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1.
Zhonghua Yu Fang Yi Xue Za Zhi ; 55(9): 1123-1128, 2021 Sep 06.
Artigo em Chinês | MEDLINE | ID: mdl-34619931

RESUMO

Objective: To investigate the role of autophagy mediated by mTOR signaling pathway in the inhibition of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) induced by cadmium. Methods: HBMSCs were divided into 0, 2.5 or 5.0 µmol/L groups according to the exposure dose of cadmium chloride (CdCl2), and each group was treated for 1 day, 4 days and (or) 7 days. The ALP activity and mRNA and protein expression levels of osteogenesis markers (ALP, RUNX2 and OSTERIX), autophagy-related proteins (LC3 and Beclin-1) and mTOR signaling pathway related proteins (mTOR, p-mTOR and p-p70S6K) expression, alkaline phosphatase staining and alizarin red staining were detected. MHY 1485 was selected as the signaling pathway activator. The control group, CdCl2 group (5.0 µmol/L), MHY 1485 group and CdCl2+MHY 1485 combined treatment group were set. After 7 days of treatment, the expression levels of autophagy related proteins and mTOR signaling pathway related proteins of hBMSCs in each group were detected. Results: There was no significant difference in ALP activity between 0, 2.5 and 5.0 µmol/L groups on day 1 and 4 (P>0.05); On day 7, compared with the 0 µmol/L group, the ALP activity, expression of osteogenic markers (ALP, RUNX2, OSTERIX) and mTOR signaling pathway related proteins (mTOR, p-mTOR, p-p70S6K) expression decreased in the 2.5 and 5.0 µmol/L group (P<0.05). Compared with the 0 µmol/L group, the staining of the 2.5 and 5.0 µmol/L groups became lighter, and the formation of ALP and mineralized nodules was reduced. Compared with the CdCl2 group, the autophagy related protein expression in the CdCl2+MHY 1485 combined treatment group decreased, and the mTOR signaling pathway related protein expression increased. The difference was statistically significant (P<0.05). Conclusion: The inhibition of osteogenic differentiation of hBMSCs by cadmium may be related to autophagy mediated by mTOR signaling pathway.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Autofagia , Cádmio , Diferenciação Celular , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
2.
Nat Commun ; 12(1): 5315, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493720

RESUMO

Human PAK4 is an ubiquitously expressed p21-activated kinase which acts downstream of Cdc42. Since PAK4 is enriched in cell-cell junctions, we probed the local protein environment around the kinase with a view to understanding its location and substrates. We report that U2OS cells expressing PAK4-BirA-GFP identify a subset of 27 PAK4-proximal proteins that are primarily cell-cell junction components. Afadin/AF6 showed the highest relative biotin labelling and links to the nectin family of homophilic junctional proteins. Reciprocally >50% of the PAK4-proximal proteins were identified by Afadin BioID. Co-precipitation experiments failed to identify junctional proteins, emphasizing the advantage of the BioID method. Mechanistically PAK4 depended on Afadin for its junctional localization, which is similar to the situation in Drosophila. A highly ranked PAK4-proximal protein LZTS2 was immuno-localized with Afadin at cell-cell junctions. Though PAK4 and Cdc42 are junctional, BioID analysis did not yield conventional cadherins, indicating their spatial segregation. To identify cellular PAK4 substrates we then assessed rapid changes (12') in phospho-proteome after treatment with two PAK inhibitors. Among the PAK4-proximal junctional proteins seventeen PAK4 sites were identified. We anticipate mammalian group II PAKs are selective for the Afadin/nectin sub-compartment, with a demonstrably distinct localization from tight and cadherin junctions.


Assuntos
Junções Intercelulares/metabolismo , Proteínas dos Microfilamentos/genética , Nectinas/genética , Proteômica/métodos , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21/genética , Biotina/química , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Linhagem Celular Tumoral , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Junções Intercelulares/genética , Junções Intercelulares/ultraestrutura , Marcação por Isótopo , Espectrometria de Massas , Proteínas dos Microfilamentos/metabolismo , Nectinas/metabolismo , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Ligação Proteica , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo
3.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502120

RESUMO

Diabetes mellitus is a main risk factor for delayed fracture healing and fracture non-unions. Successful fracture healing requires stimuli from different immune cells, known to be affected in diabetics. Especially, application of mononuclear cells has been proposed to promote wound and fracture healing. Thus, aim was to investigate the effect of pre-/diabetic conditions on mononuclear cell functions essential to promote osteoprogenitor cell function. We here show that pre-/diabetic conditions suppress the expression of chemokines, e.g., CCL2 and CCL8 in osteoprogenitor cells. The associated MCP-1 and MCP-2 were significantly reduced in serum of diabetics. Both MCPs chemoattract mononuclear THP-1 cells. Migration of these cells is suppressed under hyperglycemic conditions, proposing that less mononuclear cells invade the site of fracture in diabetics. Further, we show that the composition of cytokines secreted by mononuclear cells strongly differ between diabetics and controls. Similar is seen in THP-1 cells cultured under hyperinsulinemia or hyperglycemia. The altered secretome reduces the positive effect of the THP-1 cell conditioned medium on migration of osteoprogenitor cells. In summary, our data support that factors secreted by mononuclear cells may support fracture healing by promoting migration of osteoprogenitor cells but suggest that this effect might be reduced in diabetics.


Assuntos
Meios de Cultivo Condicionados/metabolismo , Diabetes Mellitus/metabolismo , Consolidação da Fratura , Monócitos/metabolismo , Animais , Biomarcadores , Movimento Celular , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL8/metabolismo , Quimiocinas/metabolismo , Quimiotaxia de Leucócito/imunologia , Humanos , Hiperglicemia/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Sistema de Sinalização das MAP Quinases , Monócitos/imunologia , Osteoblastos/metabolismo , Osteogênese , Células THP-1
4.
FASEB J ; 35(10): e21851, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34547121

RESUMO

It has been known that moderate mechanical loading, like that caused by exercise, promotes bone formation. However, its underlying mechanisms remain elusive. Here we showed that moderate running dramatically improved trabecular bone in mice tibias with an increase in bone volume fraction and trabecular number and a decrease in trabecular pattern factor. Results of immunohistochemical and histochemical staining revealed that moderate running mainly increased the number of osteoblasts but had no effect on osteoclasts. In addition, we observed a dramatic increase in the number of colony forming unit-fibroblast in endosteal bone marrow and the percentage of CD45- Leptin receptor+ (CD45- LepR+ ) endosteal mesenchymal progenitors. Bioinformatics analysis of the transcriptional data from gene expression omnibus (GEO) database identified chemokine c-c-motif ligands (CCL2) as a critical candidate induced by mechanical loading. Interestingly, we found that CCL2 was up-regulated mainly in osteoblastic cells in the tibia of mice after moderate running. Further, we found that mechanical loading up-regulated the expression of CCL2 by activating ERK1/2 pathway, thereby stimulating migration of endosteal progenitors. Finally, neutralizing CCL2 abolished the recruitment of endosteal progenitors and the increased bone formation in mice after 4 weeks running. These results therefore uncover an unknown connection between osteoblasts and endosteal progenitors recruited in the increased bone formation induced by mechanical loading.


Assuntos
Osso Esponjoso/citologia , Quimiocina CCL2/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteogênese , Condicionamento Físico Animal , Animais , Osso Esponjoso/metabolismo , Movimento Celular , Quimiocina CCL2/genética , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo
5.
BMC Cancer ; 21(1): 1059, 2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34565336

RESUMO

BACKGROUND: Extracellular matrix metalloproteinase inducer (EMMPRIN), a cell-surface glycoprotein, is overexpressed in several cancer types. EMMPRIN induces a metastatic phenotype by triggering the production of matrix metalloproteinase proteins (MMPs) such as MMP1 and MMP2, and vascular endothelial growth factor (VEGF) in cancer cells and the surrounding stromal cells. The purpose of this study was to investigate the expression and role of EMMPRIN in osteosarcoma. METHODS: The level of EMMPRIN expression was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) in 6 tumor-derived osteosarcoma cell lines and compared with that in normal osteoblasts. To study the prognostic significance of EMMPRIN expression, immunohistochemistry was carried out in prechemotherapy biopsies of 54 patients. siRNA knockdown of EMMPRIN in SaOS-2 cells was conducted to explore the role of EMMPRIN. To study the role of EMMPRIN in tumor-stromal interaction in MMP production and invasion, co-culture of SaOS-2 cells with osteoblasts and fibroblasts was performed. Osteosarcoma 143B cells were injected into the tail vein of BALB/c mice and lung metastasis was analyzed. RESULTS: EMMRIN mRNA expression was significantly higher in 5 of 6 (83%) tumor-derived cells than in MG63 cells. 90% of specimens (50/54) stained positive for EMMPRIN by immunohistochemistry, and higher expression of EMMPRIN was associated with shorter metastasis-free survival (p = 0.023). Co-culture of SaOS-2 with osteoblasts resulted in increased production of pro-MMP2 and VEGF expression, which was inhibited by EMMPRIN-targeting siRNA. siRNA knockdown of EMMPRIN resulted in decreased invasion. EMMPRIN shRNA-transfected 143B cells showed decreased lung metastasis in vivo. CONCLUSIONS: Our data suggest that EMMPRIN acts as a mediator of osteosarcoma metastasis by regulating MMP and VEGF production in cancer cells as well as stromal cells. EMMPRIN could serve as a therapeutic target in osteosarcoma.


Assuntos
Basigina/metabolismo , Neoplasias Ósseas/metabolismo , Osteossarcoma/metabolismo , Animais , Basigina/antagonistas & inibidores , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Progressão da Doença , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/secundário , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Osteoblastos/metabolismo , Osteossarcoma/patologia , Osteossarcoma/secundário , Intervalo Livre de Progressão , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Fatores de Crescimento do Endotélio Vascular/metabolismo
6.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502045

RESUMO

Bone is a highly dynamic tissue that is constantly adapting to micro-changes to facilitate movement. When the balance between bone building and resorption shifts more towards bone resorption, the result is reduced bone density and mineralization, as seen in osteoporosis or osteopenia. Current treatment strategies aimed to improve bone homeostasis and turnover are lacking in efficacy, resulting in the search for new preventative and nutraceutical treatment options. The myokine irisin, since its discovery in 2012, has been shown to play an important role in many tissues including muscle, adipose, and bone. Evidence indicate that irisin is associated with increased bone formation and decreased bone resorption, leading to reduced risk of osteoporosis in post-menopausal women. In addition, low serum irisin levels have been found in individuals with osteoporosis and osteopenia. Irisin targets key signaling proteins, promoting osteoblastogenesis and reducing osteoclastogenesis. The present review summarizes the existing evidence regarding the effects of irisin on bone homeostasis.


Assuntos
Fibronectinas/metabolismo , Homeostase , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Animais , Humanos
7.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502056

RESUMO

Skeletal tissue involves systemic adipose tissue metabolism and energy expenditure. MicroRNA signaling controls high-fat diet (HFD)-induced bone and fat homeostasis dysregulation remains uncertain. This study revealed that transgenic overexpression of miR-29a under control of osteocalcin promoter in osteoblasts (miR-29aTg) attenuated HFD-mediated body overweight, hyperglycemia, and hypercholesterolemia. HFD-fed miR-29aTg mice showed less bone mass loss, fatty marrow, and visceral fat mass together with increased subscapular brown fat mass than HFD-fed wild-type mice. HFD-induced O2 underconsumption, respiratory quotient repression, and heat underproduction were attenuated in miR-29aTg mice. In vitro, miR-29a overexpression repressed transcriptomic landscapes of the adipocytokine signaling pathway, fatty acid metabolism, and lipid transport, etc., of bone marrow mesenchymal progenitor cells. Forced miR-29a expression promoted osteogenic differentiation but inhibited adipocyte formation. miR-29a signaling promoted brown/beige adipocyte markers Ucp-1, Pgc-1α, P2rx5, and Pat2 expression and inhibited white adipocyte markers Tcf21 and Hoxc9 expression. The microRNA also reduced peroxisome formation and leptin expression during adipocyte formation and downregulated HFD-induced leptin expression in bone tissue. Taken together, miR-29a controlled leptin signaling and brown/beige adipocyte formation of osteogenic progenitor cells to preserve bone anabolism, which reversed HFD-induced energy underutilization and visceral fat overproduction. This study sheds light on a new molecular mechanism by which bone integrity counteracts HFD-induced whole-body fat overproduction.


Assuntos
Gordura Intra-Abdominal/metabolismo , Leptina/genética , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteoporose/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Dieta Hiperlipídica/efeitos adversos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Leptina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Osteoblastos/citologia , Osteoporose/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Peroxissomos/metabolismo , Receptores Purinérgicos P2X5/genética , Receptores Purinérgicos P2X5/metabolismo , Simportadores/genética , Simportadores/metabolismo , Termogênese , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
8.
Nat Commun ; 12(1): 5438, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521831

RESUMO

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.


Assuntos
Proteínas de Arabidopsis/metabolismo , Técnicas Biossensoriais , Proteínas Intrinsicamente Desordenadas/metabolismo , Chaperonas Moleculares/metabolismo , Pressão Osmótica , Água/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Linhagem Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Cinética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Concentração Osmolar , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Termodinâmica
9.
Nat Commun ; 12(1): 5296, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489478

RESUMO

The vascular endothelium is present within metabolic organs and actively regulates energy metabolism. Here we show osteocalcin, recognized as a bone-secreted metabolic hormone, is expressed in mouse primary endothelial cells isolated from heart, lung and liver. In human osteocalcin promoter-driven green fluorescent protein transgenic mice, green fluorescent protein signals are enriched in endothelial cells lining aorta, small vessels and capillaries and abundant in aorta, skeletal muscle and eye of adult mice. The depletion of lipoprotein receptor-related protein 1 induces osteocalcin through a Forkhead box O -dependent pathway in endothelial cells. Whereas depletion of osteocalcin abolishes the glucose-lowering effect of low-density lipoprotein receptor-related protein 1 depletion, osteocalcin treatment normalizes hyperglycemia in multiple mouse models. Mechanistically, osteocalcin receptor-G protein-coupled receptor family C group 6 member A and insulin-like-growth-factor-1 receptor are in the same complex with osteocalcin and required for osteocalcin-promoted insulin signaling pathway. Therefore, our results reveal an endocrine/paracrine role of endothelial cells in regulating insulin sensitivity, which may have therapeutic implications in treating diabetes and insulin resistance through manipulating vascular endothelium.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Glucose/metabolismo , Hiperglicemia/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Osteocalcina/genética , Animais , Células Endoteliais/patologia , Endotélio Vascular/patologia , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Teste de Tolerância a Glucose , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteocalcina/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
10.
Zhongguo Zhong Yao Za Zhi ; 46(15): 3934-3942, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34472270

RESUMO

The present study aimed to explore the effect of Erxian Decoction on proteomics of osteoblasts stimulated by hydrogen peroxide(H_2O_2) and its protective mechanism with the H_2O_2-induced cell model of oxidative stress. The primary osteoblasts were cultured from the skulls of newborn rats(within 24 hours) and divided into a control group, a model group, a Fosamax group, and an Erxian Decoction group. Blank serum was added in the control group and model group, and the drug-containing serum was added correspondingly to the remaining two groups. After 45 hours, H_2O_(2 )stimulation was conducted for three hours except for the control group, followed by protein extraction. Nano-LC-LTQ-Orbitrap system was used for protein detection, Protein Discovery for protein identification, and SIEVE for quantitative and qualitative analysis. Furthermore, following the blocking of PI3 K signaling pathway by LY294002(10 µmol·L~(-1)), a control group, a model group, an LY294002 group, an Erxian Decoction group, and an Erxian Decoction + LY294002 group were set up to observe the effect of Erxian Decoction on cell proliferation, alkaline phosphatase(ALP) activity, and the relative expression of BMP-2, OPG, p-Akt, p-FoxO1 of osteoblasts stimulated by H_2O_2 under LY294002 intervention. The results revealed that 78 differential proteins were discovered between the Erxian Decoction group and model group, which were involved in the regulation of PI3 K/Akt, glucagon, estrogen, insulin, and other signaling pathways. LY294002 blunted the promoting effect of Erxian Decoction on osteoblast proliferation and significantly down-regulated the expression of OPG and p-FoxO1, whereas its down-regulation on the expression of BMP-2 and p-Akt was not significant. Both LY294002 and Erxian Decoction increased the ALP activity of osteoblasts, which may be related to the cell state and the cell differentiation. The above results suggest that Erxian Decoction can protect osteoblasts stimulated by H_2O_2, with the PI3 K/Akt signaling pathway as one of the internal mechanisms.


Assuntos
Peróxido de Hidrogênio , Fosfatidilinositol 3-Quinases , Animais , Medicamentos de Ervas Chinesas , Osteoblastos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais
11.
Am J Physiol Renal Physiol ; 321(4): F424-F430, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34396788

RESUMO

Chronic metabolic acidosis stimulates cell-mediated net Ca2+ efflux from bone mediated by increased osteoblastic cyclooxygenase 2, leading to prostaglandin E2-induced stimulation of receptor activator of NF-κB ligand-induced osteoclastic bone resorption. Ovarian cancer G protein-coupled receptor-1 (OGR1), an osteoblastic H+-sensing G protein-coupled receptor, is activated by acidosis and leads to increased bone resorption. As regulator of G protein signaling (RGS) proteins limit GPCR signaling, we tested whether RGS proteins themselves are regulated by metabolic acidosis. Primary osteoblasts were isolated from neonatal mouse calvariae and incubated in physiological neutral or acidic (MET) medium. Cells were collected, and RNA was extracted for real-time PCR analysis with mRNA levels normalized to ribosomal protein L13a. RGS1, RGS2, RGS3, RGS4, RGS10, RGS11, and RGS18 mRNA did not differ between MET and neutral medium; however, by 30 min, MET decreased RGS16, which persisted for 60 min and 3 h. Incubation of osteoblasts with the OGR1 inhibitor CuCl2 inhibited the MET-induced increase in RGS16 mRNA. Gallein, a specific inhibitor of Gßγ signaling, was used to determine if downstream signaling by the ßγ-subunit was critical for the response to acidosis. Gallein decreased net Ca2+ efflux from calvariae and cyclooxygenase 2 and receptor activator of NF-κB ligand gene expression from isolated osteoblasts. These results indicate that regulation of RGS16 plays an important role in modulating the response of the osteoblastic GPCR OGR1 to metabolic acidosis and subsequent stimulation of osteoclastic bone resorption.NEW & NOTEWORTHY The results presented in this study indicate that regulation of regulator of G protein signaling 16 and G protein signaling in the osteoblast plays an important role in modulating the response of osteoblastic ovarian cancer G protein-coupled receptor 1 (OGR1) to metabolic acidosis and the subsequent stimulation of osteoclastic bone resorption. Further characterization of the regulation of OGR1 in metabolic acidosis-induced bone resorption will help in understanding bone loss in acidotic patients with chronic kidney disease.


Assuntos
Acidose/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/metabolismo , Proteínas RGS/metabolismo , Animais , Bicarbonatos/administração & dosagem , Bicarbonatos/farmacologia , Dióxido de Carbono , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Concentração de Íons de Hidrogênio , Camundongos , Proteínas RGS/genética , RNA/genética , RNA/metabolismo , Xantenos/farmacologia
12.
Nat Commun ; 12(1): 4923, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389713

RESUMO

With increasing age of the population, countries across the globe are facing a substantial increase in osteoporotic fractures. Genetic association signals for fractures have been reported at the RSPO3 locus, but the causal gene and the underlying mechanism are unknown. Here we show that the fracture reducing allele at the RSPO3 locus associate with increased RSPO3 expression both at the mRNA and protein levels, increased trabecular bone mineral density and reduced risk mainly of distal forearm fractures in humans. We also demonstrate that RSPO3 is expressed in osteoprogenitor cells and osteoblasts and that osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and an important regulator of vertebral trabecular bone mass and bone strength in adult mice. Mechanistic studies revealed that RSPO3 in a cell-autonomous manner increases osteoblast proliferation and differentiation. In conclusion, RSPO3 regulates vertebral trabecular bone mass and bone strength in mice and fracture risk in humans.


Assuntos
Osso Esponjoso/metabolismo , Fraturas Ósseas/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Trombospondinas/genética , Animais , Densidade Óssea , Osso Esponjoso/lesões , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Humanos , Análise da Randomização Mendeliana/métodos , Camundongos Knockout , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Fatores de Risco , Trombospondinas/deficiência
13.
Nutrients ; 13(8)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34444982

RESUMO

Several natural compounds, such as vitamin K2, have been highlighted for their positive effects on bone metabolism. It has been proposed that skeletal disorders, such as osteoporosis, may benefit from vitamin K2-based therapies or its regular intake. However, further studies are needed to better clarify the effects of vitamin K2 in bone disorders. To this aim, we developed in vitro a three-dimensional (3D) cell culture system one step closer to the bone microenvironment based on co-culturing osteoblasts and osteoclasts precursors obtained from bone specimens and peripheral blood of the same osteoporotic patient, respectively. Such a 3-D co-culture system was more informative than the traditional 2-D cell cultures when responsiveness to vitamin K2 was analyzed, paving the way for data interpretation on single patients. Following this approach, the anabolic effects of vitamin K2 on the osteoblast counterpart were found to be correlated with bone turnover markers measured in osteoporotic patients' sera. Overall, our data suggest that co-cultured osteoblasts and osteoclast precursors from the same osteoporotic patient may be suitable to generate an in vitro 3-D experimental model that potentially reflects the individual's bone metabolism and may be useful to predict personal responsiveness to nutraceutical or drug molecules designed to positively affect bone health.


Assuntos
Osso e Ossos/efeitos dos fármacos , Nutrientes/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoporose , Medicina de Precisão/métodos , Vitamina K 2/farmacologia , Biomarcadores/sangue , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/metabolismo , Células Cultivadas , Técnicas de Cocultura/métodos , Feminino , Humanos , Masculino , Modelos Biológicos , Nutrientes/uso terapêutico , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Modelagem Computacional Específica para o Paciente , Vitamina K 2/uso terapêutico , Vitaminas/farmacologia , Vitaminas/uso terapêutico
14.
Biomed Res Int ; 2021: 1340281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34336999

RESUMO

The purpose of this study was to develop an efficient vitrification system for cryopreservation of dog skin tissues as a source of stable autologous stem cells. In this study, we performed vitrification using four different cryoprotectants, namely, ethylene glycol (EG), dimethyl-sulfoxide (Me2SO), EG plus Me2SO, and EG plus Me2SO plus sucrose, and analyzed the behaviors of cells established from warmed tissues. Tissues vitrified with 15% EG, 15% Me2SO, and 0.5 M sucrose had a normal histological appearance and the highest cell viability after cell isolation, and thus, this cocktail of cryoprotectants was used in subsequent experiments. We evaluated proliferation and apoptosis of cells derived from fresh and vitrified tissues. These cells had a normal spindle-like morphology after homogenization through subculture. Dog dermal skin stem cells (dDSSCs) derived from fresh and vitrified tissues had similar proliferation capacities, and similar percentages of these cells were positive for mesenchymal stem cell markers at passage 3. The percentage of apoptotic cell did not differ between dDSSCs derived from fresh and vitrified tissues. Real-time PCR analysis revealed that dDSSCs at passage 3 derived from fresh and vitrified tissues had similar expression levels of pluripotency (OCT4, SOX2, and NANOG), proapoptotic (BAX), and antiapoptotic (BCL2 and BIRC5) genes. Both types of dDSSCs successfully differentiated into the mesenchymal lineage (adipocytes and osteocytes) under specific conditions, and their differentiation potentials did not significantly differ. Furthermore, the mitochondrial membrane potential of dDSSCs derived from vitrified tissues was comparable with that of dDSSCs derived from fresh tissues. We conclude that vitrification of dog skin tissues using cocktail solution in combination of 15% EG, 15% Me2SO, and 0.5 M sucrose allows efficient banking of these tissues for regenerative stem cell therapy and conservation of genetic resources.


Assuntos
Células-Tronco Mesenquimais/citologia , Pele/citologia , Vitrificação , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Derme/citologia , Cães , Feminino , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo
15.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360734

RESUMO

Biomimetic design provides novel opportunities for enhancing and functionalizing biomaterials. Here we created a zirconia surface with cactus-inspired meso-scale spikes and bone-inspired nano-scale trabecular architecture and examined its biological activity in bone generation and integration. Crisscrossing laser etching successfully engraved 60 µm wide, cactus-inspired spikes on yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) with 200-300 nm trabecular bone-inspired interwoven structures on the entire surface. The height of the spikes was varied from 20 to 80 µm for optimization. Average roughness (Sa) increased from 0.10 µm (polished smooth surface) to 18.14 µm (80 µm-high spikes), while the surface area increased by up to 4.43 times. The measured dimensions of the spikes almost perfectly correlated with their estimated dimensions (R2 = 0.998). The dimensional error of forming the architecture was 1% as a coefficient of variation. Bone marrow-derived osteoblasts were cultured on a polished surface and on meso- and nano-scale hybrid textured surfaces with different spike heights. The osteoblastic differentiation was significantly promoted on the hybrid-textured surfaces compared with the polished surface, and among them the hybrid-textured surface with 40 µm-high spikes showed unparalleled performance. In vivo bone-implant integration also peaked when the hybrid-textured surface had 40 µm-high spikes. The relationships between the spike height and measures of osteoblast differentiation and the strength of bone and implant integration were non-linear. The controllable creation of meso- and nano-scale hybrid biomimetic surfaces established in this study may provide a novel technological platform and design strategy for future development of biomaterial surfaces to improve bone integration and regeneration.


Assuntos
Materiais Biomiméticos , Diferenciação Celular/efeitos dos fármacos , Nanoestruturas/química , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Zircônio , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Cactaceae , Masculino , Nanoestruturas/ultraestrutura , Osteoblastos/citologia , Ratos , Ratos Sprague-Dawley , Zircônio/química , Zircônio/farmacologia
16.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360745

RESUMO

A review of the available literature was performed in order to summarize the existing evidence between osteoblast dysfunction and clinical features in non-hereditary sclerosing bone diseases. It has been known that proliferation and migration of osteoblasts are concerted by soluble factors such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), bone morphogenetic protein (BMP) but also by signal transduction cascades such as Wnt signaling pathway. Protein kinases play also a leading role in triggering the activation of osteoblasts in this group of diseases. Post-zygotic changes in mitogen-activated protein kinase (MAPK) have been shown to be associated with sporadic cases of Melorheostosis. Serum levels of FGF and PDGF have been shown to be increased in myelofibrosis, although studies focusing on Sphingosine-1-phosphate receptor was shown to be strongly expressed in Paget disease of the bone, which may partially explain the osteoblastic hyperactivity during this condition. Pathophysiological mechanisms of osteoblasts in osteoblastic metastases have been studied much more thoroughly than in rare sclerosing syndromes: striking cellular mechanisms such as osteomimicry or complex intercellular signaling alterations have been described. Further research is needed to describe pathological mechanisms by which rare sclerosing non hereditary diseases lead to osteoblast dysfunction.


Assuntos
Sistema de Sinalização das MAP Quinases , Melorreostose/metabolismo , Osteoblastos/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Melorreostose/patologia , Osteoblastos/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo
17.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445573

RESUMO

Concentrated Growth Factors (CGF) represent new autologous (blood-derived biomaterial), attracting growing interest in the field of regenerative medicine. In this study, the chemical, structural, and biological characterization of CGF was carried out. CGF molecular characterization was performed by GC/MS to quantify small metabolites and by ELISA to measure growth factors and matrix metalloproteinases (MMPs) release; structural CGF characterization was carried out by SEM analysis and immunohistochemistry; CGF has been cultured, and its primary cells were isolated for the identification of their surface markers by flow cytometry, Western blot, and real-time PCR; finally, the osteogenic differentiation of CGF primary cells was evaluated through matrix mineralization by alizarin red staining and through mRNA quantification of osteogenic differentiation markers by real-time PCR. We found that CGF has a complex inner structure capable of influencing the release of growth factors, metabolites, and cells. These cells, which could regulate the production and release of the CGF growth factors, show stem features and are able to differentiate into osteoblasts producing a mineralized matrix. These data, taken together, highlight interesting new perspectives for the use of CGF in regenerative medicine.


Assuntos
Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Osteoblastos/citologia , Osteogênese , Células-Tronco/citologia , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Masculino , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
18.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445787

RESUMO

Mechanical stress is an important factor affecting bone tissue homeostasis. We focused on the interactions among mechanical stress, glucose uptake via glucose transporter 1 (Glut1), and the cellular energy sensor sirtuin 1 (SIRT1) in osteoblast energy metabolism, since it has been recognized that SIRT1, an NAD+-dependent deacetylase, may function as a master regulator of the mechanical stress response as well as of cellular energy metabolism (glucose metabolism). In addition, it has already been demonstrated that SIRT1 regulates the activity of the osteogenic transcription factor runt-related transcription factor 2 (Runx2). The effects of mechanical loading on cellular activities and the expressions of Glut1, SIRT1, and Runx2 were evaluated in osteoblasts and chondrocytes in a 3D cell-collagen sponge construct. Compressive mechanical loading increased osteoblast activity. Mechanical loading also significantly increased the expression of Glut1, significantly decreased the expression of SIRT1, and significantly increased the expression of Runx2 in osteoblasts in comparison with non-loaded osteoblasts. Incubation with a Glut1 inhibitor blocked mechanical stress-induced changes in SIRT1 and Runx2 in osteoblasts. In contrast with osteoblasts, the expressions of Glut1, SIRT1, and Runx2 in chondrocytes were not affected by loading. Our present study indicated that mechanical stress induced the upregulation of Glut1 following the downregulation of SIRT1 and the upregulation of Runx2 in osteoblasts but not in chondrocytes. Since SIRT1 is known to negatively regulate Runx2 activity, a mechanical stress-induced downregulation of SIRT1 may lead to the upregulation of Runx2, resulting in osteoblast differentiation. Incubation with a Glut1 inhibitor the blocked mechanical stress-induced downregulation of SIRT1 following the upregulation of Runx2, suggesting that Glut1 is necessary to mediate the responses of SIRT1 and Runx2 to mechanical loading in osteoblasts.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Sirtuína 1/metabolismo , Idoso , Diferenciação Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Humanos , NAD/metabolismo , Estresse Mecânico
19.
Phytomedicine ; 91: 153721, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34461423

RESUMO

BACKGROUND: The bark and petal of Hibiscus syriacus L. (Malvaceae) have been used to relieve pain in traditional Korean medicine. Recently, we identified anthocyanin-enriched polyphenols from the petal of H. syriacus L. (AHs) and determined its anti-melanogenic, anti-inflammatory, and anti-oxidative properties. Nevertheless, the osteogenic potential of AHs remains unknown. PURPOSE: This study was aimed to investigating the effect of AHs on osteoblast differentiation and osteogenesis in osteoblastic cell lines and zebrafish larvae. Furthermore, we investigated whether AHs ameliorates prednisolone (PDS)-induced osteoporosis. STUDY DESIGN AND METHODS: Cell viability was assessed by cellular morphology, MTT assay, and flow cytometry analysis, and osteoblast differentiation was measured alizarin red staining, alkaline phosphatase (ALP) activity, and osteoblast-specific marker expression. Osteogenic and anti-osteoporotic effects of AHs were determined in zebrafish larvae. RESULTS: AHs enhanced calcification and ALP activity concomitant with the increased expression of osterix (OSX), runt-related transcription factor 2 (RUNX2), and ALP in MC3T3-E1 preosteoblast and MG-63 osteosarcoma cells. Additionally, AHs accelerated vertebral formation and mineralization in zebrafish larvae, concurrent with the increased expression of OSX, RUNX2a, and ALP. Furthermore, PDS-induced loss of osteogenic activity and vertebral formation were restored by treatment with AHs, accompanied by a significant recovery of calcification, ALP activity, and osteogenic marker expression. Molecular docking studies showed that 16 components in AHs fit to glucagon synthase kinase-3ß (GSK-3ß); particularly, isovitexin-4'-O-glucoside most strongly binds to the peptide backbone of GSK-3ß at GLY47(O), GLY47(N), and ASN361(O), with a binding score of -7.3. Subsequently, AHs phosphorylated GSK-3ß at SER9 (an inactive form) and released ß-catenin into the nucleus. Pretreatment with FH535, a Wnt/ß-catenin inhibitor, significantly inhibited AH-induced vertebral formation in zebrafish larvae. CONCLUSION: AHs stimulate osteogenic activities through the inhibition of GSK-3ß and subsequent activation of ß-catenin, leading to anti-osteoporosis effects.


Assuntos
Antocianinas , Hibiscus , Osteogênese/efeitos dos fármacos , Osteoporose , Polifenóis , Animais , Antocianinas/farmacologia , Glicogênio Sintase Quinase 3 beta , Hibiscus/química , Simulação de Acoplamento Molecular , Osteoblastos/metabolismo , Osteoporose/tratamento farmacológico , Polifenóis/farmacologia , Via de Sinalização Wnt , Peixe-Zebra/metabolismo , beta Catenina/metabolismo
20.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361085

RESUMO

A novel aptamer-based competitive drug screening platform for osteoporosis was devised in which fluorescence-labeled, sclerostin-specific aptamers compete with compounds from selected chemical libraries for the binding of immobilized recombinant human sclerostin to achieve high-throughput screening for potential small-molecule sclerostin inhibitors and to facilitate drug repurposing and drug discovery. Of the 96 selected inhibitors and FDA-approved drugs, six were shown to result in a significant decrease in the fluorescence intensity of the aptamer, suggesting a higher affinity toward sclerostin compared with that of the aptamer. The targets of these potential sclerostin inhibitors were correlated to lipid or bone metabolism, and several of the compounds have already been shown to be potential osteogenic activators, indicating that the aptamer-based competitive drug screening assay offered a potentially reliable strategy for the discovery of target-specific new drugs. The six potential sclerostin inhibitors suppressed the level of both intracellular and/or extracellular sclerostin in mouse osteocyte IDG-SW3 and increased alkaline phosphatase activity in IDG-SW3 cells, human bone marrow-derived mesenchymal stem cells and human fetal osteoblasts hFOB1.19. Potential small-molecule drug candidates obtained in this study are expected to provide new therapeutics for osteoporosis as well as insights into the structure-activity relationship of sclerostin inhibitors for rational drug design.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Aptâmeros de Nucleotídeos/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Aptâmeros de Nucleotídeos/isolamento & purificação , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Osteoporose/metabolismo , Osteoporose/patologia
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