RESUMO
Ras signaling is tightly regulated during neural stem cell (NSC) differentiation, and defects in this pathway result in aberrant brain development. However, the mechanism regulating Ras signaling during NSC differentiation was unknown. Here, we show that stabilized HRas specifically induces neuronal differentiation of NSCs. Lentivirus-mediated HRas overexpression and knockdown resulted in stimulation and inhibition, respectively, of NSC differentiation into neuron in the ex vivo embryo. Retinoic acid, an active metabolite of vitamin A, promoted neuronal differentiation of NSCs by stabilizing HRas, and HRas knockdown blocked the retinoic acid effect. Vitamin-A-deficient mice displayed abnormal brain development with reduced HRas levels and a reduced thickness of the postmitotic region containing differentiated neurons. All of these abnormal phenotypes were rescued with the restoration of HRas protein levels achieved upon feeding with a retinoic-acid-supplemented diet. In summary, this study shows that retinoic acid stabilizes HRas protein during neurogenesis, and that this is required for NSC differentiation into neurons and murine brain development.
Assuntos
Encéfalo/embriologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Neurais/citologia , Neurônios/citologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tretinoína/farmacologia , Animais , Diferenciação Celular/genética , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Deficiência de Vitamina A/patologiaRESUMO
CXXC5 is a member of a small subset of proteins containing CXXC-type zinc-finger domain. Here, we show that CXXC5 is a transcription factor activating Flk-1, a receptor for vascular endothelial growth factor. CXXC5 and Flk-1 were accumulated in nucli and membrane of mouse embryonic stem cells (mESCs), respectively, during their endothelial differentiation. CXXC5 overexpression induced Flk-1 transcription in both endothelium-differentiated mESCs and human umbilical vein endothelial cells (HUVECs). In vitro DNA binding assay showed direct interaction of CXXC5 on the Flk-1 promoter region, and mutation on its DNA-binding motif abolished transcriptional activity. We showed that bone morphorgenetic protein 4 (BMP4) induced CXXC5 transcription in the cells, and inhibitors of BMP signaling suppressed the CXXC5 induction and the consequent Flk-1 induction by BMP4 treatment. CXXC5 knockdown resulted in suppression of BMP4-induced stress fiber formation (56.8 ± 1.3% decrease, P<0.05) and migration (54.6 ± 1.9% decrease, P<0.05) in HUVECs. The in vivo roles of CXXC5 in BMP-signaling-specific vascular development and angiogenesis were shown by specific defect of caudal vein plex vessel formation (57.9 ± 11.8% decrease, P<0.05) in cxxc5 morpholino-injected zebrafish embryos and by suppression of BMP4-induced angiogenesis in subcutaneously injected Matrigel plugs in CXXC5(-/-) mice. Overall, CXXC5 is a transcriptional activator for Flk-1, mediating BMP signaling for differentiation and migration of endothelial cell and vessel formation.
Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/metabolismo , Indutores da Angiogênese/farmacologia , Animais , Proteínas de Transporte/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proteínas de Ligação a DNA , Humanos , Camundongos , Fatores de Transcrição , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/genéticaRESUMO
For the purpose of development of orally active peptide therapeutics targeting NFκB for treatment of inflammatory bowel disease (IBD), two major barriers in oral delivery of therapeutic peptides, metabolic lability and tissue impermeability, were circumvented by introduction of a colon-targeted delivery system and cell permeable peptides (CPP) to NFκB inhibitory peptides (NIP). Suppression of NFκB activation was compared following treatment with various CPP conjugated NIPs (CPP-NIP). The most potent CPP-NIP was loaded in a capsule coated with a colon specific polymer, which was administered orally to colitic rats. The anti-inflammatory activity of the colon-targeted CPP-NIP was evaluated by measuring inflammatory indices in the inflamed colonic tissue. For confirmation of the local action of the CPP-NIP, the same experiment was done after rectal administration. Tissue permeability of the CPP-NIP was examined microscopically and spectrophotometrically using FITC-labeled CPP-NIP (CPP-NIP-FITC). NEMO binding domain peptide (NBD, TALDWSWLQTE) fused with a cell permeable peptide CTP (YGRRARRRARR), CTP-NBD, was most potent in inhibiting NFκB activity in cells. Colon-targeted CTP-NBD, but not colon-targeted NBD and CTP-NBD in an enteric capsule, ameliorated the colonic injury, which was in parallel with decrease in MPO activity and the levels of inflammatory mediators. Intracolonic treatment with CTP-NBD alleviated rat colitis and improved all the inflammatory indicators. CTP-NBD-FITC was detected at much greater level in the inflamed tissue than was NBD-FITC. Taken together, introduction of cell permeability and colon targetability to NIP may be a feasible strategy for an orally active peptide therapy for treatment of IBD.
Assuntos
Peptídeos Penetradores de Células/química , Colo/metabolismo , Síndrome do Intestino Irritável/tratamento farmacológico , Síndrome do Intestino Irritável/metabolismo , NF-kappa B/antagonistas & inibidores , Peptídeos/química , Peptídeos/uso terapêutico , Administração Oral , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Western Blotting , Células CACO-2 , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Síndrome do Intestino Irritável/induzido quimicamente , Masculino , Ratos , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
This study was conducted to determine the functional role of phospholipase D (PLD) involved in testicular Leydig cell damage caused by di (2-ethylhexyl) phthalate (DEHP) in Sprague-Dawley rats. DEHP (500 mg/kg/day) was administered orally to prepubertal rats for 1, 7, 14, 21 or 28 days. After 7 days of exposure, DEHP produced morphological changes in the testis, including alterations in seminiferous tubule diameters and loss of spermatogenic cells. Immunohistochemistry (IHC) analyses revealed that DEHP increased Leydig cell number in the testes as well as significantly increased the expression of PLD1/2 in Leydig cells after 7 days of exposure. Furthermore, the protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) increased in a similar manner to the PLD1/2 expression patterns. DEHP significantly reduced the expression of sperm-associated antigen 4 (Spag4) and lactate dehydrogenase A (LDHA) mRNA. In contrast, there was a significant increase in the expression of steroidogenic acute regulatory (StAR) mRNA against DEHP in a time-dependent manner, but serum testosterone concentration was decreased. These findings demonstrate that DEHP induces PLD expression in the testicular Leydig cells; this plays a key role in hyperplasia of Leydig cells and steroidogenic pathway via pERK1/2 activation.
Assuntos
Dietilexilftalato/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Fosfolipase D/efeitos dos fármacos , Testículo/efeitos dos fármacos , Administração Oral , Animais , Dietilexilftalato/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperplasia , Células Intersticiais do Testículo/patologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo , Fosforilação/efeitos dos fármacos , Plastificantes/administração & dosagem , Plastificantes/toxicidade , Ratos , Ratos Sprague-Dawley , Testículo/patologia , Fatores de TempoRESUMO
Since detergent-resistant lipid rafts play important roles in the signal transduction for myogenesis, their comprehensive proteomic analysis could provide new insights to understand their function in myotubes. Here, the detergent-resistant lipid rafts were isolated from C2C12 myotubes and analyzed by capillary RPLC/MS/MS. Among the 327 proteins (or protein groups) identified, 28% were categorized to the plasma membrane or raft proteins, 29% to mitochondria, 20% to microsomal proteins, 10% to other proteins, and 13% to unknown proteins. The localization of oxidative phosphorylation (OXPHOS) complexes in the sarcolemma lipid rafts was further confirmed from C2C12 myotubes by cellular fractionation, surface-biotin labeling, immunofluorescence, and lipid raft fractionation. After adding exogenous cytochrome c, the sarcolemma isolated from myotubes had an ability to consume oxygen in the presence of NADH or succinate. The generation of NADH-dependent extracellular superoxide was increased by inhibiting or downregulating OXPHOS I, III, and IV in myotubes, indicating that OXPHOS proteins are major sources for extracellular ROS in skeletal muscle. With all these data, we can conclude that OXPHOS proteins are associated with the sarcolemma lipid rafts during C2C12 myogenesis to generate extracellular ROS.
Assuntos
Detergentes/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Mitocôndrias/metabolismo , Desenvolvimento Muscular , Oxigênio/metabolismo , Animais , Linhagem Celular , Camundongos , Fosforilação , ProteômicaRESUMO
Bioactive scaffolds inducing cell adhesion, differentiation have been premise for optimal formation of target tissue. Collagen has been employed as a tissue regenerative scaffold especially for bone regeneration and has been chemically surface-modified to present bioactivity. Herein, we show that peptide, denoted as collagen-binding motif (CBM, GLRSKSKKFRRPDIQYPDATDEDITSHM) identified from osteopontin (OPN) protein, was able to specifically bind collagen without chemical conjugation, while presenting apatite forming capability in vitro and in vivo. Collagen surface alone was not able to induce noticeable apatite nucleation however, mineralization was evident when assembled with CBM peptide, implying that the collagen-CBM assembly played a pivotal role in biomineralization. In vivo result further demonstrated that the CBM peptide in complex with material was able to induce bone formation by helping mineralization in the bone defect. Taken together, the CBM peptide herein and its assembly with collagen can be applied as an inducer of biomineralization as well as a bioactive scaffold for bone regeneration.
Assuntos
Substitutos Ósseos/química , Colágeno/química , Transplante de Células-Tronco Mesenquimais/métodos , Osteogênese , Osteopontina/química , Fraturas Cranianas/fisiopatologia , Fraturas Cranianas/cirurgia , Animais , Materiais Biocompatíveis/química , Masculino , Coelhos , Fraturas Cranianas/patologia , Resultado do TratamentoRESUMO
Fibroblast growth factor (FGF)-2 regulates a variety of cellular functions, such as proliferation and differentiation, by binding to cell surface FGF receptors (FGFRs) in the presence of heparin proteoglycans. FGF-2 is known as a heparin-binding growth factor, but the localization of the heparin binding site has not been fully investigated until now. We used two potential heparin binding domains of FGF-2, the residues 105-111 (F105, YKRSRYT) and 119-135 (F119, KRTGQYKLGSKTGPGQK). Peptides could be stably immobilized onto the surface of tissue culture plates. Using solid phase binding assays, we demonstrated that both peptides had higher binding affinity toward heparin compared with nonbinding control sequence. The biological significance of these sites was tested by cell attachment and osteoblast differentiation studies. Cell attachment to the peptides F105 and F119 increased in a dose-dependent manner. Heparin and heparinase treatments decreased cell adhesion to both F105 and F119. This demonstrates that both F105 and F119 interact with cell-surface heparan sulfate proteoglycans, suggesting that FGF-2 has two heparin binding sites. In addition, osteoblast differentiation, confirmed by ALPase activity and mineralization, was increased by surface immobilized peptide F105 and F119. Taken together, these heparin binding peptides could be applied as biological agents enhancing osteoblast differentiation as well as surface modification tools in the tissue regeneration area, especially for bone regeneration.
Assuntos
Diferenciação Celular , Fator 2 de Crescimento de Fibroblastos/química , Heparina/metabolismo , Osteoblastos/citologia , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
The effects of fluoxetine were studied on cloned K+ channel Kv1.4 stably expressed in Chinese hamster ovary (CHO) cells using the whole-cell configuration of the patch-clamp technique. Extracellular application of various concentrations of fluoxetine inhibited the amplitude of the peak current of Kv1.4 and accelerated its inactivation time course in a concentration-dependent manner. Thus, fluoxetine decreased Kv1.4 (the integral of the outward current) in a concentration-dependent manner; the IC50 was 33.1 +/- 2.5 microM. The inhibitory effect of fluoxetine was time-dependent. The apparent association (k) and dissociation (l) rate constants measured at +40 mV were 3.5 +/- 0.7 microM-1s-1 and 132.5 +/- 13.3 s-1, respectively. The Kd (= l/k) was 37.9 microM, which was close to the value obtained from the concentration-response curve. The block produced by fluoxetine increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. The fluoxetine block was constant at more depolarized potentials, suggesting that the block by fluoxetine was not voltage dependent. Our data indicate that fluoxetine blocks Kv1.4 channels by preferentially binding to open state.
Assuntos
Clonagem Molecular , Fluoxetina/farmacologia , Neurônios/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Animais , Células CHO , Clonagem Molecular/efeitos dos fármacos , Clonagem Molecular/métodos , Cricetinae , Relação Dose-Resposta a Droga , Canal de Potássio Kv1.4 , Neurônios/fisiologia , Canais de Potássio/genética , RatosRESUMO
Osteoclastogenesis plays an important role in joint destruction in rheumatoid arthritis (RA). IL-15 is a pleiotropic proinflammatory cytokine that appears to help mediate the pathological bone loss. This study was undertaken to explore the signaling molecules essential for osteoclastogenesis mediated by IL-15 in rheumatoid synovial fibroblasts. Expression of phospholipase D1 (PLD1) and osteoclast-related gene expression in synovial tissues and their modulation by treatment with IL-15 and different inhibitors in synovial fibroblasts of RA patients were evaluated using immunohistochemistry and quantitative polymerase chain reaction. The levels of IL-15 in serum and synovial fluid were measured by ELISA. The effects of IL-15 and phosphatidic acid (PA) on osteoclast formation were evaluated in cocultures of rheumatoid synovial fibroblasts and peripheral blood monocytes or monocytes alone in the presence of M-CSF and RANKL. The levels of RANKL and PLD1 but not PLD2 were upregulated significantly by IL-15, and the RANKL level was significantly upregulated by PA in rheumatoid synovial fibroblasts. Blocking PA production with 1-butanol and siRNA against PLD1 significantly inhibited the IL-15-stimulated expression of RANKL and PLD1. IL-15 levels were significantly higher in serum and synovial fluid from patients with RA than in osteoarthritis patients and healthy controls. IL-15 and PA induced osteoclast formation through the mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathways. Activation of PLD1 contributes to IL-15-mediated osteoclastogenesis via the MAPKs and NF-κB signaling pathways in rheumatoid synovial fibroblasts. Our data suggest that PLD1 might be an efficient therapeutic strategy for preventing bone destruction in rheumatoid arthritis.
Assuntos
Artrite Reumatoide/metabolismo , Interleucina-15/metabolismo , Osteoclastos/metabolismo , Fosfolipase D/metabolismo , Transdução de Sinais , Artrite Reumatoide/sangue , Artrite Reumatoide/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-15/sangue , Interleucina-15/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Ácidos Fosfatídicos/farmacologia , Fosfolipase D/genética , Ligante RANK/genética , Ligante RANK/metabolismo , Transdução de Sinais/genética , Líquido Sinovial/química , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
HERV-M (human endogenous retrovirus M), related to the super family of HERV-K, has a methionine (M) tRNA primer-binding site, and is located within the periphilin gene on human chromosome 12q12. HERV-M has been integrated into the periphilin gene as the truncated form, 5'LTR-gag-pol-3'LTR. Polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) approaches were conducted to investigate its evolutionary origins. Interestingly, the insertion of retroelements in a common ancestor genome can make different transcript variants in different species. In the case of the periphilin gene, human (10 variants) and mouse (2 variants) lineages show different transcript variants. Insertion of HERV-M (variant 1-3) could affect the protein-coding region. Also, Alusq/x (variant 4-9) and L1ME4a (mammalian-wide subfamilies of LINE-1) (variant 10) in humans and SINE (short interspersed repetitive element) and RLTR15 (the mouse putative long terminal repeat) (variant 2) in mice could be driving forces in transcript diversification of the periphilin gene during mammalian evolution. The HERV-M derived transcripts (variant 1-3) were expressed in different human tissues, whereas they were not detected in crab-eating monkey and squirrel monkey tissues by RT-PCR amplification. Taken together, HERV-M seems to have been integrated into our common ancestor genome after the divergence of simians and prosimians, and then was actively expressed during hominoid evolution.