RESUMEN
Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia.
Asunto(s)
Anemia de Células Falciformes , Hemoglobina Falciforme , 2,3-Difosfoglicerato/metabolismo , 2,3-Difosfoglicerato/farmacología , Adenosina Trifosfato/metabolismo , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/metabolismo , Animales , Eritrocitos/metabolismo , Hemoglobina Falciforme/metabolismo , Hemoglobina Falciforme/farmacología , Hemoglobina Falciforme/uso terapéutico , Hemoglobinas/metabolismo , Humanos , Oxígeno/metabolismo , Piruvato Quinasa/metabolismo , Piruvato Quinasa/farmacología , Piruvato Quinasa/uso terapéutico , Ácido Pirúvico/farmacologíaRESUMEN
Adipocytes occupy 70% of the cellular volume within the bone marrow (BM) wherein multiple myeloma (MM) originates and resides. However, the nature of the interaction between MM cells and adipocytes remains unclear. Cancer-associated adipocytes support tumor cells through various mechanisms, including metabolic reprogramming of cancer cells. We hypothesized that metabolic interactions mediate the dependence of MM cells on BM adipocytes. Here we show that BM aspirates from precursor states of MM, including monoclonal gammopathy of undetermined significance and smoldering MM, exhibit significant upregulation of adipogenic commitment compared with healthy donors. In vitro coculture assays revealed an adipocyte-induced increase in MM cell proliferation in monoclonal gammopathy of undetermined significance/smoldering MM compared with newly diagnosed MM. Using murine MM cell/BM adipocyte coculture assays, we describe MM-induced lipolysis in adipocytes via activation of the lipolysis pathway. Upregulation of fatty acid transporters 1 and 4 on MM cells mediated the uptake of secreted free fatty acids (FFAs) by adjacent MM cells. The effect of FFAs on MM cells was dose dependent and revealed increased proliferation at lower concentrations vs induction of lipotoxicity at higher concentrations. Lipotoxicity occurred via the ferroptosis pathway. Exogenous treatment with arachidonic acid, a very-long-chain FFA, in a murine plasmacytoma model displayed a reduction in tumor burden. Taken together, our data reveal a novel pathway involving MM cell-induced lipolysis in BM adipocytes and suggest prevention of FFA uptake by MM cells as a potential target for myeloma therapeutics.
Asunto(s)
Adipocitos/metabolismo , Proteínas de Transporte de Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Lipólisis , Mieloma Múltiple/metabolismo , Adipocitos/citología , Adipocitos/patología , Animales , Línea Celular , Técnicas de Cocultivo , Humanos , Masculino , Ratones SCID , Mieloma Múltiple/patología , Células Tumorales CultivadasRESUMEN
MYC upregulation is associated with multidrug refractory disease in patients with multiple myeloma (MM). We, isolated patient-derived MM cells with high MYC expression and discovered that NCOR2 was down-regulated in these cells. NCOR2 is a transcriptional coregulatory protein and its role in MM remains unknown. To define the role of NCOR2 in MM, we created NCOR2 knockout human myeloma cell lines and demonstrated that NCOR2 knockout led to high MYC expression. Furthermore, NCOR2 knockout conferred resistance to pomalidomide, BET and HDAC inhibitors, independent of Cereblon (CRBN), indicating high MYC expression as a cause of multidrug resistance. Moreover, NCOR2 interacted with the nucleosome remodeling and deacetylase (NuRD) complex and repressed the expression of CD180 by directly binding to its promoter and inducing MYC expression. Next, we generated lenalidomide-resistant and pomalidomide-resistant human myeloma cell lines. Whole-exome sequencing revealed that these cell lines acquired the same exonic mutations of NCOR2. These cell lines showed NCOR2 downregulation and MYC upregulation independent of CRBN and demonstrated resistance to BET and HDAC inhibitors. Our findings reveal a novel CRBN independent molecular mechanism associated with drug resistance. Low NCOR2 expression can serve as a potential biomarker for drug resistance and needs further validation in larger prospective studies.
Asunto(s)
Resistencia a Antineoplásicos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Co-Represor 2 de Receptor Nuclear/genética , Proteínas Proto-Oncogénicas c-myc/genética , Antineoplásicos/farmacología , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Técnicas de Inactivación de Genes , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Talidomida/análogos & derivados , Talidomida/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Sickle cell anemia (SCA) results from an abnormal sickle hemoglobin (HbS). HbS polymerizes upon deoxygenation, resulting in red blood cell (RBC) sickling and membrane damage that cause vaso-occlusions and hemolysis. Sickle RBCs contain less adenosine triphosphate and more 2,3-diphosphoglycerate than normal RBCs, which allosterically reduces hemoglobin (Hb) oxygen (O2) affinity (ie, increases the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen [P50]), potentiating HbS polymerization. Herein, we tested the effect of investigational agent FT-4202, an RBC pyruvate kinase (PKR) activator, on RBC sickling and membrane damage by administering it to Berkeley SCA mice. Two-week oral FT-4202 administration was well tolerated, decreasing HbS P50 to levels similar to HbA and demonstrating beneficial biological effects. In FT-4202-treated animals, there was reduced sickling in vivo, demonstrated by fewer irreversibly sickled cells, and improved RBC deformability, assessed at varying shear stress. Controlled deoxygenation followed by reoxygenation of RBCs obtained from the blood of FT-4202-treated mice showed a shift in the point of sickling to a lower partial pressure of oxygen (pO2). This led to a nearly 30% increase in RBC survival and a 1.7g/dL increase in Hb level in the FT-4202-treated SCA mice. Overall, our results in SCA mice suggest that FT-4202 might be a potentially useful oral antisickling agent that warrants investigation in patients with SCA.
Asunto(s)
Anemia de Células Falciformes , Hemoglobina Falciforme , Anemia de Células Falciformes/tratamiento farmacológico , Animales , Antidrepanocíticos , Eritrocitos Anormales , Humanos , Ratones , Piruvato QuinasaRESUMEN
Bone loss is a major health concern for astronauts during long-term spaceflight and for patients during prolonged bed rest or paralysis. Growing evidence suggests that osteocytes, the most abundant cells in the mineralized bone matrix, play a key role in sensing mechanical forces applied to the skeleton and integrating the orchestrated response into subcellular biochemical signals to modulate bone homeostasis. However, the precise molecular mechanisms underlying both mechanosensation and mechanotransduction in late-osteoblast-to-osteocyte cells under microgravity (µG) have yet to be elucidated. To unravel the mechanisms by which late osteoblasts and osteocytes sense and respond to mechanical unloading, we exposed the osteocytic cell line, Ocy454, to 2, 4, or 6 days of µG on the SpaceX Dragon-6 resupply mission to the International Space Station. Our results showed that µG impairs the differentiation of osteocytes, consistent with prior osteoblast spaceflight experiments, which resulted in the downregulation of key osteocytic genes. Importantly, we demonstrate the modulation of critical glycolysis pathways in osteocytes subjected to microgravity and discovered a set of mechanical sensitive genes that are consistently regulated in multiple cell types exposed to microgravity suggesting a common, yet to be fully elucidated, genome-wide response to microgravity. Ground-based simulated microgravity experiments utilizing the NASA rotating-wall-vessel were unable to adequately replicate the changes in microgravity exposure highlighting the importance of spaceflight missions to understand the unique environmental stress that microgravity presents to diverse cell types. In summary, our findings demonstrate that osteocytes respond to µG with an increase in glucose metabolism and oxygen consumption.
Asunto(s)
Regulación de la Expresión Génica , Glucosa/metabolismo , Osteocitos/metabolismo , Consumo de Oxígeno , Vuelo Espacial/métodos , Transcriptoma , Animales , Mecanotransducción Celular , Ratones , Osteocitos/citologíaRESUMEN
Vitamin K antagonists (VKAs) have been used in 1% of the world's population for prophylaxis or treatment of thromboembolic events for 64 years. Impairment of osteoblast function and osteoporosis has been described in patients receiving VKAs. Given the involvement of cells of the bone marrow microenvironment (BMM), such as mesenchymal stem cells (MSCs) and macrophages, as well as other factors such as the extracellular matrix for the maintenance of normal hematopoietic stem cells (HSCs), we investigated a possible effect of VKAs on hematopoiesis via the BMM. Using various transplantation and in vitro assays, we show here that VKAs alter parameters of bone physiology and reduce functional HSCs 8-fold. We implicate impairment of the functional, secreted, vitamin K-dependent, γ-carboxylated form of periostin by macrophages and, to a lesser extent, MSCs of the BMM and integrin ß3-AKT signaling in HSCs as at least partly causative of this effect, with VKAs not being directly toxic to HSCs. In patients, VKA use associates with modestly reduced leukocyte and monocyte counts, albeit within the normal reference range. VKAs decrease human HSC engraftment in immunosuppressed mice. Following published examples that alteration of the BMM can lead to hematological malignancies in mice, we describe, without providing a causal link, that the odds of VKA use are higher in patients with vs without a diagnosis of myelodysplastic syndrome (MDS). These results demonstrate that VKA treatment impairs HSC function via impairment of the BMM and the periostin/integrin ß3 axis, possibly associating with increased MDS risk.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Microambiente Celular/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Vitamina K/antagonistas & inhibidores , Animales , Anticoagulantes/farmacología , Biomarcadores , Moléculas de Adhesión Celular/metabolismo , Relación Dosis-Respuesta a Droga , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/etiología , Síndromes Mielodisplásicos/metabolismo , Vitamina K/farmacología , Warfarina/farmacologíaAsunto(s)
Antineoplásicos/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Compuestos Macrocíclicos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/uso terapéutico , Microambiente Celular , Resistencia a Antineoplásicos , HumanosRESUMEN
The stimulatory subunit of G-protein, Gsα, acts as a secondary messenger of G-protein coupled receptors (GPCRs) that primarily activates cAMP-induced signaling. GPCRs, such as the parathyroid hormone receptor (PTHR), are critical regulators of bone formation as shown by number of genetic manipulation studies targeting early osteoblast lineage cells. In this study, we have examined the role of Gsα in osteocytes, the terminally differentiated and most abundant cells of the osteoblast lineage. Mice lacking the stimulatory subunit of G-proteins (Gsα) in osteocytes (DMP1-GsαKO) have significant decrease of both trabecular and cortical bone, as assessed by µCT. Histomorphometric analysis showed that the osteopenia was mostly driven by more than 90% decrease in osteoblast numbers and activity whereas osteoclasts were only slightly decreased. The decrease in osteoblast number was associated with a striking lack of endocortical osteoblasts. We have previously shown that loss of the stimulatory subunit of G-proteins (Gsα) in osteocytes in vitro or in vivo induces high expression of sclerostin. To determine if the increased sclerostin levels contributed to the decreased endosteal bone lining cells and osteopenia, we treated wild-type mice with recombinant sclerostin and the DMP1-GsαKO mice with anti-sclerostin antibody. Treatment of wild-type mice with 100⯵g/kg sclerostin for 3-weeks significantly reduced the numbers of bone lining cells and led to osteopenia. Next, the DMP1-GsαKO and control littermates were treated with the anti-sclerostin antibody (25â¯mg/kg, 2 times per week) for 4-weeks. Upon the antibody treatment, the endocortical osteoblasts reappeared in the DMP1-GsαKO mice to a comparable level to that of the vehicle treated control littermates. In control mice, E11/gp38 positive osteocytes were observed in parallel with the endocortical osteoblasts with higher dendrite density towards the endocortical osteoblasts. In DMP1-GsαKO mice, E11/gp38 positive osteocytes were lacking dendrites and were randomly scattered throughout the bone matrix. After treatment with anti-sclerostin antibody, DMP1-GsαKO mice showed increased E11/gp38 positive osteocytes near the endosteal bone surface and endosteal osteoblasts. The anti-sclerostin antibody treatment proportionally increased the bone volume but it could not completely rescue the osteopenia in the DMP1-GsαKO mice. Taken together, this data suggests that Gsα signaling in osteocytes leads to osteopenia driven, at least in part, by increased secretion of sclerostin.
Asunto(s)
Enfermedades Óseas Metabólicas/metabolismo , Enfermedades Óseas Metabólicas/patología , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Glicoproteínas/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patología , Osteocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos Neutralizantes/metabolismo , Enfermedades Óseas Metabólicas/diagnóstico por imagen , Hueso Esponjoso/diagnóstico por imagen , Hueso Esponjoso/metabolismo , Hueso Cortical/diagnóstico por imagen , Hueso Cortical/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fémur/diagnóstico por imagen , Fémur/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Osteogénesis , Microtomografía por Rayos XRESUMEN
Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.
Asunto(s)
Anhidrasa Carbónica III/metabolismo , Osteocitos/metabolismo , Estrés Oxidativo , Proteínas Adaptadoras Transductoras de Señales , Animales , Remodelación Ósea/genética , Remodelación Ósea/fisiología , Anhidrasa Carbónica III/genética , Línea Celular , Supervivencia Celular , Glicoproteínas/genética , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Ratones , Osteocitos/citología , Osteocitos/efectos de los fármacos , Teriparatido/farmacología , TranscriptomaRESUMEN
Cells of the osteoblast lineage are increasingly identified as participants in whole-body metabolism by primarily targeting pancreatic insulin secretion or consuming energy. Osteocytes, the most abundant bone cells, secrete a Wnt-signaling inhibitor called sclerostin. Here we examined three mouse models expressing high sclerostin levels, achieved through constitutive or inducible loss of the stimulatory subunit of G-proteins (Gsα in mature osteoblasts and/or osteocytes). These mice showed progressive loss of white adipose tissue (WAT) with tendency toward increased energy expenditure but no changes in glucose or insulin metabolism. Interestingly beige adipocytes were increased extensively in both gonadal and inguinal WAT and had reduced canonical ß-catenin signaling. To determine if sclerostin directly contributes to the increased beige adipogenesis, we engineered an osteocytic cell line lacking Gsα which has high sclerostin secretion. Conditioned media from these cells significantly increased expression of UCP1 in primary adipocytes, and this effect was partially reduced after depletion of sclerostin from the conditioned media. Similarly, treatment of Gsα-deficient animals with sclerostin-neutralizing antibody partially reduced the increased UCP1 expression in WAT. Moreover, direct treatment of sclerostin to wild-type mice significantly increased UCP1 expression in WAT. These results show that osteocytes and/or osteoblasts secrete factors regulating beige adipogenesis, at least in part, through the Wnt-signaling inhibitor sclerostin. Further studies are needed to assess metabolic effects of sclerostin on adipocytes and other metabolic tissues. © 2016 American Society for Bone and Mineral Research.
Asunto(s)
Adipogénesis , Tejido Adiposo Beige/metabolismo , Adiposidad , Glicoproteínas/metabolismo , Osteocitos/metabolismo , Vía de Señalización Wnt , Proteínas Adaptadoras Transductoras de Señales , Tejido Adiposo Blanco , Animales , Animales Recién Nacidos , Linaje de la Célula , Metabolismo Energético , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Ratones Noqueados , Tamaño de los Órganos , Osteoblastos/metabolismo , Fenotipo , Delgadez/metabolismoRESUMEN
Cells of the osteoblast lineage provide critical support for B lymphopoiesis in the bone marrow (BM). Parathyroid hormone (PTH) signaling in osteoblastic cells through its receptor (PPR) is an important regulator of hematopoietic stem cells; however, its role in regulation of B lymphopoiesis is not clear. Here we demonstrate that deletion of PPR in osteoprogenitors results in a significant loss of trabecular and cortical bone. PPR signaling in osteoprogenitors, but not in mature osteoblasts or osteocytes, is critical for B-cell precursor differentiation via IL-7 production. Interestingly, despite a severe reduction in B-cell progenitors in BM, mature B-lymphocytes were increased 3.5-fold in the BM of mice lacking PPR in osteoprogenitors. This retention of mature IgD(+) B cells in the BM was associated with increased expression of vascular cell adhesion molecule 1 (VCAM1) by PPR-deficient osteoprogenitors, and treatment with VCAM1 neutralizing antibody increased mobilization of B lymphocytes from mutant BM. Our results demonstrate that PPR signaling in early osteoblasts is necessary for B-cell differentiation via IL-7 secretion and for B-lymphocyte mobilization via VCAM1.
Asunto(s)
Linfocitos B/citología , Hormona Paratiroidea/metabolismo , Transducción de Señal , Células Madre/citología , Animales , Anticuerpos Neutralizantes/química , Apoptosis , Huesos/metabolismo , Diferenciación Celular , Quimiocina CXCL12/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Inmunohistoquímica , Interleucina-7/metabolismo , Ratones , Ratones Noqueados , Osteoblastos/citología , Osteocitos/citología , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Microtomografía por Rayos XRESUMEN
In humans, aging and glucocorticoid treatment are associated with reduced bone mass and increased marrow adiposity, suggesting that the differentiation of osteoblasts and adipocytes may be coordinately regulated. Within the bone marrow, both osteoblasts and adipocytes are derived from mesenchymal progenitor cells, but the mechanisms guiding the commitment of mesenchymal progenitors into osteoblast versus adipocyte lineages are not fully defined. The heterotrimeric G protein subunit Gs α activates protein kinase A signaling downstream of several G protein-coupled receptors including the parathyroid hormone receptor, and plays a crucial role in regulating bone mass. Here, we show that targeted ablation of Gs α in early osteoblast precursors, but not in differentiated osteocytes, results in a dramatic increase in bone marrow adipocytes. Mutant mice have reduced numbers of mesenchymal progenitors overall, with an increase in the proportion of progenitors committed to the adipocyte lineage. Furthermore, cells committed to the osteoblast lineage retain adipogenic potential both in vitro and in vivo. These findings have clinical implications for developing therapeutic approaches to direct the commitment of mesenchymal progenitors into the osteoblast lineage.
Asunto(s)
Adipocitos/metabolismo , Adipogénesis , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Adipocitos/citología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , Ratones Noqueados , Osteoblastos/citologíaRESUMEN
Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM) and may be the cause of relapse following chemotherapy. Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. CD44 (refs. 3,4) and interleukin-6 (ref. 5) have been implicated previously in the LSC niche. Transforming growth factor-ß1 (TGF-ß1) is released during bone remodeling and plays a part in maintenance of CML LSCs, but a role for TGF-ß1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor attenuates BCR-ABL1 oncogene-induced CML-like myeloproliferative neoplasia (MPN) but enhances MLL-AF9 oncogene-induced AML in mouse transplantation models, possibly through opposing effects of increased TGF-ß1 on the respective LSCs. PTH treatment caused a 15-fold decrease in LSCs in wild-type mice with CML-like MPN and reduced engraftment of immune-deficient mice with primary human CML cells. These results demonstrate that LSC niches in CML and AML are distinct and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSCs, a prerequisite for the cure of CML.
Asunto(s)
Médula Ósea/metabolismo , Médula Ósea/patología , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Animales , Femenino , Genes abl , Humanos , Leucemia Mieloide/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Proteínas de Fusión Oncogénica/genética , Hormona Paratiroidea/metabolismo , Transducción de Señal , Nicho de Células Madre , Factor de Crecimiento Transformador beta1/metabolismo , Microambiente TumoralRESUMEN
Parathyroid hormone (PTH) is the only Food and Drug Administration-approved anabolic agent to treat osteoporosis; however, the cellular targets of PTH action in bone remain controversial. PTH modulates bone turnover by binding to the PTH/PTH-related peptide (PTHrP) type 1 receptor (PPR), a G-protein-coupled receptor highly expressed in bone and kidneys. Osteocytes, the most abundant cells in adult bone, also express PPR. However, the physiological relevance of PPR signaling in osteocytes remains to be elucidated. Toward this goal, we generated mice with PPR deletion in osteocytes (Ocy-PPRKO). Skeletal analysis of these mice revealed a significant increase in bone mineral density and trabecular and cortical bone parameters. Osteoblast activities were reduced in these animals, as demonstrated by decreased collagen type I α1 mRNA and receptor activator of NF-κB ligand (RANKL) expression. Importantly, when subjected to an anabolic or catabolic PTH regimen, Ocy-PPRKO animals demonstrated blunted skeletal responses. PTH failed to suppress SOST/Sclerostin or induce RANKL expression in Ocy-PPRKO animals compared with controls. In vitro, osteoclastogenesis was significantly impaired in Ocy-PPRKO upon PTH administration, indicating that osteocytes control osteoclast formation through a PPR-mediated mechanism. Taken together, these data indicate that PPR signaling in osteocytes is required for bone remodeling, and receptor signaling in osteocytes is needed for anabolic and catabolic skeletal responses.
Asunto(s)
Huesos/efectos de los fármacos , Osteocitos/efectos de los fármacos , Hormona Paratiroidea/farmacología , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Transducción de Señal , Animales , Peso Corporal , Densidad Ósea , Huesos/citología , Huesos/metabolismo , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Femenino , Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteocitos/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hematopoietic progenitors are regulated in their respective niches by cells of the bone marrow microenvironment. The bone marrow microenvironment is composed of a variety of cell types, and the relative contribution of each of these cells for hematopoietic lineage maintenance has remained largely unclear. Osteocytes, the most abundant yet least understood cells in bone, are thought to initiate adaptive bone remodeling responses via osteoblasts and osteoclasts. Here we report that these cells regulate hematopoiesis, constraining myelopoiesis through a Gsα-mediated mechanism that affects G-CSF production. Mice lacking Gsα in osteocytes showed a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. This hematopoietic phenomenon was neither intrinsic to the hematopoietic cells nor dependent on osteoblasts but was a consequence of an altered bone marrow microenvironment imposed by Gsα deficiency in osteocytes. Conditioned media from osteocyte-enriched bone explants significantly increased myeloid colony formation in vitro, which was blocked by G-CSFneutralizing antibody, indicating a critical role of osteocyte-derived G-CSF in the myeloid expansion.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Mielopoyesis , Osteocitos/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Células de la Médula Ósea/metabolismo , Proliferación Celular , Células Cultivadas , Microambiente Celular/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Células Mieloides/metabolismo , Osteocitos/citología , Osteocitos/ultraestructura , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/metabolismoRESUMEN
The insulin-like growth factors (IGF) evolved in lower animals to enable a wide range of physiologic processes, including smell, food consumption, metabolism, growth, reproduction, and dormancy. These functions were accomplished by the actions of multiple related ligands that activated a common transmembrane receptor protein. In higher organisms, including mammals, the insulin and IGF ligands and their receptors evolved to function in a more circumscribed fashion. The contemporary model assigns IGFs as central regulators of cell proliferation, survival, and organism growth, whereas insulin's action dominates at the level of regulation of fuel accumulation, storage, and energy expenditure. Such a simplistic paradigm, however, obscures the fact that insulin and IGF-1 continue to exert overlapping roles in several physiologic processes. Indeed, recent studies have identified previously unappreciated skeletal actions of insulin, which suggests that insulin-responsive bone cells participate in the regulation of global energy homeostasis. These findings raise intriguing questions on the nature of the fuel sensing and processing mechanisms in bone and their relative importance to overall energy homeostasis in mammals. Answers to these questions should ultimately improve the ability to diagnose and manage patients with metabolic diseases such as diabetes and osteoporosis.
Asunto(s)
Huesos/metabolismo , Insulina/metabolismo , Animales , Humanos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Receptor de Insulina/metabolismo , Transducción de SeñalRESUMEN
The heterotrimeric G protein subunit Gsα stimulates cAMP-dependent signaling downstream of G protein-coupled receptors. In this study, we set out to determine the role of Gsα signaling in cells of the early osteoblast lineage in vivo by conditionally deleting Gsα from osterix-expressing cells. This led to severe osteoporosis with fractures at birth, a phenotype that was found to be the consequence of impaired bone formation rather than increased resorption. Osteoblast number was markedly decreased and osteogenic differentiation was accelerated, resulting in the formation of woven bone. Rapid differentiation of mature osteoblasts into matrix-embedded osteocytes likely contributed to depletion of the osteoblast pool. In addition, the number of committed osteoblast progenitors was diminished in both bone marrow stromal cells (BMSCs) and calvarial cells of mutant mice. In the absence of Gsα, expression of sclerostin and dickkopf1 (Dkk1), inhibitors of canonical Wnt signaling, was markedly increased; this was accompanied by reduced Wnt signaling in the osteoblast lineage. In summary, we have shown that Gsα regulates bone formation by at least two distinct mechanisms: facilitating the commitment of mesenchymal progenitors to the osteoblast lineage in association with enhanced Wnt signaling; and restraining the differentiation of committed osteoblasts to enable production of bone of optimal mass, quality, and strength.
Asunto(s)
Diferenciación Celular/fisiología , Linaje de la Célula , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Células Madre Mesenquimatosas/fisiología , Osteoblastos/fisiología , Animales , Huesos/anatomía & histología , Huesos/patología , Huesos/fisiología , Células Cultivadas , Femenino , Subunidades alfa de la Proteína de Unión al GTP/genética , Eliminación de Gen , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Osteoblastos/citología , Osteogénesis/fisiología , Osteoporosis/patología , Osteoporosis/fisiopatología , Transducción de Señal/fisiología , Factor de Transcripción Sp7 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Global energy balance in mammals is controlled by the actions of circulating hormones that coordinate fuel production and utilization in metabolically active tissues. Bone-derived osteocalcin, in its undercarboxylated, hormonal form, regulates fat deposition and is a potent insulin secretagogue. Here, we show that insulin receptor (IR) signaling in osteoblasts controls osteoblast development and osteocalcin expression by suppressing the Runx2 inhibitor Twist2. Mice lacking IR in osteoblasts have low circulating undercarboxylated osteocalcin and reduced bone acquisition due to decreased bone formation and deficient numbers of osteoblasts. With age, these mice develop marked peripheral adiposity and hyperglycemia accompanied by severe glucose intolerance and insulin resistance. The metabolic abnormalities in these mice are improved by infusion of undercarboxylated osteocalcin. These results indicate the existence of a bone-pancreas endocrine loop through which insulin signaling in the osteoblast ensures osteoblast differentiation and stimulates osteocalcin production, which in turn regulates insulin sensitivity and pancreatic insulin secretion.
Asunto(s)
Osteoblastos/metabolismo , Osteogénesis , Receptor de Insulina/metabolismo , Adiposidad , Animales , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Resistencia a la Insulina , Masculino , Ratones , Osteoblastos/citología , Osteocalcina/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
A spontaneous mouse mutant, designated 'small' (sml), was recognized by reduced body size suggesting a defect in the IGF1/GH axis. The mutation was mapped to the chromosome 1 region containing Irs1, a viable candidate gene whose sequence revealed a single nucleotide deletion resulting in a premature stop codon. Despite normal mRNA levels in mutant and control littermate livers, western blot analysis revealed no detectable protein in mutant liver lysates. When compared with the control littermates, Irs1(sml)/Irs1(sml) (Irs1(sml/sml)) mice were small, lean, hearing impaired; had 20% less serum IGF1; were hyperinsulinemic; and were mildly insulin resistant. Irs1(sml/sml) mice had low bone mineral density, reduced trabecular and cortical thicknesses, and low bone formation rates, while osteoblast and osteoclast numbers were increased in the females but not different in the males compared with the Irs1(+/+) controls. In vitro, Irs1(sml/sml) bone marrow stromal cell cultures showed decreased alkaline phosphatase-positive colony forming units (pre-osteoblasts; CFU-AP+) and normal numbers of tartrate-resistant acid phosphatase-positive osteoclasts. Irs1(sml/sml) stromal cells treated with IGF1 exhibited a 50% decrease in AKT phosphorylation, indicative of defective downstream signaling. Similarities between engineered knockouts and the spontaneous mutation of Irs1(sml) were identified as well as significant differences with respect to heterozygosity and gender. In sum, we have identified a spontaneous mutation in the Irs1 gene associated with a major skeletal phenotype. Changes in the heterozygous Irs1(+)(/sml) mice raise the possibility that similar mutations in humans are associated with short stature or osteoporosis.