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1.
Elife ; 122024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38747706

RESUMO

Quiescence (G0) maintenance and exit are crucial for tissue homeostasis and regeneration in mammals. Here, we show that methyl-CpG binding protein 2 (Mecp2) expression is cell cycle-dependent and negatively regulates quiescence exit in cultured cells and in an injury-induced liver regeneration mouse model. Specifically, acute reduction of Mecp2 is required for efficient quiescence exit as deletion of Mecp2 accelerates, while overexpression of Mecp2 delays quiescence exit, and forced expression of Mecp2 after Mecp2 conditional knockout rescues cell cycle reentry. The E3 ligase Nedd4 mediates the ubiquitination and degradation of Mecp2, and thus facilitates quiescence exit. A genome-wide study uncovered the dual role of Mecp2 in preventing quiescence exit by transcriptionally activating metabolic genes while repressing proliferation-associated genes. Particularly disruption of two nuclear receptors, Rara or Nr1h3, accelerates quiescence exit, mimicking the Mecp2 depletion phenotype. Our studies unravel a previously unrecognized role for Mecp2 as an essential regulator of quiescence exit and tissue regeneration.


Assuntos
Proteína 2 de Ligação a Metil-CpG , Animais , Proteína 2 de Ligação a Metil-CpG/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Knockout , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Ciclo Celular , Regeneração Hepática/genética , Regulação da Expressão Gênica
3.
Curr Res Food Sci ; 6: 100424, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36618100

RESUMO

Lipase is a very important digestive enzyme for triglyceride absorption in vivo. The inhibitory activities of 26 dietary flavonoids, including flavone, flavanone, isoflavone and flavanol, on lipase were determined. Flavone exhibited stronger inhibitory activity than other types of flavonoids. Among them, luteolin exhibited the strongest inhibitory activity with IC50 value of 99 ± 11 µM, followed by quercetin and baicalein. The binding affinity of these flavonoids with lipase was investigated by fluorescence titration method. The binding affinity of flavones was stronger than flavanones, and was linearly positively correlated with their inhibitory activity. The binding of flavones on lipase caused the blue-shift of fluorescence, while flavanones caused red-shift. The analysis of structure-activity relationship of flavonoids on lipase revealed that the structure of C ring is very crucial. The hydrogenation of C2=C3 bond and the absence of C=O group in C ring both caused significant decrease of inhibitory activity. Besides, the hydroxylation on ring A and B of flavones increased the activity, while glycosylation weakened the activity. Molecular docking analysis confirmed that C2=C3 bond in C ring of flavones increases the π-conjugation and contributes to maintaining the planarity of flavonoid structure, which favour its Pi-Pi interaction with lipase.

4.
Stem Cells ; 40(11): 1008-1019, 2022 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-35977539

RESUMO

p53 plays a pivotal role in maintaining the genomic stability of mouse embryonic stem cells (mESCs) through transcriptionally activating and repressing target genes. However, how p53 recognizes its repressed targets remains largely unknown. Herein, we demonstrate that Sall4 negatively regulates DNA damage induced apoptosis (DIA) of mESCs through mediating p53 recruitment to enhancers of ESC-associated genes repressed by p53 from promoters of p53-activated genes. Upon DNA damage, Sall4 is transcriptionally repressed by p53 and plays an anti-apoptotic role without altering p53 activation. Moreover, Sall4 is identified as a novel p53-interacting partner. Consistently, Sall4 exerts its anti-apoptotic function in a p53-dependent manner. Intriguingly, Sall4 depletion not only promotes the transcriptional activation of several p53-regulated pro-apoptotic genes but also compromises p53-mediated repression of ESC master transcription factors in response to DNA damage. Mechanistically, Sall4 balances p53-binding affinity between p53-activated and -repressed genes through tethering p53 to ESC enhancers. In light of our study, Sall4 may contribute to tumorigenesis by antagonizing p53-mediated apoptosis.


Assuntos
Proteínas de Ligação a DNA , Células-Tronco Embrionárias Murinas , Fatores de Transcrição , Proteína Supressora de Tumor p53 , Animais , Camundongos , Dano ao DNA/genética , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo
5.
Theranostics ; 12(7): 3057-3078, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35547766

RESUMO

Rationale: Spinal cord injury (SCI) remains an incurable neurological disorder leading to permanent and profound neurologic deficits and disabilities. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are particularly appealing in SCI treatment to curtail damage, restore homeostasis and possible neural relay. However, the detailed mechanisms underlying hUC-MSC-mediated functional recovery of SCI have not been fully elucidated. The purpose of our current study is to identify novel therapeutic targets and depict the molecular mechanisms underlying the hUC-MSC-mediated recovery of subacute SCI. Methods: Adult female rats suffering from subacute incomplete thoracic SCI were treated with intrathecal transplantation of hUC-MSCs. The beneficial effects of hUC-MSCs on SCI repair were evaluated by a series of behavioral analyses, motor evoked potentials (MEPs) recording of hindlimb and immunohistochemistry. We carried out extensive transcriptome comparative analyses of spinal cord tissues at the lesion site from the subacute phase of SCI (sub-SCI) either treated without (+PBS) or with hUC-MSCs (+MSC) at 0 (sub-SCI), 1, 2, and 4 weeks post-transplantation (wpt), as well as normal spinal cord segments of intact/sham rats (Intact). Adeno-associated virus (AAV)-mediated neuron-specific expression system was employed to functionally screen specific γ-aminobutyric acid type A receptor (GABAAR) subunits promoting the functional recovery of SCI in vivo. The mature cortical axon scrape assay and transplantation of genetically modified MSCs with either overexpression or knockdown of brain-derived neurotrophic factor (BDNF) were employed to demonstrate that hUC-MSCs ameliorated the reduction of GABAAR subunits in the injured spinal cord via BDNF secretion in vitro and in vivo, respectively. Results: Comparative transcriptome analysis revealed the GABAergic synapse pathway is significantly enriched as a main target of hUC-MSC-activated genes in the injured spinal cord. Functional screening of the primary GABAAR subunits uncovered that Gabrb3 and Garbg2 harbored the motor and electrophysiological recovery-promoting competence. Moreover, targeting either of the two pivotal subunits ß3 or γ2 in combination with/without the K+/Cl- cotransporter 2 (KCC2) reinforced the therapeutic effects. Mechanistically, BDNF secreted by hUC-MSCs contributed to the upregulation of GABAAR subunits (ß3 & γ2) and KCC2 in the injured neurons. Conclusions: Our study identifies a novel mode for hUC-MSC-mediated locomotor recovery of SCI through synergistic upregulation of GABAAR ß3 and γ2 along with KCC2 by BDNF secretion, indicating the significance of restoring the excitation/inhibition balance in the injured neurons for the reestablishment of neuronal circuits. This study also provides a potential combinatorial approach by targeting the pivotal subunit ß3 or γ2 and KCC2, opening up possibilities for efficacious drug design.


Assuntos
Transplante de Células-Tronco Mesenquimais , Traumatismos da Medula Espinal , Simportadores , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Ratos , Receptores de GABA-A , Traumatismos da Medula Espinal/patologia , Cordão Umbilical/metabolismo , Ácido gama-Aminobutírico
6.
Neural Regen Res ; 17(8): 1785-1794, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35017439

RESUMO

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a promising candidate for spinal cord injury (SCI) repair owing to their advantages of low immunogenicity and easy accessibility over other MSC sources. However, modest clinical efficacy hampered the progression of these cells to clinical translation. This discrepancy may be due to many variables, such as cell source, timing of implantation, route of administration, and relevant efficacious cell dose, which are critical factors that affect the efficacy of treatment of patients with SCI. Previously, we have evaluated the safety and efficacy of 4 × 106 hUC-MSCs/kg in the treatment of subacute SCI by intrathecal implantation in rat models. To search for a more accurate dose range for clinical translation, we compared the effects of three different doses of hUC-MSCs - low (0.25 × 106 cells/kg), medium (1 × 106 cells/kg) and high (4 × 106 cells/kg) - on subacute SCI repair through an elaborate combination of behavioral analyses, anatomical analyses, magnetic resonance imaging-diffusion tensor imaging (MRI-DTI), biotinylated dextran amine (BDA) tracing, electrophysiology, and quantification of mRNA levels of ion channels and neurotransmitter receptors. Our study demonstrated that the medium dose, but not the low dose, is as efficient as the high dose in producing the desired therapeutic outcomes. Furthermore, partial restoration of the γ-aminobutyric acid type A (GABAA) receptor expression by the effective doses indicates that GABAA receptors are possible candidates for therapeutic targeting of dormant relay pathways in injured spinal cord. Overall, this study revealed that intrathecal implantation of 1 × 106 hUC-MSCs/kg is an alternative approach for treating subacute SCI.

7.
EMBO Rep ; 22(6): e52122, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33950553

RESUMO

Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.


Assuntos
Células-Tronco Embrionárias , Metabolismo dos Lipídeos , Ácidos Graxos , Oxirredução , Estresse Fisiológico
8.
Exp Cell Res ; 395(2): 112184, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32707134

RESUMO

Functional multipotency renders human umbilical cord mesenchymal stem cell (hUC-MSC) a promising candidate for the treatment of spinal cord injury (SCI). However, its safety and efficacy have not been fully understood for clinical translation. In this study, we performed cellular, kinematic, physiological, and anatomical analyses, either in vitro or in vivo, to comprehensively evaluate the safety and efficacy associated with subarachnoid transplantation of hUC-MSCs in rats with subacute incomplete SCI. Concerning safety, hUC-MSCs were shown to have normal morphology, excellent viability, steady proliferation, typical biomarkers, stable karyotype in vitro, and no tumorigenicity both in vitro and in vivo. Following subarachnoid transplantation of hUC-MSCs in the subject rodents, the biodistribution of hUC-MSCs was restricted to the spinal cord, and no toxicity to immune system or organ function was observed. Body weight, organ weight, and the ratio of the latter upon the former between stem cell-transplanted rats and placebo-injected rats revealed no statistical differences. Regarding efficacy, hUC-MSCs could differentiate into osteoblasts, chondrocytes, adipocytes and neural progenitor cells in vitro. While in vivo studies revealed that subarachnoid transplantation of stem cells resulted in significant improvement in locomotion, earlier automatic micturition recovery and reduced lesion size, which correlated with increased regeneration of tracking fiber and reduced parenchymal inflammation. In vivo luminescence imaging showed that a few of the transplanted luciferase-labeled hUC-MSCs tended to migrate towards the lesion epicenter. Shortened latency and enhanced amplitude were also observed in both motor and sensory evoked potentials, indicating improved signal conduction in the damaged site. Immunofluorescent staining confirmed that a few of the administrated hUC-MSCs integrated into the spinal cord parenchyma and differentiated into astrocytes and oligodendrocytes, but not neurons. Moreover, decreased astrogliosis, increased remyelination, and neuron regeneration could be observed. To the best of our knowledge, this preclinical study provides detailed safety and efficacy evidence regarding intrathecal transplantation of hUC-MSCs in treating SCI for the first time and thus, supports its initiation in the following clinical trial.


Assuntos
Células-Tronco Mesenquimais/citologia , Células-Tronco Neurais/citologia , Neurônios/patologia , Traumatismos da Medula Espinal/patologia , Cordão Umbilical/citologia , Astrócitos/patologia , Diferenciação Celular/fisiologia , Células Cultivadas , Condrócitos/patologia , Humanos , Transplante de Células-Tronco Mesenquimais/métodos
9.
Plant Physiol Biochem ; 146: 315-328, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31785518

RESUMO

Chrysanthemum [Dendranthema morifolium Tzvel.] is an ornamental plant grown under long-term artificial cultivation conditions. In production, early Chrysanthemum blossoms are often promoted by artificial short-day treatment. However, we found that the flower colour of Chrysanthemum blossoms induced by artificial short-day treatment was lighter than those induced by the natural photoperiod. To explore the intrinsic mechanism of colour fading in flowers, we performed full-length transcriptome sequencing of Chrysanthemum morifolium cv. 'Jinbeidahong' using single-molecule real-time sequencing and RNA-sequencing under natural daylight (ND) and short daylight (SD) conditions. The clustered transcriptome sequences were assigned to various databases, such as NCBI, Swiss-Prot, Gene Ontology and so on. The comparative results of digital gene expression analysis revealed that there were differentially expressed transcripts (DETs) in the four stages under ND and SD conditions. In addition, the expression patterns of anthocyanin biosynthesis structural genes were verified by quantitative real-time PCR. The major regulators of the light signalling ELONGATED HYPOCOTYL5 genes were markedly upregulated under ND conditions. The patterns of anthocyanin accumulation were consistent with the expression patterns of CHI1 and 3GT1. The results showed that the anthocyanin synthesis is tightly regulated by the photoperiod, which will be useful for molecular breeding of Chrysanthemum.


Assuntos
Chrysanthemum , Fotoperíodo , Flores , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Transcriptoma
10.
Molecules ; 24(22)2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31739407

RESUMO

Poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS)-based organic electrochemical transistors (OECTs) are widely utilized to construct highly sensitive biosensors. However, the PSS phase exhibits insulation, weak acidity, and aqueous instability. In this work, we fabricated PEDOT OECT by alternating current electrodeposition in protic ionic liquids. The steady-state characteristics were demonstrated to be stable in long-term tests. In detail, the maximum transconductance, the on/off current ratio, and the hysteresis were stable at 2.79 mS, 504, and 0.12 V, respectively. Though the transient behavior was also stable, the time constant could reach 218.6 ms. Thus, the trade-off between switching speed and stability needs to be considered in applications that require a rapid response.


Assuntos
Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Líquidos Iônicos/química , Galvanoplastia
11.
Open Biol ; 9(5): 180262, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31088250

RESUMO

Osteocytes secrete the glycoprotein sclerostin to inhibit bone formation by osteoblasts, but how sclerostin production is regulated in osteocytes remains unclear. Here, we show that tuberous sclerosis complex 1 (TSC1) in osteocytes promotes sclerostin secretion through inhibition of mechanistic target of rapamycin complex 1 (mTORC1) and downregulation of Sirt1. We generated mice with DMP1-Cre-directed Tsc1 gene deletion ( Tsc1 CKO) to constitutively activate mTORC1 in osteocytes. Although osteocyte TSC1 disruption increased RANKL expression and osteoclast formation, it markedly reduced sclerostin production in bone, resulting in severe osteosclerosis with enhanced bone formation in mice. Knockdown of TSC1 activated mTORC1 and decreased sclerostin, while rapamycin inhibited mTORC1 and increased sclerostin mRNA and protein expression levels in MLO-Y4 osteocyte-like cells. Furthermore, mechanical loading activated mTORC1 and prevented sclerostin expression in osteocytes. Mechanistically, TSC1 promotes sclerostin production and prevents osteogenesis through inhibition of mTORC1 and downregulation of Sirt1, a repressor of the sclerostin gene Sost. Our findings reveal a role of TSC1/mTORC1 signalling in the regulation of osteocyte sclerostin secretion and bone formation in response to mechanical loading in vitro. Targeting TSC1 represents a potential strategy to increase osteogenesis and prevent bone loss-related diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Deleção de Genes , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Osteócitos/citologia , Osteosclerose/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Osteócitos/metabolismo , Osteogênese , Ligante RANK/metabolismo , Sirolimo/farmacologia , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo
12.
Mucosal Immunol ; 11(6): 1663-1673, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30082707

RESUMO

The functional role of colonic epithelium in the pathogenesis of ulcerative colitis (UC) remains unclear. Here, we reveal a novel mechanism by which colonic epithelia recruit T helper-17 (Th17) cells during the onset of UC. mTOR complex 1 (mTORC1) was hyper-activated in colonic epithelia of UC mice. While colonic epithelial TSC1 (mTORC1 negative regulator) disruption induced constitutive mTORC1 activation in the colon epithelia and aggravated UC, RPTOR (essential mTORC1 component) depletion inactivated mTORC1 and ameliorated UC. TSC1 deficiency enhanced, whereas RPTOR ablation reduced the expression of cyclooxygenase 2 (COX-2), interleukin-1 (IL-1), IL-6, and IL-23, as well as Th17 infiltration in the colon. Importantly, inhibition of COX-2 reversed the elevation in the expression of these proinflammatory mediators induced by TSC1 deficiency, and subsequently reduced the symptoms and pathological characteristics of UC in mouse models. Mechanistically, mTORC1 activates COX-2 transcription via phosphorylating STAT3 and enhancing it's binding to the COX-2 promoter. Consistently, enhanced mTORC1 activity and COX2 expression, as well as strong positive correlation between each other, were observed in colonic epithelial tissues of UC patients. Collectively, our study demonstrates an essential role of epithelial mTORC1 in UC pathogenesis and establishes a novel link between colonic epithelium, Th17 responses, and UC development.


Assuntos
Colite Ulcerativa/imunologia , Colo/patologia , Mucosa Intestinal/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Células Th17/imunologia , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Animais , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Proteína Regulatória Associada a mTOR/genética , Fator de Transcrição STAT3/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética
13.
Cell Death Dis ; 9(5): 441, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29670094

RESUMO

DEP domain containing mTOR-interacting protein (DEPTOR) was originally identified as an in vivo dual inhibitor of mechanistic target of rapamycin (mTOR). It was recently reported to be involved in renal physiology and pathology in vitro; however, its detailed roles and mechanisms in vivo are completely unknown. We observed that DEPTOR expression in the kidney was markedly increased on day 3 after cisplatin treatment, at which time cell apoptosis peaked, implicating DEPTOR in cisplatin-induced acute kidney injury (AKI). We then used the Cre-LoxP system to generate mutant mice in which the DEPTOR gene was specifically deleted in the proximal tubule cells. DEPTOR deficiency did not alter the renal histology or functions in the saline-treated group, indicating that DEPTOR is not essential for kidney function under physiological conditions. Interestingly, DEPTOR deletion extensively preserved the renal histology and maintained the kidney functions after cisplatin treatment, suggesting that the absence of DEPTOR ameliorates cisplatin-induced AKI. Mechanistically, DEPTOR modulated p38 MAPK signaling and TNFα production in vivo and in vitro, rather than mTOR signaling, thus moderating the inflammatory response and cell apoptosis induced by cisplatin. Collectively, our findings demonstrate the roles and mechanisms of DEPTOR in the regulation of the renal physiology and pathology, and demonstrate that the loss of DEPTOR in the proximal tubules protects against cisplatin-induced AKI.


Assuntos
Injúria Renal Aguda , Cisplatino/efeitos adversos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Túbulos Renais Proximais/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Animais , Cisplatino/farmacologia , Túbulos Renais Proximais/patologia , Camundongos , Camundongos Knockout
14.
Ann Rheum Dis ; 77(6): 935-943, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29555825

RESUMO

OBJECTIVES: To investigate the role of tyrosine kinase Fyn in the development of osteoarthritis (OA) and the underlying mechanisms, and to define whether targeting Fyn could prevent OA in mice. METHODS: Cartilage samples from normal and aged mice were analysed with proteome-wide screening. Fyn expression was examined with immunofluorescence in human and age-dependent or experimental mouse OA cartilage samples. Experimental OA in Fyn-knockout mice was induced by destabilisation of the medial meniscus. Primary cultured mouse chondrocytes were treated with proinflammatory cytokine interleukin-1ß. The inhibitor of Src kinase family, AZD0530 (saracatinib), and inhibitor of Fyn, PP1, were used to treat experimental OA in mice. RESULTS: Fyn expression was markedly upregulated in human OA cartilage and in cartilage from aged mice and those with post-traumatic OA. Fyn accumulates in articular chondrocytes and interacts directly with and phosphorylates ß-catenin at Tyr142, which stabilises ß-catenin and promotes its nuclear translocation. The deletion of Fyn effectively delayed the development of post-traumatic and age-dependent OA in mice. Fyn inhibitors AZD0530 and PP1 significantly attenuated OA progression by blocking the ß-catenin pathway and reducing the levels of extracellular matrix catabolic enzymes in the articular cartilage. CONCLUSIONS: Fyn accumulates and activates ß-catenin signalling in chondrocytes, accelerating the degradation of the articular cartilage and OA development. Targeting Fyn is a novel and potentially therapeutic approach to the treatment of OA.


Assuntos
Artrite Experimental/enzimologia , Osteoartrite/enzimologia , Proteínas Proto-Oncogênicas c-fyn/fisiologia , beta Catenina/metabolismo , Envelhecimento/metabolismo , Animais , Artrite Experimental/prevenção & controle , Benzodioxóis/farmacologia , Benzodioxóis/uso terapêutico , Cartilagem Articular/enzimologia , Células Cultivadas , Condrócitos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Técnicas de Inativação de Genes , Humanos , Camundongos Knockout , Terapia de Alvo Molecular/métodos , Osteoartrite/prevenção & controle , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fyn/deficiência , Proteínas Proto-Oncogênicas c-fyn/genética , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
15.
Cell Death Differ ; 25(9): 1549-1566, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29358671

RESUMO

Reorganization of the podosome into the sealing zone is crucial for osteoclasts (OCLs) to resorb bone, but the underlying mechanisms are unclear. Here, we show that tuberous sclerosis complex 1 (TSC1) functions centrally in OCLs to promote podosome organization and bone resorption through mechanistic target of rapamycin complex 1 (mTORC1) and the small GTPases Rac1/Cdc42. During osteoclastogenesis, enhanced expression of TSC1 downregulates mTORC1 activity. TSC1 deletion in OCLs reduced podosome belt formation in vitro and sealing zone formation in vivo, leading to bone resorption deficiency and osteopetrosis. Mechanistically, TSC1 promoted podosome superstructure assembly by releasing mTORC1-dependent negative feedback inhibition of Rac1/Cdc42. Rapamycin and active Rac1/Cdc42 restore podosome organization and bone resorption and alleviate osteopetrotic phenotypes in mutant mice. Our findings reveal an essential role of TSC1 signaling in the regulation of bone resorption. Targeting TSC1 represents a novel strategy to inhibit bone resorption and prevent bone loss-related diseases.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neuropeptídeos/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular , Regulação para Baixo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/genética , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Podossomos/efeitos dos fármacos , Podossomos/patologia , Podossomos/ultraestrutura , Células RAW 264.7 , Transdução de Sinais , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Proteína 1 do Complexo Esclerose Tuberosa/deficiência , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética
16.
J Bone Miner Res ; 33(4): 732-742, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29206332

RESUMO

Osteoblasts provide a microenvironmental niche for B-cell commitment and maturation in the bone marrow (BM). Any abnormity of osteoblasts function may result in the defect of B lymphopoiesis. Signaling from mechanistic target of rapamycin complex 1 (mTORC1) has been implicated in regulating the expansion and differentiation of osteoblasts. Thus, we raise a hypothesis that mTORC1 signaling in osteoblasts plays a vital role in B-cell development. Inactivation of mTORC1 in osterix-expressing cells (mainly osteoblast lineage) through Osx-Cre-directed deletion of Raptor (an mTORC1-specific component) resulted in a reduction in the total B-cell population in the BM, which was due to a block in early B-cell development from the pro-B to pre-B cell stage. Further mechanistic studies revealed that this defect was the result of reduction of interleukin-7 (IL-7) expression in osterix-expressing immature osteoblasts, which caused the abnormality of IL-7/Stat5 signaling in early B lymphocytes, leading to an increased apoptosis of pre-B plus immature B cells. In vitro and in vivo studies demonstrated that the addition of exogenous IL-7 partially restored B lymphopoiesis in the BM of Raptor mutant mice. Furthermore, total BM cells cultured in conditioned media from Raptor null immature osteoblasts or media with anti-IL-7 neutralizing antibody failed to differentiate into pre-B and immature B cells, indicating that inactivation of mTORC1 in immature osteoblast cannot fully support normal B-cell development. Taken together, these findings demonstrate a novel role for mTORC1 in the regulation of bone marrow environments that support B-cell differentiation via regulating IL-7 expression. © 2017 American Society for Bone and Mineral Research.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Transdução de Sinais/imunologia , Fator de Transcrição Sp7/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Linfócitos B/citologia , Diferenciação Celular/genética , Interleucina-7/genética , Interleucina-7/imunologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de Sinais/genética , Fator de Transcrição Sp7/genética
17.
J Bone Miner Res ; 32(9): 1829-1840, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28520214

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) is a critical sensor for bone homeostasis and bone formation; however, the role of mTORC1 in osteoclast development and the underlying mechanisms have not yet been fully established. Here, we found that mTORC1 activity declined during osteoclast precursors differentiation in vitro and in vivo. We further targeted deletion of Raptor (mTORC1 key component) or Tsc1 (mTORC1 negative regulator) to constitutively inhibit or activate mTORC1 in osteoclast precursors (monocytes/macrophages), using LyzM-cre mice. Osteoclastic formation was drastically increased in cultures of Raptor deficient bone marrow monocytes/macrophages (BMMs), and Raptor-deficient mice displayed osteopenia with enhanced osteoclastogenesis. Conversely, BMMs lacking Tsc1 exhibited a severe defect in osteoclast-like differentiation and absorptive function, both of which were restored following rapamycin treatment. Importantly, expression of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), transcription factors that are essential for osteoclast differentiation was negatively regulated by mTORC1 in osteoclast lineages. These results provide evidence that mTORC1 plays as a critical role as an osteoclastic differentiation-limiting signal and suggest a potential drawback in treating bone loss-related diseases with mTOR inhibitors clinically. © 2017 American Society for Bone and Mineral Research.


Assuntos
Diferenciação Celular , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Transdução de Sinais , Animais , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Knockout , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Osteoclastos/patologia , Células RAW 264.7 , Proteína Regulatória Associada a mTOR/deficiência , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência
18.
Blood ; 129(24): 3196-3209, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28450306

RESUMO

Severe thrombocytopenia is a significant challenge in patients undergoing myelosuppressive chemotherapy for malignancies. Understanding the biology of platelet-producing megakaryocytes development in the bone marrow microenvironment may facilitate the development of novel therapies to stimulate platelet production and prevent thrombocytopenia. We report here that osteoblasts supported megakaryopoiesis by secreting interleukin-9 (IL-9), which stimulated IL-9 receptor (IL-9R)/Stat3 signaling in promoting megakaryopoiesis. IL-9 production in osteoblasts was negatively regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signaling in a NF-κB-dependent manner. Constitutive activation of mTORC1 inhibited IL-9 production in osteoblasts and suppressed megakaryocytic cells expansion, whereas mTORC1 inactivation increased IL-9 production and enhanced megakaryocyte and platelet numbers in mice. In mouse models, we showed that IL-9 administration stimulated megakaryopoiesis, whereas neutralizing endogenous IL-9 or IL-9R depletion inhibited the process. Importantly, we found that low doses of IL-9 efficiently prevented chemotherapy-induced thrombocytopenia (CIT) and accelerated platelet recovery after CIT. These data indicate that IL-9 is an essential regulator of megakaryopoiesis and a promising therapeutic agent for treatment of thrombocytopenia such as CIT.


Assuntos
Interleucina-9/metabolismo , Megacariócitos/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais/fisiologia , Trombopoese/fisiologia , Animais , Células Endoteliais da Veia Umbilical Humana , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Megacariócitos/citologia , Camundongos , Complexos Multiproteicos/metabolismo , Osteoblastos/citologia , Células RAW 264.7 , Receptores de Interleucina-9/metabolismo , Fator de Transcrição STAT3/metabolismo , Serina-Treonina Quinases TOR/metabolismo
19.
Nat Commun ; 7: 13885, 2016 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-27966526

RESUMO

Communication between osteoblasts and endothelial cells (ECs) is essential for bone turnover, but the molecular mechanisms of such communication are not well defined. Here we identify Cxcl9 as an angiostatic factor secreted by osteoblasts in the bone marrow microenvironment. We show that Cxcl9 produced by osteoblasts interacts with vascular endothelial growth factor and prevents its binding to ECs and osteoblasts, thus abrogating angiogenesis and osteogenesis both in mouse bone and in vitro. The mechanistic target of rapamycin complex 1 activates Cxcl9 expression by transcriptional upregulation of STAT1 and increases binding of STAT1 to the Cxcl9 promoter in osteoblasts. These findings reveal the essential role of osteoblast-produced Cxcl9 in angiogenesis and osteogenesis in bone, and Cxcl9 can be targeted to elevate bone angiogenesis and prevent bone loss-related diseases.


Assuntos
Quimiocina CXCL9/fisiologia , Neovascularização Fisiológica , Osteoblastos/metabolismo , Animais , Desenvolvimento Ósseo/genética , Quimiocina CXCL9/metabolismo , Camundongos , Osteogênese , Regiões Promotoras Genéticas , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/fisiologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Cell Death Dis ; 7(10): e2408, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27735936

RESUMO

Osteoblast dysfunction is a major cause of age-related bone loss, but the mechanisms underlying changes in osteoblast function with aging are poorly understood. This study demonstrates that osteoblasts in aged mice exhibit markedly impaired adhesion to the bone formation surface and reduced mineralization in vivo and in vitro. Rictor, a specific component of the mechanistic target of rapamycin complex 2 (mTORC2) that controls cytoskeletal organization and cell survival, is downregulated with aging in osteoblasts. Mechanistically, we found that an increased level of reactive oxygen species with aging stimulates the expression of miR-218, which directly targets Rictor and reduces osteoblast bone surface adhesion and survival, resulting in a decreased number of functional osteoblasts and accelerated bone loss in aged mice. Our findings reveal a novel functional pathway important for age-related bone loss and support for miR-218 and Rictor as potential targets for therapeutic intervention for age-related osteoporosis treatment.


Assuntos
Envelhecimento/metabolismo , Proteínas de Transporte/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoporose/metabolismo , Osteoporose/patologia , Animais , Sequência de Bases , Calcificação Fisiológica/genética , Adesão Celular/genética , Sobrevivência Celular/genética , Regulação para Baixo/genética , Sequestradores de Radicais Livres/metabolismo , Deleção de Genes , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Proteína Companheira de mTOR Insensível à Rapamicina , Espécies Reativas de Oxigênio/metabolismo
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