RESUMO
BACKGROUND: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. HYPOTHESIS/PURPOSE: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. METHODS: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. RESULTS: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. CONCLUSIONS: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proteínas Inibidoras de Diferenciação , Neoplasias Pulmonares , Proteínas de Neoplasias , Silibina , Silibina/farmacologia , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Humanos , Animais , Linhagem Celular Tumoral , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Nus , Receptores de Ativinas Tipo I/metabolismo , Receptores de Ativinas Tipo I/genética , Silimarina/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Morfogenética Óssea 6 , Silybum marianum/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , FemininoRESUMO
Bone morphogenetic proteins (BMPs) were first described over 50 years ago as potent inducers of ectopic bone formation when administrated subcutaneously. Preclinical studies have extensively examined the osteoinductive properties of BMPs in vitro and new bone formation in vivo. BMPs (BMP-2, BMP-7) have been used in orthopedics over 15 years. While osteogenic function of BMPs has been widely accepted, our previous studies demonstrated that loss-of-function of BMP receptor type IA (BMPR1A), a potent receptor for BMP-2, increased net bone mass by significantly inhibiting bone resorption in mice, indicating a positive role of BMP signaling in bone resorption. The physiological role of BMPs (i.e. osteogenic vs. osteoclastogenic) is still largely unknown. The purpose of this study was to investigate the physiological role of BMP signaling in endogenous long bones during adult stages. For this purpose, we conditionally and constitutively activated the Smad-dependent canonical BMP signaling thorough BMPR1A in osteoblast lineage cells using the mutant mice (Col1CreER™:caBmpr1a). Because trabecular bones were largely increased in the loss-of-function mouse study for BMPR1A, we hypothesized that the augmented BMP signaling would affect endogenous trabecular bones. In the mutant bones, the Smad phosphorylation was enhanced within physiological level three-fold while the resulting gross morphology, bodyweights, bone mass/shape/length, serum calcium/phosphorus levels, collagen cross-link patterns, and healing capability were all unchanged. Interestingly, we found; 1) increased expressions of both bone formation and resorption markers in femoral bones, 2) increased osteoblast and osteoclast numbers together with dynamic bone formation parameters by trabecular bone histomorphometry, 3) modest bone architectural phenotype with reduced bone quality (i.e. reduced trabecular bone connectivity, larger diametric size but reduced cortical bone thickness, and reduced bone mechanical strength), and 4) increased expression of SOST, a downstream target of the Smad-dependent BMPR1A signaling, in the mutant bones. This study is clinically insightful because gain-of-function of BMP signaling within a physiological window does not increase bone mass while it alters molecular and cellular aspects of osteoblast and osteoclast functions as predicted. These findings help explain the high-doses of BMPs (i.e. pharmacological level) in clinical settings required to substantially induce a bone formation, concurrent with potential unexpected side effects (i.e. bone resorption, inflammation) presumably due to a broader population of cell-types exposed to the high-dose BMPs rather than osteoblastic lineage cells.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Osteogênese , Transdução de Sinais , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas , Camundongos , Osteoblastos/metabolismoRESUMO
Background/aim: Drynaria fortunei (Gusuibu; GSB) is a popular traditional Chinese medicine used for bone repair. An increasing number of studies have reported that GSB induces osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). These results provide insight into the application of GSB for bone tissue engineering techniques used to repair large bone defects. However, few studies have described the molecular mechanisms of GSB. Materials and methods: In the present study, the effects of GSB and naringin, a marker compound, on the binding of BMP-2 to BMPR and BMP-2-derived signal transduction were investigated using surface plasmon resonance (SPR) and coculturing with BMPR- expressed cell line, C2C12, respectively. Furthermore, naringin was also used to prepare naringin contained scaffolds for bone tissue engineering. The physical and chemical properties of these scaffolds were analysed using scanning electron microscopy (SEM) and highperformance liquid chromatography (HPLC). These scaffolds were cocultured with rabbit BMSCs in vitro and implanted into rabbit calvarial defects for bone repair assessment. Results: The results showed that GSB and naringin affect the binding of BMP and BMPR in SPR experiments. GSB is a subtle BMP modulator that simultaneously inhibits the binding of BMP-2 to BMPR-1A and enhances its binding to BMPR-1B. In contrast, naringin inhibited BMP-2 binding to BMPR-1A. In vitro studies involving the phosphorylation of signals downstream of BMPR and Smad showed that GSB and naringin affected stem cell differentiation by inhibiting BMPR-1A signalling. When using GSB for bone tissue engineering, naringin exhibited a higher capacity for slow and gradual release from the scaffold, which promotes bone formation via osteoinduction. Moreover, control and naringin scaffolds were implanted into rabbit calvarial defects for 4 weeks, and naringin enhanced bone regeneration in vivo significantly. Conclusions: GSB and its marker compound (naringin) could inhibit the binding of BMP-2 and BMPR-1A to control cell differentiation by blocked BMPR-1A signalling and enhanced BMPR-1B signalling. GSB and naringin could be good natural BMP regulators for bone tissue engineering.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Flavanonas/farmacologia , Polypodiaceae/química , Engenharia Tecidual/métodos , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Células Cultivadas , Masculino , Osteogênese/efeitos dos fármacos , Coelhos , Transdução de Sinais/efeitos dos fármacosRESUMO
Progression of chronic kidney disease associated with progressive fibrosis and impaired tubular epithelial regeneration is still an unmet biomedical challenge because, once chronic lesions have manifested, no effective therapies are available as of yet for clinical use. Prompted by various studies across multiple organs demonstrating that preconditioning regimens to induce endogenous regenerative mechanisms protect various organs from later incurring acute injuries, we here aimed to gain insights into the molecular mechanisms underlying successful protection and to explore whether such pathways could be utilized to inhibit progression of chronic organ injury. We identified a protective mechanism controlled by the transcription factor ARNT that effectively inhibits progression of chronic kidney injury by transcriptional induction of ALK3, the principal mediator of antifibrotic and proregenerative bone morphogenetic protein-signaling (BMP-signaling) responses. We further report that ARNT expression itself is controlled by the FKBP12/YY1 transcriptional repressor complex and that disruption of such FKBP12/YY1 complexes by picomolar FK506 at subimmunosuppressive doses increases ARNT expression, subsequently leading to homodimeric ARNT-induced ALK3 transcription. Direct targeting of FKBP12/YY1 with in vivo morpholino approaches or small molecule inhibitors, including GPI-1046, was equally effective for inducing ARNT expression, with subsequent activation of ALK3-dependent canonical BMP-signaling responses and attenuated chronic organ failure in models of chronic kidney disease, and also cardiac and liver injuries. In summary, we report an organ-protective mechanism that can be pharmacologically modulated by immunophilin ligands FK506 and GPI-1046 or therapeutically targeted by in vivo morpholino approaches.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/biossíntese , Falência Renal Crônica/tratamento farmacológico , Falência Renal Crônica/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Linhagem Celular , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Falência Renal Crônica/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pirrolidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tacrolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/antagonistas & inibidores , Proteína 1A de Ligação a Tacrolimo/metabolismo , Fator de Transcrição YY1/antagonistas & inibidores , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
Heterotopic ossification (HO), the pathological extraskeletal formation of bone, can arise from blast injuries, severe burns, orthopedic procedures and gain-of-function mutations in a component of the bone morphogenetic protein (BMP) signaling pathway, the ACVR1/ALK2 receptor serine-threonine (protein) kinase, causative of Fibrodysplasia Ossificans Progressiva (FOP). All three ALKs (-2, -3, -6) that play roles in bone morphogenesis contribute to trauma-induced HO, hence are well-validated pharmacological targets. That said, development of inhibitors, typically competitors of ATP binding, is inherently difficult due to the conserved nature of the active site of the 500+ human protein kinases. Since these enzymes are regulated via inherent plasticity, pharmacological chaperone-like drugs binding to another (allosteric) site could hypothetically modulate kinase conformation and activity. To test for such a mechanism, a surface pocket of ALK2 kinase formed largely by a key allosteric substructure was targeted by supercomputer docking of drug-like compounds from a virtual library. Subsequently, the effects of docked hits were further screened in vitro with purified recombinant kinase protein. A family of compounds with terminal hydrogen-bonding acceptor groups was identified that significantly destabilized the protein, inhibiting activity. Destabilization was pH-dependent, putatively mediated by ionization of a histidine within the allosteric substructure with decreasing pH. In vivo, nonnative proteins are degraded by proteolysis in the proteasome complex, or cellular trashcan, allowing for the emergence of therapeutics that inhibit through degradation of over-active proteins implicated in the pathology of diseases and disorders. Because HO is triggered by soft-tissue trauma and ensuing hypoxia, dependency of ALK destabilization on hypoxic pH imparts selective efficacy on the allosteric inhibitors, providing potential for safe prophylactic use.
Assuntos
Receptores de Ativinas/metabolismo , Hipóxia/metabolismo , Ossificação Heterotópica/tratamento farmacológico , Ossificação Heterotópica/prevenção & controle , Receptores de Ativinas/química , Regulação Alostérica , Animais , Sítios de Ligação , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Humanos , Concentração de Íons de Hidrogênio , Fosforilação , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Proteína 1A de Ligação a Tacrolimo/metabolismo , TemperaturaRESUMO
The regulation of energy balance involves complex processes in the brain, including coordination by hypothalamic neurons that contain pro-opiomelanocortin (POMC). We previously demonstrated that central bone morphogenetic protein (BMP) 7 reduced appetite. Now we show that a type 1 BMP receptor, BMPR1A, is colocalized with POMC neurons and that POMC-BMPR1A-knockout (KO) mice are hyperphagic, revealing physiological involvement of BMP signaling in anorectic POMC neurons in the regulation of appetite. Surprisingly, the hyperphagic POMC-BMPR1A-KO mice exhibited a lack of obesity, even on a 45% high-fat diet. This is because the brown adipose tissue (BAT) of KO animals exhibited increased sympathetic activation and greater thermogenic capacity owing to a reestablishment of energy balance, most likely stemming from a compensatory increase of BMPR1A in the whole hypothalamus of KO mice. Indeed, control animals given central BMP7 displayed increased energy expenditure and a specific increase in sympathetic nerve activity (SNA) in BAT. In these animals, pharmacological blockade of BMPR1A-SMAD signaling blunted the ability of BMP7 to increase energy expenditure or BAT SNA. Together, we demonstrated an important role for hypothalamic BMP signaling in the regulation of energy balance, including BMPR1A-mediated appetite regulation in POMC neurons as well as hypothalamic BMP-SMAD regulation of the sympathetic drive to BAT for thermogenesis.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Metabolismo Energético , Neurônios/metabolismo , Pró-Opiomelanocortina/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Regulação do Apetite/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Dieta Hiperlipídica , Hipotálamo/metabolismo , Masculino , Camundongos Knockout , Camundongos Transgênicos , Obesidade/genética , Obesidade/metabolismo , Pró-Opiomelanocortina/genética , Termogênese/genéticaRESUMO
Galectin-3 (Gal-3) has been implicated in pancreatic ductal adenocarcinoma (PDAC), and its candidacy as a therapeutic target has been evaluated. Gal-3 is widely upregulated in tumors, and its expression is associated with the development and malignancy of PDAC. In the present study, we demonstrate that a polysaccharide, RN1, purified from the flower of Panax notoginseng binds to Gal-3 and suppresses its expression. In addition, RN1 markedly inhibits PDAC cells growth in vitro, in vivo and in patient-derived xenografts. Mechanistically, RN1 binds to epidermal growth factor receptor (EGFR) and Gal-3, thereby disrupting the interaction between Gal-3 and EGFR and downregulating extracellular-related kinase (ERK) phosphorylation and the transcription factor of Gal-3, Runx1 expression. Inhibiting the expression of Runx1 by RN1, suppresses Gal-3 expression and inactivates Gal-3-associated signaling pathways, including the EGFR/ERK/Runx1, BMP/smad/Id-3 and integrin/FAK/JNK signaling pathways. In addition, RN1 can also bind to bone morphogenetic protein receptors (BMPR1A and BMPR2) and block the interaction between Gal-3 and the BMPRs. Thus, our results suggest that a novel Gal-3 inhibitor RN1 may be a potential candidate for human PDAC treatment via multiple targets and multiple signaling pathways.
Assuntos
Carcinoma Ductal Pancreático/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Galectina 3/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pancreáticas/prevenção & controle , Rodaminas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/farmacologia , Tiofenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Sambucus Williamsii Hance (SWH) is a plant from a family of Caprifoliaceae, which has a long medical history of use as an effective folk treatment for fracture bruises. AIM OF THE STUDY: To evaluate the effects of 50% ethanol extracts of root-bark of Sambucus Williamsii Hance(EE-rbSWH) on fracture healing of rats and explore its mechanism of actions related to the BMP-2 signaling pathway. MATERIALS AND METHODS: EE-rbSWH was orally administered at the doses of 340 and 680mg/kg to adult Sprague-Dawley rats with operation of open femur fracture completely for 2, 4 and 8 weeks. And the rats of sham operation and Model groups were administered Vehicle (distilled water 0.8mL/200g/day). Firstly, the bone X-ray morphology and bone mineral density(BMD) of the fracture site were observed and measured after anesthesia the rats at weeks 2, 4, and 8 after surgery, then the serum levels of alkaline phosphatase(ALP) and osteocalcin (BGP) were measured; Secondly, the tissue morphology of the fracture site was observed after sacrificed the rats; Thirdly, the formation of mineralized nodules in bone marrow stromal cells(BMSC) were evaluated at week 2; Lastly, the genes levels of BMP-2 and Runx2 in the femur were detected at week 2 and 4, and the proteins expression of BMP-2 signaling pathway (BMP-2, BMPRIB, BMPRII and Runx2) in the femur also were detected at week 2. RESULTS: EE-rbSWH remarkably accelerated fracture healing by promoting bone formation at all the time points of fracture healing. Mainly by increasing the BMD level at the fracture site, the levels of serum ALP and BGP, and also the numbers increasing of calcified nodules in BMSC. The mechanism studies, EE-rbSWH can promote fracture healing by enhancing the expressions of BMP-2 and Runx2 mRNA, and also the proteins of BMP-2, BMPRIB, BMPRII and Runx2 at the fracture site of rats. CONCLUSIONS: Our results suggested that 50% ethanol extracts of root-bark of Sambucus Williamsii Hance can accelerate fracture healing by recruitment of osteoblasts at the fracture site and through up-regulation of the BMP-2 signaling pathway.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fraturas do Fêmur/tratamento farmacológico , Fêmur/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Casca de Planta/química , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Sambucus/química , Transdução de Sinais/efeitos dos fármacos , Administração Oral , Fosfatase Alcalina/sangue , Animais , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/isolamento & purificação , Proteína Morfogenética Óssea 2/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Modelos Animais de Doenças , Etanol/química , Feminino , Fraturas do Fêmur/metabolismo , Fraturas do Fêmur/patologia , Fêmur/metabolismo , Fêmur/patologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Osteocalcina/sangue , Fitoterapia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Ratos Sprague-Dawley , Solventes/química , Fatores de Tempo , Regulação para CimaRESUMO
Objective To observe the effects of Bushen Tiaojing Recipe (BTR) on the counts of survival preantral follicles and the bone morphogenetic protein receptor II (BMPR II )/activin receptor- like kinase 6-drosophila mothers against decapentaplegic proteins (ALK6-Smads) signal pathway in oocytes cultured in vitro, and to study its mechanism for improving the quality of oocytes. Methods Prean- tral follicles were mechanically isolated from 65 female 12-day old healthy Kunming mice, which were inoculated by normal rats' serum (as the control group) , high, medium, low dose BTR containing serums (as Shen-supplementing groups) , high dose BTR containing serum + K02288 (as the inhibitor group) , respectively. All were cultured by common method in vitro. On the 6th day the counts of survival preantral follicles were compared between each Shen-supplementing group and the control group respectively. mR- NA expressions of BMPR II, ALK6, Smad1 , Smad5, and Smad8 were detected by Real-time fluorescence quantitative PCR. The protein expressions of indices mentioned above and phospho-Smadl/5/8 (p- Smadl/5/8) were detected by cellular immunofluorescence test. Results Compared with the control group, the quantity of survival preantral follicles increased in the high dose BTR containing serum group; mRNA expressions of BMPR II, ALK6, Smad5, and Smad8 were elevated, protein expressions of indi- ces mentioned above and p-Smadl/5/8 were increased in the 3 Shen-supplementing groups (P <0. 05) ; mRNA and protein expressions of Smad1 were increased in high and medium dose BTR containing serum groups (P<0.05). Compared with the high dose BTR containing serum group, protein expressions of Smad1/5/8 were reduced in the inhibitor group (P <0.05). Conclusion BTR could elevate the quantity of survival preantral follicles cultured in vitroand improve the quality of oocytes, which might be possibly as- sociated to regulating the BMPR II/ALK6-Smads signal pathway in oocytes.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II , Medicamentos de Ervas Chinesas , Oócitos , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Folículo Ovariano , Ratos , Transdução de Sinais , Proteínas Smad/efeitos dos fármacos , Proteínas Smad/metabolismoRESUMO
Efficient methods for the preparation of 5'-substituted 5'-amino-5'-deoxy-N(6)-ureidoadenosine derivatives are described. Compounds were screened for antiproliferative activity against a panel of murine and human cell lines (L1210, CEM, and HeLa) and/or against the NCI-60. The most potent derivative inhibited the lung adenocarcinoma cell line NCI-H522 at low nanomolar concentrations (GI50 = 9.7 nM).
Assuntos
Adenosina/análogos & derivados , Antineoplásicos/química , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adenosina/farmacologia , Antineoplásicos/farmacologia , Sítios de Ligação , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Relação Estrutura-AtividadeRESUMO
Hypothalamic neural circuits are known to regulate energy homeostasis and feeding behavior, but how these circuits are established during development is not well understood. Here we report that embryonic neural progenitors that express the transcription factor OLIG1 contribute neurons to the ventral hypothalamus including the arcuate nucleus (ARH), a center that regulates feeding behavior. Ablation of bone morphogenetic protein receptor 1a (BMPR1A) in the OLIG1 lineage resulted in hypophagia, hypoglycemia, and weight loss after the second postnatal week with death by week 4. Differentiation and specification of inhibitory hypothalamic neurons contributing to melanocortin and dopaminergic systems were abnormal in the BMPR1A-deficient ARH. Although the hypophagia promoted expression of the orexigenic neuropeptide agouti related protein (AgRP) in the BMPR1A-deficient ARH, there was a profound decrease of AgRP(+) axonal terminals in the mutant ARH targets including dorsomedial and paraventricular hypothalamic nuclei. Projection of AgRP(+) neurons to these nuclei is known to be regulated by leptin. Leptin injection in neonatal mice increased bone morphogenic protein (BMP) signaling in the ventral hypothalamus, and blocking BMP signaling prevented leptin-induced neurite outgrowth in ARH explant cultures. These findings suggest that BMPR1A signaling is critical for postnatal establishment of leptin-responsive orexigenic fibers from ARH to multiple hypothalamic nuclei. More generally these observations indicate that BMPR1A signaling regulates postnatal establishment of OLIG1 lineage-derived ARH neuronal circuits that are critical for leptin-mediated feeding behavior.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Comportamento Alimentar/fisiologia , Hipotálamo/metabolismo , Rede Nervosa/metabolismo , Neurônios/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proliferação de Células , Comportamento Alimentar/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Leptina/farmacologia , Camundongos , Camundongos Knockout , Rede Nervosa/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Neurônios/efeitos dos fármacos , Neuropeptídeo Y/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We recently identified that cholesterol myristate in traditional Chinese medicine (TCM) is the active compound that increases proliferation of mesenchymal stem cell (MSCs). The present study is further to determine what signal pathway involves in effect of cholesterol myristate. Reverse transcription-PCR, Western blot and ELISA analysis show that cholesterol myristate increases the release of bone morphogenetic protein 4 (BMP4) from MSCs and the expression in the intracellular levels of BMP4 in a time- and dose dependent manner. However, structurally related steroids such as cholesterol and cholesten presented in TCM, both lack of the myristate, did not affect the secretion and expression of BMP4 on MSCs. These finds suggest that myristate is essential for the effects of cholesterol myristate. Furthermore, cholesterol myristate significantly increase BMPRIB levels of MSCs and the number of BMPRIB positive cells in a time- and dose dependent manner, but not BMPR IA or BMPR II. Our results indicate that action of cholesterol myristate may activate the BMP4-BMPRIB autocrine. Moreover, a blocking antibody against BMP4 or the BMP4 antagonist, noggin, partially reduced the effects of cholesterol myristate on MSCs proliferation. Thus, this study is to provide evidence that autocrine BMP4 signaling involves effect of cholesterol myristate on MSCs proliferation.
Assuntos
Comunicação Autócrina/efeitos dos fármacos , Proteína Morfogenética Óssea 4/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proliferação de Células/efeitos dos fármacos , Ésteres do Colesterol/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/imunologia , Comunicação Autócrina/fisiologia , Proteína Morfogenética Óssea 4/agonistas , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/agonistas , Proteínas de Transporte/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
In response to nutrient stimuli, the mediobasal hypothalamus (MBH) drives multiple neuroendocrine and behavioral mechanisms to regulate energy balance. While central leucine reduces food intake and body weight, the specific neuroanatomical sites of leucine sensing, downstream neural substrates, and neurochemical effectors involved in this regulation remain largely unknown. Here we demonstrate that MBH leucine engages a neural energy regulatory circuit by stimulating POMC (proopiomelanocortin) neurons of the MBH, oxytocin neurons of the paraventricular hypothalamus, and neurons within the brainstem nucleus of the solitary tract to acutely suppress food intake by reducing meal size. We identify central p70 S6 kinase and Erk1/2 pathways as intracellular effectors required for this response. Activation of endogenous leucine intracellular metabolism produced longer-term reductions in meal number. Our data identify a novel, specific hypothalamus-brainstem circuit that links amino acid availability and nutrient sensing to the control of food intake.
Assuntos
Tronco Encefálico/fisiologia , Ingestão de Alimentos/fisiologia , Hipotálamo/metabolismo , Leucina/administração & dosagem , Animais , Anorexia/metabolismo , Peso Corporal/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Butadienos/farmacologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Inibidores Enzimáticos/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Proteínas de Fluorescência Verde/genética , Hipotálamo/anatomia & histologia , Técnicas In Vitro , Injeções Intraventriculares/métodos , Cetoácidos/farmacologia , Leucina/sangue , Leucina/líquido cefalorraquidiano , Masculino , Melanocortinas/metabolismo , Hormônios Estimuladores de Melanócitos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nitrilas/farmacologia , Ocitocina/antagonistas & inibidores , Ocitocina/metabolismo , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Treonina/metabolismo , Fatores de Tempo , Tirosina/metabolismo , Vasotocina/farmacologiaRESUMO
Although the multilineage potential of human adipose-derived adult stromal cells (ADAS) has been well described, few published studies have investigated the biological and molecular mechanisms underlying osteogenic differentiation of mouse ADAS. We report here that significant osteogenesis, as determined by gene expression and histological analysis, is induced only when mouse ADAS are cultured in the presence of retinoic acid with or without recombinant human bone morphogenetic protein (BMP)-2 supplementation. Furthermore, a dynamic expression profile for the BMP receptor (BMPR) isoform IB was observed, with dramatic up-regulation during osteogenesis. Western blot analysis revealed that retinoic acid enhanced levels of BMPR-IB protein during the first 7 days of osteogenic differentiation and that RNAi-mediated suppression of BMPR-IB dramatically impaired the ability of ADAS to form bone in vitro. In contrast, absence of BMPR-IA did not significantly diminish ADAS osteogenesis. Our data therefore demonstrate that the osteogenic commitment of multipotent mouse ADAS requires retinoic acid, which enhances expression of the critical BMPR-IB isoform.
Assuntos
Tecido Adiposo/citologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Diferenciação Celular/fisiologia , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Células Estromais/fisiologia , Tretinoína/metabolismo , Células 3T3 , Animais , Antineoplásicos/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Células Estromais/citologiaRESUMO
Endocytosis is important for a variety of functions in eukaryotic cells, including the regulation of signaling cascades via transmembrane receptors. The internalization of bone morphogenetic protein (BMP) receptor type I (BRI) and type II (BRII) and its relation to signaling were largely unexplored. Here, we demonstrate that both receptor types undergo constitutive endocytosis via clathrin-coated pits (CCPs) but that only BRII undergoes also caveola-like internalization. Using several complementary approaches, we could show that (i) BMP-2-mediated Smad1/5 phosphorylation occurs at the plasma membrane in nonraft regions, (ii) continuation of Smad signaling resulting in a transcriptional response requires endocytosis via the clathrin-mediated route, and (iii) BMP signaling leading to alkaline phosphatase induction initiates from receptors that fractionate into cholesterol-enriched, detergent-resistant membranes. Furthermore, we show that BRII interacts with Eps15R, a constitutive component of CCPs, and with caveolin-1, the marker protein of caveolae. Taken together, the localization of BMP receptors in distinct membrane domains is prerequisite to their taking different endocytosis routes with specific impacts on Smad-dependent and Smad-independent signaling cascades.