RESUMO
This chapter describes the procedures for immunofluorescence microscopy (IFM) and quantitative PCR (qPCR) analyses of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol is provided outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IFM and qPCR anlysis. The techniques are illustrated with results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, PDGF, and TGFß signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment is experimentally shown illustrating that these techniques can be important tools in answering basic questions about hESC biology.
Assuntos
Cílios/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Microscopia de Fluorescência/métodos , Transdução de Sinais/genética , Biomarcadores/metabolismo , Forma Celular , Células Cultivadas , Eletroforese em Gel de Ágar , Células Alimentadoras/citologia , Fibroblastos/citologia , Humanos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Development of the cerebellum occurs postnatally and is marked by a rapid proliferation of cerebellar granule neuron precursors (CGNPs). CGNPs are the cells of origin for SHH-driven medulloblastoma, the most common malignant brain tumor in children. Here, we investigated the role of ERK, JNK, and p38 mitogen-activated protein kinases in CGNP proliferation. We found high levels of p38α in proliferating CGNPs. Concomitantly, members of the p38 pathway, such as ASK1, MKK3 and ATF-2, were also elevated. Inhibition of the Shh pathway or CGNP proliferation blunts p38α levels, irrespective of Shh treatment. Strikingly, p38α levels were high in vivo in the external granule layer of the postnatal cerebellum, Shh-dependent mouse medulloblastomas and human medulloblastomas of the SHH subtype. Finally, knocking down p38α by short hairpin RNA-carrying lentiviruses as well as the pharmacologically inhibiting of its kinase activity caused a marked decrease in CGNP proliferation, underscoring its requirement for Shh-dependent proliferation in CGNPs. The inhibition of p38α also caused a decrease in Gli1 and N-myc transcript levels, consistent with reduced proliferation. These findings suggest p38 inhibition as a potential way to increase the efficacy of treatments available for malignancies associated with deregulated SHH signaling, such as basal cell carcinoma and medulloblastoma.
Assuntos
Proliferação de Células/fisiologia , Cerebelo/enzimologia , Células-Tronco Neurais/enzimologia , Neurônios/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Encéfalo/enzimologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/enzimologia , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
BACKGROUND: Ovarian cancer is the fourth leading cause of cancer-related deaths among women in Denmark, largely due to the advanced stage at diagnosis in most patients. Approximately 90% of ovarian cancers originate from the single-layered ovarian surface epithelium (OSE). Defects in the primary cilium, a solitary sensory organelle in most cells types including OSE, were recently implicated in tumorigenesis, mainly due to deregulation of ciliary signaling pathways such as Hedgehog (Hh) signaling. However, a possible link between primary cilia and epithelial ovarian cancer has not previously been investigated. METHODS: The presence of primary cilia was analyzed in sections of fixed human ovarian tissue as well as in cultures of normal human ovarian surface epithelium (OSE) cells and two human OSE-derived cancer cell lines. We also used immunofluorescence microscopy, western blotting, RT-PCR and siRNA to investigate ciliary signaling pathways in these cells. RESULTS: We show that ovarian cancer cells display significantly reduced numbers of primary cilia. The reduction in ciliation frequency in these cells was not due to a failure to enter growth arrest, and correlated with persistent centrosomal localization of aurora A kinase (AURA). Further, we demonstrate that ovarian cancer cells have deregulated Hh signaling and platelet-derived growth factor receptor alpha (PDGFRα) expression and that promotion of ciliary formation/stability by AURA siRNA depletion decreases Hh signaling in ovarian cancer cells. Lastly, we show that the tumor suppressor protein and negative regulator of AURA, checkpoint with forkhead-associated and ring finger domains (CHFR), localizes to the centrosome/primary cilium axis. CONCLUSIONS: Our results suggest that primary cilia play a role in maintaining OSE homeostasis and that the low frequency of primary cilia in cancer OSE cells may result in part from over-expression of AURA, leading to aberrant Hh signaling and ovarian tumorigenesis.
RESUMO
This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment will be shown illustrating that these techniques can be important tools in answering basic questions about hESC biology.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Microscopia de Fluorescência/métodos , RNA Mensageiro/análise , Sequência de Bases , Biomarcadores/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Cílios/metabolismo , Cílios/ultraestrutura , Primers do DNA/genética , Marcadores Genéticos , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de SinaisRESUMO
Although first described as early as 1898 and long considered a vestigial organelle of little functional importance, the primary cilium has become one of the hottest research topics in modern cell biology and physiology. Primary cilia are nonmotile sensory organelles present in a single copy on the surface of most growth-arrested or differentiated mammalian cells, and defects in their assembly or function are tightly coupled to many developmental defects, diseases and disorders. In normal tissues, the primary cilium coordinates a series of signal transduction pathways, including Hedgehog, Wnt, PDGFRalpha and integrin signaling. In the kidney, the primary cilium may function as a mechano-, chemo- and osmosensing unit that probes the extracellular environment and transmits signals to the cell via, e.g., polycystins, which depend on ciliary localization for appropriate function. Indeed, hypomorphic mutations in the mouse ift88 (previously called Tg737) gene, which encodes a ciliogenic intraflagellar transport protein, result in malformation of primary cilia, and in the collecting ducts of kidney tubules this is accompanied by development of autosomal recessive polycystic kidney disease (PKD). While PKD was one of the first diseases to be linked to dysfunctional primary cilia, defects in this organelle have subsequently been associated with many other phenotypes, including cancer, obesity, diabetes as well as a number of developmental defects. Collectively, these disorders of the cilium are now referred to as the ciliopathies. In this review, we provide a brief overview of the structure and function of primary cilia and some of their roles in coordinating signal transduction pathways in mammalian development, health and disease.
Assuntos
Cílios/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Animais , Cílios/patologia , Desenvolvimento Embrionário , Humanos , Rim/metabolismo , Rim/patologia , Mutação , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Células-Tronco/metabolismo , Células-Tronco/patologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality.
Assuntos
Técnicas de Silenciamento de Genes , Proteínas de Protozoários/genética , Interferência de RNA , Tetrahymena thermophila/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Imunofluorescência , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Tetrahymena thermophila/metabolismoRESUMO
Hedgehog (Hh) signaling controls pancreatic development and homeostasis; aberrant Hh signaling is associated with several pancreatic diseases. Here we investigated the link between Hh signaling and primary cilia in the human developing pancreatic ducts and in cultures of human pancreatic duct adenocarcinoma cell lines, PANC-1 and CFPAC-1. We show that the onset of Hh signaling from human embryogenesis to fetal development is associated with accumulation of Hh signaling components Smo and Gli2 in duct primary cilia and a reduction of Gli3 in the duct epithelium. Smo, Ptc, and Gli2 localized to primary cilia of PANC-1 and CFPAC-1 cells, which may maintain high levels of nonstimulated Hh pathway activity. These findings indicate that primary cilia are involved in pancreatic development and postnatal tissue homeostasis.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Pâncreas/citologia , Pâncreas/embriologia , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Feto/citologia , Proteínas de Fluorescência Verde/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores Patched , Receptor Patched-1 , Gravidez , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Receptor Smoothened , Transfecção , Proteína Gli2 com Dedos de ZincoRESUMO
Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.
Assuntos
Diferenciação Celular/genética , Cílios/ultraestrutura , Células-Tronco Embrionárias/ultraestrutura , Proteínas Hedgehog/metabolismo , Transdução de Sinais/genética , Axonema/genética , Axonema/metabolismo , Axonema/ultraestrutura , Linhagem Celular , Linhagem da Célula/genética , Cílios/genética , Cílios/metabolismo , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Embrionárias/metabolismo , Feminino , Imunofluorescência , Proteínas Hedgehog/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Two Tetrahymena kinesin-like proteins (klps) of the kinesin II subfamily, Kin1 and Kin2, were first identified by Brown et al. [1999: Mol Biol Cell 10: 3081-3096] and shown to be involved in ciliary morphogenesis probably as molecular motors in intraciliary transport (ICT). Using Tetrahymena genomic DNA as a template, we cloned Kin5, another kinesin II subfamily member. Kin5 is upregulated upon deciliation, suggesting that Kin5 is a ciliary protein. Kin5 is most closely related to Osm3, a Caenorhabditis elegans kinesin II; Osm3 and Kin5 have a 56% identity, which rises to 60.4% in the motor domain and a 45% identity in a 60 amino acid region of the C-terminal FERM (4.1, Ezrin, Radixin, Moesin) domain, not present in Kin1 or Kin2, which we hypothesize to be a critical domain either for dimerization or for cargo recognition in ICT. An antibody to a peptide sequence from the tail region of Kin5 localizes in a punctate pattern along the ciliary axoneme, colocalizing with an antibody to the raft protein IFT139. These findings suggest that Kin5 is an ICT motor like Osm3. Osm3 orthologs apparently transport membrane proteins and Kin5 may be the homodimeric kinesin II that performs this function in Tetrahymena cilia.