RESUMO
Bone metastases are frequent complications of malignant melanoma leading to reduced quality of life and significant morbidity. Regulation of immune cells by the gut microbiome influences cancer progression, but the role of the microbiome in tumor growth in bone is unknown. Using intracardiac or intratibial injections of B16-F10 melanoma cells into mice, we showed that gut microbiome depletion by broad-spectrum antibiotics accelerated intraosseous tumor growth and osteolysis. Microbiome depletion blunted melanoma-induced expansion of intestinal NK cells and Th1 cells and their migration from the gut to tumor-bearing bones. Demonstrating the functional relevance of immune cell trafficking from the gut to the bone marrow (BM) in bone metastasis, blockade of S1P-mediated intestinal egress of NK and Th1 cells, or inhibition of their CXCR3/CXCL9-mediated influx into the BM, prevented the expansion of BM NK and Th1 cells and accelerated tumor growth and osteolysis. Using a mouse model, this study revealed mechanisms of microbiota-mediated gut-bone crosstalk that are relevant to the immunological restraint of melanoma metastasis and tumor growth in bone. Microbiome modifications induced by antibiotics might have negative clinical consequences in patients with melanoma.
Assuntos
Microbioma Gastrointestinal , Melanoma Experimental , Osteólise , Animais , Antibacterianos/farmacologia , Desenvolvimento Ósseo , Humanos , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Qualidade de Vida , Células Th1/patologiaRESUMO
Adiponectin and insulin resistance creates a vicious cycle that exacerbates type 2 diabetes. Earlier, we observed that female leptin receptor-deficient BLKS mice (BKS-db/db) were more sensitive to an adiponectin mimetic GTDF than males, which led us to explore if E2 plays a crucial role in modulation of adiponectin-sensitivity. Male but not female BKS-db/db mice were resistant to metabolic effects of globular adiponectin treatment. Male BKS-db/db displayed reduced skeletal muscle AdipoR1 protein expression, which was consequent to elevated polypyrimidine tract binding protein 1 (PTB) and miR-221. E2 treatment in male BKS-db/db, and ovariectomized BALB/c mice rescued AdipoR1 protein expression via downregulation of PTB and miR-221, and also directly increased AdipoR1 mRNA by its classical nuclear receptors. Estrogen receptor regulation via dietary or pharmacological interventions may improve adiponectin resistance and consequently ameliorate insulin resistance in type 2 diabetes.
Assuntos
Adiponectina/metabolismo , Diabetes Mellitus Experimental , Estradiol/farmacologia , Receptores de Adiponectina/genética , Animais , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Resistência a Medicamentos/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Receptores de Adiponectina/metabolismo , Receptores para Leptina/genética , Caracteres SexuaisRESUMO
Previously, we established adiponectin receptors (AdipoRs) as osteoanabolic target. To discover small molecule agonists of AdipoRs, we studied apigenin and apigenin-6C-glucopyranose (isovitexin) that induced osteoblast differentiation. In-silico, in vitro and omics-based studies were performed. Molecular docking using the crystal structures of AdipoRs showed different interaction profiles of isovitexin and apigenin. In osteoblasts, isovitexin but not apigenin rapidly phosphorylated AMP-activated protein kinase (pAMPK) which is downstream of AdipoRs and a master regulator of cellular energy metabolism, and upregulated expression of AdipoRs. Blocking AMPK abolished the osteogenic effect of isovitexin and its effect on AdipoR expression. Isovitexin upregulated the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the mitochondrial biogenesis factor in osteoblasts, and the effect was blocked by AMPK inhibition. Upregulation of PGC-1α by isovitexin was accompanied by increased mitochondrial membrane proteins and mitochondrial DNA (mtDNA). Isovitexin via AdipoRs and PGC-1α induced oxidative phosphorylation (OxPhos) and ATP synthesis that resulted in osteoblast differentiation. Isovitexin had no agonistic/antagonistic activity and stimulatory/inhibitory effect in screening platforms for G protein-coupled receptors and kinases, respectively. In vivo, isovitexin upregulated AdipoRs and osteogenic genes, and increased mtDNA in rat calvarium. We conclude that isovitexin selectively via AdipoRs induced osteoblast differentiation that was fuelled by mitochondrial respiration.
Assuntos
Apigenina/farmacologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Receptores de Adiponectina/agonistas , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Metabolismo Energético/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Osteoblastos/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Cultura Primária de Células , Receptores de Adiponectina/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
A technology for systemic and repeated administration of osteogenic factors for orthopedic use is an unmet medical need. Lactoferrin (â¼80 kDa), present in milk, is known to support bone growth. We discovered a lactoferrin-mimetic peptide, LP2 (an 18-residue fragment from the N-terminus of the N-lobe of human lactoferrin), which self-assembles into a nano-globular assembly with a ß-sheet structure in an aqueous environment. LP2 is non-hemolytic and non-cytotoxic against human red blood cells and 3T3 fibroblasts, respectively, and appreciably stable in the human serum. LP2 through the bone morphogenetic protein-dependent mechanism stimulates osteoblast differentiation more potently than the full-length protein as well as the osteoblastic production of osteoprotegerin (an anti-osteoclastogenic factor). Consequently, daily subcutaneous administration of LP2 to rats and rabbits with osteotomy resulted in faster bone healing and stimulated bone formation in rats with a low bone mass more potently than that with teriparatide, the standard-of-care osteogenic peptide for osteoporosis. LP2 has skeletal bioavailability and is safe at the 15× osteogenic dose. Thus, LP2 is a novel peptide that can be administered systemically for the medical management of hard-to-heal fractures.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Lactoferrina/química , Nanoestruturas/química , Procedimentos Ortopédicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Células 3T3 , Animais , Disponibilidade Biológica , Diferenciação Celular/efeitos dos fármacos , Estabilidade de Medicamentos , Humanos , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/farmacocinética , SegurançaRESUMO
Estrogen deficiency causes a gut microbiome-dependent expansion of BM Th17 cells and TNF-α-producing T cells. The resulting increased BM levels of IL-17a (IL-17) and TNF stimulate RANKL expression and activity, causing bone loss. However, the origin of BM Th17 cells and TNF+ T cells is unknown. Here, we show that ovariectomy (ovx) expanded intestinal Th17 cells and TNF+ T cells, increased their S1P receptor 1-mediated (S1PR1-mediated) egress from the intestine, and enhanced their subsequent influx into the BM through CXCR3- and CCL20-mediated mechanisms. Demonstrating the functional relevance of T cell trafficking, blockade of Th17 cell and TNF+ T cell egress from the gut or their influx into the BM prevented ovx-induced bone loss. Therefore, intestinal T cells are a proximal target of sex steroid deficiency relevant for bone loss. Blockade of intestinal T cell migration may represent a therapeutic strategy for the treatment of postmenopausal bone loss.
Assuntos
Movimento Celular/imunologia , Intestinos , Osteoporose Pós-Menopausa , Ovariectomia , Células Th17/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Quimiocina CCL20/genética , Quimiocina CCL20/imunologia , Feminino , Humanos , Intestinos/imunologia , Intestinos/microbiologia , Camundongos , Camundongos Knockout , Osteoporose Pós-Menopausa/imunologia , Osteoporose Pós-Menopausa/microbiologia , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
A major limitation in the bio-medical sector is the availability of materials suitable for bone tissue engineering using stem cells and methodology converting the stochastic biological events towards definitive as well as efficient bio-mineralization. We show that osteoblasts and Bone Marrow-derived Mesenchymal Stem Cell Pools (BM-MSCP) express TRPM8, a Ca2+-ion channel critical for bone-mineralization. TRPM8 inhibition triggers up-regulation of key osteogenesis factors; and increases mineralization by osteoblasts. We utilized CMT:HEMA, a carbohydrate polymer-based hydrogel that has nanofiber-like structure suitable for optimum delivery of TRPM8-specific activators or inhibitors. This hydrogel is ideal for proper adhesion, growth, and differentiation of osteoblast cell lines, primary osteoblasts, and BM-MSCP. CMT:HEMA coated with AMTB (TRPM8 inhibitor) induces differentiation of BM-MSCP into osteoblasts and subsequent mineralization in a dose-dependent manner. Prolonged and optimum inhibition of TRPM8 by AMTB released from the gels results in upregulation of osteogenic markers. We propose that AMTB-coated CMT:HEMA can be used as a tunable surface for bone tissue engineering. These findings may have broad implications in different bio-medical sectors.
Assuntos
Osteoblastos/metabolismo , Canais de Cátion TRPM/metabolismo , Engenharia Tecidual/métodos , Animais , Benzamidas/metabolismo , Benzamidas/farmacologia , Células da Medula Óssea/citologia , Osso e Ossos/metabolismo , Diferenciação Celular , Células Cultivadas , Feminino , Hidrogéis/química , Hidrogéis/farmacologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Osteogênese , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPM/antagonistas & inibidores , Tiofenos/metabolismo , Tiofenos/farmacologiaRESUMO
Calcipenic rickets is prevalent in underprivileged children in developing countries. Calcipenic rickets resulting from dietary calcium (Ca) deficiency decreases bone mass and deteriorates bone microstructure in humans. The effect of dietary Ca replenishment (CaR) on rachitic bones in animal models depends on the amount, critical period and duration of replenishment, however, the extent of recovery in various bone parameters including bone quality remains unclear. We investigated the effect of CaR in rat skeleton after inducing calcipenic rickets. Female SD rats (postnatal 28 days/P28) were rendered calcipenic by feeding calcium deficient (CaD) diet (0.1% Ca) till P70 while control SD rats were fed Ca sufficient diet (0.8% Ca). At P70, calcipenic rats were switched to 0.8% Ca diet till P150 for one group and P210 for another group (endpoint). The CaD groups received 0.1% Ca diet throughout the study (P210). In the CaD groups, serum Ca and phosphate, and bone mineral density (BMD) were significantly decreased whereas serum alkaline phosphatase (ALP), iPTH and CTX-1 were increased compared to age-matched controls. Moreover, at the endpoint, the CaD group had reduced bone mass, surface referent bone formation parameters, tissue mineralization and strength accompanied by the increased osteoid thickness and microarchitectural decay (measured by trabecular geometric parameters) with poor crystal packing. The CaR group showed complete recovery in serum Ca, iPTH, ALP and CTX-1, and BMD, however, the bone quality parameters including bone strength, microarchitectural decay, tissue mineralization, and crystallinity were incompletely restored. Decreased surface referent bone formation and increased unmineralized bones (osteoid) indicative of osteomalacia were also observed in the CaR group at P210 compared with control despite prolonged replenishment. We conclude that a prolonged Ca repletion following the induction of calcipenic rickets in rats although shows the recovery of biochemical measures of bone metabolism and bone mass, however, the bone quality remains compromised. This suggests that a "memory" of calcipenia occurring at the early growth stage persists in the skeleton of adult rats despite a prolonged Ca replenishment.
Assuntos
Cálcio da Dieta , Raquitismo , Animais , Densidade Óssea , Osso e Ossos , Cálcio , Feminino , Ratos , Ratos Sprague-Dawley , Raquitismo/tratamento farmacológicoRESUMO
Anabolic therapies for osteoporosis including dietary polyphenols promote osteoblast function by influencing its energy metabolism. Among the dietary polyphenols, the beneficial skeletal effects of genistein (an isoflavone), kaempferol (a flavone), resveratrol (RES, a stilbenoid) and epigallocatechin gallate (EGCG, a catechin) have been reported in preclinical studies. We studied the action mechanism of these nutraceuticals on osteoblast bioenergetics. All stimulated differentiation of human fetal osteoblasts (hFOB). However, only EGCG and RES stimulated mitochondrial parameters including basal and maximum respiration, spare respiratory capacity and ATP production (a measure of the activity of electron transport chain/ETC). Increases in these parameters were due to increased mitochondrial biogenesis and consequent upregulation of several mitochondrial proteins including those involved in ETC. Rotenone blocked the osteogenic effect of EGCG and RES suggesting the mediatory action of mitochondria. Both compounds rapidly activated AMPK, and dorsomorphin (an AMPK inhibitor) abolished ATP production stimulated by these compounds. Moreover, EGCG and RES upregulated the mitochondrial biogenesis factor, PGC-1α which is downstream of AMPK activation, and silencing PGC-1α blocked their stimulatory effects on ATP production and hFOB differentiation. Adiponectin receptor 1 (AdipoR1) is an upstream regulator of PGC-1α, and both compounds increased the expression of AdipoR1 but not AdipoR2. Silencing AdipoR1 blocked the upregulation of EGCG/RES-induced PGC-1α and hFOB differentiation. In rat calvarium, both compounds increased AdipoR1, PGC-1α, and RunX2 (the osteoblast transcription factor) with a concomitant increase in mitochondrial copy number and ATP levels. We conclude that EGCG and RES display osteogenic effects by reprogramming osteoblastic bioenergetics by acting as the AdipoR1 agonists.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Polifenóis/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Suplementos Nutricionais , Humanos , Mitocôndrias/metabolismo , Biogênese de Organelas , Osteoblastos/citologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Adiponectina/metabolismoRESUMO
Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides and thereby regulate diverse cellular functions. The reports on the skeletal effects of PDE inhibitors are conflicting. Here, we screened 17 clinically used non-xanthine PDE inhibitors (selective and non-selective) using mouse calvarial osteoblasts (MCO) where the readout was osteoblast differentiation. From this screen, we identified sildenafil and vardenafil (both PDE5 inhibitors) having the least osteogenic EC50. Both drugs significantly increased vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expressions in MCO and the nitric oxide synthase inhibitor L-NAME completely blocked VEGF expression induced by these drugs. Sunitinib, a tyrosine receptor kinase inhibitor that also blocks VEGFR2 blocked sildenafil-/vardenafil-induced osteoblast differentiation. At half of their human equivalent doses, i.e. 6.0 mg/kg sildenafil and 2.5 mg/kg vardenafil, the maximum bone marrow level of sildenafil was 32% and vardenafil was 21% of their blood levels. At these doses, both drugs enhanced bone regeneration at the femur osteotomy site and completely restored bone mass, microarchitecture, and strength in OVX mice. Furthermore, both drugs increased surface referent bone formation and serum bone formation marker (P1NP) without affecting the resorption marker (CTX-1). Both drugs increased the expression of VEGF and VEGFR2 in bones and osteoblasts and increased skeletal vascularity. Sunitinib completely blocked the bone restorative and vascular effects of sildenafil and vardenafil in OVX mice. Taken together, our study suggested that sildenafil and vardenafil at half of their adult human doses completely reversed osteopenia in OVX mice by an osteogenic mechanism that was associated with enhanced skeletal vascularity.
Assuntos
Inibidores da Fosfodiesterase 5 , Fator A de Crescimento do Endotélio Vascular , Animais , Imidazóis/farmacologia , Camundongos , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Citrato de Sildenafila/farmacologia , Citrato de Sildenafila/uso terapêutico , Sulfonas/farmacologia , Sunitinibe , Triazinas/farmacologia , Dicloridrato de Vardenafila/farmacologiaRESUMO
Estrogen deficiency reduces estrogen receptor-alpha (ERα) and promotes apoptosis in the hippocampus, inducing learning-memory deficits; however, underlying mechanisms remain less understood. Here, we explored the molecular mechanism in an ovariectomized (OVX) rat model, hypothesizing participation of autophagy and growth factor signaling that relate with apoptosis. We observed enhanced hippocampal autophagy in OVX rats, characterized by increased levels of autophagy proteins, presence of autophagosomes and inhibition of AKT-mTOR signaling. Investigating upstream effectors of reduced AKT-mTOR signaling revealed a decrease in hippocampal heparin-binding epidermal growth factor (HB-EGF) and p-EGFR. Moreover, 17ß-estradiol and HB-EGF treatments restored hippocampal EGFR activation and alleviated downstream autophagy process and neuronal loss in OVX rats. In vitro studies using estrogen receptor (ERα)-silenced primary hippocampal neurons further corroborated the in vivo observations. Additionally, in vivo and in vitro studies suggested the participation of an attenuated hippocampal neuronal HB-EGF and enhanced autophagy in apoptosis of hippocampal neurons in estrogen- and ERα-deficient conditions. Subsequently, we found evidence of mitochondrial loss and mitophagy in hippocampal neurons of OVX rats and ERα-silenced cells. The ERα-silenced cells also showed a reduction in ATP production and an HB-EGF-mediated restoration. Finally in concordance with molecular studies, inhibition of autophagy and treatment with HB-EGF in OVX rats restored cognitive performances, assessed through Y-Maze and passive avoidance tasks. Overall, our study, for the first time, links neuronal HB-EGF/EGFR signaling and autophagy with ERα and memory performance, disrupted in estrogen-deficient condition.
Assuntos
Receptor alfa de Estrogênio/deficiência , Estrogênios/deficiência , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Transtornos da Memória/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Hipocampo/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Ovariectomia , Ratos , Transdução de SinaisRESUMO
Mycobacterium leprae infection causes bone lesions and osteoporosis, however, the effect of antileprosy drugs on the bone is unknown. We, therefore, set out to address it by investigating osteogenic differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs). Out of 7 antileprosy drugs, only clofazimine (CFZ) reduced MSCs viability (IC50 â¼ 1 µM) and their osteogenic differentiation but increased adipogenic differentiation on a par with rosiglitazone, and this effect was blocked by a peroxisome proliferator-activated receptor gamma antagonist, GW9662. CFZ also decreased osteoblast viability and resulted in impaired bone regeneration in a rat femur osteotomy model at one-third human drug dose owing to increased callus adipogenesis as GW9662 prevented this effect. CFZ treatment decreased BM MSC population and homing of MSC to osteotomy site despite drug levels in BM being much less than its in vitro IC50 value. In adult rats, CFZ caused osteopenia in long bones marked by suppressed osteoblast function due to enhanced adipogenesis and increased osteoclast functions. A robust increase in marrow adipose tissue (MAT) by CFZ did not alter the hematologic parameters but likely reduced BM vascular bed leading to osteonecrosis (ON) characterized by empty osteocyte lacunae. However, CFZ had no effect on visceral fat content and was not associated with any metabolic and hematologic changes. Levels of unsaturated fatty acids in MAT were higher than saturated fatty acids and CFZ further increased the former. From these data, we conclude that CFZ has adverse skeletal effects and could be used for creating a rodent ON model devoid of extraskeletal effects.
RESUMO
A combination of diosmin and hesperidin (9:1 ratio) is marketed as a dietary supplement/nutraceutical for cardiovascular health. We studied the skeletal effect of this combination (90% diosmin and 10% hesperidin, henceforth named as DH). We showed that a) in rats with femur osteotomy, DH stimulated callus bone regeneration, b) in growing rats, DH promoted peak bone mass achievement and c) in OVX rats rendered osteopenic, DH completely restored femur trabecular bones and strength along with the increases in surface referent bone formation and serum osteogenic marker. Furthermore, DH suppressed bone resorption in OVX rats as well as in OVX rats treated with teriparatide (human parathyroid hormone 1-34) but did not affect the osteoanabolic effect of teriparatide. These data suggested that DH could prolong the anabolic window of teriparatide. To understand the mechanism of DH action, we performed pharmacokinetic studies and observed that upon its oral administration the only circulating metabolites was diosmetin (the aglycone form of diosmin) while none of the two input flavanones were detectable. Accordingly, subsequent experiments with diosmetin revealed that it was a selective estrogen receptor-ß agonist that stimulated osteoblast differentiation and suppressed sclerostin the anti-osteoblastogenic Wnt antagonist. Taken together, our study defined a positive skeletal effect of DH.
Assuntos
Doenças Ósseas Metabólicas/prevenção & controle , Regeneração Óssea/efeitos dos fármacos , Diosmina/farmacologia , Hesperidina/farmacologia , Osteogênese/efeitos dos fármacos , Teriparatida/farmacologia , Animais , Animais Recém-Nascidos , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/metabolismo , Suplementos Nutricionais , Diosmina/administração & dosagem , Feminino , Fêmur/efeitos dos fármacos , Fêmur/crescimento & desenvolvimento , Fêmur/metabolismo , Hesperidina/administração & dosagem , Ratos Sprague-Dawley , Teriparatida/administração & dosagem , Tíbia/efeitos dos fármacos , Tíbia/crescimento & desenvolvimento , Tíbia/metabolismoRESUMO
Abaloparatide is an analog of human parathyroid hormone-related protein (PTHrP) that has recently been approved for the treatment of post-menopausal osteoporosis. Abaloparatide is a stimulator of bone formation similar to teriparatide (1-34 PTH/TPTD), the first-in-class osteoanabolic drug. Both PTH and PTHrP signal via the type 1 PTH receptor (PTH1R) however, the downstream signaling varies between the two ligands. Both ligands have a similar affinity for the RG (GTPγS-sensitive) state of PTH1R, but, TPTD has a four-fold higher affinity for R0 (GTPγS-insensitive) than PTHrP that results in a prolonged cAMP signaling. Consequently, a greater production from osteoblastic cells of a potent resorption inducer, receptor activator of nuclear factor κB ligand (RANKL) is caused by TPTD than PTHrP. TPTD causes an excess formation over resorption early on producing an anabolic "window" which is lost with time due to increased RANKL production causing resorption to catch up with the formation. Although highly labile, PTHrP has an osteogenic effect accompanied by lesser resorptive and hypercalcemic effects than TPTD because of faster PTHrP-PTH1R dissociation than PTH-PTH1R complex. Engineered from PTHrP (1-34), abaloparatide was made stable and overcame the loss of the anabolic window and hypercalcemia associated with TPTD. The receptor activating domain (1-21 amino acids) of both ligands is same but multiple substitutions between amino acids 22-34 of PTHrP were made to enhance the peptide's stability. In, women with osteoporosis, abaloparatide increased BMD faster than TPTD and decreased fracture risk at both vertebral and non-vertebral sites but unlike TPTD/PTH did not increase resorption or hypercalcemia.
Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Densidade Óssea/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Proteína Relacionada ao Hormônio Paratireóideo/uso terapêutico , Teriparatida/uso terapêutico , Sequência de Aminoácidos , Anabolizantes/farmacologia , Anabolizantes/uso terapêutico , Animais , Densidade Óssea/fisiologia , Conservadores da Densidade Óssea/farmacologia , Humanos , Osteoporose/genética , Osteoporose/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Teriparatida/farmacologiaRESUMO
Oncostatin M (OSM), an inflammatory cytokine belonging to the interleukin-6 (IL-6) superfamily, plays a vital role in multitude of physiological and pathological processes. Its role in breast tumor progression and metastasis to distant organs is well documented. Recent reports implicate OSM in macrophage M2 polarization, a key pro-tumoral phenomenon. M2 polarization of macrophages is believed to promote tumor progression by potentiating metastasis and angiogenesis. In the current study, we delineated the mechanism underlying OSM induced macrophage M2 polarization. The findings revealed that OSM skews macrophages towards an M2 polarized phenotype via mTOR signaling complex 2 (mTORC2). mTORC2 relays signals through two effector kinases i.e. PKC-α and Akt. Our results indicated that mTORC2 mediated M2 polarization of macrophages is not dependent on PKC-α and is primarily affected via Akt, particularly Akt1. In vivo studies conducted on 4T1/BALB/c mouse orthotropic model of breast cancer further corroborated these observations wherein i.v. reintroduction of mTORC2 abrogated monocytes into orthotropic mouse model resulted in diminished acquisition of M2 specific attributes by tumor associated macrophages. Metastasis to distant organs like lung, liver and bone was reduced as evident by decrease in formation of focal metastatic lesions in mTORC2 abrogated monocytes mice. Our study pinpoints key role of mTORC2-Akt1 axis in OSM induced macrophage polarization and suggests for possible usage of Oncostatin-M blockade and/or selective mTORC2 inhibition as a potential anti-cancer strategy particularly with reference to metastasis of breast cancer to distant organs such as lung, liver and bone.
Assuntos
Proliferação de Células/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Metástase Neoplásica/tratamento farmacológico , Oncostatina M/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Interleucina-6/metabolismo , Células MCF-7 , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Metástase Neoplásica/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1RESUMO
Leishmania donovani is a protozoan parasite that infects mammalian macrophages, wherein the parasite resides and replicates as amastigotes, inflicting the potentially fatal disease visceral leishmaniasis. The disease is characterized by severe immunosuppression and hypocholesterolemia implying metabolic changes in L. donovani infection; whether such metabolic changes are also linked to susceptibility to the infection is not known. Herein, four inbred mouse strains were first characterized for their resistance or susceptibility profile to L. donovani infection. It was observed that these four mouse strains were differentially susceptible to L. donovani infection. Splenic expression of four key cytokines- IL-10, IL-12, IFN-γ and IL-4- revealed that the differential susceptibility of these four mouse strains to L. donovani was partially associated with these cytokines. The association was further correlated with the expression of different enzymes of the glycolytic pathway in the spleen of these L. donovani-infected mice. Thus, the observations reported here suggest an association between host metabolism, cytokine secretion profile and L. donovani susceptibility. As the chemotherapeutic choices are extremely limited and a vaccine for human use is yet to be discovered for the neglected tropical disease that is prevalent in 88 countries affecting 320 million people, this metabolic study is a significant research area that has potentials to develop a new target for anti-leishmanial chemotherapy.
Assuntos
Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Animais , Citocinas/imunologia , Citocinas/metabolismo , Leishmaniose Visceral/parasitologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/imunologia , Baço/metabolismo , Baço/parasitologiaRESUMO
Secondary osteoporosis is the major concern associated with long term intake of antiepileptic drugs (AEDs). Women are the vulnerable targets owing to post-menopausal bone loss. In the present work, we evaluated the effect of 10â¯weeks of treatment with AED therapy (carbamazepine, CBZ, 75â¯mg/kg; sodium valproate, SVP, 300â¯mg/kg; levetiracetam, LTM, 150â¯mg/kg) on bone mineral density and microarchitecture at femoral epiphysis, lumbar vertebrae and proximal tibia of normal and ovariectomised Wistar rats. In addition, we measured serum levels of vitamin D, receptor activator of nuclear factor kappa ß-ligand (RANKL), procollagen type 1 amino-terminal propeptide (P1NP) and wnt inhibitors (sclerostin and DKK-1) following AED therapy. Micro-computed tomography analysis of bones revealed significant reduction in BMD at femur epiphysis and lumbar vertebrae with all the three AEDs evaluated. At proximal tibia, only CBZ showed a significant decline. The reduction in BMD was more pronounced in ovariectomised rats. AEDs also resulted in alteration of micro-CT parameters. These changes were accompanied by an increased serum RANKL with all AEDs while vitamin D levels were reduced only with CBZ treatment and P1NP levels were reduced with SVP and CBZ. Serum sclerostin levels were elevated following all AEDs in normal and ovariectomised rats except with CBZ in normal rats. However, increase in DKK-1 levels was observed with only LTM. Ovariectomy itself resulted in increased RANKL, sclerostin and DKK-1 and reduced vitamin D and P1NP levels. Significant differences were discernible between normal and ovariectomised rats treated with AEDs in all the parameters. However, while sclerostin increased further upon AEDs treatment, P1NP decreased with SVP and CBZ and serum DKK-1 levels showed a declining trend with all the three AEDs studied. We confirm adverse effects on bone following AEDs in female rats. Further, our results demonstrate for the first time that these effects are more pronounced in ovariectomised rats as compared to normal rats and that this could be related to estrogen deficiency which in turn enhances bone resorption via increased RANKL and reduces bone formation via increased sclerostin and reduced P1NP. Finally, our study demonstrated for the first time that AED treatment displayed changes in the serum levels of wnt inhibitors and hence modulation of wnt inhibitors might be partly involved in their adverse effects on bone.
Assuntos
Anticonvulsivantes/toxicidade , Osteoporose Pós-Menopausa/induzido quimicamente , Osteoporose Pós-Menopausa/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Carbamazepina/toxicidade , Feminino , Humanos , Levetiracetam/toxicidade , Ovariectomia , Ratos , Ratos Wistar , Ácido Valproico/toxicidadeRESUMO
OBJECTIVES: Postmenopausal osteoporosis is one of most commonly occurring skeletal diseases leading to bone loss and fragility. Probiotics have been associated with various immunomodulatory properties and thus can be exploited to enhance bone health. In the present study, we report, to our knowledge for the first time, that oral administration of Bacillus clausii (BC) in postmenopausal osteoporotic (OVX) mice model enhances bone health. METHODS: BC was selected as probiotic of choice due to its established immunomodulatory properties. BC skews the Treg-Th17 cell balance in vivo by inhibiting osteoclastogenic Th17 cells and promoting antiosteoclastogenic Treg cell development in postmenopausal osteoporotic mice. Mice were divided into three groups (sham, OVX, and OVX + BC), and BC was administered orally in drinking water for 6 wk post-ovariectomy. At the end of experiment, mice were sacrificed and bones were analyzed for various parameters, along with lymphoid tissues for Treg-Th17 cells and serum cytokines. RESULTS: We observed that BC administration enhanced bone health. This effect of BC administration was found due to skewing of Treg-Th17 cell balance (enhanced Treg and decreased Th17 cells) in vivo. BC administration reduced levels of proinflammatory cytokines (interleukin [IL]-6, IL-17, IFN-γ and tumor necrosis factor-α) and increased levels of anti-inflammatory cytokine (IL-10). CONCLUSIONS: The present study strongly supports and establishes the osteoprotective potential of BC leading to enhanced bone health in postmenopausal osteoporotic mice model.
Assuntos
Bacillus clausii , Osteoporose Pós-Menopausa/terapia , Probióticos/uso terapêutico , Linfócitos T Reguladores/microbiologia , Células Th17/microbiologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Osteogênese/imunologia , Osteoporose Pós-Menopausa/imunologia , Osteoporose Pós-Menopausa/microbiologiaRESUMO
Modafinil is primarily prescribed for treatment of narcolepsy and other sleep-associated disorders. However, its off-prescription use as a cognition enhancer increased considerably, specially among youths. Given its increasing use in young adults the effect of modafinil on peak bone accrual is an important issue but has never been investigated. Modafinil treatment to young male rats caused trabecular and cortical bone loss in tibia and femur, and reduction in biomechanical strength. Co-treatment of modafinil with alendronate (a drug that suppresses bone resorption) reversed the trabecular bone loss but failed to prevent cortical loss. Modafinil increased serum type 1 pro-collagen N-terminal protein (P1NP) and collagen type 1 cross-linked C-telopeptide (CTX-1) indicating a high turnover bone loss. The drug also increased receptor activator of nuclear factor κB ligand (RANKL) to osteoprotegerin (OPG) ratio in serum which likely resulted in increased osteoclast number per bone surface. Furthermore, conditioned medium from modafinil treated osteoblasts increased the expression of osteoclastogenic genes in bone marrow-derived macrophages and the effect was blocked by RANKL neutralizing antibody. In primary osteoblasts, modafinil stimulated cAMP production and using pharmacological approach, we showed that modafinil signalled via adenosine receptors (A2AR and A2BR) which resulted in increased RANKL expression. ZM-241,385 (an A2AR inhibitor) and MRS 1754 (an A2BR inhibitor) suppressed modafinil-induced upregulation of RANKL/OPG ratio in the calvarium of new born rat pups. Our data suggests that by activating osteoblast adenosine receptors modafinil increases the production of osteoclastogenic cytokine, RANKL that in turn results in high turnover bone loss in young rats.
Assuntos
Agonistas do Receptor A2 de Adenosina/toxicidade , Compostos Benzidrílicos/toxicidade , Remodelação Óssea/efeitos dos fármacos , Osso Esponjoso/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoporose/induzido quimicamente , Ligante RANK/metabolismo , Receptor A2A de Adenosina/efeitos dos fármacos , Receptor A2B de Adenosina/efeitos dos fármacos , Promotores da Vigília/toxicidade , Animais , Fenômenos Biomecânicos , Osso Esponjoso/metabolismo , Osso Esponjoso/patologia , Osso Esponjoso/fisiopatologia , Células Cultivadas , Osso Cortical/efeitos dos fármacos , Osso Cortical/metabolismo , Osso Cortical/patologia , Osso Cortical/fisiopatologia , AMP Cíclico/metabolismo , Masculino , Modafinila , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Osteoporose/metabolismo , Osteoporose/patologia , Osteoporose/fisiopatologia , Osteoprotegerina/metabolismo , Ligante RANK/genética , Ratos Sprague-Dawley , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Transdução de Sinais , Fatores de Tempo , Regulação para CimaRESUMO
Promising news in the treatment of osteoporosis is that sequestering sclerostin from circulation with antibodies stimulates robust bone formation. Pre-clinical studies on rodents and monkeys have confirmed that treatment with anti-sclerostin monoclonal antibody (Scl-Ab) increases bone mass, improves bone strength and enhances fracture repair. Clinical trials show that bone gain (anabolic effect) is transient and are primarily at central (spine and hips) than peripheral (wrist) sites. Interestingly Scl-Ab also inhibited bone resorption. Thus Scl-Ab is being regarded as the pharmacologic agent with dual properties - stimulating bone formation and decreasing bone resorption. Sclerostin neutralization transiently increases bone formation markers in post-menopausal women and like parathyroid hormone (PTH) activates osteoblasts and lining cells resulting in bone anabolic effect. However, unlike PTH, sclerostin antibody also decreases bone resorption (anti-catabolic). Although, the U.S. Food and Drug Administration have accepted the Biologics License Application for one of the monoclonal antibodies against sclerostin (romosozumab) for review, many questions remain before romosozumab can be introduced as a skeletal anabolic agent to clinical practice. For example, neutralizing sclerostin alters calcium homeostasis and increases PTH. In addition, sclerostin depletion in preclinical studies has been reported to severely compromises B cell depletion in bone marrow. We have reviewed the currently available evidences that support the use of sclerostin antibody in treating osteoporosis and compare its efficacy and mechanism of action with the currently available anabolic drug, human PTH.
Assuntos
Anticorpos Monoclonais/farmacologia , Conservadores da Densidade Óssea/farmacologia , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Reabsorção Óssea/tratamento farmacológico , Osteoporose Pós-Menopausa/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais/uso terapêutico , Biomarcadores/sangue , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/sangue , Reabsorção Óssea/patologia , Cálcio/metabolismo , Modelos Animais de Doenças , Feminino , Marcadores Genéticos , Humanos , Osteoblastos/efeitos dos fármacos , Osteoporose Pós-Menopausa/patologia , Hormônio Paratireóideo/sangue , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/uso terapêutico , Teriparatida/uso terapêutico , Resultado do TratamentoRESUMO
Adiponectin regulates various metabolic processes including glucose flux, lipid breakdown and insulin response. We recently reported that adiponectin receptor1 (adipoR1) activation by a small molecule reverses osteopenia in leptin receptor deficient db/db (diabetic) mice. However, the role of adiponectin in bone metabolism under the setting of post-menopausal (estrogen-deficiency) osteopenia and associated metabolic derangements has not been studied. Here, we studied the therapeutic effect of the globular form of adiponectin (gAd), which is predominantly an adipoR1 agonist, in aged ovariectomized (OVX) rats and compared it with standard-of-care anti-osteoporosis drugs. In OVX rats with established osteopenia, gAd completely restored BMD and load bearing capacity and improved bone quality. Skeletal effects of gAd were comparable to PTH (osteoanabolic) but better than alendronate (anti-catabolic). Both osteoanabolic and anti-catabolic mechanisms led to the anti-osteoporosis effect of gAd. In cultured osteoblasts and bones, gAd increased a) adipoR1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) expression to promote mitochondrial respiration, which likely fueled osteoblast differentiation, b) suppressed sclerostin (a wnt antagonist) in a sirtuin1-dependent manner and c) decreased receptor-activator of nuclear factor κB ligand (RANKL) to achieve its anti-catabolic effect. The OVX-induced sarcopenia and insulin resistance were also improved by gAd. We conclude that gAd has therapeutic efficacy in estrogen deficiency-induced osteoporosis, sarcopenia and insulin resistance and hold metabolic disease modifying potential in postmenopausal women.