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1.
Arterioscler Thromb Vasc Biol ; 40(5): 1220-1230, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32160775

RESUMO

OBJECTIVE: Sickle cell anemia (SCA) causes chronic inflammation and multiorgan damage. Less understood are the arterial complications, most evident by increased strokes among children. Proteolytic mechanisms, biomechanical consequences, and pharmaceutical inhibitory strategies were studied in a mouse model to provide a platform for mechanistic and intervention studies of large artery damage due to sickle cell disease. Approach and Results: Townes humanized transgenic mouse model of SCA was used to test the hypothesis that elastic lamina and structural damage in carotid arteries increased with age and was accelerated in mice homozygous for SCA (sickle cell anemia homozygous genotype [SS]) due to inflammatory signaling pathways activating proteolytic enzymes. Elastic lamina fragmentation observed by 1 month in SS mice compared with heterozygous littermate controls (sickle cell trait heterozygous genotype [AS]). Positive immunostaining for cathepsin K, a powerful collagenase and elastase, confirmed accelerated proteolytic activity in SS carotids. Larger cross-sectional areas were quantified by magnetic resonance angiography and increased arterial compliance in SS carotids were also measured. Inhibiting JNK (c-jun N-terminal kinase) signaling with SP600125 significantly reduced cathepsin K expression, elastin fragmentation, and carotid artery perimeters in SS mice. By 5 months of age, continued medial thinning and collagen degradation was mitigated by treatment of SS mice with JNK inhibitor. CONCLUSIONS: Arterial remodeling due to SCA is mediated by JNK signaling, cathepsin proteolytic upregulation, and degradation of elastin and collagen. Demonstration in Townes mice establishes their utility for mechanistic studies of arterial vasculopathy, related complications, and therapeutic interventions for large artery damage due to SCA.


Assuntos
Anemia Falciforme/tratamento farmacológico , Antracenos/farmacologia , Artérias Carótidas/efeitos dos fármacos , Doenças das Artérias Carótidas/prevenção & controle , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Remodelação Vascular/efeitos dos fármacos , Anemia Falciforme/enzimologia , Anemia Falciforme/genética , Anemia Falciforme/fisiopatologia , Animais , Artérias Carótidas/enzimologia , Artérias Carótidas/fisiopatologia , Doenças das Artérias Carótidas/enzimologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/fisiopatologia , Catepsina K/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , Elastina/metabolismo , Hemoglobinas/genética , Homozigoto , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos Transgênicos , Mutação , Proteólise , Transdução de Sinais , Fatores de Tempo
2.
FASEB J ; 34(1): 1052-1064, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914701

RESUMO

The past decade, it has become evident that circadian rhythms within metabolically active tissues are very important for physical health. However, although shift work has also been associated with an increased risk of fractures, circadian rhythmicity has not yet been extensively studied in bone. Here, we investigated which genes are rhythmically expressed in bone, and whether circadian disruption by shifts in light-dark cycle affects bone turnover and structure in mice. Our results demonstrate diurnal expression patterns of clock genes (Rev-erbα, Bmal1, Per1, Per2, Cry1, Clock), as well as genes involved in osteoclastogenesis, osteoclast proliferation and function (Rankl, Opg, Ctsk), and osteocyte function (c-Fos) in bone. Weekly alternating light-dark cycles disrupted rhythmic clock gene expression in bone and caused a reduction in plasma levels of procollagen type 1 amino-terminal propeptide (P1NP) and tartrate-resistant acidic phosphatase (TRAP), suggestive of a reduced bone turnover. These effects coincided with an altered trabecular bone structure and increased cortical mineralization after 15 weeks of light-dark cycles, which may negatively affect bone strength in the long term. Collectively, these results show that a physiological circadian rhythm is important to maintain bone health, which stresses the importance of further investigating the association between shift work and skeletal disorders.


Assuntos
Densidade Óssea , Osso e Ossos/fisiologia , Ritmo Circadiano , Regulação da Expressão Gênica , Luz , Fatores de Transcrição ARNTL/metabolismo , Animais , Comportamento Animal , Proteínas CLOCK/metabolismo , Catepsina K/metabolismo , Relógios Circadianos , Criptocromos/metabolismo , Feminino , Lipídeos/química , Camundongos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Osteogênese , Osteoprotegerina/metabolismo , Proteínas Circadianas Period/metabolismo , Fotoperíodo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ligante RANK/metabolismo , Microtomografia por Raio-X
3.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140318, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31740411

RESUMO

Human cathepsin K (hCatK), which is highly expressed in osteoclasts, has the noteworthy ability to cleave type I and II collagens in their helical domain. Its collagenase potency depends strictly on the formation of an oligomeric complex with chondroitin 4-sulfate (C4-S). Accordingly, hCatK is a pivotal protease involved in bone resorption and is an attractive target for the treatment of osteoporosis. As rat is a common animal model for the evaluation of hCatK inhibitors, we conducted a comparative analysis of rat CatK (rCatK) and hCatK, which share a high degree of identity (88%) and similarity (93%). The pH activity profile of both enzymes displayed a similar bell-shaped curve (optimal pH: 6.4). Presence of Ser134 and Val160 in the S2 pocket of rCatK instead of Ala and Leu residues, respectively, in hCatK, led to a weaker peptidase activity, as observed for mouse CatK. Also, regardless of the presence of C4-S, rCatK cleaved in the nonhelical telopeptide regions of both type I (tail) and type II (articular joint) rat collagens. Structure-based computational analyses (electrostatic potential, molecular docking, molecular dynamics, free energy calculations) sustained that the C4-S mediated collagenolytic activity of rCatK obeys distinct molecular interactions from those of hCatK. Additionally, T-kininogen (a.k.a. thiostatin), a unique rat serum acute phase molecule, acted as a tight-binding inhibitor of hCatK (Ki = 0.11 ± 0.05 nM). Taken into account the increase of T-Kininogen level in inflamed rat sera, this may raise the question of the appropriateness to evaluate pharmacological hCatK inhibitors in this peculiar animal model.


Assuntos
Catepsina K/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catepsina K/antagonistas & inibidores , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Alinhamento de Sequência , Especificidade por Substrato , Termodinâmica
4.
Int J Mol Sci ; 20(19)2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31546645

RESUMO

Abdominal aortic aneurysm (AAA) is among the top 20 causes of death in the United States. Surgical repair is the gold standard for AAA treatment, therefore, there is a need for non-invasive therapeutic interventions. Aneurysms are more closely associated with the osteoclast-like catabolic degradation of the artery, rather than the osteoblast-like anabolic processes of arterial calcification. We have reported the presence of osteoclast-like cells (OLCs) in human and mouse aneurysmal tissues. The aim of this study was to examine OLCs from aneurysmal tissues as a source of degenerative proteases. Aneurysmal and control tissues from humans, and from the mouse CaPO4 and angiotensin II (AngII) disease models, were analyzed via flow cytometry and immunofluorescence for the expression of osteoclast markers. We found higher expression of the osteoclast markers tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), and cathepsin K, and the signaling molecule, hypoxia-inducible factor-1α (HIF-1α), in aneurysmal tissue compared to controls. Aneurysmal tissues also contained more OLCs than controls. Additionally, more OLCs from aneurysms express HIF-1α, and produce more MMP-9 and cathepsin K, than myeloid cells from the same tissue. These data indicate that OLCs are a significant source of proteases known to be involved in aortic degradation, in which the HIF-1α signaling pathway may play an important role. Our findings suggest that OLCs may be an attractive target for non-surgical suppression of aneurysm formation due to their expression of degradative proteases.


Assuntos
Aneurisma da Aorta Abdominal/enzimologia , Osteoclastos/enzimologia , Animais , Aneurisma da Aorta Abdominal/metabolismo , Catepsina K/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/enzimologia , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Osteoclastos/metabolismo , Proteólise , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/metabolismo
5.
J Biol Regul Homeost Agents ; 33(4): 1105-1111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332987

RESUMO

The adapter protein myeloid differentiation primary response gene 88 (MyD88) links the intracellular domains of interleukin receptors 1 and 18, and most Toll-like receptors (TLRs) to interleukin 1 receptor associated kinase (IRAK) signaling and subsequent NF-κB-mediated transcription. Previous work showed that mice with global deficiency of MyD88 (MyD88-/-) have osteopenic cancellous bone along with a reduction in osteoblastic but also osteoclastic surfaces. To further elucidate the role of MyD88 in bone, we utilized mice with osteoclast-restricted MyD88 expression in bone (MyD88OC). Bones of MyD88OC and wild type (wt) mice were examined by microCT analysis. Mechanical properties of bones were tested by three-point bending, and gene expression measured using quantitative real-time polymerase chain reaction. In MyD88OC mice, no osteopenic traits were observed, however, a drastic reduction in geometric parameters was detected. In trabecular bone a loss of connectivity density (-44%, p less than 0.0001) was measured and in cortical bone Imax (-31%, p less than 0.0001), Imin (-20%, p less than 0.001), J (-26%, p less than 0.0001) were reduced. Mechanical testing showed increased load to failure (77%, p less than 0.01) and decreased deflection at failure (-68%, p less than 0.01) of the femur. On the molecular level, relative gene expression analysis showed a (-29%, p less than 0.01) reduction in receptor activator of nuclear factor κ B ligand (RANKL) and no difference in osteoprotegerin (OPG) or RANK. Further, the bone resorption markers cathepsin K (CTSK) and tartrate-resistant acid phosphatase 5 (TRAP) were unchanged. In contrast, the bone formation markers collagen type 1 (COL1A1) and osteocalcin (OC) were decreased by -72% (p less than 0.0001) and -82% (p less than 0.0001), respectively. Together, our data suggests that the function of MyD88 in osteoclasts is sufficient to maintain bone mass, while it fails to preserve bone geometry, likely through dysfunctions in osteoblasts.


Assuntos
Reabsorção Óssea , Osso e Ossos/patologia , Fator 88 de Diferenciação Mieloide/metabolismo , Osteoclastos/citologia , Animais , Catepsina K/metabolismo , Diferenciação Celular , Colágeno Tipo I/metabolismo , Camundongos , Osteoblastos , Osteocalcina/metabolismo , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fosfatase Ácida Resistente a Tartarato/metabolismo
6.
Arch Oral Biol ; 105: 59-64, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31276939

RESUMO

OBJECTIVE: This study aimed to investigate the effect of risedronate on orthodontic tooth movement in ovariectomized rats. METHODS: Forty-five female Sprague-Dawley rats were divided into three groups. Bilateral ovariectomies were performed on the rats in the ovariectomy and ovariectomy + risedronate groups. Two weeks after ovariectomy, risedronate was administered intraperitoneally every three days in the ovariectomy + risedronate group, while rats in the ovariectomy and sham groups were administered saline until they were sacrificed. One month after ovariectomy, nickel-titanium alloy closed-coil springs were placed between the maxillary left first molar and the maxillary left incisor. On days 3, 7 and 14, five rats from each group were sacrificed. The distance of orthodontic tooth movement was measured by a digital caliper, and slides were obtained for histological analysis. RESULTS: The distance of orthodontic tooth movement and the number of tartar-resistant acid phosphatase (TRAP)-positive cells were the highest in the ovariectomy group, followed by those in the ovariectomy + risedronate group, on days 3, 7 and 14. The positive expression levels of receptor activator of nuclear factor-kappa ß (RANK) ligand and cathepsin K were the strongest while the positive expression of osteoprotegerin in the ovariectomy group was the weakest, followed by the corresponding expression levels in the ovariectomy + risedronate group, on days 3, 7 and 14. CONCLUSIONS: Risedronate can inhibit orthodontic tooth movement in ovariectomized rats and may function by regulating the RANK/RANK ligand/osteoprotegerin pathway.


Assuntos
Ácido Risedrônico/farmacologia , Técnicas de Movimentação Dentária , Animais , Catepsina K/metabolismo , Feminino , Osteoclastos , Osteoprotegerina/metabolismo , Ovariectomia , Ligante RANK/metabolismo , Ratos , Ratos Sprague-Dawley
7.
J Vasc Res ; 56(3): 139-151, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31064000

RESUMO

BACKGROUND: It has been reported that smoking is one of the strongest positive risk factors for abdominal aortic aneurysms (AAAs). Although many studies have been directed to decipher the effect of smoking on AAA, its effect on macrophage activation has not yet been explored. OBJECTIVES: We have reported the importance of osteoclastogenesis (OCG) in aneurysm formation. Therefore, we examined the effect of cigarette smoking on OCG and arterial aneurysmal formation by using cigarette smoke extract (CSE) in this study. METHODS: Macrophage cell lines were stimulated with CSE, and their activation and differentiation were examined in vitro. Since macrophages activated through the OCG pathway are identified by tartrate-resistant acid phosphatase (TRAP) expression, these cells are referred to as TRAP-positive macrophages (TPMs) in this study. We also applied CSE-contained PBS in the calcium chloride-induced mouse carotid aneurysm model in vivo. RESULTS: Macrophages stimulated with CSE expressed significantly higher levels of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), TRAP, cathepsin K, matrix metalloproteinase-9 and membrane-type metalloproteinase (MT1-MMP). CSE-treated mouse aneurysms showed increased aneurysm size with increased TPM infiltration and protease expression compared to non-CSE-treated mouse aneurysms. CONCLUSIONS: These results suggest that CSE intensifies OCG in macrophages and promotes arterial aneurysmal progression.


Assuntos
Aneurisma/induzido quimicamente , Doenças das Artérias Carótidas/induzido quimicamente , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fumaça/efeitos adversos , Fosfatase Ácida Resistente a Tartarato/metabolismo , Produtos do Tabaco/efeitos adversos , Aneurisma/enzimologia , Aneurisma/patologia , Animais , Cloreto de Cálcio , Doenças das Artérias Carótidas/enzimologia , Doenças das Artérias Carótidas/patologia , Catepsina K/metabolismo , Modelos Animais de Doenças , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/enzimologia , Osteoclastos/patologia , Células RAW 264.7 , Transdução de Sinais
8.
Cell Mol Neurobiol ; 39(6): 823-831, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31065924

RESUMO

Severe haemorrhagic transformation (HT), a common complication of recombinant tissue plasminogen activator (rtPA) treatment, predicts poor clinical outcomes in acute ischaemic stroke. The search for agents to mitigate this effect includes investigating biomolecules involved in neovascularization. This study examines the role of Cathepsin K (Ctsk) in rtPA-induced HT after focal cerebral ischaemia in mice. After knockout of Ctsk, the gene encoding Ctsk, the outcomes of Ctsk+/+ and Ctsk-/- mice were compared 24 h after rtPA-treated cerebral ischaemia with respect to HT severity, neurological deficits, brain oedema, infarct volume, number of apoptotic neurons and activated microglia/macrophage, blood-brain barrier integrity, vascular endothelial growth factor (VEGF) expression and Akt-mTOR pathway activation. We observed that haemoglobin levels, brain oedema and infarct volume were significantly greater and resulted in more severe neurological deficits in Ctsk-/- than in Ctsk+/+ mice. Consistent with our hypothesis, the number of NeuN-positive neurons was lower and the number of TUNEL-positive apoptotic neurons and activated microglia/macrophage was higher in Ctsk-/- than in Ctsk+/+ mice. Ctsk knockout mice exhibited more severe blood-brain barrier (BBB) disruption, with microvascular endothelial cells exhibiting greater VEGF expression and lower ratios of phospo-Akt/Akt and phospo-mTOR/mTOR than in Ctsk+/+ mice. This study is the first to provide molecular insights into Ctsk-regulated HT after cerebral ischaemia, suggesting that Ctsk deficiency may disrupt the BBB via Akt/mTOR/VEGF signalling, resulting in neurological deficits and neuron apoptosis. Ctsk administration has the potential as a novel modality for improving the safety of rtPA treatment following stroke.


Assuntos
Isquemia Encefálica/complicações , Catepsina K/deficiência , Hemorragia Cerebral/etiologia , Animais , Apoptose , Barreira Hematoencefálica/patologia , Catepsina K/metabolismo , Infarto da Artéria Cerebral Média/patologia , Macrófagos/patologia , Masculino , Camundongos Knockout , Microglia/patologia , Neurônios/patologia , Permeabilidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Ativador de Plasminogênio Tecidual , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Med Sci Monit ; 25: 3902-3909, 2019 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-31129676

RESUMO

BACKGROUND Osteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor kappaB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force. MATERIAL AND METHODS Cells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm² compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates. RESULTS Multinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm² force than 0.3 g/cm². Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force. CONCLUSIONS Our study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.


Assuntos
Reabsorção Óssea/genética , Força Compressiva/fisiologia , Osteoclastos/metabolismo , Animais , Reabsorção Óssea/metabolismo , Anidrase Carbônica II/metabolismo , Catepsina K/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , NF-kappa B/metabolismo , Osteoclastos/fisiologia , Ligante RANK/metabolismo , Células RAW 264.7 , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais
10.
Phytomedicine ; 59: 152910, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30978650

RESUMO

BACKGROUND: The receptor activator of nuclear factor-kappa B ligand (RANKL)-induced nuclear factor-kappa B (NF-κB) signaling pathway plays essential roles in osteoclast differentiation and may serve as an attractive target for the development of therapeutics for osteoporosis. PURPOSE: This study aimed to identify plant extracts that attenuated RANKL-induced NF-κB signaling pathway and examine their anti-osteoporotic effects in animal model systems. METHODS: Osteoclast differentiation was determined by western blot analysis, RT-PCR, and tartrate-resistant acid phosphatase (TRAP) assay. The effect of Longan (Dimocarpus longan Lour.) fruit extract (LFE) on bone mineral density was evaluated by calcein staining in zebrafish and micro-CT analysis in ovariectomized (OVX) rat. RESULTS: LFE nullified RANKL-induced down-regulation of inhibitor of NF-κB, which keeps NF-κB sequestered in the cytosol, thereby inhibiting translocation of NF-κB to the nucleus, in RAW264.7 cells. In addition, LFE decreased the nuclear levels of c-Fos and nuclear factor of activated T-cells c1, which play crucial roles in RANKL-induced osteoclast differentiation, in RAW264.7 cells. LFE repressed RANKL-activated cathepsin K and TRAP expression in RAW264.7 cells, resulting in a reduction of the number of TRAP-positive multinucleated cells, without cytotoxicity. Furthermore, LFE increased bone mineralization in zebrafish and prevented bone loss in OVX rat. CONCLUSION: Collectively, our findings suggest that LFE exerts its anti-osteoporotic activity through inhibition of osteoclast differentiation and may have potential as a herbal therapeutic or preventive agent for the treatment of osteoporosis.


Assuntos
Densidade Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Frutas , Osteoclastos/efeitos dos fármacos , Ligante RANK/metabolismo , Animais , Reabsorção Óssea/tratamento farmacológico , Catepsina K/metabolismo , Regulação para Baixo/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Osteoclastos/fisiologia , Osteoporose/prevenção & controle , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7 , Ratos , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismo
11.
Biomed Pharmacother ; 114: 108803, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30951949

RESUMO

Physiological root resorption of deciduous teeth is a normal phenomenon, however, the potential mechanisms underlying this process remain unclear. This study aimed to investigate ability of stem cells from human exfoliated deciduous teeth (SHED) on promoting the osteoclastic differentiation of osteoclast precursors and clarify mechanisms underlying this process in vitro. SHED and dental pulp stem cells (DPSCs) were obtained from deciduous teeth and healthy permanent teeth. An indirect co-culture system of SHED or DPSCs were used. The osteoclast precursor peripheral blood mononuclear cells (PBMCs) were established. Ability of SHED and DPSCs in promoting osteoclastogenesis was determined using triiodothyronine receptor auxiliary protein (TRAP) staining, real-time real-time PCR (RT-PCR) and western blotting. The effect of inflammation on the pro-osteoclastogenesis ability of SHED was determined using enzyme linked immunosorbent assay (ELISA), RT-PCR and western blotting. The function of the nuclear factor-κB (NF-κB) pathway in promoting the osteoclastogenesis ability of SHED was determined using RT-PCR and western blotting. SHED exhibited an increased ability to promote osteoclastic differentiation. Expression of tumor necrosis factor-α (TNF-α) was significantly higher in SHED than in DPSCs. Expression of cathepsin K (CTSK), TRAP, and receptor-activator of nuclear-factor-κ B ligand (RANKL), RANKL/osteoprotegerin (OPG) ratio, and expression of cytoplasmic phosphorylated inhibitor of NF-κB α (p-IκBα) and nuclear p65 were markedly up-regulated in SHED post the TNF-α treatment but decreased following NF-κB inhibition. In conclusion, inflammatory cytokine TNF-α appeared to activate NF-κB pathway to up-regulate expression of NF-κB, enhancing ability of SHED in promoting osteoclastogenesis via regulating RANKL/OPG expression.


Assuntos
Osteogênese/fisiologia , Reabsorção da Raiz/metabolismo , Dente Decíduo/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Catepsina K/metabolismo , Diferenciação Celular/fisiologia , Criança , Técnicas de Cocultura/métodos , Citocinas/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Células-Tronco/metabolismo , Adulto Jovem
12.
Bioorg Med Chem ; 27(6): 1034-1042, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30773420

RESUMO

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC50 values and inhibition constants (Ki) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.


Assuntos
Catepsina K/antagonistas & inibidores , Desdiferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Nitrilos/farmacologia , Inibidores de Proteases/farmacologia , Animais , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Catepsina K/metabolismo , Linhagem Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/enzimologia , Desenho de Fármacos , Humanos , Camundongos , Nitrilos/síntese química , Nitrilos/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química
13.
Blood ; 133(21): 2320-2324, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-30745304

RESUMO

Bone marrow (BM) sclerosis is commonly found in patients with late-stage myelofibrosis (MF). Because osteoclasts (OCs) and osteoblasts play a key role in bone remodeling, and MF monocytes, the OC precursors, are derived from the neoplastic clone, we wondered whether decreased OC numbers or impairment in their osteolytic function affects the development of osteosclerosis. Analysis of BM biopsies from 50 MF patients showed increased numbers of multinucleated tartrate-resistant acid phosphatase (TRAP)/cathepsin K+ OCs expressing phosphorylated Janus kinase 2 (JAK2). Randomly microdissected TRAP+ OCs from 16 MF patients harbored JAK2 or calreticulin (CALR) mutations, confirming MF OCs are clonal. To study OC function, CD14+ monocytes from MF patients and healthy individuals were cultured and differentiated into OCs. Unlike normal OCs, MF OCs appeared small and round, with few protrusions, and carried the mutations and chromosomal abnormalities of neoplastic clones. In addition, MF OCs lacked F-actin-rich ring-like structures and had fewer nuclei and reduced colocalization signals, compatible with decreased fusion events, and their mineral resorption capacity was significantly reduced, indicating impaired osteolytic function. Taken together, our data suggest that, although the numbers of MF OCs are increased, their impaired osteolytic activity distorts bone remodeling and contributes to the induction of osteosclerosis.


Assuntos
Remodelação Óssea , Osteoclastos , Osteólise , Mielofibrose Primária , Calreticulina/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Feminino , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Mutação , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteólise/genética , Osteólise/metabolismo , Osteólise/patologia , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , Fosfatase Ácida Resistente a Tartarato/genética , Fosfatase Ácida Resistente a Tartarato/metabolismo
14.
Biochem J ; 476(3): 499-512, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30622151

RESUMO

Cathepsin K (CatK) is a cysteine protease and drug target for skeletal disorders that is known for its potent collagenase and elastase activity. The formation of oligomeric complexes of CatK in the presence of glycosaminoglycans has been associated with its collagenase activity. Inhibitors that disrupt these complexes can selectively block the collagenase activity without interfering with the other regulatory proteolytic activities of the enzyme. Here, we have developed a fluorescence polarization (FP) assay to screen 4761 compounds for substrate-specific ectosteric collagenase inhibitors of CatK. A total of 38 compounds were identified that block the collagenase activity without interfering with the hydrolysis of active site substrates such as the synthetic peptide substrate, benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, and gelatin. The identified inhibitors can be divided into two main classes, negatively charged and polyaromatic compounds which suggest the binding to different ectosteric sites. Two of the inhibitors were highly effective in preventing the bone-resorption activity of CatK in osteoclasts. Interestingly, some of the ectosteric inhibitors were capable of differentiating between the collagenase and elastase activity of CatK depending on the ectosteric site utilized by the compound. Owing to their substrate-specific selectivity, ectosteric inhibitors represent a viable alternative to side effect-prone active site-directed inhibitors.


Assuntos
Catepsina K/antagonistas & inibidores , Peptídeos/química , Inibidores de Proteases/química , Animais , Catepsina K/química , Catepsina K/metabolismo , Bovinos , Humanos , Osteoclastos/enzimologia , Especificidade por Substrato
15.
J Cell Physiol ; 234(7): 10432-10444, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30652303

RESUMO

Chronic periodontitis (CP) is one of the most common oral diseases, which is characterized by the loss of connective tissue and alveolar bone in adults. AZD8835, a novel dual phosphoinositide-3-kinase (PI3K) inhibitor, is currently in phase 1 clinical evaluation to treat breast cancer. However, whether AZD8835 has any effect on teeth and alveolar bone health remains unclear. In the current study, we aimed to investigate the potential effect of AZD8835 in treating CP in vitro and in vivo. We found that AZD8835 could inhibit osteoclast differentiation, bone resorption, and downregulate the expression of osteoclast marker genes, such as tartrate-resistant acid phosphatase (Trap), cathepsin K (Ctsk), V-ATPase d2 (Atp6v0d2), and calcitonin receptor (Ctr). In addition, AZD8835 suppressed osteoclastogenesis by inhibiting receptor activator of nuclear factor kappa B ligand (RANKL)-induced PI3K/protein kinase B (AKT), extracellular signal-regulated kinase, and nuclear factor-κB signaling in BMMs. In vivo, AZD8835 greatly ameliorated alveolar bone (ABL) loss in rats with CP. Meanwhile, histological examination showed fewer osteoclasts in the treatment group. In conclusion, these results indicated that AZD8835 is a promising agent to reduce ABL in CP.


Assuntos
Perda do Osso Alveolar/tratamento farmacológico , Osteogênese/efeitos dos fármacos , Oxidiazóis/farmacologia , Periodontite/tratamento farmacológico , Piperidinas/farmacologia , Perda do Osso Alveolar/metabolismo , Animais , Biomarcadores/metabolismo , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Catepsina K/metabolismo , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Periodontite/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos
16.
Br J Clin Pharmacol ; 85(6): 1072-1083, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30663085

RESUMO

Cathepsin K (CatK) is a cysteine protease abundantly expressed by osteoclasts and localized in the lysosomes and resorption lacunae of these cells. CatK is the principal enzyme responsible for the degradation of bone collagen. Odanacatib is a selective, reversible inhibitor of CatK at subnanomolar potency. The pharmacokinetics of odanacatib have been extensively studied and are similar in young healthy men, postmenopausal women and elderly men, and were qualitatively similar throughout Phase 1 development and in-patient studies. Following 3 weeks of 50 mg once weekly dosing the geometric mean area under the curve from 0 to 168 hours was 41.1 µM h, the concentration at 168 hours was 126 nM and the harmonic mean apparent terminal half-life was 84.8 hr. Odanacatib exposure increased in a less than dose proportional manner due to solubility limited absorption. It is estimated that approximately 70% of the absorbed dose of odanacatib is eliminated via metabolism, 20% is excreted as unchanged drug in the bile or faeces, and 10% is excreted as unchanged drug in the urine. The systemic clearance was low (approximately 13 mL/min). Odanacatib decreases the degradation of bone matrix proteins and reduces the efficiency of bone resorption with target engagement confirmed by a robust decrease in serum C-telopeptides of type 1 collagen (approximately 60%), urinary aminoterminal crosslinked telopeptides of type 1 collagen to creatinine ratio (approximately 50%) and total urine deoxypyridinoline/Cr (approximately 30%), with an increase in serum cross-linked carboxy-terminal telopeptide of type 1 collagen (approximately 55%). The 50-mg weekly dosing regimen evaluated in Phase 3 achieved near maximal reduction in bone resorption throughout the treatment period. The extensive clinical programme for odanacatib, together with more limited clinical experience with other CatK inhibitors (balicatib and ONO-5334), provides important insights into the clinical pharmacology of CatK inhibition and the potential role of CatK in bone turnover and mineral homeostasis. Key findings include the ability of this mechanism to: (i) provide sustained reductions in resorption markers, increases in bone mineral density, and demonstrated fracture risk reduction; (ii) be associated with relative formation-sparing effects such that sustained resorption reduction is achieved without accompanying meaningful reductions in bone formation; and (iii) lead to increases in osteoclast number as well as other osteoclast activity (including build-up of CatK enzyme), which may yield transient increases in resorption following treatment discontinuation and the potential for nonmonotonic responses at subtherapeutic doses.


Assuntos
Compostos de Bifenilo/uso terapêutico , Conservadores da Densidade Óssea/uso terapêutico , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Catepsina K/antagonistas & inibidores , Inibidores de Cisteína Proteinase/uso terapêutico , Osteoporose/tratamento farmacológico , Animais , Compostos de Bifenilo/efeitos adversos , Compostos de Bifenilo/farmacocinética , Conservadores da Densidade Óssea/efeitos adversos , Conservadores da Densidade Óssea/farmacocinética , Osso e Ossos/enzimologia , Osso e Ossos/fisiopatologia , Catepsina K/metabolismo , Inibidores de Cisteína Proteinase/efeitos adversos , Inibidores de Cisteína Proteinase/farmacocinética , Feminino , Humanos , Masculino , Osteoporose/enzimologia , Osteoporose/patologia , Transdução de Sinais , Pesquisa Médica Translacional , Resultado do Tratamento
17.
Bone ; 120: 166-175, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30409757

RESUMO

High-bone-mass (HBM)-causing missense mutations in the low density lipoprotein receptor-related protein-5 (Lrp5) are associated with increased osteoanabolic action and protection from disuse- and ovariectomy-induced osteopenia. These mutations (e.g., A214V and G171V) confer resistance to endogenous secreted Lrp5/6 inhibitors, such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). Cells in the osteoblast lineage are responsive to canonical Wnt stimulation, but recent work has indicated that osteoclasts exhibit both indirect and direct responsiveness to canonical Wnt. Whether Lrp5-HBM receptors, expressed in osteoclasts, might alter osteoclast differentiation, activity, and consequent net bone balance in the skeleton, is not known. To address this, we bred mice harboring heterozygous Lrp5 HBM-causing conditional knock-in alleles to Ctsk-Cre transgenic mice and studied the phenotype using DXA, µCT, histomorphometry, serum assays, and primary cell culture. Mice with HBM alleles induced in Ctsk-expressing cells (TG) exhibited higher bone mass and architectural properties compared to non-transgenic (NTG) counterparts. In vivo and in vitro measurements of osteoclast activity, population density, and differentiation yielded significant reductions in osteoclast-related parameters in female but not male TG mice. Droplet digital PCR performed on osteocyte enriched cortical bone tubes from TG and NTG mice revealed that ~8-17% of the osteocyte population (depending on sex) underwent recombination of the conditional Lrp5 allele in the presence of Ctsk-Cre. Further, bone formation parameters in the midshaft femur cortex show a small but significant increase in anabolic action on the endocortical but not periosteal surface. These findings suggest that Wnt/Lrp5 signaling in osteoclasts affects osteoclastogenesis and activity in female mice, but also that some of the changes in bone mass in TG mice might be due to Cre expression in the osteocyte population.


Assuntos
Osso e Ossos/metabolismo , Catepsina K/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Mutação/genética , Absorciometria de Fóton , Alelos , Animais , Biomarcadores/sangue , Células da Medula Óssea/metabolismo , Reabsorção Óssea/sangue , Reabsorção Óssea/patologia , Osso e Ossos/diagnóstico por imagem , Diferenciação Celular , Feminino , Integrases/metabolismo , Masculino , Camundongos Transgênicos , Tamanho do Órgão/genética , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/genética , Periósteo/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética/genética , Transgenes , Microtomografia por Raio-X
18.
Pharmacology ; 103(1-2): 101-109, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30522105

RESUMO

It has been reported that taxifolin inhibit osteoclastogenesis in RAW264.7 cells. In our research, the inhibition effects of taxifolin on the osteoclastogenesis of human bone marrow-derived macrophages (BMMs) induced by receptor activator of NF-κB ligand (RANKL) as well as the protection effects in lipopolysaccharide-induced bone lysis mouse model have been demonstrated. In vitro, taxifolin inhibited RANKL-induced osteoclast differentiation of human BMMs without cytotoxicity. Moreover, taxifolin significantly suppressed RANKL-induced gene expression, including tartrate-resistant acid phosphatase, matrix metalloproteinase-9 nuclear factor of activated T cells 1 and cathepsin K, and F-actin ring formation. Further studies showed that taxifolin inhibit osteoclastogenesis via the suppression of the NF-κB signaling pathway. In vivo, taxifolin prevented bone loss in mouse calvarial osteolysis model. In conclusion, the results suggested that taxifolin has a therapeutic potential for osteoclastogenesis-related diseases such as osteoporosis, osteolysis, and rheumatoid arthritis.


Assuntos
Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Quercetina/análogos & derivados , Ligante RANK/antagonistas & inibidores , Actinas/metabolismo , Animais , Catepsina K/metabolismo , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Quinase I-kappa B/metabolismo , Macrófagos/citologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteólise/induzido quimicamente , Osteólise/tratamento farmacológico , Osteólise/patologia , Quercetina/farmacologia , Ligante RANK/farmacologia , Células RAW 264.7 , Transdução de Sinais , Fator de Transcrição RelA/metabolismo
19.
Bioorg Chem ; 84: 226-238, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30502634

RESUMO

A series of novel 7-aminoalkyl substituted pyrazolo[1,5-a]pyrimidine derivatives were synthesized and tested for inhibition of cathepsin K. The synthetic methodology comprises cyclization of 5-aminopyrazoles with N-Boc-α-amino acid-derived ynones followed by transformation of the ester and the Boc-amino functions. It allows for easy diversification of the pyrazolo[1,5-a]pyrimidine scaffold at various positions. Molecular docking studies with pyrazolo[1,5-a]pyrimidine derivatives were also performed to elucidate the binding mode in the active site of cathepsin K. The synthesized compounds exhibited moderate inhibition activity (Ki ≥ 77 µM).


Assuntos
Catepsina K/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Pirazóis/química , Pirimidinas/química , Sítios de Ligação , Domínio Catalítico , Catepsina K/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Pirazóis/metabolismo , Pirimidinas/metabolismo , Relação Estrutura-Atividade
20.
J Thromb Haemost ; 17(1): 183-194, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30394658

RESUMO

Essentials During contact system activation, factor XII is progressively cleaved by plasma kallikrein. We investigated the role of factor XII truncation in biochemical studies. Factor XII contains naturally occurring truncating cleavage sites for a variety of enzymes. Truncation of factor XII primes it for activation in solution through exposure of R353. SUMMARY: Background The contact activation system and innate immune system are interlinked in inflammatory pathology. Plasma kallikrein (PKa) is held responsible for the stepwise processing of factor XII (FXII). A first cleavage activates FXII (into FXIIa); subsequent cleavages truncate it. This truncation eliminates its surface-binding domains, which negatively regulates surface-dependent coagulation. Objectives To investigate the influence of FXII truncation on its activation and downstream kallikrein-kinin system activation. Methods We study activation of recombinant FXII variants by chromogenic assays, by FXIIa ELISA and western blotting. Results We demonstrate that FXII truncation primes it for activation by PKa in solution. We demonstrate this phenomenon in three settings. (i) Truncation at a naturally occurring PKa-sensitive cleavage site, R334, accelerates FXIIa formation in solution. A site-directed mutant FXII-R334A displays ~50% reduced activity when exposed to PKa. (ii) A pathogenic mutation in FXII that causes hereditary angioedema, introduces an additional plasmin-sensitive cleavage site. Truncation at this site synergistically accelerates FXII activation in solution. (iii) We identify new, naturally occurring cleavage sites in FXII that have so far not been functionally linked to contact system activation. As examples, we show that non-activating truncation of FXII by neutrophil elastase and cathepsin K primes it for activation by PKa in solution. Conclusions FXII truncation, mediated by either pathogenic mutations or naturally occurring cleavage sites, primes FXII for activation in solution. We propose that the surface-binding domains of FXII shield its activating cleavage site, R353. This may help to explain how the contact system contributes to inflammatory pathology.


Assuntos
Coagulação Sanguínea , Fator XII/metabolismo , Fator XIIa/metabolismo , Calicreína Plasmática/metabolismo , Catepsina K/metabolismo , Ativação Enzimática , Fator XII/genética , Fator XIIa/genética , Células HEK293 , Humanos , Elastase de Leucócito/metabolismo , Mutação , Domínios Proteicos Ricos em Prolina , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato , Fatores de Tempo
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