RESUMO
Primary cilia are dynamic signaling organelles that project from the cell surface to sense diverse chemical, physical and morphogenetic cues. Ciliary defects therefore cause diseases (ciliopathies) that affect multiple organs in developing and adult organisms. Cilia-mediated signaling involves the orchestrated movement of signaling proteins in and out of the ciliary compartment, including movement of receptors such as the Sonic Hedgehog (Shh) receptor Patched 1 (PTCH1), Smoothened (SMO), and various other G protein-coupled receptors (GPCRs), as well as transforming growth factor ß (TGF-ß) receptors I and II (TGF-ß-RI/II). We provide here a current understanding of trafficking events associated with cilia-mediated signaling, with emphasis on the involvement of clathrin-dependent receptor-mediated endocytosis in regulating ciliary Shh and TGF-ß signaling.
Assuntos
Cílios/metabolismo , Endocitose , Transdução de Sinais , Proteínas Hedgehog/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Recent evidence has indicated that caveolins are localized at the base of primary cilia, which are microtubule-based sensory organelles present on the cell surface, and that Caveolin-1 (CAV1) plays important roles in regulating ciliary membrane composition and function. Here we describe methods to analyze the localization and function of CAV1 in primary cilia of cultured mammalian cells. These include methods for culturing and transfecting mammalian cells with a CAV1-encoding plasmid or small interfering RNA (siRNA), analysis of mammalian cells by immunofluorescence microscopy (IFM) with antibodies against ciliary markers and CAV1, as well as methods for analyzing ciliary CAV1 function in siRNA-treated cells by IFM and cell-based signaling assays.
Assuntos
Caveolina 1/metabolismo , Técnicas de Cultura de Células/métodos , Cílios/metabolismo , Microscopia de Fluorescência/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Caveolina 1/genética , Linhagem Celular , Células Cultivadas , Humanos , RNA Interferente Pequeno , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Patients with haematological malignancies are often vitamin C deficient, and vitamin C is essential for the TET-induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), the first step in active DNA demethylation. Here, we investigate whether oral vitamin C supplementation can correct vitamin C deficiency and affect the 5hmC/5mC ratio in patients with myeloid cancers treated with DNA methyltransferase inhibitors (DNMTis). RESULTS: We conducted a randomized, double-blinded, placebo-controlled pilot trial (NCT02877277) in Danish patients with myeloid cancers performed during 3 cycles of DNMTi-treatment (5-azacytidine, 100 mg/m2/d for 5 days in 28-day cycles) supplemented by oral dose of 500 mg vitamin C (n = 10) or placebo (n = 10) daily during the last 2 cycles. Fourteen patients (70%) were deficient in plasma vitamin C (< 23 µM) and four of the remaining six patients were taking vitamin supplements at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (< 11.4 µM; 4.997 vs 4.656% 5mC relative to deoxyguanosine, 95% CI [0.126, 0.556], P = 0.004). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (mean increase 34.85 ± 7.94 µM, P = 0.0004). We show for the first time that global 5hmC/5mC levels were significantly increased in mononuclear myeloid cells from patients receiving oral vitamin C compared to placebo (0.037% vs - 0.029%, 95% CI [- 0.129, - 0.003], P = 0.041). CONCLUSIONS: Normalization of plasma vitamin C by oral supplementation leads to an increase in the 5hmC/5mC ratio compared to placebo-treated patients and may enhance the biological effects of DNMTis. The clinical efficacy of oral vitamin C supplementation to DNMTis should be investigated in a large randomized, placebo-controlled clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02877277 . Registered on 9 August 2016, retrospectively registered.
Assuntos
Ácido Ascórbico/administração & dosagem , Azacitidina/administração & dosagem , Metilação de DNA/efeitos dos fármacos , Leucemia Mieloide/terapia , Síndromes Mielodisplásicas/terapia , Administração Oral , Idoso , Idoso de 80 Anos ou mais , Ácido Ascórbico/sangue , Ácido Ascórbico/farmacologia , Azacitidina/farmacologia , Ilhas de CpG/efeitos dos fármacos , Dinamarca , Método Duplo-Cego , Epigênese Genética/efeitos dos fármacos , Feminino , Humanos , Leucemia Mieloide/sangue , Leucemia Mieloide/genética , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/genética , Projetos PilotoRESUMO
The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-ß/bone morphogenetic protein (TGF-ß/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-ß1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Centríolos/metabolismo , Cílios/metabolismo , Proteínas dos Microtúbulos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Centrossomo/metabolismo , Humanos , Transporte Proteico , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Since the beginning of the millennium, research in primary cilia has revolutionized our way of understanding how cells integrate and organize diverse signaling pathways during vertebrate development and in tissue homeostasis. Primary cilia are unique sensory organelles that detect changes in their extracellular environment and integrate and transmit signaling information to the cell to regulate various cellular, developmental, and physiological processes. Many different signaling pathways have now been shown to rely on primary cilia to function properly, and mutations that lead to ciliary dysfunction are at the root of a pleiotropic group of diseases and syndromic disorders called ciliopathies. In this review, we present an overview of primary cilia-mediated regulation of receptor tyrosine kinase (RTK) and transforming growth factor ß (TGF-ß) signaling. Further, we discuss how defects in the coordination of these pathways may be linked to ciliopathies.
Assuntos
Cílios/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Células 3T3-L1 , Animais , Centríolos/metabolismo , Endocitose , Genoma Humano , Complexo de Golgi/metabolismo , Homeostase , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Microscopia de Fluorescência , Mutação , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Ciliary membrane composition is controlled by transition zone (TZ) proteins such as RPGRIP1, RPGRIPL and NPHP4, which are vital for balanced coordination of diverse signalling systems like the Sonic hedgehog (Shh) pathway. Activation of this pathway involves Shh-induced ciliary accumulation of Smoothened (SMO), which is disrupted by disease-causing mutations in TZ components. Here we identify kinesin-3 motor protein KIF13B as a novel member of the RPGRIP1N-C2 domain-containing protein family and show that KIF13B regulates TZ membrane composition and ciliary SMO accumulation. KIF13B is upregulated during ciliogenesis and is recruited to the ciliary base by NPHP4, which binds to two distinct sites in the KIF13B tail region, including an RPGRIP1N-C2 domain. KIF13B and NPHP4 are both essential for establishment of a CAV1 membrane microdomain at the TZ, which in turn is required for Shh-induced ciliary SMO accumulation. Thus KIF13B is a novel regulator of ciliary TZ configuration, membrane composition and Shh signalling.