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1.
PLoS Genet ; 17(4): e1009275, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33819267

RESUMO

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.


Assuntos
Artrite Reumatoide/genética , Proteínas de Drosophila/genética , Músculo Esquelético/metabolismo , Receptor Smoothened/genética , Ubiquitina-Proteína Ligases/genética , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster/genética , Proteínas Hedgehog/genética , Homeostase/genética , Humanos , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Camundongos , Músculo Esquelético/patologia , Osteogênese/genética , Transdução de Sinais/genética , Tendões/metabolismo , Tendões/patologia
2.
Trends Biochem Sci ; 44(7): 616-628, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30799235

RESUMO

ENPP1 is well known for its role in regulating skeletal and soft tissue mineralization. It primarily exerts its function through the generation of pyrophosphate, a key inhibitor of hydroxyapatite formation. Several previous studies have suggested that ENPP1 also contributes to a range of human diseases including diabetes, cancer, cardiovascular disease, and osteoarthritis. In this review, we summarize the pathological roles of ENPP1 in mineralization and these soft tissue disorders. We also discuss the underlying mechanisms through which ENPP1 exerts its pathological effects. A fuller understanding of the pathways through which ENPP1 acts may help to develop novel therapeutic strategies for these commonly diagnosed morbidities.


Assuntos
Neoplasias/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Animais , Doenças Cardiovasculares/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Osteoartrite/metabolismo
3.
Calcif Tissue Int ; 113(4): 449-468, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37470794

RESUMO

Bisphosphonates prevent bone loss in glucocorticoid (GC)-treated boys with Duchenne muscular dystrophy (DMD) and are recommended as standard of care. Targeting receptor activator of nuclear factor kappa-B ligand (RANKL) may have advantages in DMD by ameliorating dystrophic skeletal muscle function in addition to their bone anti-resorptive properties. However, the potential effects of anti-RANKL treatment upon discontinuation in GC-induced animal models of DMD are unknown and need further investigation prior to exploration in the clinical research setting. In the first study, the effects of anti-RANKL and deflazacort (DFZ) on dystrophic skeletal muscle function and bone microstructure were assessed in mdx mice treated with DFZ or anti-RANKL, or both for 8 weeks. Anti-RANKL and DFZ improved grip force performance of mdx mice but an additive effect was not noted. However, anti-RANKL but not DFZ improved ex vivo contractile properties of dystrophic muscles. This functional improvement was associated with a reduction in muscle damage and fibrosis, and inflammatory cell number. Anti-RANKL treatment, with or without DFZ, also improved trabecular bone structure of mdx mice. In a second study, intravenous zoledronate (Zol) administration (1 or 2 doses) following 2 months of discontinuation of anti-RANKL treatment was mostly required to record an improvement in bone microarchitecture and biomechanical properties in DFZ-treated mdx mice. In conclusion, the ability of anti-RANKL therapy to restore muscle function has profound implications for DMD patients as it offers the possibility of improving skeletal muscle function without the steroid-related skeletal side effects.


Assuntos
Doenças Ósseas Metabólicas , Distrofia Muscular de Duchenne , Animais , Masculino , Camundongos , Doenças Ósseas Metabólicas/tratamento farmacológico , Modelos Animais de Doenças , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne/tratamento farmacológico , NF-kappa B
4.
Cell Biochem Funct ; 41(2): 189-201, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36540015

RESUMO

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28-day iPTH (80 µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1-/- mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in the femora of WT mice following iPTH administration but remained unchanged in the femora of Phospho1-/- mice. After 28 days of iPTH administration, the anabolic response in the femora of WT was greater than that noted in Phospho1-/- mice. Specifically, cortical and trabecular bone volume/total volume, as well as cortical thickness, were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1-/- mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1-/-  mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO1, this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.


Assuntos
Fosfatase Alcalina , Hormônio Paratireóideo , Masculino , Camundongos , Animais , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/farmacologia , Osso e Ossos/metabolismo , Osteoblastos/metabolismo , Osteogênese , Densidade Óssea , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielina Fosfodiesterase/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo
5.
J Cell Physiol ; 236(6): 4614-4624, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33305372

RESUMO

Supraphysiological levels of the osteoblast-enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase-1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast-specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6-week-old mice lacking osteoblast NPP1 expression (osteoblast-specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.


Assuntos
Osso e Ossos/enzimologia , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina , Osteoblastos/enzimologia , Osteogênese , Diester Fosfórico Hidrolases/deficiência , Pirofosfatases/deficiência , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Osso e Ossos/patologia , Osso Esponjoso/enzimologia , Osso Esponjoso/patologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fêmur/enzimologia , Fêmur/patologia , Insulina/sangue , Masculino , Camundongos Knockout , Osteoblastos/patologia , Osteocalcina/sangue , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Fatores Sexuais , Crânio/enzimologia , Crânio/patologia , Tíbia/enzimologia , Tíbia/patologia
6.
J Cell Biochem ; 122(7): 696-715, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33529442

RESUMO

MicroRNAs are small noncoding RNAs that can bind to the target sites in the 3'-untranslated region of messenger RNA to regulate posttranscriptional gene expression. Increasing evidence has identified the miR-29 family, consisting of miR-29a, miR-29b-1, miR-29b-2, and miR-29c, as key regulators of a number of biological processes. Moreover, their abnormal expression contributes to the etiology of numerous diseases. In the current review, we aimed to summarize the differential expression patterns and functional roles of the miR-29 family in the etiology of diseases including osteoarthritis, osteoporosis, cardiorenal, and immune disease. Furthermore, we highlight the therapeutic potential of targeting members of miR-29 family in these diseases. We present miR-29s as promoters of osteoblast differentiation and apoptosis but suppressors of chondrogenic and osteoclast differentiation, fibrosis, and T cell differentiation, with clear avenues for therapeutic manipulation. Further research will be crucial to identify the precise mechanism of miR-29 family in these diseases and their full potential in therapeutics.


Assuntos
Doença/etiologia , MicroRNAs/genética , Humanos
7.
Calcif Tissue Int ; 109(6): 696-705, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34213594

RESUMO

Proton pump inhibitors (PPIs) have been associated with an increased risk of fragility fractures in pharmaco-epidemiological studies. The mechanism is unclear, but it has been speculated that by neutralising gastric acid, they may reduce intestinal calcium absorption, causing secondary hyperparathyroidism and bone loss. Here we investigated that hypothesis that the skeletal effects of PPI might be mediated by inhibitory effects on the bone-specific phosphatase PHOSPHO1. We found that the all PPIs tested inhibited the activity of PHOSPHO1 with IC50 ranging between 0.73 µM for esomeprazole to 19.27 µM for pantoprazole. In contrast, these PPIs did not inhibit TNAP activity. We also found that mineralisation of bone matrix in primary osteoblast cultures was inhibited by several PPIs in a concentration dependent manner. In contrast, the histamine-2 receptor antagonists (H2RA) nizatidine, famotidine, cimetidine and ranitidine had no inhibitory effects on PHOSPHO1 activity. Our experiments show for the first time that PPIs inhibit PHOSPHO1 activity and matrix mineralisation in vitro revealing a potential mechanism by which these widely used drugs are associated with the risk of fractures.


Assuntos
Antagonistas dos Receptores H2 da Histamina , Inibidores da Bomba de Prótons , Calcificação Fisiológica , Pantoprazol , Monoéster Fosfórico Hidrolases , Inibidores da Bomba de Prótons/farmacologia
8.
BMC Biol ; 18(1): 149, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33092598

RESUMO

BACKGROUND: The classical functions of the skeleton encompass locomotion, protection and mineral homeostasis. However, cell-specific gene deletions in the mouse and human genetic studies have identified the skeleton as a key endocrine regulator of metabolism. The bone-specific phosphatase, Phosphatase, Orphan 1 (PHOSPHO1), which is indispensable for bone mineralisation, has been recently implicated in the regulation of energy metabolism in humans, but its role in systemic metabolism remains unclear. Here, we probe the mechanism underlying metabolic regulation by analysing Phospho1 mutant mice. RESULTS: Phospho1-/- mice exhibited improved basal glucose homeostasis and resisted high-fat-diet-induced weight gain and diabetes. The metabolic protection in Phospho1-/- mice was manifested in the absence of altered levels of osteocalcin. Osteoblasts isolated from Phospho1-/- mice were enriched for genes associated with energy metabolism and diabetes; Phospho1 both directly and indirectly interacted with genes associated with glucose transport and insulin receptor signalling. Canonical thermogenesis via brown adipose tissue did not underlie the metabolic protection observed in adult Phospho1-/- mice. However, the decreased serum choline levels in Phospho1-/- mice were normalised by feeding a 2% choline rich diet resulting in a normalisation in insulin sensitivity and fat mass. CONCLUSION: We show that mice lacking the bone mineralisation enzyme PHOSPHO1 exhibit improved basal glucose homeostasis and resist high-fat-diet-induced weight gain and diabetes. This study identifies PHOSPHO1 as a potential bone-derived therapeutic target for the treatment of obesity and diabetes.


Assuntos
Metabolismo Energético , Resistência à Insulina/genética , Obesidade/genética , Monoéster Fosfórico Hidrolases/genética , Animais , Colina/metabolismo , Glucose/metabolismo , Homeostase , Masculino , Camundongos , Monoéster Fosfórico Hidrolases/metabolismo
9.
Blood ; 131(26): 2955-2966, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29712634

RESUMO

Red cells contain a unique constellation of membrane lipids. Although much is known about regulated protein expression, the regulation of lipid metabolism during erythropoiesis is poorly studied. Here, we show that transcription of PHOSPHO1, a phosphoethanolamine and phosphocholine phosphatase that mediates the hydrolysis of phosphocholine to choline, is strongly upregulated during the terminal stages of erythropoiesis of both human and mouse erythropoiesis, concomitant with increased catabolism of phosphatidylcholine (PC) and phosphocholine as shown by global lipidomic analyses of mouse and human terminal erythropoiesis. Depletion of PHOSPHO1 impaired differentiation of fetal mouse and human erythroblasts, and, in adult mice, depletion impaired phenylhydrazine-induced stress erythropoiesis. Loss of PHOSPHO1 also impaired phosphocholine catabolism in mouse fetal liver progenitors and resulted in accumulation of several lipids; adenosine triphosphate (ATP) production was reduced as a result of decreased oxidative phosphorylation. Glycolysis replaced oxidative phosphorylation in PHOSPHO1-knockout erythroblasts and the increased glycolysis was used for the production of serine or glycine. Our study elucidates the dynamic changes in lipid metabolism during terminal erythropoiesis and reveals the key roles of PC and phosphocholine metabolism in energy balance and amino acid supply.


Assuntos
Eritroblastos/metabolismo , Eritropoese , Fosforilcolina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Eritroblastos/citologia , Deleção de Genes , Glicólise , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação Oxidativa , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo
10.
Arch Biochem Biophys ; 667: 14-21, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-30998909

RESUMO

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (-Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.


Assuntos
Biomineralização/fisiologia , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Microscopia de Força Atômica/métodos , Animais , Fenômenos Biomecânicos , Cartilagem/química , Cartilagem/metabolismo , Cartilagem/ultraestrutura , Embrião de Galinha , Vesículas Extracelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Espectroscopia de Infravermelho com Transformada de Fourier
11.
FASEB J ; 32(1): 20-25, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28864658

RESUMO

Mineralization is a key process in the formation of bone and cartilage in vertebrates, involving the deposition of calcium- and phosphate-containing hydroxyapatite (HA) mineral within a collagenous matrix. Inorganic phosphate (Pi) accumulation within matrix vesicles (MVs) is a fundamental stage in the precipitation of HA, with PHOSPHO1 being identified as the principal enzyme acting to produce Pi PHOSPHO1 is a dual-specific phosphocholine/phosphoethanolamine phosphatase enriched in mineralizing cells and within MVs. However, the source and mechanism by which PHOSPHO1 substrates are formed before mineralization have not been determined. Here, we propose that 2 enzymes-phospholipase A2 (PLA2) and ectonucleotide pyrophophatase/phosphodiesterase 6 (ENPP6)-act in sequence upon phosphatidylcholine found in MV membranes to produce phosphocholine, which PHOSPHO1 can hydrolyze to liberate Pi This hypothesis is supported by evidence that both enzymes are expressed in mineralizing cells and data showing that phosphatidylcholine is broken down in MVs during mineralization. Therefore, PLA2 and ENPP6 activities may represent a key step in the mineralization process. Further functional studies are urgently required to examine their specific roles in the initiation of skeletal mineralization.-Stewart, A. J., Leong, D. T. K., Farquharson, C. PLA2 and ENPP6 may act in concert to generate phosphocholine from the matrix vesicle membrane during skeletal mineralization.


Assuntos
Calcificação Fisiológica/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Fosfolipases A2/metabolismo , Fosforilcolina/metabolismo , Pirofosfatases/metabolismo , Animais , Osso e Ossos/metabolismo , Cartilagem , Matriz Extracelular/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , Redes e Vias Metabólicas , Modelos Biológicos , Fosfolipídeos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
12.
BMC Musculoskelet Disord ; 20(1): 344, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31351471

RESUMO

BACKGROUND: Subchondral bone (SCB) thickening is one of the earliest detectable changes in osteoarthritic joints and is considered a potential trigger for subsequent articular cartilage degeneration. In this manuscript, we examine whether disruption to the SCB osteocyte network contributes to the initiation and pathogenesis of osteoarthritis. METHODS: We examined expression patterns of the glycoprotein E11/podoplanin by immunohistochemical labelling in murine, human and canine osteoarthritis models. We also examined the effects of twice-weekly administration of Bortezomib, a proteasome inhibitor which stabilises osteocyte E11 levels, to C57/BL6 wild-type male mice (1 mg/kg/day) for 8 weeks after surgical destabilisation of the medial meniscus. By inducing osteoarthritis-like changes in the right knee joint of 12-week-old male E11 hypomorphic mice (and corresponding controls) using a post-traumatic joint loading model, we also investigated whether a bone-specific E11 deletion in mice increases joint vulnerability to osteoarthritis. Articular cartilage degradation and osteophyte formation were assessed by histology and in line with the OARSI grading system. RESULTS: Our studies reveal increased E11 expression in osteocytes of human and canine osteoarthritic SCB. We found that Bortezomib administration had no effect on surgically-induced osteoarthritis, potentially due to a lack of the expected stabilisation of E11 in the SCB. We also found, in concordance with our previous work, wild-type mice exhibited significant load-induced articular cartilage lesions on the lateral femoral condyle (p < 0.01) and osteophyte formation. In contrast, E11 hypomorphic mice did not develop osteophytes or any corresponding articular lesions. CONCLUSIONS: Overall, these data suggest that an intact osteocyte network in the SCB contributes to the development of mechanically-driven osteoarthritis. Further, the data presented here indicate that the molecular pathways that preserve the osteocyte network, such as those driven by E11, may be targeted to limit osteoarthritis pathogenesis.


Assuntos
Cartilagem Articular/patologia , Glicoproteínas de Membrana/metabolismo , Osteoartrite/patologia , Osteófito/patologia , Animais , Bortezomib/administração & dosagem , Modelos Animais de Doenças , Cães , Humanos , Masculino , Glicoproteínas de Membrana/genética , Meniscos Tibiais/patologia , Camundongos , Camundongos Knockout , Osteoartrite/tratamento farmacológico , Osteoartrite/etiologia , Osteócitos/efeitos dos fármacos , Osteócitos/patologia , Osteófito/tratamento farmacológico , Suporte de Carga
13.
Int J Mol Sci ; 20(23)2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31816823

RESUMO

Patients with inflammatory bowel disease (IBD) often present poor bone health and are 40% more at risk of bone fracture. Studies have implicated autophagy in IBD pathology and drugs used to treat IBD stimulate autophagy in varying degrees, however, their effect on the skeleton is currently unknown. Here, we have utilised the dextran sulphate sodium (DSS) model of colitis in mice to examine the effects of the thiopurine drug azathioprine on the skeleton. Ten-week-old male mice (n = 6/group) received 3.0% DSS in their drinking water for four days, followed by a 14-day recovery period. Mice were treated with 10 mg/kg/day azathioprine or vehicle control. Histopathological analysis of the colon from DSS mice revealed significant increases in scores for inflammation severity, extent, and crypt damage (p < 0.05). Azathioprine provided partial protection to the colon, as reflected by a lack of significant difference in crypt damage and tissue regeneration with DSS treatment. MicroCT of vehicle-treated DSS mice revealed azathioprine treatment had a significant detrimental effect on the trabecular bone microarchitecture, independent of DSS treatment. Specifically, significant decreases were observed in bone volume/tissue volume (p < 0.01), and trabecular number (p < 0.05), with a concurrent significant increase in trabecular pattern factor (p < 0.01). Immunohistochemical labelling for LC3 revealed azathioprine to induce autophagy in the bone marrow. Together these data suggest that azathioprine treatment may have a deleterious effect on IBD patients who may already be at increased risk of osteoporotic bone fractures and thus will inform on future treatment strategies for patient stratification.


Assuntos
Azatioprina/efeitos adversos , Doenças Inflamatórias Intestinais/patologia , Tíbia/patologia , Animais , Autofagia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/patologia , Colo/patologia , Sulfato de Dextrana , Doenças Inflamatórias Intestinais/induzido quimicamente , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Tíbia/efeitos dos fármacos
14.
J Cell Physiol ; 233(7): 5334-5347, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29215722

RESUMO

E11/podoplanin is critical in the early stages of osteoblast-to-osteocyte transitions (osteocytogenesis), however, the upstream events which regulate E11 expression are unknown. The aim of this study was to examine the effects of FGF-2 on E11-mediated osteocytogenesis and to reveal the nature of the underlying signaling pathways regulating this process. Exposure of MC3T3 osteoblast-like cells and murine primary osteoblasts to FGF-2 (10 ng/ml) increased E11 mRNA and protein expression (p < 0.05) after 4, 6, and 24 hr. FGF-2 induced changes in E11 expression were also accompanied by significant (p < 0.01) increases in Phex and Dmp1 (osteocyte markers) expression and decreases in Col1a1, Postn, Bglap, and Alpl (osteoblast markers) expression. Immunofluorescent microscopy revealed that FGF-2 stimulated E11 expression, facilitated the translocation of E11 toward the cell membrane, and subsequently promoted the formation of osteocyte-like dendrites in MC3T3 and primary osteoblasts. siRNA knock down of E11 expression achieved >70% reduction of basal E11 mRNA expression (p < 0.05) and effectively abrogated FGF-2-related changes in E11 expression and dendrite formation. FGF-2 strongly activated the ERK signaling pathway in osteoblast-like cells but inhibition of this pathway did not block the ability of FGF-2 to enhance E11 expression or to promote acquisition of the osteocyte phenotype. The results of this study highlight a novel mechanism by which FGF-2 can regulate osteoblast differentiation and osteocyte formation. Specifically, the data suggests that FGF-2 promotes osteocytogenesis through increased E11 expression and further studies will identify if this regulatory pathway is essential for bone development and maintenance in health and disease.


Assuntos
Diferenciação Celular/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glicoproteínas de Membrana/genética , Osteogênese/efeitos dos fármacos , Células 3T3 , Animais , Fator 2 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteogênese/genética
15.
J Cell Physiol ; 232(11): 3006-3019, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28488815

RESUMO

The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone-specific Pdpn knockdown on osteocyte form and function in the post-natal mouse. Extensive skeletal phenotyping of male and female 6-week-old Oc-cre;Pdpnflox/flox (cKO) mice and their Pdpnflox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (p < 0.05). Consistent with this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (p < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (p < 0.001) and length (p < 0.001) in cKO mice in which deletion of Pdpn also modifies the bone anabolic loading response (p < 0.05) in comparison to age-matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodeling, this highlights an essential role for Pdpn in bone homeostasis.


Assuntos
Diferenciação Celular , Forma Celular , Deleção de Genes , Glicoproteínas de Membrana/metabolismo , Osteócitos/metabolismo , Osteogênese , Tíbia/metabolismo , Animais , Feminino , Genótipo , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos Knockout , Osteócitos/patologia , Fenótipo , Transdução de Sinais , Tíbia/diagnóstico por imagem , Tíbia/patologia , Microtomografia por Raio-X
16.
J Cell Physiol ; 232(11): 2985-2995, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28369848

RESUMO

Patients with end-stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4-fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8-fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP-1, and LAMP-2 and a concomitant up-regulation of the Annexin family of calcium-binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC-derived MVs (51.9-fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up-regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC-derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co-localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC-derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD.


Assuntos
Anexina A6/metabolismo , Estenose da Valva Aórtica/metabolismo , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Calcinose/metabolismo , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Hipercalcemia/etiologia , Falência Renal Crônica/complicações , Idoso , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Valva Aórtica/ultraestrutura , Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Calcinose/etiologia , Calcinose/genética , Calcinose/patologia , Cálcio/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Matriz Extracelular/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hipercalcemia/diagnóstico , Falência Renal Crônica/diagnóstico , Masculino , Microscopia Eletrônica de Transmissão , Mapas de Interação de Proteínas , Proteômica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Regulação para Cima
17.
J Anat ; 231(2): 298-308, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28737011

RESUMO

The phosphatase PHOSPHO1 is involved in the initiation of biomineralisation. Bones in Phospho1 knockout (KO) mice show histological osteomalacia with frequent bowing of long bones and spontaneous fractures: they contain less mineral, with smaller mineral crystals. However, the consequences of Phospho1 ablation on the microscale structure of bone are not yet fully elucidated. Tibias and femurs obtained from wild-type and Phospho1 null (KO) mice (25-32 weeks old) were embedded in PMMA, cut and polished to produce near longitudinal sections. Block surfaces were studied using 20 kV backscattered-electron (BSE) imaging, and again after iodine staining to reveal non-mineralised matrix and cellular components. For 3D characterisation, we used X-ray micro-tomography. Bones opened with carbide milling tools to expose endosteal surfaces were macerated using an alkaline bacterial pronase enzyme detergent, 5% hydrogen peroxide and 7% sodium hypochlorite solutions to produce 3D surfaces for study with 3D BSE scanning electron microscopy (SEM). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral and/or showed arrested mineralisation fronts, characterised by a failure in the fusion of the calcospherite-like, separately mineralising, individual micro-volumes within bone. Osteoclastic resorption of the uncalcified matrix in Phospho1 KO mice was attenuated compared with surrounding normally mineralised bone. The extent and position of this aberrant biomineralisation varied considerably between animals, contralateral limbs and anatomical sites. The most frequent manifestation lay, however, in the nearly complete failure of mineralisation in the bone surrounding the numerous transverse blood vessel canals in the cortices. In conclusion, SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which has previously not been recognised. These data further confirm the role of this phosphatase in physiological skeletal mineralisation.


Assuntos
Osso e Ossos/patologia , Osso e Ossos/ultraestrutura , Osteomalacia/patologia , Monoéster Fosfórico Hidrolases/deficiência , Animais , Calcificação Fisiológica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
18.
J Cell Physiol ; 231(6): 1392-404, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26639105

RESUMO

The transmembrane glycoprotein E11 is considered critical in early osteoblast-osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO-A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post-translational degradation. We found that exogenous treatment of MLO-A5 and osteocytic IDG-SW3 cells with 10 µM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome-selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin-A) produced similar dose-dependent increases in E11 protein levels in MLO-A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO-A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome-mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Osteoblastos/enzimologia , Osteócitos/enzimologia , Fenótipo , Estabilidade Proteica , Proteólise , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , Ubiquitinação , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP
19.
Int J Mol Sci ; 18(1)2016 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-28035954

RESUMO

Mice harbouring a dentin matrix protein 1 (Dmp1) promoter-driven human diphtheria toxin (DT) receptor (HDTR) transgene (Tg) have recently been used to attain targeted ablation of osteocytes by diphtheria toxin (DT) treatment in order to define osteocyte function. Use of these Tg mice has asserted mechano- and novel paracrine regulatory osteocyte functions. To explore osteocyte roles fully, we sought to confirm the selectivity of DT effects in these transgenic mice. However, our findings revealed incomplete DT-induced osteocyte ablation, prevalent HDTR misexpression, as well as more prominent histopathological DT-induced changes in multiple organs in Tg than in wild-type (WT) littermate mice. Mechanistic evidence for DT action, via prominent regulation of phosphorylation status of elongation factor-2 (EF-2), was also found in many non-skeletal tissues in Tg mice; indicative of direct "off-target" DT action. Finally, very rapid deterioration in health and welfare status in response to DT treatment was observed in these Tg when compared to WT control mice. Together, these data lead us to conclude that alternative models for osteocyte ablation should be sought and caution be exercised when drawing conclusions from experiments using these Tg mice alone.


Assuntos
Proteínas da Matriz Extracelular/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Transgenes , Animais , Osso e Ossos/metabolismo , Encéfalo/metabolismo , Toxina Diftérica/toxicidade , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Rim/metabolismo , Camundongos , Miocárdio/metabolismo , Especificidade de Órgãos , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Regiões Promotoras Genéticas
20.
J Cell Mol Med ; 19(1): 165-74, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25297851

RESUMO

The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP-9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP-9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre-dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP-9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP-9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP-9-induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5-Dimethoxy-N-(quinolin-3-yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP-9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Calcificação Fisiológica , Diferenciação Celular , Fator 2 de Diferenciação de Crescimento/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Osteoblastos/citologia , Receptores de Activinas Tipo II , Fosfatase Alcalina/metabolismo , Animais , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diálise , Fator 2 de Diferenciação de Crescimento/sangue , Fator 2 de Diferenciação de Crescimento/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Fosfatos/farmacologia , Insuficiência Renal Crônica/sangue , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
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