RESUMO
BACKGROUND: Clear cell renal cell carcinoma (ccRCC) has gender differences, with the androgen receptor (AR) linked positively with metastasis to the lung. Its linkage to ccRCC bone metastases (RBMs), however, remains unclear. METHODS: In the current study, five human RCC and five RCC bone metastasis tissues were deeply sequenced using Arraystar human circRNA V2.0 microarray. We conducted gain-of-function screening in vitro and in vivo to elucidate the AR's role in the RBM. Loss/gain-of-function was also implemented to verify the roles of related non-coding RNAs and proteins. RESULTS: We uncovered that RBM also has a gender difference showing higher AR expression may be linked to fewer RBMs, which might involve suppressing osteolytic formation. Mechanism dissection indicates that AR can decrease the circular RNA EXOC7 (circEXOC7), expression via enhancing transcription of DHX9, a regulatory protein in circRNA biogenesis. The circEXOC7 can sponge/suppress miR-149-3p resulting in suppressing the CSF1 expression by directly binding to the 3'UTR region of CSF1 mRNA. Results from clinical epidemiological surveys also found that AR has a positive correlation with miR-149-3p and a negative correlation with CSF1 in AR-positive ccRCC tissues. Preclinical studies with Balb/c nude mouse model also validated that targeting this newly verified AR/DHX9/circEXOC7/miR-149-3p/CSF1 signaling via altering circEXOC7 or AR could lead to suppressing the RBM progression. CONCLUSIONS: These data showed that AR/DHX9/circEXOC7/miR-149-3p/CSF1 signaling acts as a valuable feature in the bone metastasis of renal cancer, which may benefit in suppressing the RBM progression.
Assuntos
Neoplasias Ósseas/secundário , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , RNA Circular/genética , Receptores Androgênicos/genética , Proteínas de Transporte Vesicular/genética , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/prevenção & controle , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Osteólise/genética , Osteólise/metabolismo , RNA Circular/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismoRESUMO
Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remains to be addressed. Here, we demonstrated, after a survey of various actin-binding proteins, that the post-translational up-regulation of cofilin-1 was essential for the reduced rate of actin depolymerization morphological enlargement in senescent cells. Additionally, up-regulated cofilin-1 mainly existed in the serine-3 phosphorylated form, according to the 2D gel immunoblotting assay. The up-regulation of cofilin-1 was also detected in aged mammalian tissues. The over-expression of wild-type cofilin-1 and constitutively phosphorylated cofilin-1 promoted cell senescence with an increased cell size. Additionally, senescent phenotypes were also reduced by knockdown of total cofilin-1, which led to a decrease in phosphorylated cofilin-1. The senescence induced by the over-expression of cofilin-1 was dependent on p27Kip1 , but not on the p53 and p16INK4 expressions. The knockdown of p27Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that the over-expression of cofilin-1 induced the expression of p27Kip1 through transcriptional suppression of the transcriptional enhancer factors domain 1 (TEAD1) transcription factor. The TEAD1 transcription factor played a transrepressive role in the p27Kip1 gene promoter, as determined by the promoter deletion reporter gene assay. Interestingly, the down-regulation of TEAD1 was accompanied by the up-regulation of cofilin-1 in senescence. The knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27Kip1 and senescent phenotypes, respectively. Taken together, the current data suggest that cofilin-1/TEAD1/p27Kip1 signaling is involved in senescence-related morphological change and growth arrest.
Assuntos
Cofilina 1/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Senescência Celular , Humanos , Regulação para CimaRESUMO
Bone fracture is accompanied by trauma, mechanical stresses, and inflammation - conditions known to induce the mitochondrial permeability transition. This phenomenon occurs due to opening of the mitochondrial permeability transition pore (MPTP) promoted by cyclophilin D (CypD). MPTP opening leads to more inflammation, cell death and potentially to disruption of fracture repair. Here we performed a proof-of-concept study and tested a hypothesis that protecting mitochondria from MPTP opening via inhibition of CypD improves fracture repair. First, our in vitro experiments indicated pro-osteogenic and anti-inflammatory effects in osteoprogenitors upon CypD knock-out or pharmacological inhibition. Using a bone fracture model in mice, we observed that bone formation and biomechanical properties of repaired bones were significantly increased in CypD knock-out mice or wild type mice treated with a CypD inhibitor, NIM811, when compared to controls. These effects were evident in young male but not female mice, however in older (13 month-old) female mice bone formation was also increased during fracture repair. In contrast to global CypD knock-out, mesenchymal lineage-specific (Prx1-Cre driven) CypD deletion did not result in improved fracture repair. Our findings implicate MPTP in bone fracture and suggest systemic CypD inhibition as a modality to promote fracture repair.
Assuntos
Fraturas Ósseas , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria , Animais , Peptidil-Prolil Isomerase F , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteínas de Transporte da Membrana MitocondrialRESUMO
The androgen receptor (AR) has been linked to bladder cancer (BCa) progression, but if this involves circular RNAs (circRNAs) remains unclear. Here, we find that AR alters the levels of circRNA-FNTA (circFNTA) to increase BCa cell invasion and chemo-resistance. Mechanistically, AR represses the RNA editing gene ADAR2 via direct binding to its 5' promoter region to increase circFNTA levels, which then sponges the microRNA miR-370-3p to increase the expression of its host gene FNTA. This AR-mediated ADAR2/circFNTA/miR-370-3p/FNTA pathway then activates KRAS signaling to alter BCa cell invasion and chemo-sensitivity to cisplatin. A clinical BCa sample survey shows that circFNTA expression is elevated in BCa tissues, and results from a BCa mouse model indicate that depletion of circFNTA leads to the suppression of BCa metastases and increased cisplatin chemo-sensitivity. Together, based on our results using multiple BCa cell lines and an in vivo mouse model we suggest that targeting this newly identified AR/ADAR2/circFNTA/miR-370-3p/FNTA/KRAS axis may lead to the development of therapies to suppress BCa metastasis and to increase its chemo-sensitivity.
Assuntos
Alquil e Aril Transferases/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Circular/genética , Receptores Androgênicos/metabolismo , Neoplasias da Bexiga Urinária , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores Androgênicos/genética , Neoplasias da Bexiga Urinária/genéticaRESUMO
Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.
Assuntos
Proteínas de Homeodomínio/metabolismo , Tecido Parenquimatoso/fisiologia , Dente/citologia , Dente/embriologia , Adolescente , Animais , Feminino , Proteínas de Homeodomínio/genética , Humanos , Incisivo/citologia , Incisivo/embriologia , Camundongos Endogâmicos , Dente Serotino/citologia , Técnicas de Cultura de Órgãos , Tecido Parenquimatoso/citologia , Gravidez , Regiões Promotoras Genéticas , Regeneração , Células Estromais/fisiologia , Suínos , Fator A de Crescimento do Endotélio Vascular/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismoRESUMO
AIMS/HYPOTHESIS: We previously reported that exposure to antibodies neutralising serpin B13, a protease inhibitor expressed in exocrine pancreatic ducts, promotes beta cell proliferation, underscoring the importance of a functional relationship between exocrine and endocrine pancreas. The aim of the present study was to identify the molecular events that link inhibition of serpin B13 to islet cell proliferation. METHODS: We used an in vitro culture system consisting of isolated pancreatic islets, an extract of pancreatic ductal epithelium and a monoclonal antibody (mAb) to serpin B13 or IgG isotype control. In vivo studies involved treatment of mice with these mAbs. RESULTS: The catalytic activity of cathepsin L (CatL), a cysteine protease target of serpin B13, was augmented in the pancreas of mice injected with serpin B13 mAb. Furthermore, the addition of serpin B13 mAb to the islets, together with the pancreatic ductal epithelium lysate, caused CatL-dependent cleavage of E-cadherin and concomitant upregulation of REG genes, ultimately leading to beta cell proliferation. Direct blockade of E-cadherin with mAb also markedly enhanced REG gene induction, while chemical inhibition of ß-catenin, a binding target of E-cadherin, prevented the serpin B13 mAb-induced upregulation of REG genes. CONCLUSIONS/INTERPRETATION: Our work implicates the CatL-E-cadherin-REG pathway in the regulation of islet cell proliferation in response to signals generated in exocrine pancreatic tissue and demonstrates that protease activity may promote adaptive changes in the islets. DATA AVAILABILITY: Microarray data that support the findings of this study have been deposited in Gene Expression Omnibus (GEO) with the accession no. GSE125151.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Catepsina L/metabolismo , Ilhotas Pancreáticas/metabolismo , Serpinas/metabolismo , Animais , Anticorpos Monoclonais , Proliferação de Células , Feminino , Expressão Gênica , Células HEK293 , Humanos , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Pâncreas Exócrino/metabolismo , Ductos Pancreáticos/citologia , Transdução de Sinais , alfa 1-Antitripsina/metabolismoRESUMO
Drug delivery to bone is challenging, whereby drug distribution is commonly <1% of injected dose, despite development of several bone-targeted drug delivery systems specific to hydroxyapatite. These bone-targeted drug delivery systems still suffer from poor target cell localization within bone, as at any given time overall bone volume is far greater than acutely remodeling bone volume, which harbors relevant cell targets (osteoclasts or osteoblasts). Thus, there exists a need to target bone-acting drugs specifically to sites of bone remodeling. To address this need, this study synthesized oligo(ethylene glycol) copolymers based on a peptide with high affinity to tartrate-resistant acid phosphatase (TRAP), an enzyme deposited by osteoclasts during the bone resorption phase of bone remodeling, which provides greater specificity relevant for bone cell drugging. Gradient and random peptide orientations, as well as polymer molecular weights, were investigated. TRAP-targeted, high molecular weight (Mn) random copolymers exhibited superior accumulation in remodeling bone, where fracture accumulation was observed for at least 1 week and accounted for 14% of tissue distribution. Intermediate and low Mn random copolymer accumulation was lower, indicating residence time depends on Mn. High Mn gradient polymers were cleared, with only 2% persisting at fractures after 1 week, suggesting TRAP binding depends on peptide density. Peptide density and Mn are easily modified in this versatile targeting platform, which can be applied to a range of bone drug delivery applications.
Assuntos
Sistemas de Liberação de Medicamentos , Peptídeos/metabolismo , Polímeros/farmacocinética , Acrilamida/química , Animais , Remodelação Óssea , Células Cultivadas , Feminino , Corantes Fluorescentes/química , Humanos , Masculino , Camundongos Endogâmicos C57BL , Peso Molecular , Osteoclastos/enzimologia , Peptídeos/química , Polímeros/química , Fosfatase Ácida Resistente a Tartarato/metabolismo , Distribuição TecidualRESUMO
Despite several decades of progress, bone-specific drug delivery is still a major challenge. Current bone-acting drugs require high-dose systemic administration which decreases therapeutic efficacy and increases off-target tissue effects. Here, a bone-targeted nanoparticle (NP) delivery system for a ß-catenin agonist, 3-amino-6-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide, a glycogen synthase kinase 3 beta (GSK-3ß) inhibitor, was developed to enhance fracture healing. The GSK-3ß inhibitor loading capacity was found to be 15 wt % within highly stable poly(styrene-alt-maleic anhydride)-b-poly(styrene) NPs, resulting in â¼50 nm particles with â¼â¯-30 mV surface charge. A peptide with high affinity for tartrate-resistant acid phosphatase (TRAP), a protein deposited by osteoclasts on bone resorptive surfaces, was introduced to the NP corona to achieve preferential delivery to fractured bone. Targeted NPs showed improved pharmacokinetic profiles with greater accumulation at fractured bone, accompanied by significant uptake in regenerative cell types (mesenchymal stem cells (MSCs) and osteoblasts). MSCs treated with drug-loaded NPs in vitro exhibited 2-fold greater ß-catenin signaling than free drug that was sustained for 5 days. To verify similar activity in vivo, TOPGAL reporter mice bearing fractures were treated with targeted GSK-3ß inhibitor-loaded NPs. Robust ß-galactosidase activity was observed in fracture callus and periosteum treated with targeted carriers versus controls, indicating potent ß-catenin activation during the healing process. Enhanced bone formation and microarchitecture were observed in mice treated with GSK-3ß inhibitor delivered via TRAP-binding peptide-targeted NPs. Specifically, increased bone bridging, â¼4-fold greater torsional rigidity, and greater volumes of newly deposited bone were observed 28 days after treatment, indicating expedited fracture healing.
Assuntos
Portadores de Fármacos/química , Consolidação da Fratura/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Nanopartículas/química , Peptídeos/química , Inibidores de Proteínas Quinases/administração & dosagem , beta Catenina/agonistas , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Células Cultivadas , Sistemas de Liberação de Medicamentos , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , beta Catenina/metabolismoRESUMO
Organ development requires complex signaling by cells in different tissues. Epithelium and mesenchyme interactions are crucial for the development of skin, hair follicles, kidney, lungs, prostate, major glands, and teeth. Despite myriad literature on cell-cell interactions and ligand-receptor binding, the roles of extracellular vesicles in epithelium-mesenchyme interactions during organogenesis are poorly understood. Here, we discovered that â¼100 nm exosomes were secreted by the epithelium and mesenchyme of a developing tooth organ and diffused through the basement membrane. Exosomes were entocytosed by epithelium or mesenchyme cells with preference by reciprocal cells rather than self-uptake. Exosomes reciprocally evoked cell differentiation and matrix synthesis: epithelium exosomes induce mesenchyme cells to produce dentin sialoprotein and undergo mineralization, whereas mesenchyme exosomes induce epithelium cells to produce basement membrane components, ameloblastin and amelogenenin. Attenuated exosomal secretion by Rab27a/b knockdown or GW4869 disrupted the basement membrane and reduced enamel and dentin production in organ culture and reduced matrix synthesis and the size of the cervical loop, which harbors epithelium stem cells, in Rab27aash/ash mutant mice. We then profiled exosomal constituents including miRNAs and peptides and further crossed all epithelium exosomal miRNAs with literature-known miRNA Wnt regulators. Epithelium exosome-derived miR135a activated Wnt/ß-catenin signaling and escalated mesenchymal production of dentin matrix proteins, partially reversible by Antago-miR135a attenuation. Our results suggest that exosomes may mediate epithelium-mesenchyme crosstalk in organ development, suggesting that these vesicles and/or the molecular contents they are transporting may be interventional targets for treatment of diseases or regeneration of tissues.
Assuntos
Epitélio/metabolismo , Exossomos/metabolismo , Mesoderma/metabolismo , Animais , Western Blotting , Diferenciação Celular , Exossomos/genética , Imunofluorescência , Camundongos , MicroRNAs/genética , Microscopia Eletrônica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Stress during prenatal development is correlated with detrimental cognitive and behavioral outcomes in offspring. However, the long-term impact of prenatal stress (PS) and disrupted glucocorticoid signaling on bone mass and strength is not understood. In contrast, the detrimental effect of lead (Pb) on skeletal health is well documented. As stress and Pb act on common biological targets via glucocorticoid signaling pathways and co-occur in the environment, this study first sought to assess the combined effect of stress and Pb on bone quality in association with alterations in glucocorticoid signaling. Bone parameters were evaluated using microCT, histomorphometry, and strength determination in 8-month-old male mouse offspring subjected to PS on gestational days 16 and 17, lifetime Pb exposure (100 p.p.m. Pb in drinking water), or to both. Pb reduced trabecular bone mass and, when combined with PS, Pb unmasked an exaggerated decrement in bone mass and tensile strength. Next, to characterize a mechanism of glucocorticoid effect on bone, prednisolone was implanted subcutaneously (controlled-release pellet, 5 mg·kg-1 per day) in 5-month-old mice that decreased osteoblastic activity and increased sclerostin and leptin levels. Furthermore, the synthetic glucocorticoid dexamethasone alters the anabolic Wnt signaling pathway. The Wnt pathway inhibitor sclerostin has several glucocorticoid response elements, and dexamethasone administration to osteoblastic cells induces sclerostin expression. Dexamethasone treatment of isolated bone marrow cells decreased bone nodule formation, whereas removal of sclerostin protected against this decrement in mineralization. Collectively, these findings suggest that bone loss associated with steroid-induced osteoporosis is a consequence of sclerostin-mediated restriction of Wnt signaling, which may mechanistically facilitate glucocorticoid toxicity in bone.
RESUMO
Tissue regeneration using stem cell-based transplantation faces many hurdles. Alternatively, therapeutically exploiting endogenous stem cells to regenerate injured or diseased tissue may circumvent these challenges. Here we show resident fibrocartilage stem cells (FCSCs) can be used to regenerate and repair cartilage. We identify FCSCs residing within the superficial zone niche in the temporomandibular joint (TMJ) condyle. A single FCSC spontaneously generates a cartilage anlage, remodels into bone and organizes a haematopoietic microenvironment. Wnt signals deplete the reservoir of FCSCs and cause cartilage degeneration. We also show that intra-articular treatment with the Wnt inhibitor sclerostin sustains the FCSC pool and regenerates cartilage in a TMJ injury model. We demonstrate the promise of exploiting resident FCSCs as a regenerative therapeutic strategy to substitute cell transplantation that could be beneficial for patients suffering from fibrocartilage injury and disease. These data prompt the examination of utilizing this strategy for other musculoskeletal tissues.
Assuntos
Fibrocartilagem/citologia , Regeneração , Transplante de Células-Tronco , Células-Tronco/citologia , Articulação Temporomandibular/patologia , Articulação Temporomandibular/fisiopatologia , Cicatrização , Proteínas Adaptadoras de Transdução de Sinal , Animais , Osso e Ossos/patologia , Diferenciação Celular , Condrócitos/patologia , Glicoproteínas/metabolismo , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos Nus , Modelos Biológicos , Coelhos , Ratos Sprague-Dawley , Nicho de Células-Tronco , Proteínas Wnt/metabolismo , Via de Sinalização WntRESUMO
The suture mesenchyme serves as a growth centre for calvarial morphogenesis and has been postulated to act as the niche for skeletal stem cells. Aberrant gene regulation causes suture dysmorphogenesis resulting in craniosynostosis, one of the most common craniofacial deformities. Owing to various limitations, especially the lack of suture stem cell isolation, reconstruction of large craniofacial bone defects remains highly challenging. Here we provide the first evidence for an Axin2-expressing stem cell population with long-term self-renewing, clonal expanding and differentiating abilities during calvarial development and homeostastic maintenance. These cells, which reside in the suture midline, contribute directly to injury repair and skeletal regeneration in a cell autonomous fashion. Our findings demonstrate their true identity as skeletal stem cells with innate capacities to replace the damaged skeleton in cell-based therapy, and permit further elucidation of the stem cell-mediated craniofacial skeletogenesis, leading to revealing the complex nature of congenital disease and regenerative medicine.
Assuntos
Desenvolvimento Ósseo/fisiologia , Ossos Faciais/citologia , Células-Tronco Mesenquimais/fisiologia , Regeneração/fisiologia , Crânio/fisiologia , Animais , Diferenciação Celular , Ossos Faciais/crescimento & desenvolvimento , Transplante de Células-Tronco Mesenquimais , CamundongosRESUMO
BACKGROUND: TGF-ß1 regulates bone metabolism and mediates bone turnover during postmenopause. Sclerostin negatively regulates Wnt signaling pathway and also has an important role in postmenopausal bone loss. Little is known about the relationship between serum TGF-ß1 and sclerostin during menopause. METHODS: We compared serum levels of TGF-ß1 and sclerostin in pre- and postmenopausal women and assessed the potential correlations of these levels with each other and with serum levels of bone turnover markers and bone mineral density. RESULTS: A total of 176 women (58 premenopausal, 62 early postmenopausal, and 56 late postmenopausal) were included in this study. Serum TGF-ß1 level was significantly higher in early postmenopausal women compared with premenopausal (32.0±7.19 vs. 26.55±6.67 ng/ml, p=0.01) and late postmenopausal (32.0±7.19 vs. 28.65±7.70 pg/ml, p=0.031) women, and no significant differences in serum sclerostin levels were observed among the 3 groups. There was a significant negative correlation between TGF-ß1 and sclerostin in early postmenopausal women, but not in other groups of women. Based on multiple regression analysis, only TGF-ß1 (ß=-0.362; p=0.007) was an independent predictor of sclerostin during early postmenopause. CONCLUSIONS: Our findings suggest that serum TGF-ß1 level increases during postmenopause and declines in old age. Sclerostin production is inhibited by TGF-ß1 during early postmenopause.
Assuntos
Proteínas Morfogenéticas Ósseas/sangue , Pós-Menopausa , Fator de Crescimento Transformador beta1/sangue , Proteínas Adaptadoras de Transdução de Sinal , Estudos de Coortes , Estudos Transversais , Feminino , Marcadores Genéticos , Humanos , Pessoa de Meia-IdadeRESUMO
WNT/ß-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of ß-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which ß-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/ß-CATENIN signaling in chondrocytes. Specifically, we found that ß-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis.
Assuntos
Cartilagem Articular/crescimento & desenvolvimento , Diferenciação Celular , Condrócitos/metabolismo , Condrócitos/patologia , Proteína Rica em Cisteína 61/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Cartilagem Articular/patologia , Condrogênese , Epífises/patologia , Menisco/patologia , Camundongos , Membrana Sinovial/patologia , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
The heavy metal lead (Pb) has a deleterious effect on skeletal health. Because bone mass is maintained through a balance of bone formation and resorption, it is important to understand the effect of Pb levels on osteoblastic and osteoclastic activity. Pb exposure is associated with low bone mass in animal models and human populations; however, the correlation between Pb dosing and corresponding bone mass has been poorly explored. Thus, mice were exposed to increasing Pb and at higher levels (500 ppm), there was unexpectedly an increase in femur-tibial bone mass by 3 months of age. This is contrary to several studies alluded to earlier. Increased bone volume (BV) was accompanied by a significant increase in cortical thickness of the femur and trabecular bone that extended beyond the epiphyseal area into the marrow cavity. Subsequent evaluations revealed an increase in osteoclast numbers with high Pb exposure, but a deficiency in osteoclastic activity. These findings were substantiated by observed increases in levels of the resorption-altering hormones calcitonin and estrogen. In addition we found that pro-osteoclastic nuclear factor-kappa beta (NF-κB) pathway activity was dose dependently elevated with Pb, both in vivo and in vitro. However, the ability of osteoclasts to resorb bone was depressed in the presence of Pb in media and within test bone wafers. These findings indicate that exposure to high Pb levels disrupts early life bone accrual that may involve a disruption of osteoclast activity. This study accentuates the dose dependent variation in Pb exposure and consequent effects on skeletal health.
Assuntos
Densidade Óssea/efeitos dos fármacos , Chumbo/toxicidade , Osteoclastos/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Envelhecimento , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/fisiologia , Osteoclastos/fisiologia , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Resistência à Tração/efeitos dos fármacosRESUMO
Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/ß-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the ß-catenin pathway using a pharmacological inhibitor of GSK-3ß ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFß canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/ß-catenin pathway.
Assuntos
Poluentes Ambientais/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Chumbo/toxicidade , Osteogênese/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Osso e Ossos/anatomia & histologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Linhagem Celular , Células Cultivadas , Exposição Ambiental/efeitos adversos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND: Lead (Pb) exposure and obesity are co-occurring risk factors for decreased bone mass in the young, particularly in low socioeconomic communities. OBJECTIVES: The goal of this study was to determine whether the comorbidities of Pb exposure and high-fat diet-induced obesity amplify skeletal deficits independently associated with each of these risk factors, and to explore associated mechanisms of the observed deficiencies. METHODS: Five-week-old male C57BL/6J mice were placed on low-fat (10% kcal, LFD) or high-fat (60% kcal, HFD) diets for 12 weeks. Mice were exposed to lifetime Pb (50 ppm) through drinking water. RESULTS: HFD was associated with increased body mass and glucose intolerance. Both HFD and Pb increased fasting glucose and serum leptin levels. Pb and HFD each reduced trabecular bone quality and together had a further detrimental effect on these bone parameters. Mechanical bone properties of strength were depressed in Pb-exposed bones, but HFD had no significant effect. Both Pb and HFD altered progenitor cell differentiation, promoting osteoclastogenesis and increasing adipogenesis while suppressing osteoblastogenesis. In support of this lineage shift being mediated through altered Wnt signaling, Pb and non-esterified fatty acids in MC3T3 cells increased in vitro PPAR-γ activity and inhibited ß-catenin activity. Combining Pb and non-esterified fatty acids enhanced these effects. CONCLUSIONS: Pb and HFD produced selective deficits in bone accrual that were associated with alterations in progenitor cell activity that may involve reduced Wnt signaling. This study emphasizes the need to assess toxicants together with other risk factors relevant to human health and disease.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Poluentes Ambientais/toxicidade , Chumbo/toxicidade , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/induzido quimicamente , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
PGE2 exerts anabolic and catabolic effects on bone through the discrete actions of four prostanoid receptors (EP1-4). We have previously demonstrated that loss EP1 accelerates fracture repair by enhancing bone formation. In the present study we defined the role of EP1 in bone maintenance and homeostasis during aging and in response to ovariectomy. The femur and L4 vertebrae of wild type (WT) and EP1(-/-) mice were examined at 2-months, 6-months, and 1-year of age, and in WT and EP1(-/-) mice following ovariectomy (OVX) or sham surgery. Bone volume fraction, trabecular architecture and mechanical properties were maintained during aging in EP1(-/-) mice to a greater degree than age-matched WT mice. Moreover, significant increases in bone formation rate (BFR) (+60%) and mineral apposition rate (MAR) (+50%) were observed in EP1(-/-), relative to WT, while no change in osteoclast number and osteoclast surface were observed. Following OVX, loss of EP1 was protective against bone loss in both femur and L4 vertebrae, with increased bone volume/total volume (BV/TV) (+32% in femur) and max load at failure (+10% in femur) relative to WT OVX, likely resulting from the increased bone formation rate that was observed in these mice. Taken together these studies identify inhibition of EP1 as a potential therapeutic approach to suppress bone loss in aged or post-menopausal patients.
Assuntos
Osso e Ossos/metabolismo , Receptores de Prostaglandina E Subtipo EP1/genética , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Envelhecimento , Animais , Diferenciação Celular , Feminino , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Osteoclastos/citologia , Ovariectomia , Transdução de Sinais , Estresse Mecânico , Microtomografia por Raio-XRESUMO
There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb-exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb-treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased ß-catenin levels. Reduction of ß-catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb-exposed mice throughout fracture healing. ß-catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in ß-catenin signaling, suggesting the Wnt/ß-catenin as a pivotal pathway in the influence of Pb on fracture repair.