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Quantification of in vitro osteoclast cultures (e.g. cell number) often relies on manual counting methods. These approaches are labour intensive, time consuming and result in substantial inter- and intra-user variability. This study aimed to develop and validate an automated workflow to robustly quantify in vitro osteoclast cultures. Using ilastik, a machine learning-based image analysis software, images of tartrate resistant acid phosphatase-stained mouse osteoclasts cultured on dentine discs were used to train the ilastik-based algorithm. Assessment of algorithm training showed that osteoclast numbers strongly correlated between manual- and automatically quantified values (r = 0.87). Osteoclasts were consistently faithfully segmented by the model when visually compared to the original reflective light images. The ability of this method to detect changes in osteoclast number in response to different treatments was validated using zoledronate, ticagrelor, and co-culture with MCF7 breast cancer cells. Manual and automated counting methods detected a 70% reduction (p < 0.05) in osteoclast number, when cultured with 10 nM zoledronate and a dose-dependent decrease with 1-10 µM ticagrelor (p < 0.05). Co-culture with MCF7 cells increased osteoclast number by ≥ 50% irrespective of quantification method. Overall, an automated image segmentation and analysis workflow, which consistently and sensitively identified in vitro osteoclasts, was developed. Advantages of this workflow are (1) significantly reduction in user variability of endpoint measurements (93%) and analysis time (80%); (2) detection of osteoclasts cultured on different substrates from different species; and (3) easy to use and freely available to use along with tutorial resources.
Assuntos
Reabsorção Óssea , Osteoclastos , Camundongos , Animais , Ácido Zoledrônico , Ticagrelor , Técnicas de Cocultura , Células Cultivadas , Fosfatase Ácida/análise , Fosfatase Ácida Resistente a Tartarato , Diferenciação CelularRESUMO
Romosozumab (EVENITY™ [romosozumab-aqqg in the US]) is a humanized monoclonal antibody that inhibits sclerostin and has been approved in several countries for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Sclerostin is expressed in bone and aortic vascular smooth muscle (AVSM). Its function in AVSM is unclear but it has been proposed to inhibit vascular calcification, atheroprogression, and inflammation. An increased incidence of positively adjudicated serious cardiovascular adverse events driven by an increase in myocardial infarction and stroke was observed in romosozumab-treated subjects in a clinical trial comparing alendronate with romosozumab (ARCH; NCT01631214) but not in a placebo-controlled trial (FRAME; NCT01575834). To investigate the effects of sclerostin inhibition with sclerostin antibody on the cardiovascular system, a comprehensive nonclinical toxicology package with additional cardiovascular studies was conducted. Although pharmacodynamic effects were observed in the bone, there were no functional, morphological, or transcriptional effects on the cardiovascular system in animal models in the presence or absence of atherosclerosis. These nonclinical studies did not identify evidence that proves the association between sclerostin inhibition and adverse cardiovascular function, increased cardiovascular calcification, and atheroprogression.
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Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Conservadores da Densidade Óssea/farmacologia , Sistema Cardiovascular/efeitos dos fármacos , Animais , Anticorpos Monoclonais/uso terapêutico , Conservadores da Densidade Óssea/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Feminino , Fraturas Ósseas/prevenção & controle , Humanos , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Osteoporose/tratamento farmacológico , Ratos Sprague-Dawley , RiscoRESUMO
Dickkopf (Dkk) family proteins are important regulators of Wnt signaling pathways, which play key roles in many essential biological processes. Here, we report the first detailed structural and dynamics study of a full-length mature Dkk protein (Dkk4, residues 19-224), including determination of the first atomic-resolution structure for the N-terminal cysteine-rich domain (CRD1) conserved among Dkk proteins. We discovered that CRD1 has significant structural homology to the Dkk C-terminal cysteine-rich domain (CRD2), pointing to multiple gene duplication events during Dkk family evolution. We also show that Dkk4 consists of two independent folded domains (CRD1 and CRD2) joined by a highly flexible, nonstructured linker. Similarly, the N-terminal region preceding CRD1 and containing a highly conserved NXI(R/K) sequence motif was shown to be dynamic and highly flexible. We demonstrate that Dkk4 CRD2 mediates high-affinity binding to both the E1E2 region of low-density lipoprotein receptor-related protein 6 (LRP6 E1E2) and the Kremen1 (Krm1) extracellular domain. In contrast, the N-terminal region alone bound with only moderate affinity to LRP6 E1E2, consistent with binding via the conserved NXI(R/K) motif, but did not interact with Krm proteins. We also confirmed that Dkk and Krm family proteins function synergistically to inhibit Wnt signaling. Insights provided by our integrated structural, dynamics, interaction, and functional studies have allowed us to refine the model of synergistic regulation of Wnt signaling by Dkk proteins. Our results indicate the potential for the formation of a diverse range of ternary complexes comprising Dkk, Krm, and LRP5/6 proteins, allowing fine-tuning of Wnt-dependent signaling.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Ligação Proteica , Domínios Proteicos , Alinhamento de Sequência , Via de Sinalização WntRESUMO
LRP5 and LRP6 are proteins predicted to contain four six-bladed ß-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the ß-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another ß-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína Wnt1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Proteínas Morfogenéticas Ósseas/genética , Calorimetria , Linhagem Celular , Cristalografia , DNA Complementar , Marcadores Genéticos/genética , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Proteína Wnt1/genéticaRESUMO
The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects.
Assuntos
Interleucina-6 , Osteogênese , Colinérgicos , Fibras Colinérgicas , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologiaRESUMO
Sclerostin antibody romosozumab (EVENITY™, romosozumab-aqqg) has a dual mechanism of action on bone, increasing bone formation and decreasing bone resorption, leading to increases in bone mass and strength, and a decreased risk of fracture, and has been approved for osteoporosis treatment in patients with high risk of fragility fractures. The bone formation aspect of the response to sclerostin antibody treatment has thus far been best described as having two phases: an immediate and robust phase of anabolic bone formation, followed by a long-term response characterized by attenuated bone accrual. We herein test the hypothesis that following the immediate pharmacologic anabolic response, the changes in bone morphology result in altered (lesser) mechanical stimulation of the resident osteocytes, initiating a negative feedback signal quantifiable by a reduced osteocyte signaling response to load. This potential desensitization of the osteocytic network is probed via a novel ex vivo assessment of intracellular calcium (Ca2+) oscillations in osteocytes below the anteromedial surface of murine tibiae subjected to load after short-term (2 weeks) or long-term (8 weeks) treatment with sclerostin antibody or vehicle control. We found that for both equivalent load levels and equivalent strain levels, osteocyte Ca2+ dynamics are maintained between tibiae from the control mice and the mice that received long-term sclerostin antibody treatment. Furthermore, under matched strain environments, we found that short-term sclerostin antibody treatment results in a reduction of both the number of responsive cells and the speed of their responses, which we attribute largely to the probability that the observed cells in the short-term group are relatively immature osteocytes embedded during initial pharmacologic anabolism. Within this study, we demonstrate that osteocytes embedded following long-term sclerostin antibody treatment exhibit localized Ca2+ signaling akin to those of mature osteocytes from the vehicle group, and thus, systemic attenuation of responses such as circulating P1NP and bone formation rates likely occur as a result of processes downstream of osteocyte Ca2+ signaling.
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Proteínas Morfogenéticas Ósseas , Osteócitos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Densidade Óssea , Proteínas Morfogenéticas Ósseas/metabolismo , Marcadores Genéticos , Humanos , Camundongos , Osteócitos/metabolismoRESUMO
BACKGROUND: Sclerosteosis, a severe autosomal recessive sclerosing skeletal dysplasia characterised by excessive bone formation, is caused by absence of sclerostin, a negative regulator of bone formation that binds LRP5/6 Wnt co-receptors. Current treatment is limited to surgical management of symptoms arising from bone overgrowth. This study investigated the effectiveness of sclerostin replacement therapy in a mouse model of sclerosteosis. METHODS: Recombinant wild type mouse sclerostin (mScl) and novel mScl fusion proteins containing a C-terminal human Fc (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), were produced and purified using mammalian expression and standard chromatography methods. In vitro functionality and efficacy of the recombinant proteins were evaluated using three independent biophysical techniques and an in vitro bone nodule formation assay. Pharmacokinetic properties of the proteins were investigated in vivo following a single administration to young female wild type (WT) or SOST knock out (SOST-/-) mice. In a six week proof-of-concept in vivo study, young female WT or SOST-/- mice were treated with 10â¯mg/kg mScl hFc or mScl hFc PD (weekly), or 4.4â¯mg/kg mScl (daily). The effect of recombinant sclerostin on femoral cortical and trabecular bone parameters were assessed by micro computed tomography (µCT). RESULTS: Recombinant mScl proteins bound to the extracellular domain of the Wnt co-receptor LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in vitro. Pharmacokinetic assessment following a single dose administered to WT or SOST-/- mice indicated the presence of hFc increased protein half-life from less than 5â¯min to at least 1.5 days. Treatment with mScl hFc PD over a six week period resulted in modest but significant reductions in trabecular volumetric bone mineral density (vBMD) and bone volume fraction (BV/TV), of 20% and 15%, respectively. CONCLUSION: Administration of recombinant mScl hFc PD partially corrected the high bone mass phenotype in SOST-/- mice, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/- mouse model of sclerosteosis. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: These findings support the concept that exogenous sclerostin can reduce bone mass, however the modest efficacy suggests that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored.
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Irradiation therapy causes bone deterioration and increased risk for skeletal-related events. Irradiation interferes with trabecular architecture through increased osteoclastic activity, decreased osteoblastic activity, and increased adipocyte expansion in the bone marrow (BM), which further compounds bone-related disease. Neutralizing antibodies to sclerostin (Scl-Ab) increase bone mass and strength by increasing bone formation and reducing bone resorption. We hypothesized that treatment with Scl-Ab would attenuate the adverse effects of irradiation by increasing bone volume and decreasing BM adipose tissue (BMAT), resulting in better quality bone. In this study, 12-week-old female C57BL/6J mice were exposed to 6 Gy whole-body irradiation or were non-irradiated, then administered Scl-Ab (25 mg/kg) or vehicle weekly for 5 weeks. Femoral µCT analysis confirmed that the overall effect of IR significantly decreased trabecular bone volume/total volume (Tb.BV/TV) (2-way ANOVA, p < 0.0001) with a -43.8% loss in Tb.BV/TV in the IR control group. Scl-Ab independently increased Tb.BV/TV by 3.07-fold in non-irradiated and 3.6-fold in irradiated mice (2-way ANOVA, p < 0.0001). Irradiation did not affect cortical parameters, although Scl-Ab increased cortical thickness and area significantly in both irradiated and non-irradiated mice (2-way ANOVA, p < 0.0001). Femoral mechanical testing confirmed Scl-Ab significantly increased bending rigidity and ultimate moment independently of irradiation (2-way ANOVA, p < 0.0001). Static and dynamic histomorphometry of the femoral metaphysis revealed osteoblast vigor, not number, was significantly increased in the irradiated mice treated with Scl-Ab. Systemic alterations were assessed through serum lipidomic analysis, which showed that Scl-Ab normalized lipid profiles in the irradiated group. This data supports the theory of sclerostin as a novel contributor to the regulation of osteoblast activity after irradiation. Overall, our data support the hypothesis that Scl-Ab ameliorates the deleterious effects of whole-body irradiation on bone and adipose tissue in a mouse model. Our findings suggest that future research into localized and systemic therapies after irradiation exposure is warranted.
Assuntos
Osso Esponjoso , Irradiação Corporal Total , Animais , Osso e Ossos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos , OsteogêneseRESUMO
Inhibition of sclerostin increases bone formation and decreases bone resorption, leading to increased bone mass, bone mineral density, and bone strength and reduced fracture risk. In a clinical study of the sclerostin antibody romosozumab versus alendronate in postmenopausal women (ARCH), an imbalance in adjudicated serious cardiovascular (CV) adverse events driven by an increase in myocardial infarction (MI) and stroke was observed. To explore whether there was a potential mechanistic plausibility that sclerostin expression, or its inhibition, in atherosclerotic (AS) plaques may have contributed to this imbalance, sclerostin was immunostained in human plaques to determine whether it was detected in regions relevant to plaque stability in 94 carotid and 50 femoral AS plaques surgically collected from older female patients (mean age 69.6 ± 10.4 years). Sclerostin staining was absent in most plaques (67%), and when detected, it was of reduced intensity compared with normal aorta and was located in deeper regions of the plaque/wall but was not observed in areas considered relevant to plaque stability (fibrous cap and endothelium). Additionally, genetic variants associated with lifelong reduced sclerostin expression were explored for associations with phenotypes including those related to bone physiology and CV risk factors/events in a population-based phenomewide association study (PheWAS). Natural genetic modulation of sclerostin by variants with a significant positive effect on bone physiology showed no association with lifetime risk of MI or stroke. These data do not support a causal association between the presence of sclerostin, or its inhibition, in the vasculature and increased risk of serious cardiovascular events. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
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Doenças Cardiovasculares , Placa Aterosclerótica , Idoso , Idoso de 80 Anos ou mais , Alendronato , Densidade Óssea , Doenças Cardiovasculares/genética , Regulação para Baixo , Feminino , Humanos , Pessoa de Meia-Idade , Placa Aterosclerótica/genéticaRESUMO
Destruction of the alveolar bone in the jaws can occur due to periodontitis, trauma or following tumor resection. Common reconstructive therapy can include the use of bone grafts with limited predictability and efficacy. Romosozumab, approved by the FDA in 2019, is a humanized sclerostin-neutralizing antibody (Scl-Ab) indicated in postmenopausal women with osteoporosis at high risk for fracture. Preclinical models show that Scl-Ab administration preserves bone volume during periodontal disease, repairs bone defects surrounding dental implants, and reverses alveolar bone loss following extraction socket remodeling. To date, there are no studies evaluating Scl-Ab to repair osseous defects around teeth or to identify the efficacy of locally-delivered Scl-Ab for targeted drug delivery. In this investigation, the use of systemically-delivered versus low dose locally-delivered Scl-Ab via poly(lactic-co-glycolic) acid (PLGA) microspheres (MSs) was compared at experimentally-created alveolar bone defects in rats. Systemic Scl-Ab administration improved bone regeneration and tended to increase cementogenesis measured by histology and microcomputed tomography, while Scl-Ab delivered by MSs did not result in enhancements in bone or cemental repair compared to MSs alone or control. In conclusion, systemic administration of Scl-Ab promotes bone and cemental regeneration while local, low dose delivery did not heal periodontal osseous defects in this study.
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Perda do Osso Alveolar/tratamento farmacológico , Anticorpos Monoclonais/administração & dosagem , Proteínas Morfogenéticas Ósseas/imunologia , Marcadores Genéticos/imunologia , Microesferas , Periodonto/citologia , Regeneração , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/patologia , Animais , Masculino , Periodonto/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
The discovery that two rare autosomal recessive high bone mass conditions were caused by the loss of sclerostin expression prompted studies into its role in bone homeostasis. In this article, we aim to bring together the wealth of information relating to sclerostin in bone though discussion of rare human disorders in which sclerostin is reduced or absent, sclerostin manipulation via genetic approaches and treatment with antibodies that neutralise sclerostin in animal models and in human. Together, these findings demonstrate the importance of sclerostin as a regulator of bone homeostasis and provide valuable insights into its biological mechanism of action. We summarise the current state of knowledge in the field, including the current understanding of the direct effects of sclerostin on the canonical WNT signalling pathway and the actions of sclerostin as an inhibitor of bone formation. We review the effects of sclerostin, and its inhibition, on bone at the cellular and tissue level and discuss new findings that suggest that sclerostin may also regulate adipose tissue. Finally, we highlight areas in which future research is expected to yield additional insights into the biology of sclerostin.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Osso e Ossos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Osso e Ossos/anatomia & histologia , Humanos , Modelos Biológicos , Mutação/genética , Via de Sinalização WntRESUMO
BACKGROUND: Tubulointerstitial fibrosis is a key feature of chronic kidney diseases leading to renal failure. It is characterised by the infiltration of fibroblasts and aberrant accumulation of extracellular matrix (ECM) proteins, which are associated with progressive loss of renal function. Integrins play a major role in fibrosis, but the mechanisms through which they do this are not fully understood. OBJECTIVE: Using a complex cell system, we test the hypothesis that integrins are pro-fibrotic via regulation of functional interactions between tubular epithelial cells and renal fibroblasts. METHOD: Contact co-culture of human primary renal proximal tubular epithelial cells and renal fibroblasts promoted the spontaneous accumulation of a mature ECM rich in interstitial collagens, which was considerably in excess of that seen in the individual mono-cultures. Both cell types persisted throughout the culture and were capable of expressing multiple ECM components. RESULTS: While ECM accumulation was inhibited by the clinically proven anti-fibrotic, nintedanib, and was partially abrogated by transforming growth factor ß neutralisation, its levels did not return to basal, indicating additional pathways were implicated in the pro-ECM response. Application of anti-integrin blocking antibodies and small molecules demonstrated a major role of the αV integrins in the ECM accumulation during fibroblast: epithelial cell interactions. CONCLUSION: Integrin-mediated pathways can facilitate the spontaneous accumulation of ECM during fibroblast: epithelial cell interactions, and this direct renal co-culture assay system could provide a translational in vitro assay for investigating novel pathways involved in the pro-ECM response and the screening of renal anti-fibrotic agents.
Assuntos
Matriz Extracelular/metabolismo , Fibrose/metabolismo , Integrinas/metabolismo , Nefropatias/metabolismo , Células Cultivadas , Humanos , Técnicas In VitroRESUMO
Administration of antibodies to sclerostin (Scl-Ab) has been shown to increase bone mass, bone mineral density (BMD) and bone strength by increasing bone formation and decreasing bone resorption in both animal studies and human clinical trials. In these studies, the magnitude and rate of increase in bone formation markers is attenuated upon repeat dosing with Scl-Ab despite a continuous and progressive increase in BMD. Here, we investigated whether the attenuation in the bone formation response following repeated administration of Scl-Ab was associated with increased expression of secreted antagonists of Wnt signalling and determined how the circulating marker of bone formation, P1NP, responded to single, or multiple doses, of Scl-Ab four days post-dosing. Female Balb/c mice were treated with Scl-Ab and we demonstrated that the large increase in serum P1NP observed following the first dose was reduced following administration of multiple doses of Scl-Ab. This dampening of the P1NP response was not due to a change in the kinetics of the bone formation marker response, or differences in exposure to the drug. The abundance of transcripts encoding several secreted Wnt antagonists was determined in femurs collected from mice following one or six doses of Scl-Ab, or vehicle treatment. Compared with vehicle controls, expression of SOST, SOST-DC1, DKK1, DKK2, SFRP1, SFRP2, FRZB, SFRP4 and WIF1 transcripts was significantly increased (approximately 1.5-4.2 fold) following a single dose of Scl-Ab. With the exception of SFRP1, these changes were maintained or further increased following six doses of Scl-Ab and the abundance of SFRP5 was also increased. Up-regulation of these Wnt antagonists may exert a negative feedback to increased Wnt signalling induced by repeated administration of Scl-Ab and could contribute to self-regulation of the bone formation response over time. After an antibody-free period of four weeks or more, the P1NP response was comparable to the naïve response, and a second phase of treatment with Scl-Ab following an antibody-free period elicited additional gains in BMD. Together, these data demonstrate that the rapid dampening of the bone formation response in the immediate post-dose period which occurs after repeat dosing of Scl-Ab is associated with increased expression of Wnt antagonists, and a treatment-free period can restore the full bone formation response to Scl-Ab.
Assuntos
Glicoproteínas/antagonistas & inibidores , Osteogênese/efeitos dos fármacos , Fragmentos de Peptídeos/sangue , Pró-Colágeno/sangue , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos/farmacologia , Feminino , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos BALB C , Regulação para CimaRESUMO
A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.
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Activated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM) in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-ß) blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.
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Fibrosis is a common driver of end-stage organ failure in most organs. It is characterised by excessive accumulation of extracellular matrix (ECM) proteins. Therapeutic options are limited and novel treatments are urgently required, however current cell-based high-throughput screening (HTS) models to identify molecules affecting ECM accumulation are limited in their relevance or throughput. We report a novel sensitive approach which combines in situ fluorescent staining of accumulated decellularised ECM proteins with automated high-content microscopy. Using this method to measure ECM accumulation in a kidney cell model, we demonstrated good agreement with established radiolabelled amino acid incorporation assays: TGFß1 delivered a potent pro-fibrotic stimulus, which was reduced by TGFß antibody or the anti-fibrotic nintedanib. Importantly, our method also provides information about matrix organisation: the extent of ECM accumulation was unaffected by the BMP antagonist Gremlin-1 but a pronounced effect on matrix fibrillar organisation was revealed. This rapid, straightforward endpoint provides quantitative data on ECM accumulation and offers a convenient cross-species readout that does not require antibodies. Our method facilitates discovery of novel pro- and anti-fibrotic agents in 384-well plate format and may be widely applied to in vitro cell-based models in which matrix protein deposition reflects the underlying biology or pathology.
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Matriz Extracelular/química , Fibrose/patologia , Nefropatias/patologia , Microscopia de Fluorescência/métodos , Proteínas/análise , Automação Laboratorial/métodos , Células Cultivadas , Células Epiteliais/patologia , Humanos , Modelos Biológicos , Coloração e Rotulagem/métodosRESUMO
The CHO-K1 cell line is commonly used for studies of recombinantly expressed proteins, including proteins of the G protein-coupled receptor (GPCR) family. This laboratory has used CHO-K1 cells for the functional characterization of Edg family GPCRs. However, parental CHO-K1 cells respond to lysophospholipids in in-vitro functional assays, which suggests expression of endogenous Edg family GPCRs. To determine the repertoire of Edg family receptor expression in this cell line, alignments of human and rodent sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) receptor sequences were used to design semi-redundant oligonucleotide pairs. A portion of each receptor gene coding sequence was amplified from Chinese hamster genomic DNA and the resultant gene fragments sequenced. Species-specific oligonucleotide pairs were designed using this novel sequence information and used to detect expression of S1P(1,2,4) and LPA(1) transcripts in CHO-K1 cells by RT-PCR.
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Expressão Gênica/genética , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Lisoesfingolipídeo/genética , Animais , Células CHO , Cricetinae , Cricetulus , DNA Complementar/química , DNA Complementar/genética , Humanos , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNARESUMO
The Wnt signaling pathway is of central importance in embryogenesis, development and adult tissue homeostasis, and dysregulation of this pathway is associated with cancer and other diseases. Despite the developmental and potential therapeutic significance of this pathway, many aspects of Wnt signaling, including the control of the master transcriptional co-activator ß-catenin, remain poorly understood. In order to explore this aspect, a diverse immune llama VHH phagemid library was constructed and panned against ß-catenin. VHH antibody fragments from the library were expressed intracellularly, and a number of antibodies were shown to possess function-modifying intracellular activity in a luciferase-based Wnt signaling HEK293 reporter bioassay. Further characterization of one such VHH (named LL3) confirmed that it bound endogenous ß-catenin, and that it inhibited the Wnt signaling pathway downstream of the destruction complex, while production of a control Ala-substituted complementarity-determining region (CDR)3 mutant demonstrated that the inhibition of ß-catenin activity by the parent intracellular antibody was dependent on the specific CDR sequence of the antibody.
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Regiões Determinantes de Complementaridade/biossíntese , Anticorpos de Cadeia Única/biossíntese , Via de Sinalização Wnt , beta Catenina/antagonistas & inibidores , Sítios de Ligação de Anticorpos , Regiões Determinantes de Complementaridade/genética , Células HEK293 , Humanos , Anticorpos de Cadeia Única/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
A number of proteins have been shown to modulate canonical Wnt signalling at the cell surface, including members of the Dickkopf (Dkk) family (Baron and Rawadi in J Endocrinol 148:2635-2643, 2007; Cruciat and Niehrs in Cold Spring Harb Perspect Biol 5:a015081, 2013). The Dkk family includes four secreted proteins (Dkk1-4), which are characterised by two highly conserved cysteine-rich regions corresponding to C24-C73 and C128-C201 in human Dkk4 (hDkk4). Here we report essentially complete backbone and comprehensive side chain (15)N, (13)C and (1)H NMR assignments for full length mature hDkk4 (M1-L207) containing a short C-terminal hexa-histidine tag (E208-H222). Analysis of the backbone chemical shift data obtained indicates that there is a very limited amount of regular secondary structure, with only small stretches of ß-strand identified in both cysteine-rich regions. The N-terminal region of hDkk4 (M1-G21) and the relatively long linker between the two cysteine-rich regions (E77-Q123) appear to be unstructured and relatively mobile.