RESUMO
BACKGROUND: Malfunction of astrocytes is implicated as one of the pathological factors of ALS. Thus, intrathecal injection of healthy astrocytes in ALS can potentially compensate for the diseased astrocytes. AstroRx® is an allogeneic cell-based product, composed of healthy and functional human astrocytes derived from embryonic stem cells. AstroRx® was shown to clear excessive glutamate, reduce oxidative stress, secrete various neuroprotective factors, and act as an immunomodulator. Intrathecal injection of AstroRx® to animal models of ALS slowed disease progression and extended survival. Here we report the result of a first-in-human clinical study evaluating intrathecal injection of AstroRx® in ALS patients. METHODS: We conducted a phase I/IIa, open-label, dose-escalating clinical trial to evaluate the safety, tolerability, and therapeutic effects of intrathecal injection of AstroRx® in patients with ALS. Five patients were injected intrathecally with a single dose of 100 × 106 AstroRx® cells and 5 patients with 250 × 106 cells (low and high dose, respectively). Safety and efficacy assessments were recorded for 3 months pre-treatment (run-in period) and 12 months post-treatment (follow-up period). RESULTS: A single administration of AstroRx® at either low or high doses was safe and well tolerated. No adverse events (AEs) related to AstroRx® itself were reported. Transient AEs related to the Intrathecal (IT) procedure were all mild to moderate. The study demonstrated a clinically meaningful effect that was maintained over the first 3 months after treatment, as measured by the pre-post slope change in ALSFRS-R. In the 100 × 106 AstroRx® arm, the ALSFRS-R rate of deterioration was attenuated from - 0.88/month pre-treatment to - 0.30/month in the first 3 months post-treatment (p = 0.039). In the 250 × 106 AstroRx® arm, the ALSFRS-R slope decreased from - 1.43/month to - 0.78/month (p = 0.0023). The effect was even more profound in a rapid progressor subgroup of 5 patients. No statistically significant change was measured in muscle strength using hand-held dynamometry and slow vital capacity continued to deteriorate during the study. CONCLUSIONS: Overall, these findings suggest that a single IT administration of AstroRx® to ALS patients at a dose of 100 × 106 or 250 × 106 cells is safe. A signal of beneficial clinical effect was observed for the first 3 months following cell injection. These results support further investigation of repeated intrathecal administrations of AstroRx®, e.g., every 3 months. TRIAL REGISTRATION: NCT03482050.
Assuntos
Esclerose Lateral Amiotrófica , Transplante de Células-Tronco Mesenquimais , Humanos , Esclerose Lateral Amiotrófica/terapia , Astrócitos , Injeções Espinhais , Transplante de Células-Tronco Mesenquimais/métodosRESUMO
Background: Cell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the ß-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells. Methods: Functional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice. Results: CD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A + cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26 + cells followed by enrichment for CD49A + cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26 - /CD49A + enriched ILC exhibited improved function over non-sorted ILC or CD49A + cells in diabetic mice and maintain prolonged blood C-peptide levels. Conclusions: Refining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Dipeptidil Peptidase 4/biossíntese , Integrina alfa1/biossíntese , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Peptídeo C/biossíntese , Diferenciação Celular , Separação Celular , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Endogâmicos C57BL , MicroesferasRESUMO
Cell therapies in animal models of neurobehavioral defects are normally derived from neural stem cells (NSC) of the developing cortex. However, the clinical feasibility of NSC therapies would be greatly improved by deriving transplanted cells and from a tissue culture source that is self-renewing, containing cells that potentially differentiate into the desired neuronal phenotypes. These cultures can be engineered to contain the appropriate factors to support their therapeutic action and likely evoke lesser immune reactions. In the current study, we employed our model of mice neurobehaviorally impaired via prenatal exposure to heroin, to test the therapeutic efficacy of NSC derived from murine embryonic stem cells culture (ESC). The culture contained elongated bipolar cells, 90% of which are positive for nestin, the intermediate filament protein found in neural precursors. After removal of growth factors, the NSC differentiated into neurons (34.0% +/- 3.8% NF-160 positive), including cholinergic cells (ChAT positive), oligodendrocytes (29.9% +/- 4.2% O(4)), and astrocytes (36.1% +/- 4.7% GFAP positive). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis confirmed the immunocytochemical findings. Mice made deficient in Morris maze behavior by prenatal heroin exposure (10 mg/kg heroin s.c. on gestational days 9-18) were transplanted into the hippocampus region on postnatal day 35 with the ES culture-derived NSC (ES-NSC) labeled with dialkylcarbocyanine (Dil) cell tracker. Dil+ and NF160+ cells were detected in the hippocampal region (50% +/- 8% survival). The transplantation completely restored maze performance to normal; e.g., on day 3, transplantation improved the behavior from the deficient level of 11.9-sec latency to the control of 5.6-sec latency (44.5% improvement).
Assuntos
Transtornos Cognitivos/cirurgia , Heroína/toxicidade , Entorpecentes/toxicidade , Neurônios/transplante , Efeitos Tardios da Exposição Pré-Natal/cirurgia , Transplante de Células-Tronco , Animais , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Transtornos Cognitivos/induzido quimicamente , Transtornos Cognitivos/fisiopatologia , Modelos Animais de Doenças , Células-Tronco Embrionárias/fisiologia , Feminino , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Neurogênese/fisiologia , Neurônios/fisiologia , Transtornos Relacionados ao Uso de Opioides/fisiopatologia , Transtornos Relacionados ao Uso de Opioides/cirurgia , Gravidez , Células-Tronco/fisiologiaRESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease characterized by the loss of MNs in the central nervous system. As MNs die, patients progressively lose their ability to control voluntary movements, become paralyzed and eventually die from respiratory/deglutition failure. Despite the selective MN death in ALS, there is growing evidence that malfunctional astrocytes play a crucial role in disease progression. Thus, transplantation of healthy astrocytes may compensate for the diseased astrocytes. METHODS: We developed a good manufacturing practice-grade protocol for generation of astrocytes from human embryonic stem cells (hESCs). The first stage of our protocol is derivation of astrocyte progenitor cells (APCs) from hESCs. These APCs can be expanded in large quantities and stored frozen as cell banks. Further differentiation of the APCs yields an enriched population of astrocytes with more than 90% GFAP expression (hES-AS). hES-AS were injected intrathecally into hSOD1G93A transgenic mice and rats to evaluate their therapeutic potential. The safety and biodistribution of hES-AS were evaluated in a 9-month study conducted in immunodeficient NSG mice under good laboratory practice conditions. RESULTS: In vitro, hES-AS possess the activities of functional healthy astrocytes, including glutamate uptake, promotion of axon outgrowth and protection of MNs from oxidative stress. A secretome analysis shows that these hES-AS also secrete several inhibitors of metalloproteases as well as a variety of neuroprotective factors (e.g. TIMP-1, TIMP-2, OPN, MIF and Midkine). Intrathecal injections of the hES-AS into transgenic hSOD1G93A mice and rats significantly delayed disease onset and improved motor performance compared to sham-injected animals. A safety study in immunodeficient mice showed that intrathecal transplantation of hES-AS is safe. Transplanted hES-AS attached to the meninges along the neuroaxis and survived for the entire duration of the study without formation of tumors or teratomas. Cell-injected mice gained similar body weight to the sham-injected group and did not exhibit clinical signs that could be related to the treatment. No differences from the vehicle control were observed in hematological parameters or blood chemistry. CONCLUSION: Our findings demonstrate the safety and potential therapeutic benefits of intrathecal injection of hES-AS for the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Astrócitos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Injeções Espinhais/métodos , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Ratos , Superóxido Dismutase-1/metabolismoRESUMO
Alongside their contribution to research, human embryonic stem cells (hESC) may also prove valuable for cell-based therapies. Traditionally, these cells have been grown in adhesion culture either with or without feeder cells, allowing for their continuous growth as undifferentiated cells. However, to be applicable in therapy and industry they must be produced in a scalable and controlled process. Here we present for the first time a suspension culture system for undifferentiated hESC and induced pluripotent stem cells (iPSC), based on medium supplemented with the IL6RIL6 chimera (interleukin-6 receptor fused to interleukin-6), and basic fibroblast growth factor. Four hESC lines cultured in this system maintained all ESC features after 20 passages, including stable karyotype and pluripotency. Similar results were obtained when hESC were replaced with iPSC from two different cell lines. We demonstrate that the IL6RIL6 chimera supports the self-renewal and expansion of undifferentiated hESC and iPSC in suspension, and thus present another efficient system for large-scale propagation of undifferentiated pluripotent cells for clinical and translational applications.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Apoptose/efeitos dos fármacos , Western Blotting , Diferenciação Celular/fisiologia , Citometria de Fluxo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Cariotipagem , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
The conditional knockdown of the Interleukin-6 (IL-6) family signal transducer (gp130) causes peripheral nerve demyelination and degeneration. In the present work, we investigated the effect of gp130 signaling on peripheral nerves and Schwann cells (SC). We stimulated gp130 signaling with IL6RIL6, a fusion molecule of IL-6 and IL-6R, in rat embryonic day 14 dorsal root ganglia (DRG) cell cultures. In neurons, IL6RIL6 strongly increased the axonal network. In SC, IL6RIL6 favored the appearance of elongated more mature cells versus stellar shaped cells. Gene expression profiling showed an increased expression of neuronal and glial-specific genes. mRNAs related to SC function, including myelin-specific genes, were increased by IL6RIL6 treatment of DRG cells, or of purified SCs isolated from rat sciatic nerve. In IL6RIL6-treated cells, immunostaining showed a strong nuclear signal for Krox-20, a transcription factor essential for differentiation of the SC lineage. On the contrary, we observed that IL6RIL6 inhibited the genes related to TGF-beta family as well as the production of smooth muscle actin.
Assuntos
Receptor gp130 de Citocina/metabolismo , Gânglios Espinais/embriologia , Interleucina-6/farmacologia , Bainha de Mielina/metabolismo , Neurônios/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Células de Schwann/metabolismo , Nervo Isquiático/embriologia , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Miócitos de Músculo Liso/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Interleucina-6 , Células de Schwann/efeitos dos fármacosRESUMO
In attempts to produce mature oligodendrocytes from human embryonic stem (huES) cells, we searched conditions inducing transcription factors Olig1/2, as well as Nkx2.2 and Sox10, which are needed for maturation. This was obtained by retinoic acid treatment followed by noggin, an antagonist of bone morphogenetic proteins (BMPs). We found that retinoic acid induces BMPs in huES cells. Addition of noggin at a specific step was essential to form numerous mature oligodendrocytes with ramified branches and producing myelin basic protein (MBP). We describe a procedure converting huES cells into enriched populations of oligodendrocyte precursors that can be expanded and passaged repeatedly and subsequently differentiated into mature cells. Transplantation of such precursors showed that pretreatment by noggin markedly stimulates their capacity to myelinate in the brain of MBP-deficient shiverer mice in organotypic cultures and in living animals. Arrays of numerous long MBP+ fibers were generated over extended areas in the brain, with evidence of cell migration after transplantation and with formation of compact myelin sheaths.
Assuntos
Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Proteína Básica da Mielina/metabolismo , Oligodendroglia/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Linhagem Celular , Doenças Desmielinizantes/cirurgia , Interações Medicamentosas , Células-Tronco Embrionárias/fisiologia , Feto , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos Mutantes Neurológicos , Microscopia Eletrônica de Transmissão/métodos , Proteína Básica da Mielina/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares , Oligodendroglia/ultraestrutura , Organogênese , Transplante de Células-Tronco/métodos , Fatores de Transcrição , Tretinoína/farmacologiaRESUMO
Neurosphere cells (NSc) derived from embryonic stem cells have characteristics of neural stem cells and can differentiate into oligodendrocyte precursors. Culture of NSc with IL6RIL6 chimera (soluble interleukin-6 receptor fused to interleukin-6) enhances their differentiation into oligodendrocytes with longer and more numerous branches and with peripheral accumulation of myelin basic protein (MBP) in myelin membranes indicating maturation. Gene expression profiling reveals that one of the proteins strongly induced by IL6RIL6 is a regulator of microtubule dynamics, stathmin-like 2 (SCG10/Stmn2), and gene silencing shows that Stmn2 plays an important role in the development of the mature oligodendrocyte morphology. IL6RIL6 acts as an effective stimulator of the myelinating function of ES cell-derived oligodendrocyte precursors, as observed upon transplantation of the IL6RIL6- pretreated cells into brain slices of MBP-deficient shiverer mice.
Assuntos
Diferenciação Celular/fisiologia , Interleucina-6/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Receptores de Interleucina-6/metabolismo , Células-Tronco/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Regulação para Baixo/fisiologia , Interleucina-6/genética , Interleucina-6/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , Proteína Básica da Mielina/efeitos dos fármacos , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Fatores de Crescimento Neural/efeitos dos fármacos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Oligodendroglia/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Receptores de Interleucina-6/genética , Proteínas Recombinantes de Fusão/farmacologia , Estatmina , Transplante de Células-Tronco/métodos , Células-Tronco/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos are a potential large scale source of oligodendrocytes and of their progenitors for transplantation into the central nervous system for the repair of demyelinating lesions. We found previously that interleukin-6 (IL-6) fused to its soluble receptor (IL-6R), a potent activator of the gp130 receptor, induces myelin gene expression in Schwann cells of embryonic dorsal root ganglia. Like leukemia inhibitory factor, IL-6R/IL-6 inhibits the differentiation of murine ES cells into embryoid bodies. In the present study, we show that this recombinant cytokine may be efficiently used to stimulate the differentiation of oligodendrocytes if added to ES cell-derived neural precursors. IL-6R/IL-6 leads to an increase in early chondroitin sulfate proteoglycan positive and late O4 positive progenitors and to a stimulation of maturation into O1 and myelin basic protein expressing oligodendrocytes. Expression of the genes for transcription factor genes Olig-1 and Sox10, which appear early in the oligodendrocyte lineage, was stimulated by IL-6R/IL-6 addition. We conclude that this cytokine can significantly enhance the derivation of oligodendrocytes from ES cells.
Assuntos
Diferenciação Celular , Linhagem da Célula , Interleucina-6/fisiologia , Oligodendroglia/citologia , Transdução de Sinais , Células-Tronco/citologia , Animais , Antígenos CD/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Receptor gp130 de Citocina , Proteínas de Ligação a DNA/metabolismo , Estruturas Embrionárias/citologia , Estruturas Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Grupo de Alta Mobilidade/metabolismo , Interleucina-6/farmacologia , Glicoproteínas de Membrana/fisiologia , Camundongos , Bainha de Mielina/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Receptores de Interleucina-6/metabolismo , Fatores de Transcrição SOXE , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células-Tronco/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
The microphthalmia-associated transcription factor (Mitf) is essential for melanocytic lineage development and for expression of melanogenic enzymes, such as tyrosinase. Interleukin-6 receptor/interleukin-6 chimera (IL6RIL6) induces in B16/F10.9 melanoma cells a loss of melanogenesis preceded by a sharp decrease in Mitf mRNA and gene promoter activity. In the Mitf promoter, the main cis-acting element mediating the IL6RIL6 effect is shown to be the binding site of Pax3, a paired homeodomain factor regulating among other things the development of melanocytes. Pax3 protein and mRNA levels decline steadily after IL6RIL6 treatment, and overexpression of an ectopic Pax3 cDNA suppresses the Mitf promoter inhibition. Loss of the synergism between Pax3 and Sox10, a high mobility group domain costimulatory factor, seems to be critical in the rapid decrease in Mitf gene expression. The Pax3 down-regulation in IL6RIL6-induced F10.9 cell is linked to growth arrest and transdifferentiation to a glial cell phenotype. IL6RIL6 stimulates the interleukin-6 family cytokine receptor gp130, leading to the rapid phosphorylation of Stat3 on tyrosine 705. This phosphorylation is required for Pax3 down-regulation and Mitf promoter silencing since these are inhibited in F10.9 cells overexpressing the Stat3 DN-mutant Y705F.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Melanócitos/citologia , Melanoma Experimental/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais , Fatores de Transcrição , Animais , Sequência de Bases , Linhagem da Célula , Primers do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Fenótipo , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Fatores de Transcrição SOXE , Fator de Transcrição STAT3 , Transativadores/química , Transativadores/metabolismo , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
Induction of myelin genes occurs around birth in the last stage of Schwann cells differentiation and is reactivated in case of nerve injury. Previous studies showed that activation of the gp130 receptor system, using as ligand interleukin-6 fused to its soluble receptor (IL6RIL6), causes induction of myelin genes such as myelin basic protein (MBP) and myelin protein zero (Po) in embryonic dorsal root ganglia Schwann cells. We also reported that in murine melanoma B16/F10.9 cells, IL6RIL6 causes a shut-off of melanogenesis mediated by a down-regulation of the paired-homeodomain factor Pax3. The present work demonstrates that these IL6RIL6-treated F10.9 cells undergo transdifferentiation to a myelinating glial phenotype characterized by induction of the transcriptional activities of both Po and MBP promoters and accumulation of myelin gene products. For both Po and MBP promoters, a repression by Pax3 and stimulation by Sox10 can be demonstrated. Because after IL6RIL6-treatment, Pax3 disappears from the F10.9 cells (as it does in mature myelinating Schwann cells) whereas the level of Sox10 rather increases, we modulated the relative level of these factors and show their involvement in the induction of myelin gene expression by IL6RIL6. In addition, however, we show that a C/G-rich CACC box in the Po promoter is required for activation by IL6RIL6, as well as by ectopic Sox10, and identify a Kruppel-type zinc finger factor acting through this CACC box, which stimulates Po promoter activity.
Assuntos
Melanoma/metabolismo , Proteína Básica da Mielina/metabolismo , Proteína P0 da Mielina/metabolismo , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Neuroglia/metabolismo , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Diferenciação Celular , Divisão Celular , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Doxiciclina/farmacologia , Proteína 2 de Resposta de Crescimento Precoce , Genes Reporter , Vetores Genéticos , Proteínas de Grupo de Alta Mobilidade/metabolismo , Interleucina-6/metabolismo , Melanoma/patologia , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteína Básica da Mielina/genética , Proteína P0 da Mielina/genética , Neuroglia/patologia , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXE , Fatores de Tempo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-HíbridoRESUMO
Expression of genes encoding structural myelin proteins marks the inception of the myelinating Schwann cell (SC) phenotype. Earlier embryonic SC as well as adult non-myelinating SC produce the intermediate filament glial fibrillary acid protein (GFAP), which disappears from the myelinating SC. We previously observed that triggering of the gp130 receptor system by the IL6RIL6 ligand, comprising interleukin-6 (IL-6) fused to the soluble IL-6 receptor, induces myelin gene expression in rat embryonic dorsal root ganglia (DRG) cultures as well as in the murine melanoma cell line B16/F10.9. Study of target genes regulated by IL6RIL6 indicates a strong and selective induction of the transcriptional regulator C/EBP-delta in DRG cultures and in the F10.9 cell line. As shown here, silencing of C/EBP-delta mRNA and protein expression by introduction of small interference RNA-producing plasmids in the F10.9 cells prevented the induction of myelin protein zero (P0) and myelin basic protein (MBP) mRNAs by IL6RIL6. Doxycycline-regulated overexpression of C/EBP-delta was sufficient to induce accumulation of P0 and MBP mRNAs, the effect being selective, because C/EBP-delta did not affect several other genes strongly regulated by IL6RIL6. Interestingly, GFAP was inhibited by C/EBP-delta overexpression, leading to a modulation of the ratio between myelin gene products versus GFAP and suggesting that C/EBP-delta plays a role in the switch to a myelinating phenotype. The down-regulation of Pax3, also typical of the transition to myelinating cells, was observed after C/EBP-delta expression in correlation to P0 induction and to decrease of melanogenesis and cell growth. In cultures of dissociated cells of embryonic rat DRG, where we knocked-down the C/EBP-delta mRNA, we found an inhibition of P0 mRNA induction by IL6RIL6, showing that the role of C/EBP-delta on this myelin gene is not unique to the melanoma system.
Assuntos
Antígenos CD/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Bainha de Mielina/metabolismo , Transdução de Sinais , Animais , Antibacterianos/farmacologia , Western Blotting , Proteína delta de Ligação ao Facilitador CCAAT , Divisão Celular , Linhagem Celular Tumoral , Receptor gp130 de Citocina , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Doxiciclina/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Interleucina-6/metabolismo , Ligantes , Camundongos , Fator de Transcrição Associado à Microftalmia , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Fenótipo , Ligação Proteica , RNA Complementar/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraciclina/farmacologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
A novel recombinant molecule, termed IL-6c and consisting of a chimera of interleukin 6 (IL-6) and its soluble receptor is extremely potent in stimulating proliferation of hematopoietic progenitors. We investigated the effect of the IL-6c on the proliferation and differentiation of E14 fetal hepatocytes. IL-6c, in a dose-dependent manner, stimulated proliferation of E14 fetal rat hepatocytes. Adult hepatocyte mitogens together with IL-6c showed no further effect on proliferation. Hematopoietic stem cells mitogens SCF and flt3 ligand (FL) were also mitogenic for fetal hepatocytes, but did not further enhance the effect of IL-6c on cell proliferation. IL-6c decreased expression of fetal markers alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase, and induced expression of adult enzyme glucose-6-phosphatase (Gluc-6-P) in E14 hepatocytes. On the other hand, IL-6c strongly reduced, in a dose-dependant manner, expression of albumin and tyrosine aminotransferase (TAT). However, when the cells were grown for 3 days with IL-6c, and IL-6c was removed for the next 5 days, expression of albumin and TAT returned to levels found in control cultures. In conclusion, IL-6c stimulated proliferation and affected gene expression in fetal hepatocytes in culture.