RESUMO
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
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
TAK1 is a MAP3K that mediates non-canonical TGF-ß and BMP signaling. During the embryonic period, TAK1 is essential for cartilage and joint development as deletion of Tak1 in chondro-osteo progenitor cells leads to severe chondrodysplasia with defects in both chondrocyte proliferation and maturation. We have investigated the role of TAK1 in committed chondrocytes during early postnatal development. Using the Col2a1-CreER(T2); Tak1(f/f) mouse model, we induced deletion of Tak1 at postnatal day 7 and characterized the skeletal phenotypes of these mice at 1 and 3 months of age. Mice with chondrocyte-specific Tak1 deletion exhibited severe growth retardation and reduced proteoglycan and type II collagen content in the extracellular matrix of the articular cartilage. We found reduced Col2a1 and Acan expression, but increased Mmp13 and Adamts5 expression, in Tak1-deficient chondrocytes along with reduced expression of the SOX trio of transcription factors, SOX9, SOX5 and SOX6. In vitro, BMP2 stimulated Sox9 gene expression and Sox9 promoter activity. These effects were reduced; however, following Tak1 deletion or treatment with a TAK1 kinase inhibitor. TAK1 affects both canonical and non-canonical BMP signal transduction and we found that both of these pathways contribute to BMP2-mediated Sox9 promoter activation. Additionally, we found that ATF2 directly binds the Sox9 promoter in response to BMP signaling and that this effect is dependent upon TAK1 kinase activity. These novel findings establish that TAK1 contributes to BMP2-mediated Sox9 gene expression and is essential for the postnatal development of normal growth plate and articular cartilages.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Lâmina de Crescimento/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Fatores de Transcrição SOX9/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Animais , Proteína Morfogenética Óssea 2/fisiologia , Cartilagem Articular/citologia , Cartilagem Articular/crescimento & desenvolvimento , Proliferação de Células , Células Cultivadas , Matriz Extracelular/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Lâmina de Crescimento/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ligação Proteica , Proteoglicanas/metabolismo , Fatores de Transcrição SOX9/genéticaRESUMO
Vascular invasion into the normally avascular articular surface is a hallmark of advanced osteoarthritis (OA). In this study, we demonstrated that the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), an anti-angiogenic factor, was present at high levels in normal articular chondrocytes, and was drastically decreased shortly after destabilization of the medial meniscus (DMM). We also investigated the anti-angiogenic properties of TIMP2 via knockout. We hypothesized that the loss of TIMP2 could accelerate osteoarthritis development via promotion of angiogenesis. Loss of TIMP2 led to increased periarticular vascular formation 1 month post DMM, compared to wild-type mice, and did so without altering the expression pattern of matrix metalloproteinases and vascular endothelial growth factors. The increased vascularization eventually resulted in a severe degeneration of the articular surface by 4 months post DMM. Our findings suggest that reduction of TIMP2 levels and increased angiogenesis are possible primary events in OA progression. Inhibiting or delaying angiogenesis by TIMP2 expression or other anti-angiogenic therapies could improve OA prevention and treatment.
Assuntos
Meniscos Tibiais/irrigação sanguínea , Neovascularização Patológica/metabolismo , Osteoartrite/fisiopatologia , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Animais , Modelos Animais de Doenças , Metaloproteinases da Matriz , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Inibidor Tecidual de Metaloproteinase-2/genética , Fatores de Crescimento do Endotélio VascularRESUMO
OBJECTIVE: To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor beta (TGFbeta) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation. METHODS: Joint disease in Smad3-knockout (Smad3(-/-)) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNA-binding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3(-/-) mice. RESULTS: In Smad3(-/-) mice, an end-stage OA phenotype gradually developed. TGFbeta-activated kinase 1 (TAK1)/ATF-2 signaling was disrupted in Smad3(-/-) mouse chondrocytes at the level of p38 MAP kinase (MAPK) activation, resulting in reduced ATF-2 phosphorylation and transcriptional activity. Reintroduction of Smad3 into Smad3(-/-) cells restored the normal p38 response to TGFbeta. Phosphorylated p38 formed a complex with Smad3 by binding to a portion of Smad3 containing both the MAD homology 1 and linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAPK phosphatase 1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild-type and Smad3(-/-) chondrocytes. P38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms alpha, beta, and gamma, but not delta. CONCLUSION: Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1/ATF-2 signaling, most likely by disrupting the Smad3-phosphorylated p38 complex, thereby promoting p38 dephosphorylation and inactivation by MKP-1. ATF-2 and p38 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Condrócitos/patologia , Osteoartrite/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator 2 Ativador da Transcrição/genética , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Progressão da Doença , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Osteoartrite/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
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 tumor suppressor p21WAF/CIP1 mediates the proliferation arrest via p53-dependent or -independent gene transactivation following distinct environmental stresses. In this study, we show that direct destabilization of the actin cytoskeleton by actin-targeting reagents leads to a p53-independent up-regulation of p21WAF/CIP1. The actin-targeting agent cytochalasin B (10 microM) quickly disrupted the actin cytoskeleton of p53 wild-type and p53-null cells accompanied by up-regulation of p21WAF/CIP1. Nevertheless, both total p53 and ser-15 phosphorylated p53 were not accumulated concomitantly, compared to the effect caused by ionizing irradiation. P53-independent up-regulation of p21WAF/CIP1 was also observed by two other actin-targeting agents cytochalasin D and latrunculin B, but not by the microtubule inhibitor colcemid. Furthermore, we showed that p21WAF/CIP1 mRNA level was not increased, whereas the protein degradation was delayed. A reduction of ubiquitination for p21WAF/CIP1 protein was detected using immunoprecipitation/immunoblot analysis. Up-regulation of p21WAF/CIP1 was not associated with cytotoxicity induced by cytochalasin B that influenced DNA integrity and plating efficiency only after 24 h of treatment. In addition, up-regulated p21WAF/CIP1 was accompanied by reduction of phosphorylation on retinoblastoma (Rb) protein in p53-null cells, implying that p21WAF/CIP1 might in part account for the molecular regulation of cytochalasin B induced G1 phase arrest. Together, current results suggest that p21WAF/CIP1 level can be mediated by actin organization in the absence of p53 via a post-transcriptional machinery, and it may contribute to the growth ablation by agents targeting the actin cytoskeleton.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Citoesqueleto/efeitos dos fármacos , Genes p53 , Processamento Pós-Transcricional do RNA , Actinas/metabolismo , Adenocarcinoma/genética , Neoplasias Ósseas/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Ciclo Celular , Linhagem Celular Tumoral , Citocalasina D/farmacologia , DNA de Neoplasias/genética , Genes p53/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Osteossarcoma/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Tiazolidinas/farmacologia , Ubiquitina/metabolismoRESUMO
UNLABELLED: The AHR mediates many of the toxicological effects of aromatic hydrocarbons. We show that AHR expression in osteoblasts parallels the induction of early bone-specific genes involved in maturation. The AHR may not only mediate the effects of toxicants, but with an as yet unidentified ligand, be involved in the differentiation pathways of osteoblasts. INTRODUCTION: Metabolic bone diseases arise as a result of an imbalance in bone cell activities. Recent evidence suggests that environmental toxicants may be contributing factors altering these activities. One candidate molecule implicated in mediating the toxic effects of exogenous compounds is the aryl hydrocarbon receptor (AHR). MATERIALS AND METHODS: Osteoblasts isolated from neonatal rat calvaria were analyzed for AHR expression by quantitative PCR, Western blot, and immunohistochemistry. In addition, AHR activation was evaluated by electromobility gel shift assay and fluorescence microscopy. RESULTS: Our findings showed AHR expression in mature osteoblasts in vivo. The pattern of AHR expression peaks after alkaline phosphatase and before induction of osteocalcin. We first show that AHR functions as a transactivating receptor in osteoblasts, as evidenced by its ligand-dependent migration to the nucleus and its association with known dioxin response elements. AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) mediated the induction of cytochrome p450 1A1 and cycloxygenase-2 protein levels. This effect could be inhibited by the potent AHR antagonist, 3'4 methoxynitroflavone. Furthermore, lead treatment of osteoblasts upregulates the expression of AHR mRNA and protein levels, supporting a novel mechanism whereby lead in the skeleton may increase the sensitivity of bone cells to toxicant exposure. CONCLUSIONS: These data imply that the AHR mediates the effects of aromatic toxicants on bone and that AHR expression is regulated during osteoblast differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Substâncias Perigosas/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Ativação Enzimática/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Ratos , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Ativação Transcricional/genética , Regulação para CimaRESUMO
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
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
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
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
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
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.