RESUMO
Retinoic acid (RA) exerts pleiotropic effects during neural development and regulates homeostasis in the adult human brain. The RA signal may be transduced through RXR (retinoid-X receptor)-non-permissive RA receptor/RXR heterodimers or through RXR-permissive RXR heterodimers. The significance of RA signaling in malignant brain tumors such as glioblastoma multiforme (GBM) and gliosarcoma (GS) is poorly understood. In particular, the impact RA has on the proliferation, survival, differentiation, or metabolism of GBM- or GS-derived cells with features of stem cells (SLGCs) remains elusive. In the present manuscript, six GBM- and two GS-derived SLGC lines were analyzed for their responsiveness to RAR- and RXR-selective agonists. Inhibition of proliferation and initiation of differentiation were achieved with a RAR-selective pan-agonist in a subgroup of SLGC lines, whereas RXR-selective pan-agonists (rexinoids) supported proliferation in most SLGC lines. To decipher the RAR-dependent and RAR-independent effects of RXR, the genes encoding the RAR or RXR isotypes were functionally inactivated by CRISPR/Cas9-mediated editing in an IDH1-/p53-positive SLGC line with good responsiveness to RA. Stemness, differentiation capacity, and growth behavior were preserved after editing. Taken together, this manuscript provides evidence about the positive impact of RAR-independent RXR signaling on proliferation, survival, and tumor metabolism in SLGCs.
Assuntos
Glioma , Receptores do Ácido Retinoico , Adulto , Humanos , Receptores do Ácido Retinoico/metabolismo , Retinoides/farmacologia , Tretinoína/farmacologia , Receptores X de Retinoides , Glioma/genética , Células-Tronco/metabolismoRESUMO
Glioblastoma multiforme (GBM) and the GBM variant gliosarcoma (GS) are among the tumors with the highest morbidity and mortality, providing only palliation. Stem-like glioma cells (SLGCs) are involved in tumor initiation, progression, therapy resistance, and relapse. The identification of general features of SLGCs could contribute to the development of more efficient therapies. Commercially available protein arrays were used to determine the cell surface signature of eight SLGC lines from GBMs, one SLGC line obtained from a xenotransplanted GBM-derived SLGC line, and three SLGC lines from GSs. By means of non-negative matrix factorization expression metaprofiles were calculated. Using the cophenetic correlation coefficient (CCC) five metaprofiles (MPs) were identified, which are characterized by specific combinations of 7-12 factors. Furthermore, the expression of several factors, that are associated with GBM prognosis, GBM subtypes, SLGC differentiation stages, or neural identity was evaluated. The investigation encompassed 24 distinct SLGC lines, four of which were derived from xenotransplanted SLGCs, and included the SLGC lines characterized by the metaprofiles. It turned out that all SLGC lines expressed the epidermal growth factor EGFR and EGFR ligands, often in the presence of additional receptor tyrosine kinases. Moreover, all SLGC lines displayed a neural signature and the IDH1 wildtype, but differed in their p53 and PTEN status. Pearson Correlation analysis identified a positive association between the pluripotency factor Sox2 and the expression of FABP7, Musashi, CD133, GFAP, but not with MGMT or Hif1α. Spherical growth, however, was positively correlated with high levels of Hif1α, CDK4, PTEN, and PDGFRß, whereas correlations with stemness factors or MGMT (MGMT expression and promoter methylation) were low or missing. Factors highly expressed by all SLGC lines, irrespective of their degree of stemness and growth behavior, are Cathepsin-D, CD99, EMMPRIN/CD147, Intß1, the Galectins 3 and 3b, and N-Cadherin.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Gliossarcoma , Humanos , Glioblastoma/metabolismo , Gliossarcoma/genética , Gliossarcoma/metabolismo , Gliossarcoma/patologia , Neoplasias Encefálicas/metabolismo , Recidiva Local de Neoplasia/patologia , Glioma/patologia , Células-Tronco Neoplásicas/metabolismo , Receptores ErbB/metabolismo , Linhagem Celular TumoralRESUMO
Human skin-derived Nestin+ cells serve as a convenient source for autologous, adult, pluripotent progenitor cells that offer new therapeutic possibilities in cell-based regenerative medicine. However, the isolation of human Nestin+ cells has tended to be of very low efficiency and to produce highly variable cell yields. Here we report a standardised protocol that facilitates the isolation and enrichment of Nestin+ progenitor cells from enzymatically digested adult human scalp dermis. The use of distinct media like Dulbecco's modified Eagle medium supplemented with foetal bovine serum or, alternatively, serum-free, supplemented neural stem cell medium greatly affected cell morphology, proliferation and differentiation (e.g. towards a neural versus mesenchymal phenotype). Finally, Nestin+ cells were isolated from a heterogeneous dermis-derived progenitor cell population, which proliferates within clones or floating microspheres under defined serum-free culture conditions. Supplementation of the medium with epidermal growth factor and basic fibroblast growth factor as well as coating with fibronectin allowed the highest enrichment level of Nestin+ progenitors and differentiation towards neural fate. These methodological advances should greatly facilitate the isolation, culture and targeted differentiation of primary, adult human scalp skin dermis-derived Nestin+ cells.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Separação Celular/métodos , Derme/citologia , Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Actinas/metabolismo , Adulto , Células-Tronco Adultas/efeitos dos fármacos , Idoso , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Feminino , Fibronectinas/farmacologia , Expressão Gênica/genética , Humanos , Masculino , Mesoderma/citologia , Pessoa de Meia-Idade , Nestina , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neurônios/metabolismoRESUMO
Retinoic acid (RA) is indispensable for morphogenesis and differentiation of several tissues, including the nervous system. The requirement of the RA receptor (RAR) isotypes alpha, beta, and gamma and the putative role of retinoid X receptor-(RXR) signaling in RA-induced neural differentiation, was analyzed. For this compound-selective retinoids and the murine embryonal carcinoma cell line PCC7, a model system for RA-dependent neural differentiation was used. The present paper shows that proliferating PCC7 cells primarily express RXRalpha and RARalpha, lower levels of RXRbeta, and barely detectable amounts of RARbeta, RARgamma, and RXRgamma. At receptor-selective concentrations, only a RARalpha or RARgamma agonist induced the typical tissue-like differentiation pattern consisting of neuronal and nonneuronal cells. Differentiation-associated processes, such as the down-regulation of Oct4, up-regulation of certain nuclear receptors and proneuronal genes, and the induction of neuronal markers could be triggered by receptor-selective concentrations of a RARalpha-, beta-, or gamma-selective agonist, although with distinct efficacy. The differences are only partially explained by the distinct RARalpha, beta, and gamma expression levels and the dissociation constants for the bound retinoids, suggesting differential requirement of RAR isotypes during the initial stages of neural differentiation of PCC7 cells.
Assuntos
Receptores do Ácido Retinoico/química , Animais , Apoptose , Western Blotting , Morte Celular , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Imuno-Histoquímica , Cinética , Ligantes , Camundongos , Neurônios/metabolismo , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares , Fator 3 de Transcrição de Octâmero , Plasmídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas , RNA/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Tretinoína/metabolismo , Regulação para CimaRESUMO
The germ cell nuclear factor (GCNF) is essential for normal embryonic development and gametogenesis. To test the prediction that GCNF is additionally required for neuronal differentiation, we used the mouse embryonal carcinoma cell line PCC7-Mz1, which represents an advantageous model to study neuronal cells from the stage of fate choice until the acquirement of functional competence. We generated stable transfectants that express gcnf sense or antisense RNA under the control of a tetracycline-regulated promoter. After retinoic acid-induced withdrawal from the cell cycle, sense clones developed a neuron network with changed properties, and the time course of neuron maturation was shortened. Consistent with these data, differentiation of neuronal precursor cells was impaired in antisense cultures. This involved a delay in 1) the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells and stem cells of the central nervous system, and 2) up-regulation of the somatodendritic protein microtubule-associated protein 2 and the synaptic vesicle protein synaptophysin. Neuronal cells in the antisense cultures acquired functional competence, although with a significant delay. Our data propose that the level of GCNF is critical for differentiation and maturation of neuronal precursor cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neurônios/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/genética , Polaridade Celular , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Proteína GAP-43/análise , Proteína GAP-43/biossíntese , Proteína GAP-43/genética , Expressão Gênica , Proteínas de Filamentos Intermediários/análise , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/citologia , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares , Técnicas de Patch-Clamp , RNA Antissenso/genética , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/genética , Sinaptofisina/análise , Sinaptofisina/biossíntese , Sinaptofisina/genética , Tretinoína/farmacologia , Regulação para CimaRESUMO
BACKGROUND: The therapeutic resistance of gliomas is, at least in part, due to stemlike glioma cells (SLGCs), which self-renew, generate the bulk of tumor cells, and sustain tumor growth. SLGCs from glioblastomas (GB) have been studied in cell cultures or mouse models, whereas little is known about SLGCs from lower grade gliomas. OBJECTIVE: To compare cell and organotypic slice cultures from GBs and lower grade gliomas and study the maintenance of SLGCs. METHODS: Cells and tissue slices from astrocytomas, oligodendrogliomas, oligoastrocytomas, and GBs were cultivated in (1) serum-free medium supplemented with the growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), (2) medium containing 10% serum plus EGF and bFGF (F+GF medium), or (3) medium containing 10% fetal calf serum (F medium). Maintenance of cells and cytoarchitecture was addressed, using several candidate SLGC markers (Nestin, Sox2, CD133, CD44, CD49f/integrin α6, and Notch) as well as CD31 (endothelial cells), ionized calcium-binding adapter molecule 1 (microglia), and vimentin. Cell vitality was determined. RESULTS: SLGCs were present in tissue slices from lower and higher grade gliomas. Preservation of the cytoarchitecture in slices was possible for >3 weeks. Maintenance of SLGCs required the presence of EGF/bFGF in cell and slice cultures, in which F+GF appeared superior to N medium. Constraints were observed regarding the preservation of the microglia but not of the endothelial cells. Maintenance of the microglia was improved by addition of the cytokine macrophage colony-stimulating factor. CONCLUSION: Medium supplemented with serum and growth factors EGF, bFGF, and macrophage colony-stimulating factor permits the preservation of SLGCs and non-SLGCs in the original glioma microenvironment.
Assuntos
Astrocitoma/metabolismo , Astrocitoma/patologia , Microglia/metabolismo , Microglia/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Técnicas de Cultura de Células , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Microglia/citologia , Células-Tronco Neoplásicas/citologia , Nestina/metabolismo , Oligodendroglioma/metabolismo , Oligodendroglioma/patologia , Técnicas de Cultura de Órgãos , Fatores de Transcrição SOXB1/metabolismoRESUMO
Numerous tissue niches in the human body, such as skin, are now recognized to harbour adult stem cells. In this study, we analyze multipotent human dermis-derived progenitor cell populations, isolated and propagated from mechanically and enzymatically processed adult scalp skin. The populations encompass Nestin-positive and -negative cells, which may serve as a convenient and abundant source for various therapeutic applications in regenerative medicine. Here, we show that these cultures exhibit a strong tendency to differentiate into mesodermal derivatives, particularly myofibroblasts, when maintained in media containing serum. Since undesired and excessive myofibroblast formation is a frequent postsurgical complication, we sought culture conditions that would prevent myofibroblast formation. In particular, we analyzed the effect of growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor AB (PDGF AB). Our results demonstrate that bFGF is a potent inhibitor of mesodermal differentiation, whereas PDFG AB favours myofibroblast formation and up-regulates expression of TGFbeta receptors I and II. This interesting discovery may help in the prevention and treatment of tissue fibrosis and in particular in the eradication of hypertrophic and keloid scars.
Assuntos
Cicatriz/tratamento farmacológico , Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Hipertrofia/tratamento farmacológico , Queloide/tratamento farmacológico , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Derme/citologia , Derme/fisiologia , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Humanos , Imuno-Histoquímica , Fator de Crescimento Derivado de Plaquetas/fisiologia , Regeneração , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Células-Tronco/fisiologia , Fator de Crescimento Transformador beta/genéticaRESUMO
The germ cell nuclear factor (GCNF), which is also known as RTR (retinoid receptor-related testis-associated receptor) is a member of the nuclear receptor superfamily. As a natural ligand remains to be discovered, GCNF is referred to as an orphan receptor. Owing to GCNF's unique features and its distant relation to any other known nuclear receptor it has been classified as the only member of the subgroup six and designated NR6A1 by the Receptor Nomenclature Committee (Duarte et al., 2002: Nucleic Acids Res 30: 364-368). To date, GCNF has been cloned from distinct vertebrate species, including zebrafish, Xenopus laevis, mouse, rat, and human. Cloning and characterization of the gene, domain organization and DNA binding properties of the protein, as well as the differential expression of mRNA splice variants or the protein during development and in the adult animal have been comprehensively reviewed by others (Greschik and Schüle, 1998: J Mol Med 76:800-810; Cooney et al., 1999: Am Zool 39:796-806). In this minireview I focus on the pleiotropic function of GCNF in embryogenesis and germ cell differentiation, and discuss novel concepts about its putative role in neurogenesis.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Germinativas/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , VertebradosRESUMO
The ligand-activated retinoid receptors RXR and RAR control development, homeostasis and disease by regulating transcription of retinoic acid (RA) responsive target genes or crosstalk with other signalling pathways. According to the current model ligand-binding triggers an exchange between corepressor- and coactivator-complexes that inhibit or potentiate transcription by deacetylating and acetylating nucleosomal histones, respectively. Additional cofactors may modify the transcriptional regulatory process by linking liganded retinoid receptors to structural components of chromatin or protein degradation. The desire to specifically influence defined events in RA-signalling, while others are left unaffected, motivated the synthesis of retinoid X receptors (RXR)- and retinoid acid receptors (RAR) isoform-selective retinoids. The present study investigates the potential of RARalpha isotype-specific synthetic agonists and antagonists to separate the processes of coactivator recruitment and corepressor release. The synthetic retinoids studied fall into four categories, two of which work according the above model, since they induce surfaces within the RARalpha ligand binding domain (LBD) suitable for either corepressor or coactivator interaction; these retinoids act as pure antagonists and pure agonists, respectively. In contrast, another type of retinoid induces a structure that allows for both, the interaction with corepressors and coactivators (partial RARalpha agonist), and exerts a cell context-specific (ant)agonistic activity. Finally, another type of retinoid, which cannot activate transcription itself but renders heterodimeric RARalpha permissive for signaling by RXR agonists inhibits both, corepressor and coactivator interaction (partial antagonist). Moreover, this retinoid discriminates between the nuclear corepressors SMRT and NCoR, since it efficiently dissociates SMRT but not NCoR from the RARalpha LBD.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas Repressoras/metabolismo , Retinoides/farmacologia , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Correpressor 1 de Receptor Nuclear , Correpressor 2 de Receptor Nuclear , Isoformas de Proteínas , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptor alfa de Ácido Retinoico , Ativação Transcricional/fisiologiaRESUMO
Crystal structure and co-regulator interaction studies have led to a general mechanistic view of the initial steps of nuclear receptor (NR) action. Agonist-induced transconformation of the ligand-binding domain (holo-LBD) leads to the formation of co-activator complexes, and destabilizes the co-repressor complexes bound to the ligand-free (apo) LBD. However, the molecular basis of retinoid-X receptor (RXR) 'subordination' in heterodimers, an essential mechanism to avoid signalling pathway promiscuity, has remained elusive. RXR, in contrast to its heterodimer partner, cannot autonomously induce transcription on binding of cognate agonists. Here we show that RXR can bind ligand and recruit co-activators as a heterodimer with apo-retinoic-acid receptor (apo-RAR). However, in the usual cellular environment co-repressors do not dissociate and they prohibit co-activator access because co-regulator binding is mutually exclusive. Accordingly, RXR subordination can be overcome in heterodimers that bind co-repressor weakly or in cells with a high co-activator content. We identify two types of RAR antagonists that differentially modulate co-regulator interaction, and we demonstrate that synergy between RAR ligands and RXR agonists results from increased interaction efficiency of a single p160 with the heterodimer, requiring two intact receptor-binding surfaces on the co-activator.