RESUMO
Ionizing radiation is a common therapeutic modality and following irradiation dermal changes, including fibrosis and atrophy, may lead to permanent changes. We have previously demonstrated that occupancy of A2A receptor (A2AR) stimulates collagen production, so we determined whether blockade or deletion of A2AR could prevent radiation-induced fibrosis. After targeted irradiation (40 Gy) of the skin of wild-type (WT) or A2AR knockout (A2ARKO) mice, the A2AR antagonist ZM241385 was applied daily for 28 d. In irradiated WT mice treated with the A2AR antagonist, there was a marked reduction in collagen content and skin thickness, and ZM241385 treatment reduced the number of myofibroblasts and angiogenesis. After irradiation, there is an increase in loosely packed collagen fibrils, which is significantly diminished by ZM241385. Irradiation also induced an increase in epidermal thickness, prevented by ZM241385, by increasing the number of proliferating keratinocytes. Similarly, in A2ARKO mice, the changes in collagen alignment, skin thickness, myofibroblast content, angiogenesis, and epidermal hyperplasia were markedly reduced following irradiation. Radiation-induced changes in the dermis and epidermis were accompanied by an infiltrate of T cells, which was prevented in both ZM241385-treated and A2ARKO mice. Radiation therapy is administered to a significant number of patients with cancer, and radiation reactions may limit this therapeutic modality. Our findings suggest that topical application of an A2AR antagonist prevents radiation dermatitis and may be useful in the prevention or amelioration of radiation changes in the skin.
Assuntos
Radiodermite/metabolismo , Receptor A2A de Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Animais , Colágeno/metabolismo , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Radiodermite/tratamento farmacológico , Receptor A2A de Adenosina/genética , Triazinas/farmacologia , Triazinas/uso terapêutico , Triazóis/farmacologia , Triazóis/uso terapêuticoRESUMO
Promoting bone regeneration and repair of bone defects is a need that has not been well met to date. We have previously found that adenosine, acting via A2A receptors (A2AR) promotes wound healing and inhibits inflammatory osteolysis and hypothesized that A2AR might be a novel target to promote bone regeneration. Therefore, we determined whether direct A2AR stimulation or increasing endogenous adenosine concentrations via purine transport blockade with dipyridamole regulates bone formation. We determined whether coverage of a 3 mm trephine defect in a mouse skull with a collagen scaffold soaked in saline, bone morphogenetic protein-2 (BMP-2; 200 ng), 1 µM CGS21680 (A2AR agonist, EC50 = 160 nM), or 1 µM dipyridamole (EC50 = 32 nM) promoted bone regeneration. Microcomputed tomography examination demonstrated that CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP-2 8 wk after surgery (60 ± 2%, 79 ± 2%, and 75 ± 1% bone regeneration, respectively, vs. 32 ± 2% in control, P < 0.001). Blockade by a selective A2AR antagonist (ZM241385, 1 µM) or deletion of A2AR abrogated the effect of CGS21680 and dipyridamole on bone regeneration. Both CGS21680 and dipyridamole treatment increased alkaline phosphatase-positive osteoblasts and diminished tartrate resistance acid phosphatase-positive osteoclasts in the defects. In vivo imaging with a fluorescent dye for new bone formation revealed a strong fluorescent signal in treated animals that was equivalent to BMP-2. In conclusion, stimulation of A2AR by specific agonists or by increasing endogenous adenosine levels stimulates new bone formation as well as BMP-2 and represents a novel approach to stimulating bone regeneration.
Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/fisiologia , Receptor A2A de Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Animais , Contagem de Células , Dipiridamol/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Fenetilaminas/farmacologiaRESUMO
Bisphosphonates inhibit osteoclast differentiation/function via inhibition of Rap1A isoprenylation. As Rap1 is the effector of exchange protein directly activated by cAMP (EPAC) proteins, we determined the role of EPAC in osteoclast differentiation. We examined osteoclast differentiation as the number of primary murine/human bone-marrow precursors that differentiated into multinucleated TRAP-positive cells in the presence of EPAC-selective stimulus (8-pCTP-2'-O-Me-cAMP, 100 µM; 8-pCTP-2'-O-Me-cAMP-AM, 1 µM) or inhibitor brefeldin A (BFA), ESI-05, and ESI-09 (10 µM each). Rap1 activity was assessed, and signaling events, as well as differentiation in EPAC1/2-knockdown RAW264.7 cells, were studied. Direct EPAC1/2 stimulation significantly increased osteoclast differentiation, whereas EPAC1/2 inhibition diminished differentiation (113 ± 6%, P < 0.05, and 42 ± 10%, P < 0.001, of basal, respectively). Rap1 activation was maximal 15 min after RANKL stimulation (147 ± 9% of basal, P < 0.001), whereas silencing of EPAC1/2 diminished activated Rap1 (43 ± 13 and 20 ± 15% of control, P < 0.001) and NFkB nuclear translocation. TRAP-staining revealed no osteoclast differentiation in EPAC1/2-KO cells. Cathepsin K, NFATc1, and osteopontin mRNA expression decreased in EPAC1/2-KO cells when compared to control. RhoA, cdc42, Rac1, and FAK were activated in an EPAC1/2-dependent manner, and there was diminished cytoskeletal assembly in EPAC1/2-KO cells. In summary, EPAC1 and EPAC2 are critical signaling intermediates in osteoclast differentiation that permit RANKL-stimulated NFkB nuclear translocation and actin rearrangements. Targeting this signaling intermediate may diminish bone destruction in inflammatory arthritis.
Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , NF-kappa B/metabolismo , Osteoclastos/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Hidrazonas/farmacologia , Isoxazóis/farmacologia , Camundongos , NF-kappa B/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Ligante RANK/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Activation of adenosine A2A receptor (A2AR) promotes fibrosis and collagen synthesis. However, the underlying mechanism is still unclear, not least because cAMP, its principal effector, has been found to inhibit TGFß1-induced collagen synthesis. Here, we show that in primary normal human dermal fibroblasts, A2AR stimulation with CGS21680 elicits a modest cAMP increase (150 ± 12% of control; EC50 54.8 nM), which stimulates collagen1 (Col1) and collagen3 (Col3), but maximal cAMP resulting from direct activation of adenylyl cyclase by forskolin (15,689 ± 7038% of control; EC50 360.7 nM) inhibits Col1 and increases Col3. Similar to Col1 expression, fibroblast proliferation increased following physiological cAMP increases by CGS21680 but was inhibited by cAMP increases beyond the physiological range by forskolin. The A2AR-mediated increase of Col1 and Col3 was mediated by AKT, while Col3, but not Col1, expression was dependent on p38 and repressed by ERK. TGFß1 induced phosphorylation of Smad2/3 and increased Col3 expression, which was prevented by Smad3 depletion. In contrast, CGS21680 did not activate Smad2/3, and Smad2/3 knockdown did not prevent CGS21680-induced Col1 or Col3 increases. Our results indicate that cAMP is a concentration-dependent switch for collagen production via noncanonical, AKT-dependent, Smad2/3-independent signaling. These observations explain the paradoxical effects of cAMP on collagen expression.
Assuntos
Colágeno/metabolismo , AMP Cíclico/metabolismo , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor A2A de Adenosina/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Fenetilaminas/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , Proteína Smad2/genética , Proteína Smad3/genéticaRESUMO
Adenosine has an important role in inflammation and tissue remodeling and promotes dermal fibrosis by adenosine receptor (A2AR) activation. Adenosine may be formed intracellularly from adenine nucleotides or extracellularly through sequential phosphohydrolysis of released ATP by nucleoside triphosphate diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73). Because the role of these ecto-enzymes in fibrosis appears to be tissue specific, we determined whether these ectonucleotidases were directly involved in diffuse dermal fibrosis. Wild-type and mice globally deficient in CD39 knockout (CD39KO), CD73 (CD73KO), or both (CD39/CD73DKO) were challenged with bleomycin. Extracellular adenosine levels and dermal fibrosis were quantitated. Adenosine release from skin cultured ex vivo was increased in wild-type mice after bleomycin treatment but remained low in skin from CD39KO, CD73KO, or CD39/CD73DKO bleomycin-treated mice. Deletion of CD39 and/or CD73 decreased the collagen content, and prevented skin thickening and tensile strength increase after bleomycin challenge. Decreased dermal fibrotic features were associated with reduced expression of the profibrotic mediators, transforming growth factor-ß1 and connective tissue growth factor, and diminished myofibroblast population in CD39- and/or CD73-deficient mice. Our work supports the hypothesis that extracellular adenosine, generated in tandem by ecto-enzymes CD39 and CD73, promotes dermal fibrogenesis. We suggest that biochemical or biological inhibitors of CD39 and/or CD73 may hold promise in the treatment of dermal fibrosis in diseases such as scleroderma.
Assuntos
5'-Nucleotidase , Adenosina , Antígenos CD , Apirase , Derme , Escleroderma Sistêmico , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adenosina/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/efeitos adversos , Antibióticos Antineoplásicos/farmacologia , Antígenos CD/genética , Antígenos CD/metabolismo , Apirase/genética , Apirase/metabolismo , Bleomicina/efeitos adversos , Bleomicina/farmacologia , Derme/metabolismo , Derme/patologia , Fibrose/induzido quimicamente , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Camundongos , Camundongos Knockout , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The Reconstructed Human Epidermis (RHE) model derived from epidermal keratinocytes offers an ethical and scientific alternative to animal experimentation, particularly in cutaneous toxicology and dermatological research, where the elimination of animal cruelty is of paramount importance. Thus, we compared commercially available chemically defined animal origin-free (cdAOF) supplements, designed for regenerative medicine, to the widely utilized supplement (human keratinocyte growth supplement), which contains growth factors and bovine pituitary extract. Herein we present the extended characterization of RHE derived from newborn, adult, and immortalized N/telomerase reverse transcriptase keratinocytes under cdAOF conditions. Culture of RHE in the cdAOF media produced histological features that were similar to that produced using human keratinocyte growth supplement, with the exception that the basal keratinocytes were less cylindrical. Additionally, immunolocalization of involucrin in the basal layer and increased mRNA expression of several inflammatory-proliferative markers were observed under cdAOF conditions. In RHEs cultured in cdAOF media, expression and immunolocalization of other expected markers of keratinization were similar, while monitoring of barrier function (transepithelial electrical resistance) revealed results that were statistically equal to, or lower than those observed in RHE cultured in human keratinocyte growth supplement. Our study indicates that reconstruction of RHE was accomplished under cdAOF culture conditions and that further refinement could promote an expanded use beyond regenerative medicine, for in vitro toxicology applications.
RESUMO
Adenosine A2A receptor (A2AR) stimulation promotes wound healing and is required for the development of fibrosis in murine models of scleroderma and cirrhosis. Nonetheless, the role of A2AR in the formation of scars following skin trauma has not been explored. Here, we examined the effect of pharmacological blockade of A2AR, with the selective adenosine A2AR-antagonist ZM241385 (2.5 mg/ml), in a murine model of scarring that mimics human scarring. We found that application of the selective adenosine A2AR antagonist ZM241385 decreased scar size and enhanced the tensile strength of the scar. Within the scar itself, collagen alignment and composition (marked reduction in collagen 3), but not periostin, biglycan, or fibronectin accumulation, was improved by application of ZM241385. Moreover, A2AR blockade reduced the number of myofibroblasts and angiogenesis but not macrophage infiltration in the scar. Taken together, our work strongly suggests that pharmacological A2AR blockade can be used to diminish scarring while improving the collagen composition and tensile strength of the healed wound.
Assuntos
Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Cicatriz/tratamento farmacológico , Cicatriz/metabolismo , Receptor A2A de Adenosina/metabolismo , Triazinas/uso terapêutico , Triazóis/uso terapêutico , Animais , Western Blotting , Colágeno/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor A2A de Adenosina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cicatrização/efeitos dos fármacos , Cicatrização/genéticaRESUMO
Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A2A receptor (A2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A2AR stimulation to increased collagen production is complex and obscure, not least because cAMP and its downstream molecules PKA and Epac1 have been reported to inhibit collagen production. We therefore examined A2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A2AR activation by CGS21680 was studied by western blotting. Contribution of PKA and Epac was analyzed by the PKA inhibitor PKI and by knockdowns of the PKA-Cα, -Cß, -Cγ, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-Cα and -Cß knockdown prevents A2AR-mediated Col1 increase. In contrast, A2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A2AR stimulation with CGS21680 at 0.1 µM increased Col3 expression only upon PKA blockade. A2AR activation downstream signaling for Col1 and Col3 expression proceeds via two distinct pathways with varying sensitivity to cAMP activation; more highly cAMP-sensitive PKA activation stimulates Col1 expression, and less cAMP-sensitive Epac activation promotes both Col1 and Col3 expression. These observations may explain the dramatic change in Col1:Col3 ratio in hypertrophic and immature scars, where adenosine is present in higher concentrations than in normal skin.
Assuntos
Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Receptor A2A de Adenosina/metabolismo , Transdução de Sinais/fisiologia , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , HumanosRESUMO
Adenosine A2A receptor mediates the promotion of wound healing and revascularization of injured tissue, in healthy and animals with impaired wound healing, through a mechanism depending upon tissue plasminogen activator (tPA), a component of the fibrinolytic system. In order to evaluate the contribution of plasmin generation in the proangiogenic effect of adenosine A2A receptor activation, we determined the expression and secretion of t-PA, urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) and annexin A2 by human dermal microvascular endothelial cells stimulated by the selective agonist CGS-21680. The plasmin generation was assayed through an enzymatic assay and the proangiogenic effect was studied using an endothelial tube formation assay in Matrigel. Adenosine A2A receptor activation in endothelial cells diminished the release of PAI-1 and promoted the production of annexin A2, which acts as a cell membrane co-receptor for plasminogen and its activator tPA. Annexin A2 mediated the increased cell membrane-associated plasmin generation in adenosine A2A receptor agonist treated human dermal microvascular endothelial cells and is required for tube formation in an in vitro model of angiogenesis. These results suggest a novel mechanism by which adenosine A2A receptor activation promotes angiogenesis: increased endothelial expression of annexin A2, which, in turn, promotes fibrinolysis by binding tPA and plasminogen to the cell surface.
RESUMO
BACKGROUND: Fibrosing diseases are a leading cause of morbidity and mortality worldwide and, therefore, there is a need for safe and effective antifibrotic therapies. Adenosine, generated extracellularly by the dephosphorylation of adenine nucleotides, ligates specific receptors which play a critical role in development of hepatic and dermal fibrosis. Results of recent clinical trials indicate that tenofovir, a widely used antiviral agent, reverses hepatic fibrosis/cirrhosis in patients with chronic hepatitis B infection. Belonging to the class of acyclic nucleoside phosphonates, tenofovir is an analogue of AMP. We tested the hypothesis that tenofovir has direct antifibrotic effects in vivo by interfering with adenosine pathways of fibrosis using two distinct models of adenosine and A2AR-mediated fibrosis. METHODS: Thioacetamide (100mg/kg IP)-treated mice were treated with vehicle, or tenofovir (75mg/kg, SubQ) (n = 5-10). Bleomycin (0.25U, SubQ)-treated mice were treated with vehicle or tenofovir (75mg/kg, IP) (n = 5-10). Adenosine levels were determined by HPLC, and ATP release was quantitated as luciferase-dependent bioluminescence. Skin breaking strength was analysed and H&E and picrosirus red-stained slides were imaged. Pannexin-1expression was knocked down following retroviral-mediated expression of of Pannexin-1-specific or scrambled siRNA. RESULTS: Treatment of mice with tenofovir diminished adenosine release from the skin of bleomycin-treated mice and the liver of thioacetamide-treated mice, models of diffuse skin fibrosis and hepatic cirrhosis, respectively. More importantly, tenofovir treatment diminished skin and liver fibrosis in these models. Tenofovir diminished extracellular adenosine concentrations by inhibiting, in a dose-dependent fashion, cellular ATP release but not in cells lacking Pannexin-1. CONCLUSIONS: These studies suggest that tenofovir, a widely used antiviral agent, could be useful in the treatment of fibrosing diseases.
Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Antivirais/farmacologia , Conexinas/antagonistas & inibidores , Modelos Animais de Doenças , Cirrose Hepática/prevenção & controle , Fígado/efeitos dos fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Dermatopatias/prevenção & controle , Pele/efeitos dos fármacos , Tenofovir/farmacologia , Animais , Conexinas/fisiologia , Relação Dose-Resposta a Droga , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/fisiologia , Pele/metabolismoRESUMO
BACKGROUND AND PURPOSE: Adenosine A2A receptor stimulation promotes the synthesis of collagen type I and type III (Col1 and Col3), mediators of fibrosis and scarring. The A2A receptor modulates collagen balance via cAMP/PKA/p38-MAPK/Akt pathways. Wnt signalling is important in fibrosis and the cAMP and Wnt pathways converge. Because the A2A receptor is Gs-linked and increases cAMP, we determined whether A2A receptors and Wnt signalling interact. EXPERIMENTAL APPROACH: Total ß-catenin, de-phosphorylated ß-catenin (canonical activation, de-phospho ß-catenin) and phosphorylated ß-catenin at Ser552 (non-canonical activation, p-Ser552 ß-catenin) levels were determined in primary human dermal fibroblasts, cytosol and nucleus, by western blot analysis and fluorescence microscopy, before and after stimulation by A2A receptor-selective agonist CGS21680, with/without A2A receptor-selective antagonist (SCH56261) pretreatment. ß-Catenin was knocked down by transfection with scrambled-siRNA or specific-siRNA, and Col1 and Col3 levels determined by western blots. KEY RESULTS: CGS21680 stimulation rapidly (15 min) increased cellular ß-catenin levels. Both de-phospho ß-catenin and p-Ser552 ß-catenin levels were also increased. CGS21680 stimulated the translocation of total de-phospho and p-Ser552 ß-catenin to the nucleus. A2A receptor-stimulation increased Col1 synthesis similarly in ß-catenin knockeddown and scrambled cells. However, ß-catenin knockdown abolished the increase in Col3 synthesis induced in A2A receptor-stimulated fibroblasts. CONCLUSIONS AND IMPLICATIONS: A2A receptor stimulation promotes Col3 synthesis via the activation of canonical and non-canonical ß-catenin, consistent with a role for A2A receptors in dermal fibrosis and scarring.
Assuntos
Colágeno Tipo III/biossíntese , Fibroblastos/metabolismo , Receptor A2A de Adenosina/metabolismo , beta Catenina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Western Blotting , Cicatriz/patologia , Colágeno Tipo I/biossíntese , Fibroblastos/efeitos dos fármacos , Fibrose/patologia , Técnicas de Silenciamento de Genes , Humanos , Microscopia de Fluorescência , Fenetilaminas/farmacologia , Fosforilação , RNA Interferente Pequeno/administração & dosagem , Receptor A2A de Adenosina/efeitos dos fármacos , Pele/patologia , beta Catenina/genéticaRESUMO
OBJECTIVE: Adenosine, acting at the A2A receptor, mediates the antiinflammatory effects of methotrexate (MTX) in models of inflammation. We previously reported that A2A receptor ligation diminishes wear particle-driven osteolysis. The aim of this study was to investigate whether MTX treatment could prevent bone resorption caused by inflammatory osteolysis. METHODS: C57BL/6 mice (6-8 weeks old) received intraperitoneal injections of 1 mg/kg MTX (n = 20) or 0.9% saline (n = 10), starting 2 weeks prior to surgical implantation of 3 mg of wear particles (ultrahigh molecular weight polyethylene [UHMWPE] particles). The MTX-treated mice received daily injections of vehicle or ZM241385 at the surgical site until they were killed, 14 days later. XenoLight RediJect Bone Probe 680 was injected intravenously, and fluorescence analysis of the calvaria using an IVIS imaging system was performed to assess bone formation. Micro-computed tomography (micro-CT) and immunostaining for osteoclast and osteoblast markers were performed. RESULTS: Implantation of wear particles induced bone pitting and thinning, as shown by micro-CT. MTX treatment markedly reduced osteolysis, and this effect was abrogated by treatment with the A2A receptor antagonist ZM241385. Implantation of UHMWPE reduced new bone formation, and MTX treatment restored new bone formation, an effect that was completely reversed by treatment with ZM241385. Histologic examination of particle-exposed calvariae demonstrated that MTX prevented accumulation of an inflammatory infiltrate at the site of particle implantation, increased the number of osteoblasts, and reduced the number of osteoclasts at the site of inflammation, an effect that was reversed by treatment with ZM241385. CONCLUSION: MTX reduces inflammatory osteolysis indirectly via stimulation of A2A receptor and may represent a novel approach to enhance orthopedic implant survival, delaying or eliminating the need for revision arthroplasty surgery.
Assuntos
Metotrexato/uso terapêutico , Osteoclastos/efeitos dos fármacos , Osteólise/prevenção & controle , Receptor A2A de Adenosina/metabolismo , Crânio/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Humanos , Inflamação , Masculino , Metotrexato/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Osteólise/induzido quimicamente , Osteólise/tratamento farmacológico , Osteólise/metabolismo , Polietilenos , Crânio/metabolismoRESUMO
INTRODUCTION: This work was undertaken to delineate intracellular signaling pathways for the PDE4 inhibitor apremilast and to examine interactions between apremilast, methotrexate and adenosine A2A receptors (A2AR). METHODS: After apremilast and LPS incubation, intracellular cAMP, TNF-α, IL-10, IL-6 and IL-1α were measured in the Raw264.7 monocytic murine cell line. PKA, Epac1/2 (signaling intermediates for cAMP) and A2AR knockdowns were performed by shRNA transfection and interactions with A2AR and A2BR, as well as with methotrexate were tested in vitro and in the murine air pouch model. Statistical differences were determined using one or two-way ANOVA or Student's t test. The alpha nominal level was set at 0.05 in all cases. A P value of < 0.05 was considered significant. RESULTS: In vitro, apremilast increased intracellular cAMP and inhibited TNF-α release (IC50=104nM) and the specific A2AR-agonist CGS21680 (1µM) increased apremilast potency (IC50=25nM). In this cell line, apremilast increased IL-10 production. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by apremilast. In the murine air pouch model, both apremilast and MTX significantly inhibited leukocyte infiltration, while apremilast, but not MTX, significantly inhibited TNF-α release. The addition of MTX (1 mg/kg) to apremilast (5 mg/kg) yielded no more inhibition of leukocyte infiltration or TNF-α release than with apremilast alone. CONCLUSIONS: The immunoregulatory effects of apremilast appear to be mediated by cAMP through the downstream effectors PKA, Epac1, and Epac2. A2AR agonism potentiated TNF-α inhibition by apremilast, consistent with the cAMP-elevating effects of that receptor. Because the A2AR is also involved in the anti-inflammatory effects of MTX, the mechanism of action of both drugs involves cAMP-dependent pathways and is therefore partially overlapping in nature.
Assuntos
AMP Cíclico/metabolismo , Mediadores da Inflamação/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Talidomida/análogos & derivados , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Antirreumáticos/farmacologia , Western Blotting , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Metotrexato/farmacologia , Camundongos , Fenetilaminas/farmacologia , Interferência de RNA , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Talidomida/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Bone metabolism is a vital process that involves resorption by osteoclasts and formation by osteoblasts, which is closely regulated by immune cells. The neuronal guidance protein Netrin-1 regulates immune cell migration and inflammatory reactions, but its role in bone metabolism is unknown. During osteoclast differentiation, osteoclast precursors increase expression of Netrin-1 and its receptor Unc5b. Netrin-1 binds, in an autocrine and paracrine manner, to Unc5b to promote osteoclast differentiation in vitro, and absence of Netrin-1 or antibody-mediated blockade of Netrin-1 or Unc5b prevents osteoclast differentiation of both murine and human precursors. We confirmed the functional relationship of Netrin-1 in osteoclast differentiation in vivo using Netrin-1-deficient (Ntn1(-/-) ) or wild-type (WT) bone marrow transplanted mice. Notably, Ntn1(-/-) chimeras have markedly diminished osteoclasts, as well as increased cortical and trabecular bone density and volume compared with WT mice. Mechanistic studies revealed that Netrin-1 regulates osteoclast differentiation by altering cytoskeletal assembly. Netrin-1 increases regulator of Rho-GEF subfamily (LARG) and repulsive guidance molecule (RGMa) association with Unc5b, which increases expression and activation of cytoskeletal regulators RhoA and focal adhesion kinase (FAK). Netrin-1 and its receptor Unc5b likely play a role in fusion of osteoclast precursors because Netrin-1 and DC-STAMP are tightly linked. These results identify Netrin-1 as a key regulator of osteoclast differentiation that may be a new target for bone therapies.
Assuntos
Comunicação Autócrina , Diferenciação Celular , Fatores de Crescimento Neural/metabolismo , Osteoclastos/citologia , Comunicação Parácrina , Proteínas Supressoras de Tumor/metabolismo , Animais , Comunicação Autócrina/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Osso e Ossos/anatomia & histologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Netrina , Netrina-1 , Tamanho do Órgão/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Receptores de Superfície Celular/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
ß-Adrenoceptors (ß-ARs) modulate ERK1/2 and p38 in different cells, but little is known about the contribution of these signaling pathways to the function of ß-ARs in vascular tissue. Immunoblotting analysis of rat aortic rings, primary endothelial (ECs) and smooth muscle cells (SMCs) isolated from aorta showed that ß-AR stimulation with isoprenaline activated p38 in aortic rings and in both cultured cell types, whereas it had a dual effect on ERK1/2 phosphorylation, decreasing it in ECs while increasing it in SMCs. These effects were reversed by propranolol, which by itself increased p-ERK1/2 in ECs. Isoprenaline ß-AR mediated vasodilation of aortic rings was potentiated by the ERK1/2 inhibitor, U0126, in the presence or absence of endothelium or L-NAME, whereas inhibition of p38 had no impact. Isoprenaline moderately decreased sprouting from aorta rings in the Matrigel angiogenesis assay; conversely propranolol not only prevented isoprenaline inhibition, but stimulated angiogenesis. ERK1/2 inhibition decreased angiogenesis, while a dramatic stimulation was observed by p38 blockade. Our results suggest that ERK1/2 activation after ß-ARs stimulation in the smooth muscle hinders the vasodilator effect of isoprenaline, but in the endothelium ß-ARs decreases ERK1/2 and increases p38 activity reducing therefore angiogenesis.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores Adrenérgicos beta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Propranolol/farmacologia , Ratos , Ratos Wistar , Receptores Adrenérgicos beta/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
BACKGROUND AND PURPOSE: We previously reported that adenosine, acting at adenosine A(2A) receptors (A(2A)R), inhibits osteoclast (OC) differentiation in vitro (A(2A)R activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A(2A)R stimulation regulates OC differentiation, we dissected the signalling pathways involved in A(2A)R signalling. EXPERIMENTAL APPROACH: OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A(2A)R agonist CGS21680, the A(2A)R antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR. KEY RESULTS: A(2A)R stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A(2A)R stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A(2A)R stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A(2A)R stimulation inhibited OC expression of differentiation markers by a PKA-mechanism. CONCLUSIONS AND IMPLICATIONS: A(2A)R activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like rheumatoid arthritis.
Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Núcleo Celular/efeitos dos fármacos , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Subunidade p50 de NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Receptor A2A de Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Células Cultivadas , AMP Cíclico/agonistas , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Camundongos , Camundongos Endogâmicos C57BL , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Osteoclastos/citologia , Osteoclastos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Receptor A2A de Adenosina/química , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismoRESUMO
Morning stiffness and increased symptoms of inflammatory arthritis are among the most common manifestations of rheumatoid arthritis (RA). Tumor necrosis alpha (TNF-α), an important mediator of inflammation in RA, regulates the circadian expression of clock proteins, and adenosine A(2A) receptors (A(2A)R) mediate many of the anti-inflammatory and antirheumatic actions of methotrexate, the cornerstone drug in the treatment of RA. We found that A(2A)R activation and TNF-α activated the clock core loop of the human monocytic THP-1 cell line. We further observed that interleukin (IL)-10, but not IL-12, mRNA expression fluctuates in a circadian fashion and that TNF-α and A(2A)R stimulation combined increased IL-10 expression. Interestingly, TNF-α, but not CGS21680, dramatically inhibited IL-12 mRNA expression. The demonstration that A(2A)R and TNF-α regulate the intrinsic circadian clock in immune cells provides an explanation for both the pathologic changes in circadian rhythms in RA and for the adverse circadian effects of methotrexate, such as fatigue.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/metabolismo , Relógios Circadianos , Metotrexato/farmacologia , Monócitos/fisiologia , Receptor A2A de Adenosina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Artrite Reumatoide/metabolismo , Linhagem Celular , Ritmo Circadiano , Criptocromos/biossíntese , Fadiga , Humanos , Inflamação , Interleucina-1/genética , Interleucina-10/genética , Metotrexato/uso terapêutico , Monócitos/metabolismo , Proteínas Circadianas Period/biossíntese , Fenetilaminas/farmacologia , RNA Mensageiro/biossíntese , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
AIMS: To compare the constitutive and agonist-dependent endosomal trafficking of α(1A)- and α(1B)-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. METHODS: Using CypHer5 technology and VSV-G epitope tagged α(1A)- and α(1B)-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). RESULTS AND CONCLUSIONS: Constitutive as well as agonist-induced trafficking of α(1A) and α(1B) ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α(1A)-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to ß-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α(1B)-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-ß-cyclodextrin.
Assuntos
Endossomos/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Transdução de Sinais , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Sinalização do Cálcio , Linhagem Celular , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Endossomos/efeitos dos fármacos , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
INTRODUCTION: Adenosine, acting through the A(2A) receptor, promotes tissue matrix production in the skin and the liver and induces the development of dermal fibrosis and cirrhosis in murine models. Since expression of A(2A) receptors is increased in scleroderma fibroblasts, we examined the mechanisms by which the A(2A) receptor produces its fibrogenic effects. METHODS: The effects of A(2A) receptor ligation on the expression of the transcription factor, Fli1, a constitutive repressor for the synthesis of matrix proteins, such as collagen, is studied in dermal fibroblasts. Fli1 is also known to repress the transcription of CTGF/CCN2, and the effects of A(2A) receptor stimulation on CTGF and TGF-ß1 expression are also examined. RESULTS: A(2A) receptor occupancy suppresses the expression of Fli1 by dermal fibroblasts. A(2A) receptor activation induces the secretion of CTGF by dermal fibroblasts, and neutralization of CTGF abrogates the A(2A) receptor-mediated enhancement of collagen type I production. A(2A)R activation, however, resulted in a decrease in TGF-ß1 protein release. CONCLUSIONS: Our results suggest that Fli1 and CTGF are important mediators of the fibrogenic actions of adenosine and the use of small molecules such as adenosine A(2A) receptor antagonists may be useful in the therapy of dermal fibrosis in diseases such as scleroderma.
Assuntos
Colágeno/biossíntese , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Receptor A2A de Adenosina/metabolismo , Adenosina/metabolismo , Western Blotting , Linhagem Celular , Fibrose/metabolismo , Fibrose/patologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esclerodermia Localizada/metabolismo , Esclerodermia Localizada/patologia , Pele/metabolismo , Pele/patologiaRESUMO
The objective of this study was to determine whether the different time-course characteristics of α1-adrenoceptor-mediated contraction in arteries can be related to the subtypes involved. Contractile responses to noradrenaline (NA) were compared with inositol phosphate accumulation and extracellular signal-regulated kinase (ERK)1/2 phosphorylation after α1-agonist stimuli in the same vessels in the presence or absence of α1-antagonists in rat or in α1-subtype knockout (KO) mice. Aorta, where α1D-AR is the main functional subtype, had higher sensitivity to NA (in respect of inositol phosphate [IP], pERK1/2, and contractile response) than tail artery, where the α1A-adrenoceptor subtype is predominant. Furthermore, the contraction in aorta exhibited a slower decay after agonist removal and this was consistent in all strains harboring α1D-adrenoceptors (from rat, α1B-KO, and wild-type [WT] mice) but was not observed in the absence of the α1D-adrenoceptor signal (α1D-adrenoceptor blocked rat aorta or aorta from α1D-KO). IP formation paralleled α1-adrenoceptor-mediated contraction (agonist present or postagonist) in aorta and tail artery. High sensitivity to agonist and persistence of response after agonist removal is a property of α1D-adrenoceptors. Therefore, the preponderance of this subtype in noninnervated conductance arteries such as aorta allows responsiveness to circulating catecholamines and prevents abrupt changes in vessel caliber when the stimulus fluctuates. Conversely, in innervated distributing arteries, high local concentrations of NA are required to activate α1A-adrenoceptors for a response that is rapid but short lived allowing fine adjustment of the contractile tone by perivascular sympathetic nerves.