RESUMO
Fibroblast growth factor receptor 2 (FGFR2) signaling is critical for proper craniofacial development. A gain-of-function mutation in the 2c splice variant of the receptor's gene is associated with Crouzon syndrome, which is characterized by craniosynostosis, the premature fusion of one or more of the cranial vault sutures, leading to craniofacial maldevelopment. Insight into the molecular mechanism of craniosynostosis has identified the ERK-MAPK signaling cascade as a critical regulator of suture patency. The aim of this study is to investigate the role of FGFR2c-induced ERK-MAPK activation in the regulation of coronal suture development. Loss-of-function and gain-of-function Fgfr2c mutant mice have overlapping phenotypes, including coronal synostosis and craniofacial dysmorphia. In vivo analysis of coronal sutures in loss-of-function and gain-of-function models demonstrated fundamentally different pathogenesis underlying coronal suture synostosis. Calvarial osteoblasts from gain-of-function mice demonstrated enhanced osteoblastic function and maturation with concomitant increase in ERK-MAPK activation. In vitro inhibition with the ERK protein inhibitor U0126 mitigated ERK protein activation levels with a concomitant reduction in alkaline phosphatase activity. This study identifies FGFR2c-mediated ERK-MAPK signaling as a key mediator of craniofacial growth and coronal suture development. Furthermore, our results solve the apparent paradox between loss-of-function and gain-of-function FGFR2c mutants with respect to coronal suture synostosis.
Assuntos
Suturas Cranianas/embriologia , Disostose Craniofacial/embriologia , Sistema de Sinalização das MAP Quinases/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/fisiologia , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Butadienos/farmacologia , Células Cultivadas , Suturas Cranianas/anormalidades , Ativação Enzimática/efeitos dos fármacos , Camundongos , Camundongos Knockout , Mutação , Nitrilas/farmacologia , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteogênese/fisiologia , Fenótipo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Sinostose/genética , Sinostose/patologiaRESUMO
Dietary methionine restriction (MR) increases longevity and improves healthspan in rodent models. Young male C57BL/6J mice were placed on MR to assess effects on bone structure and formation. Mice were fed diets containing 0.86% or 0.12% methionine for 5 weeks. Fasting blood plasma was analyzed for metabolic and bone-related biomarkers. Tibiae were analyzed by histomorphometry, while femurs were analyzed by micro-CT and biomechanically using 4-point bending. MR mice had reduced plasma glucose and insulin, while FGF21 and FGF23 increased. Plasma levels of osteocalcin and osteoprotegrin were unaffected, but sclerostin and procollagen I decreased. MR induced bone marrow fat accretion, antithetical to the reduced fat depots seen throughout the body. Cortical bone showed significant decreases in Bone Tissue Density (BTD). In trabecular bone, mice had decreased BTD, bone surface, trabecula and bone volume, and trabecular thickness.. Biomechanical testing showed that on MR, bones were significantly less stiff and had reduced maximum load and total work, suggesting greater fragility. Reduced expression of RUNX2 occurred in bone marrow of MR mice. These results suggest that MR alters bone remodeling and apposition. In MR mice, miR-31 in plasma and liver, and miR-133a, miR-335-5p, and miR-204 in the bone marrow was elevated. These miRNAs were shown previously to target and regulate Osterix and RUNX2 in bone, which could inhibit osteoblast differentiation and function. Therefore, dietary MR in young animals alters bone structure by increasing miRNAs in bone and liver that can target RUNX2. J. Cell. Biochem. 118: 31-42, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Alimentos Formulados/efeitos adversos , Regulação da Expressão Gênica , Metionina/deficiência , MicroRNAs/metabolismo , Tíbia/metabolismo , Animais , Glicemia/metabolismo , Densidade Óssea , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Masculino , Camundongos , Tíbia/patologiaRESUMO
The Lnk adapter protein negatively regulates the signaling of thrombopoietin (TPO), the main megakaryocyte (MK) growth factor. Lnk-deficient (-/-) mice have increased TPO signaling and increased MK number. Interestingly, several mouse models exist in which increased MK number leads to a high bone mass phenotype. Here we report the bone phenotype of these mice. MicroCT and static histomorphometric analyses at 20 weeks showed the distal femur of Lnk-/- mice to have significantly higher bone volume fraction and trabecular number compared to wild-type (WT) mice. Notably, despite a significant increase in the number of osteoclasts (OC), and decreased bone formation rate in Lnk-/- mice compared to WT mice, Lnk-/- mice demonstrated a 2.5-fold greater BV/TV suggesting impaired OC function in vivo. Additionally, Lnk-/- mouse femurs exhibited non-significant increases in mid-shaft cross-sectional area, yet increased periosteal BFR compared to WT femurs was observed. Lnk-/- femurs also had non-significant increases in polar moment of inertia and decreased cortical bone area and thickness, resulting in reduced bone stiffness, modulus, and strength compared to WT femurs. Of note, Lnk is expressed by OC lineage cells and when Lnk-/- OC progenitors are cultured in the presence of TPO, significantly more OC are observed than in WT cultures. Lnk is also expressed in osteoblast (OB) cells and in vitro reduced alkaline phosphatase activity was observed in Lnk-/- cultures. These data suggest that both direct effects on OB and OC as well as indirect effects of MK in regulating OB contributes to the observed high bone mass. J. Cell. Biochem. 118: 2231-2240, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Trombopoetina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Células da Medula Óssea/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Feminino , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Megacariócitos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , Células RAW 264.7 , Trombopoetina/genética , Microtomografia por Raio-XRESUMO
C-Mpl is the receptor for thrombopoietin (TPO), the main megakaryocyte (MK) growth factor, and c-Mpl is believed to be expressed on cells of the hematopoietic lineage. As MKs have been shown to enhance bone formation, it may be expected that mice in which c-Mpl was globally knocked out (c-Mpl(-/-) mice) would have decreased bone mass because they have fewer MKs. Instead, c-Mpl(-/-) mice have a higher bone mass than WT controls. Using c-Mpl(-/-) mice we investigated the basis for this discrepancy and discovered that c-Mpl is expressed on both osteoblasts (OBs) and osteoclasts (OCs), an unexpected finding that prompted us to examine further how c-Mpl regulates bone. Static and dynamic bone histomorphometry parameters suggest that c-Mpl deficiency results in a net gain in bone volume with increases in OBs and OCs. In vitro, a higher percentage of c-Mpl(-/-) OBs were in active phases of the cell cycle, leading to an increased number of OBs. No difference in OB differentiation was observed in vitro as examined by real-time PCR and functional assays. In co-culture systems, which allow for the interaction between OBs and OC progenitors, c-Mpl(-/-) OBs enhanced osteoclastogenesis. Two of the major signaling pathways by which OBs regulate osteoclastogenesis, MCSF/OPG/RANKL and EphrinB2-EphB2/B4, were unaffected in c-Mpl(-/-) OBs. These data provide new findings for the role of MKs and c-Mpl expression in bone and may provide insight into the homeostatic regulation of bone mass as well as bone loss diseases such as osteoporosis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Receptores de Trombopoetina/genética , Trombopoetina/genética , Animais , Animais Recém-Nascidos , Densidade Óssea , Contagem de Células , Diferenciação Celular , Divisão Celular , Efrina-B2/genética , Efrina-B2/metabolismo , Homeostase/genética , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoclastos/citologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptor EphB4/genética , Receptor EphB4/metabolismo , Receptores de Trombopoetina/deficiência , Transdução de Sinais , Crânio/citologia , Crânio/metabolismo , Trombopoetina/metabolismoRESUMO
Obesity has been associated with high bone mineral density (BMD) but a greater propensity to fracture. Some obese individuals have increased marrow adipose tissue (MAT), but the impact of MAT on bone turnover remains controversial, as do changes in BMD associated with a high fat diet (HFD). In this study we hypothesized that MAT volume would increase in response to HFD but would be independent of changes in BMD. Hence, we fed C57BL/6J (B6) male mice at 3 weeks of age either a high fat diet (60 kcal %) or regular diet (10 kcal %) for 12 weeks (n = 10/group). We measured MAT volume by osmium staining and micro-CT (µCT) as well as bone parameters by µCT, histomorphometry, and dual-energy X-ray absorptiometry. We also performed a short-term pilot study using 13-week-old B6 males and females fed a HFD (58 kcal %) for 2 weeks (n = 3/sex). Both long- and short-term HFD feedings were associated with high MAT volume, however, femoral trabecular bone volume fraction (BV/TV), bone formation rate and cortical bone mass were not altered in the long-term study. In the short-term pilot study, areal BMD was unchanged after 2 weeks of HFD. We conclude that, for B6 mice fed a HFD starting at wean or 13 weeks of age, MAT increases whereas bone mass is not altered. More studies are needed to define the mechanism responsible for the rapid storage of energy in the marrow and its distinction from other adipose depots.
Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Medula Óssea/metabolismo , Dieta Hiperlipídica/efeitos adversos , Obesidade/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Metabolismo Energético , Feminino , Fêmur/efeitos dos fármacos , Fêmur/crescimento & desenvolvimento , Humanos , Leptina/metabolismo , Masculino , Camundongos , Obesidade/patologia , Osteogênese/efeitos dos fármacosRESUMO
Emerging data suggest that megakaryocytes (MKs) play a significant role in skeletal homeostasis. Indeed, osteosclerosis observed in several MK-related disorders may be a result of increased numbers of MKs. In support of this idea, we have previously demonstrated that MKs increase osteoblast (OB) proliferation by a direct cell-cell contact mechanism and that MKs also inhibit osteoclast (OC) formation. As MKs and OCs are derived from the same hematopoietic precursor, in these osteoclastogenesis studies we examined the role of the main MK growth factor, thrombopoietin (TPO) on OC formation and bone resorption. Here we show that TPO directly increases OC formation and differentiation in vitro. Specifically, we demonstrate the TPO receptor (c-mpl or CD110) is expressed on cells of the OC lineage, c-mpl is required for TPO to enhance OC formation in vitro, and TPO activates the mitogen-activated protein kinases, Janus kinase/signal transducer and activator of transcription, and nuclear factor-kappaB signaling pathways, but does not activate the PI3K/AKT pathway. Further, we found TPO enhances OC resorption in CD14+CD110+ human OC progenitors derived from peripheral blood mononuclear cells, and further separating OC progenitors based on CD110 expression enriches for mature OC development. The regulation of OCs by TPO highlights a novel therapeutic target for bone loss diseases and may be important to consider in the numerous hematologic disorders associated with alterations in TPO/c-mpl signaling as well as in patients suffering from bone disorders.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Osteoclastos/metabolismo , Proteínas Recombinantes/administração & dosagem , Trombopoetina/administração & dosagem , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Hematopoese/genética , Humanos , Megacariócitos/metabolismo , Megacariócitos/patologia , Camundongos , Camundongos Knockout , Osteoclastos/patologia , Receptores de Trombopoetina/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Trombopoetina/metabolismoRESUMO
GATA-1(low/low) mice have an increase in megakaryocytes (MKs) and trabecular bone. The latter is thought to result from MKs directly stimulating osteoblastic bone formation while simultaneously inhibiting osteoclastogenesis. Osteoprotegerin (OPG) is known to inhibit osteoclastogenesis and OPG(-/-) mice have reduced trabecular and cortical bone due to increased osteoclastogenesis. Interestingly, GATA-1(low/low) mice have increased OPG levels. Here, we sought to determine whether GATA-1 knockdown in OPG(-/-) mice could rescue the observed osteoporotic bone phenotype. GATA-1(low/low) mice were bred with OPG(-/-) mice and bone phenotype assessed. GATA-1(low/low) × OPG(-/-) mice have increased cortical bone porosity, similar to OPG(-/-) mice. Both OPG(-/-) and GATA-1(low/low) × OPG(-/-) mice, were found to have increased osteoclasts localized to cortical bone, possibly producing the observed elevated porosity. Biomechanical assessment indicates that OPG(-/-) and GATA-1(low/low) × OPG(-/-) femurs are weaker and less stiff than C57BL/6 or GATA-1(low/low) femurs. Notably, GATA-1(low/low) × OPG(-/-) mice had trabecular bone parameters that were not different from C57BL/6 values, suggesting that GATA-1 deficiency can partially rescue the trabecular bone loss observed with OPG deficiency. The fact that GATA-1 deficiency appears to be able to partially rescue the trabecular, but not the cortical bone phenotype suggests that MKs can locally enhance trabecular bone volume, but that MK secreted factors cannot access cortical bone sufficiently to inhibit osteoclastogenesis or that OPG itself is required to inhibit osteoclastogenesis in cortical bone.
Assuntos
Fator de Transcrição GATA1/deficiência , Megacariócitos/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Animais , Reabsorção Óssea/genética , Fêmur/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteogênese/genética , Osteoprotegerina/deficiênciaRESUMO
Recent studies suggest that megakaryocytes (MKs) may play a significant role in skeletal homeostasis, as evident by the occurrence of osteosclerosis in multiple MK related diseases (Lennert et al., 1975; Thiele et al., 1999; Chagraoui et al., 2006). We previously reported a novel interaction whereby MKs enhanced proliferation of osteoblast lineage/osteoprogenitor cells (OBs) by a mechanism requiring direct cell-cell contact. However, the signal transduction pathways and the downstream effector molecules involved in this process have not been characterized. Here we show that MKs contact with OBs, via beta1 integrin, activate the p38/MAPKAPK2/p90RSK kinase cascade in the bone cells, which causes Mdm2 to neutralizes p53/Rb-mediated check point and allows progression through the G1/S. Interestingly, activation of MAPK (ERK1/2) and AKT, collateral pathways that regulate the cell cycle, remained unchanged with MK stimulation of OBs. The MK-to-OB signaling ultimately results in significant increases in the expression of c-fos and cyclin A, necessary for sustaining the OB proliferation. Overall, our findings show that OBs respond to the presence of MKs, in part, via an integrin-mediated signaling mechanism, activating a novel response axis that de-represses cell cycle activity. Understanding the mechanisms by which MKs enhance OB proliferation will facilitate the development of novel anabolic therapies to treat bone loss associated with osteoporosis and other bone-related diseases.
Assuntos
Diferenciação Celular/genética , Megacariócitos/citologia , Osteoblastos/citologia , Transdução de Sinais/genética , Ciclo Celular/genética , Linhagem da Célula , Proliferação de Células/genética , Células Cultivadas , Humanos , Sistema de Sinalização das MAP Quinases/genética , Megacariócitos/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismoRESUMO
The coordination of multiple cytokines and transcription factors with their downstream signaling pathways has been shown to be integral to nephron maturation. Here we present a completely novel role for the helix-loop-helix transcription factor Early B-cell factor 1 (Ebf1), originally identified for B-cell maturation, for the proper maturation of glomerular cells from mesenchymal progenitors. The expression of Ebf1 was both spatially and temporally regulated within the developing cortex and glomeruli. Using Ebf1-null mice, we then identified biochemical, metabolic, and histological abnormalities in renal development that arose in the absence of this transcription factor. In the Ebf1 knockout mice, the developed kidneys show thinned cortices and reduced glomerular maturation. The glomeruli showed abnormal vascularization and severely effaced podocytes. The mice exhibited early albuminuria and elevated blood urea nitrogen levels. Moreover, the glomerular filtration rate was reduced >66% and the expression of podocyte-derived vascular endothelial growth factor A was decreased compared with wild-type control mice. Thus, Ebf1 has a significant and novel role in glomerular development, podocyte maturation, and the maintenance of kidney integrity and function.
Assuntos
Glomérulos Renais/metabolismo , Transativadores/metabolismo , Fatores Etários , Albuminúria/genética , Albuminúria/metabolismo , Albuminúria/fisiopatologia , Animais , Nitrogênio da Ureia Sanguínea , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Taxa de Filtração Glomerular , Glomérulos Renais/crescimento & desenvolvimento , Glomérulos Renais/fisiopatologia , Glomérulos Renais/ultraestrutura , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organogênese , Fenótipo , Podócitos/metabolismo , Podócitos/ultraestrutura , Transdução de Sinais , Fatores de Tempo , Transativadores/deficiência , Transativadores/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Continuous methionine restriction (MR) is one of only a few dietary interventions known to dramatically extend mammalian healthspan. For example, continuously methionine-restricted rodents show less age-related pathology and are up to 45% longer-lived than controls. Intriguingly, MR is feasible for humans, andanumberofstudieshavesuggestedthatmethionine-restrictedindividualsmayreceivesimilarhealthspan benefits as rodents. However, long-term adherence to a continuously methionine-restricted diet is likely to be challenging (or even undesirable) for many individuals. To address this, we previously developed an intermittent version of MR (IMR) and demonstrated that it confers nearly identical metabolic health benefits to mice as the continuous intervention, despite having a relatively short interventional period (i.e., only three days per week). We also observed that female mice undergoing IMR show a more pronounced amelioration of diet-induced dysglycemia than continuously methionine-restricted counterparts, while male mice undergoing IMR retain more lean body mass as compared with continuously methionine-restricted controls. Prompted by such findings, we sought to determine other ways in which IMR might compare favorably with continuous MR. While it is known that continuous MR has deleterious effects on bone in mice, including loss of both trabecular and cortical bone, we considered that mice undergoing IMR might retain more bone mass. Here, we report that, as compared with continuous MR, IMR results in a preservation of both trabecular and cortical bone, as well as a dramatic reduction in the accumulation of marrow fat. Consistent with such findings, mechanical testing revealed that the bones of intermittently methionine-restricted mice are significantly stronger than those of mice subjected to the continuous intervention. Finally, static histomorphometric analyses suggest that IMR likely results in more bone mass than that produced by continuous MR, primarily by increasing the number of osteoblasts. Together, our results demonstrate that the more practicable intermittent form of MR not only confers similar metabolic health benefits to the continuous intervention but does so without markedly deleterious effects on either the amount or strength of bone. These data provide further support for the use of IMR in humans.
RESUMO
Mice deficient in GATA-1 or NF-E2, transcription factors required for normal megakaryocyte (MK) development, have increased numbers of MKs, reduced numbers of platelets, and a striking high bone mass phenotype. Here, we show the bone geometry, microarchitecture, biomechanical, biochemical, and mineral properties from these mutant mice. We found that the outer geometry of the mutant bones was similar to controls, but that both mutants had a striking increase in total bone area (up to a 35% increase) and trabecular bone area (up to a 19% increase). Interestingly, only the NF-E2 deficient mice had a significant increase in cortical bone area (21%) and cortical thickness (27%), which is consistent with the increase in bone mineral density (BMD) seen only in the NF-E2 deficient femurs. Both mutant femurs exhibited significant increases in several biomechanical properties including peak load (up to a 32% increase) and stiffness (up to a 13% increase). Importantly, the data also demonstrate differences between the two mutant mice. GATA-1 deficient femurs break in a ductile manner, whereas NF-E2 deficient femurs are brittle in nature. To better understand these differences, we examined the mineral properties of these bones. Although none of the parameters measured were different between the NF-E2 deficient and control mice, an increase in calcium (21%) and an increase in the mineral/matrix ratio (32%) was observed in GATA-1 deficient mice. These findings appear to contradict biomechanical findings, suggesting the need for further research into the mechanisms by which GATA-1 and NF-E2 deficiency alter the material properties of bone.
Assuntos
Densidade Óssea/fisiologia , Osso e Ossos/fisiologia , Fator de Transcrição GATA1/deficiência , Subunidade p45 do Fator de Transcrição NF-E2/deficiência , Animais , Fenômenos Biomecânicos , Osso e Ossos/anatomia & histologia , Cálcio/metabolismo , Feminino , Fêmur/anatomia & histologia , Fêmur/fisiologia , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subunidade p45 do Fator de Transcrição NF-E2/genética , Subunidade p45 do Fator de Transcrição NF-E2/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismoRESUMO
Nocturnin (NOC) is a circadian-regulated protein related to the yeast family of transcription factors involved in the cellular response to nutrient status. In mammals, NOC functions as a deadenylase but lacks a transcriptional activation domain. It is highly expressed in bone-marrow stromal cells (BMSCs), hepatocytes, and adipocytes. In BMSCs exposed to the PPAR-gamma (peroxisome proliferator-activated receptor-gamma) agonist rosiglitazone, Noc expression was enhanced 30-fold. Previously, we reported that Noc(-/-) mice had low body temperature, were protected from diet-induced obesity, and most importantly exhibited absence of Pparg circadian rhythmicity on a high-fat diet. Consistent with its role in influencing BMSCs allocation, Noc(-/-) mice have reduced bone marrow adiposity and high bone mass. In that same vein, NOC overexpression enhances adipogenesis in 3T3-L1 cells but negatively regulates osteogenesis in MC3T3-E1 cells. NOC and a mutated form, which lacks deadenylase activity, bind to PPAR-gamma and markedly enhance PPAR-gamma transcriptional activity. Both WT and mutant NOC facilitate nuclear translocation of PPAR-gamma. Importantly, NOC-mediated nuclear translocation of PPAR-gamma is blocked by a short peptide fragment of NOC that inhibits its physical interaction with PPAR-gamma. The inhibitory effect of this NOC-peptide was partially reversed by rosiglitazone, suggesting that effect of NOC on PPAR-gamma nuclear translocation may be independent of ligand-mediated PPAR-gamma activation. In sum, Noc plays a unique role in the regulation of mesenchymal stem-cell lineage allocation by modulating PPAR-gamma activity through nuclear translocation. These data illustrate a unique mechanism whereby a nutrient-responsive gene influences BMSCs differentiation, adipogenesis, and ultimately body composition.
Assuntos
Adipogenia , Proteínas Nucleares/metabolismo , PPAR gama/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Composição Corporal , Linhagem Celular , Linhagem da Célula , Ritmo Circadiano , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Osteoblastos/citologia , Osteoblastos/metabolismo , Fatores de Transcrição/deficiênciaRESUMO
Adipose tissue is a dynamic component of the bone marrow, regulating skeletal remodelling and secreting paracrine and endocrine factors that can affect haematopoiesis, as well as potentially nourishing the bone marrow during periods of stress. Bone marrow adipose tissue is regulated by multiple factors, but particularly nutrient status. In this Review, we examine how bone marrow adipocytes originate, their function in normal and pathological states and how bone marrow adipose tissue modulates whole-body homoeostasis through actions on bone cells, haematopoietic stem cells and extra-medullary adipocytes during nutritional challenges. We focus on both rodent models and human studies to help understand the unique marrow adipocyte, its response to the external nutrient environment and its effects on the skeleton. We finish by addressing some critical questions that to date remain unanswered.
Assuntos
Tecido Adiposo , Células da Medula Óssea , Medula Óssea , Humanos , Adipócitos/fisiologia , Medula Óssea/patologia , Medula Óssea/fisiologia , Células da Medula Óssea/fisiologia , Obesidade/patologia , Redução de PesoRESUMO
Dietary methionine restriction (MR) increases longevity by improving health. In experimental models, MR is accompanied by decreased cystathionine ß-synthase activity and increased cystathionine γ-lyase activity. These enzymes are parts of the transsulfuration pathway which produces cysteine and 2-oxobutanoate. Thus, the decrease in cystathionine ß-synthase activity is likely to account for the loss of tissue cysteine observed in MR animals. Despite this decrease in cysteine levels, these tissues exhibit increased H2S production which is thought to be generated by ß-elimination of the thiol moiety of cysteine, as catalyzed by cystathionine ß-synthase or cystathionine γ-lyase. Another possibility for this H2S production is the cystathionine γ-lyase-catalyzed ß-elimination of cysteine persulfide from cystine, which upon reduction yields H2S and cysteine. Here, we demonstrate that MR increases cystathionine γ-lyase production and activities in the liver and kidneys, and that cystine is a superior substrate for cystathionine γ-lyase catalyzed ß-elimination as compared to cysteine. Moreover, cystine and cystathionine exhibit comparable Kcat/Km values (6000 M-1 s-1) as substrates for cystathionine γ-lyase-catalyzed ß-elimination. By contrast, cysteine inhibits cystathionine γ-lyase in a non-competitive manner (Ki ~ 0.5 mM), which limits its ability to function as a substrate for ß-elimination by this enzyme. Cysteine inhibits the enzyme by reacting with its pyridoxal 5'-phosphate cofactor to form a thiazolidine and in so doing prevents further catalysis. These enzymological observations are consistent with the notion that during MR cystathionine γ-lyase is repurposed to catabolize cystine and thereby form cysteine persulfide, which upon reduction produces cysteine.
RESUMO
BACKGROUNDAdipocytes were long considered inert components of the bone marrow niche, but mouse and human models suggest bone marrow adipose tissue (BMAT) is dynamic and responsive to hormonal and nutrient cues.METHODSIn this study of healthy volunteers, we investigated how BMAT responds to acute nutrient changes, including analyses of endocrine determinants and paracrine factors from marrow aspirates. Study participants underwent a 10-day high-calorie protocol, followed by a 10-day fast.RESULTSWe demonstrate (a) vertebral BMAT increased significantly during high-calorie feeding and fasting, suggesting BMAT may have different functions in states of caloric excess compared with caloric deprivation; (b) ghrelin, which decreased in response to high-calorie feeding and fasting, was inversely associated with changes in BMAT; and (c) in response to high-calorie feeding, resistin levels in the marrow sera, but not the circulation, rose significantly. In addition, TNF-α expression in marrow adipocytes increased with high-calorie feeding and decreased upon fasting.CONCLUSIONHigh-calorie feeding, but not fasting, induces an immune response in bone marrow similar to what has been reported in peripheral adipose tissue. Understanding the immunomodulatory regulators in the marrow may provide further insight into the homeostatic function of this unique adipose tissue depot.FUNDINGNIH grant R24 DK084970, Harvard Catalyst/The Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, NIH, award UL 1TR002541), and NIH grants P30 DK040561 and U19 AG060917S1.
Assuntos
Tecido Adiposo , Medula Óssea , Jejum/fisiologia , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiologia , Adulto , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Feminino , Humanos , MasculinoRESUMO
Genome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we use Diversity Outbred (DO) mice to directly address these limitations by performing a systems genetics analysis of 55 complex skeletal phenotypes. We apply a network approach to cortical bone RNA-seq data to discover 66 genes likely to be causal for human BMD GWAS associations, including the genes SERTAD4 and GLT8D2. We also perform GWAS in the DO for a wide-range of bone traits and identify Qsox1 as a gene influencing cortical bone accrual and bone strength. In this work, we advance our understanding of the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics.
Assuntos
Densidade Óssea/genética , Osteoporose/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Animais , Diferenciação Celular/genética , Camundongos de Cruzamento Colaborativo , Conjuntos de Dados como Assunto , Feminino , Fêmur/fisiologia , Fluoresceínas/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Estudo de Associação Genômica Ampla , Glicosiltransferases/genética , Humanos , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Knockout , Osteoblastos , Osteogênese/genética , RNA-Seq , Análise de Célula ÚnicaRESUMO
Inactivating mutations in human ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) may result in early-onset osteoporosis (EOOP) in haploinsufficiency and autosomal recessive hypophosphatemic rickets (ARHR2) in homozygous deficiency. ARHR2 patients are frequently treated with phosphate supplementation to ameliorate the rachitic phenotype, but elevating plasma phosphorus concentrations in ARHR2 patients may increase the risk of ectopic calcification without increasing bone mass. To assess the risks and efficacy of conventional ARHR2 therapy, we performed comprehensive evaluations of ARHR2 patients at two academic medical centers and compared their skeletal and renal phenotypes with ENPP1-deficient Enpp1asj/asj mice on an acceleration diet containing high phosphate treated with recombinant murine Enpp1-Fc. ARHR2 patients treated with conventional therapy demonstrated improvements in rickets, but all adults and one adolescent analyzed continued to exhibit low bone mineral density (BMD). In addition, conventional therapy was associated with the development of medullary nephrocalcinosis in half of the treated patients. Similar to Enpp1asj/asj mice on normal chow and to patients with mono- and biallelic ENPP1 mutations, 5-week-old Enpp1asj/asj mice on the high-phosphate diet exhibited lower trabecular bone mass, reduced cortical bone mass, and greater bone fragility. Treating the Enpp1asj/asj mice with recombinant Enpp1-Fc protein between weeks 2 and 5 normalized trabecular bone mass, normalized or improved bone biomechanical properties, and prevented the development of nephrocalcinosis and renal failure. The data suggest that conventional ARHR2 therapy does not address low BMD inherent in ENPP1 deficiency, and that ENPP1 enzyme replacement may be effective for correcting low bone mass in ARHR2 patients without increasing the risk of nephrocalcinosis. © 2021 American Society for Bone and Mineral Research (ASBMR).
Assuntos
Terapia de Reposição de Enzimas , Fosfatos , Adolescente , Animais , Suplementos Nutricionais , Humanos , Camundongos , Fenótipo , Diester Fosfórico Hidrolases/genética , PirofosfatasesRESUMO
As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes (MKs) can induce osteoblast (OB) proliferation in vitro, but do so only when direct cell-to-cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of MKs with another cell type of mesenchymal origin-the fibroblast (FB). Our findings implicate the involvement of fibronectin/RGD-binding integrins including alpha3beta1 (VLA-3) and alpha5beta1 (VLA-5) as well as glycoprotein (gp) IIb (CD41), all of which are known to be expressed on MK membranes. Furthermore, we demonstrate that interleukin (IL)-3 can enhance MK-induced OB activation in vitro, as demonstrated in the MK-FB model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic-mesenchymal cell activation are mechanistically analogous in several ways.
Assuntos
Integrina alfa3beta1/metabolismo , Integrina alfa5beta1/metabolismo , Megacariócitos/citologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Animais , Anticorpos/farmacologia , Moléculas de Adesão Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ácido Edético/farmacologia , Interleucina-3/farmacologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oligopeptídeos/farmacologia , Osteoblastos/efeitos dos fármacosRESUMO
Recent data suggest that megakaryocytes (MKs) play a role in skeletal homeostasis. In vitro and in vivo data show that MKs stimulate osteoblast (OB) proliferation and inhibit osteoclast (OC) formation, thus favoring net bone deposition. There are several mouse models with dysregulated megakaryopoiesis and resultant high bone mass phenotypes. One such model that our group has extensively studied is GATA-1 deficient mice. GATA-1 is a transcription factor required for normal megakaryopoiesis, and mice deficient in GATA-1 have increases in immature MK number and a striking increase in bone mass. While the increased bone mass could simply be a result of increased MK number, here we take a more in depth look at the MKs of these mice to see if there is a unique factor inherent to GATA-1 deficient MKs that favors increased bone deposition. We show that increased MK number does correspond with increased OB proliferation and decreased OC formation that stage of maturation does not alter the effect of MKs on bone cell lineages beyond the megakaryoblast stage, and that GATA-1 deficient MKs survive longer than wild-type controls. So while increased MK number in GATA-1 deficient mice likely contributes to the high bone mass phenotype, we propose that the increased longevity of this lineage also plays a role. Since GATA-1 deficient MKs live longer they are able to exert both more proliferative influence on OBs and more inhibitory influence on OCs.
Assuntos
Proliferação de Células , Megacariócitos/fisiologia , Osteoblastos/citologia , Osteoclastos/citologia , Animais , Densidade Óssea , Osso e Ossos/citologia , Linhagem da Célula , Fator de Transcrição GATA1/deficiência , Camundongos , Camundongos KnockoutRESUMO
Only three decades ago adipose tissue was considered inert, with little relationship to insulin resistance. Similarly, bone has long been thought of purely in its structural context. In the last decade, emerging evidence has revealed important endocrine roles for both bone and adipose tissue. The interaction between these two tissues is remarkable. Bone marrow mesenchymal stem cells give rise to both osteoblasts and adipocytes. Leptin and adiponectin, two adipokines secreted by fat tissue, control energy homeostasis, but also have complex actions on the skeleton. In turn, the activities of bone cells are not limited to their bone remodelling activities but also to modulation of adipose cell sensitivity and insulin secretion. This review will discuss these new insights linking bone remodelling to the control of fat metabolism and the association between diabetes mellitus and osteoporosis.