de Zinc/metabolismoRESUMEN
TSC1 and TSC2 are genes mutated in the syndrome TSC (tuberous sclerosis complex). We describe a 3-generation family with 17 affected members, all presenting classic TSC features except renal manifestations. The disease segregates with a silent substitution in TSC2, c.4149C>T, p.(Ser1383Ser), which leads to the formation of an active donor splice site, resulting in three shorter alternatively spliced transcripts with premature stop codons. However a small amount of normal spliced transcript is apparently produced from the mutated allele, which might explain the milder phenotype. The gene products of TSC1/2 form a complex which at energy limiting states, down-regulates the activity of the regulator of protein synthesis, the mammalian target of rapamycin complex1 (mTORC1). As expected, in contrast to cultured control fibroblasts, starvation of cultured patient fibroblasts obtained from a hypomelanotic macule did not lead to repression of mTORC1, whereas partial repression was observed in patient fibroblasts obtained from non-lesional skin. The findings indicate that the development of hypomelanotic macules is associated with constitutive activated mTORC1, whereas mild deregulation of mTORC1 allows the maintenance of normal skin. Furthermore, the finding establishes the pathogenic effect of the "silent" c.4149C>T substitution and emphasizes the need for awareness when interpreting silent substitutions in general.
Asunto(s)
Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Enfermedades de la Piel/patología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Esclerosis Tuberosa/complicaciones , Proteínas Supresoras de Tumor/genética , Sustitución de Aminoácidos , Células Cultivadas , Regulación hacia Abajo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Linaje , Empalme del ARN , Análisis de Secuencia de ADN , Enfermedades de la Piel/genética , Enfermedades de la Piel/metabolismo , Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
The mTORC1-complex is negatively regulated by TSC1 and TSC2. Activation of Hedgehog signaling is strictly dependent on communication between Smoothened and the Hedgehog-signaling effector and transcription factor, GLI2, in the primary cilium. Details about this communication are not known, and we wanted to explore this further. Here we report that in Tsc2 -/- MEFs constitutively activated mTORC1 led to mis-localization of Smoothened to the plasma membrane, combined with increased concentration of GLI2 in the cilia and reduced Hedgehog signaling, measured by reduced expression of the Hedgehog target gene, Gli1 Inhibition of mTORC1 rescued the cellular localization of Smoothened to the cilia, reduced the cilia concentration of GLI2, and restored Hedgehog signaling. Our results reveal evidence for a two-step activation process of GLI2. The first step includes GLI2 stabilization and cilium localization, whereas the second step includes communication with cilia-localized Smoothened. We found that mTORC1 inhibits the second step. This is the first demonstration that mTORC1 is involved in the regulation of Hedgehog signaling.
Asunto(s)
Cilios , Proteínas Hedgehog , Diana Mecanicista del Complejo 1 de la Rapamicina , Transducción de Señal , Receptor Smoothened , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor , Proteína Gli2 con Dedos de Zinc , Animales , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína Gli2 con Dedos de Zinc/metabolismo , Proteína Gli2 con Dedos de Zinc/genética , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Fibroblastos/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Membrana Celular/metabolismo , Ratones NoqueadosRESUMEN
Hedgehog (Hh) signaling and mTOR signaling, essential for embryonic development and cellular metabolism, are both coordinated by the primary cilium. Observations from cancer cells strongly indicate crosstalk between Hh and mTOR signaling. This hypothesis is supported by several studies: Evidence points to a TGFß-mediated crosstalk; Increased PI3K/AKT/mTOR activity leads to increased Hh signaling through regulation of the GLI transcription factors; increased Hh signaling regulates mTORC1 activity positively by upregulating NKX2.2, leading to downregulation of negative mTOR regulators; GSK3 and AMPK are, as members of both signaling pathways, potentially important links between Hh and mTORC1 signaling; The kinase DYRK2 regulates Hh positively and mTORC1 signaling negatively. In contrast, both positive and negative regulation of Hh has been observed for DYRK1A and DYRK1B, which both regulate mTORC1 signaling positively. Based on crosstalk observed between cilia, Hh, and mTORC1, we suggest that the interaction between Hh and mTORC1 is more widespread than it appears from our current knowledge. Although many studies focusing on crosstalk have been carried out, contradictory observations appear and the interplay involving multiple partners is far from solved.
Asunto(s)
Proteínas Hedgehog/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Transducción de Señal , Animales , Autofagia , Cilios/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio , Humanos , Modelos Biológicos , Proteínas Nucleares , Factores de TranscripciónRESUMEN
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in the skin and other organs, including brain, heart, lung, kidney and bones. TSC is caused by mutations in TSC1 and TSC2. Here, we present the TSC1 and TSC2 variants identified in 168 Danish individuals out of a cohort of 327 individuals suspected of TSC. A total of 137 predicted pathogenic or likely pathogenic variants were identified: 33 different TSC1 variants in 42 patients, and 104 different TSC2 variants in 126 patients. In 40 cases (24%), the identified predicted pathogenic variant had not been described previously. In total, 33 novel variants in TSC2 and 7 novel variants in TSC1 were identified. To assist in the classification of 11 TSC2 variants, we investigated the effects of these variants in an in vitro functional assay. Based on the functional results, as well as population and genetic data, we classified 8 variants as likely to be pathogenic and 3 as likely to be benign.
Asunto(s)
Empalme Alternativo , Biomarcadores de Tumor/genética , Mutación , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Esclerosis Tuberosa/genética , Estudios de Cohortes , Análisis Mutacional de ADN , Dinamarca/epidemiología , Humanos , Esclerosis Tuberosa/epidemiología , Esclerosis Tuberosa/patologíaRESUMEN
Purpose: Cone-rod dystrophy (CRD) is a rare hereditary eye disorder that causes progressive degeneration of cone and rod photoreceptors. More than 30 genes, including RAB28, have been associated with CRD; however, only a few RAB28 variants have been reported to be associated with CRD. In this study, we describe two brothers with CRD and a homozygous missense variant, c.55G>A (p.Gly19Arg), in RAB28. Methods: The missense variant was identified as part of a study investigating underlying genetic defects in a large patient cohort (n = 667) using targeted next-generation sequencing of 125 genes associated with retinal dystrophy. Cellular localization of RAB28 and ciliogenesis in patient fibroblasts were investigated by immunofluorescence microscopy. The effect of the missense variant on RAB28 expression level was investigated by quantitative real-time PCR. Results: Two brothers of a consanguineous couple presented with CRD, postaxial polydactyly (PAP), and myopia. Both brothers had a homozygous missense RAB28 variant located in the G1 box of the guanosine triphosphate/guanosine diphosphate binding domain of RAB28. This missense variant caused a considerable reduction of RAB28 localized to the cilia, whereas ciliogenesis seemed unaffected. Conclusions: The missense variant in RAB28 is classified as likely pathogenic with functional effect on protein localization. The combination of retinal dystrophy and PAP are well known from ciliopathies; however, more data are needed to finally conclude that the RAB28 variant described here is the cause of PAP in these brothers.
Asunto(s)
Cilios/metabolismo , Distrofias de Conos y Bastones/genética , Dedos/anomalías , Mutación Missense , Polidactilia/genética , Dedos del Pie/anomalías , Proteínas de Unión al GTP rab , Niño , Humanos , Masculino , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Bardet-Biedl syndrome (BBS), an autosomal recessive disease, is associated with non-functional primary cilia. BBS5 is part of the protein complex termed the BBSome. The BBSome associates with intra flagellar transport (IFT) particles and mediates trafficking of membrane proteins in the cilium, a process important for cilia-mediated signal transduction. Here we describe the generation of three induced pluripotent stem cell (iPSC) lines, KCi003-A, KCi003-B and KCi003-C from a patient with BBS and homozygous for the disease causing variant c.214G>A, p.(Gly72Ser) in BBS5. The iPSC lines can be used for investigation of IFT in different cell types differentiated from the iPSC line.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Diferenciación Celular , Proteínas del Citoesqueleto/genética , Fibroblastos/patología , Células Madre Pluripotentes Inducidas/patología , Mutación , Proteínas de Unión a Fosfato/genética , Adulto , Células Cultivadas , Fibroblastos/metabolismo , Homocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , MasculinoRESUMEN
Bardet-Biedl syndrome (BBS) is genetically heterogeneous with at least 21 genes involved, and BBS10 encodes, together with BBS6 and BBS12, chaperonin-like proteins which are important for the assembly of the multiprotein complex, the BBSome encoded by other BBS genes. Here we describe the successful generation of an induced pluripotent stem cell (iPSC) line KCi002-A from a male with BBS, homozygous for the disease causing variant c.271insT, p.(Cys91fsX95) in BBS10. Resource table.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Células Madre Pluripotentes Inducidas/metabolismo , Homocigoto , Humanos , Masculino , MutaciónRESUMEN
Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy with a wide range of symptoms including obesity, retinal dystrophy, polycystic kidney disease, polydactyly, hypogonadism and learning difficulties. Here we describe the successful generation of an induced pluripotent stem cell (iPSC) KCi001-A from a BBS patient compound heterozygous for two disease causing BBS1 variants c.1169T>G, p. (Met390Arg)/c.1135G>C, p.(Gly370Arg). Resource table.