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Eggs of oviparous reptiles are ideal models for studying evolutionary patterns of embryonic metabolism since they allow tracking of energy allocation during development. Analyzing oxygen consumption of whole eggs throughout development indicates three patterns among reptiles. Embryos initially grow and consume oxygen exponentially, but oxygen consumption slows, or drops before hatching in some species. Turtles, crocodilians, and most lizards follow curves with initial exponential increases followed by declines, whereas embryonic snakes that have been studied exhibit a consistently exponential pattern. This study measured oxygen consumption of corn snake, Pantherophis guttatus, embryos to determine if this species also exhibits an exponential increase in oxygen consumption. Individual eggs, sampled weekly from oviposition to hatching, were placed in respirometry chambers for 24-h during which oxygen consumption was recorded. Embryos were staged and carcasses and yolk were weighed separately. Results indicate steady inclines in oxygen consumption during early stages of development, with a rapid increase prior to hatching. The findings support the hypothesis that embryonic oxygen consumption of snakes differs from most other non-avian reptiles. Total energy required for development was determined based on calorimetry of initial yolk compared to hatchlings and residual yolk and by integration of the area under the curve plotting oxygen consumption versus age of embryos. The cost of development estimates based on these two methods were 6.4 and 10.0 kJ, respectively. Our results emphasize the unique physiological aspects of snake embryogenesis and illustrate how the study of physiological characteristics can contribute to the broader understanding of reptilian evolution.
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Colubridae , Oviparidad , Zea mays , Femenino , Animales , Oviparidad/fisiología , Embrión no Mamífero/fisiología , SerpientesRESUMEN
Severe osteoporosis is often treated with one of three Food and Drug Administration (FDA)-approved osteoanabolics. These drugs act by (1) parathyroid hormone (PTH) receptor stimulation using analogues to PTH (teriparatide) or PTH-related peptide (abaloparatide) or by (2) monoclonal antibody neutralization of sclerostin, an innate Wnt inhibitor (Scl-mAb, romosozumab-aqqg). The efficacies of both strategies wane over time. The transcription factor Nmp4 (Nuclear Matrix Protein 4) is expressed in all tissues yet mice lacking this gene are healthy and exhibit enhanced PTH-induced bone formation. Conditional deletion of Nmp4 in mesenchymal stem progenitor cells (MSPCs) phenocopies the elevated response to PTH in global Nmp4-/- mice. However, targeted deletion in later osteoblast stages does not replicate this response. In this study we queried whether loss of Nmp4 improves Scl-mAb potency. Experimental cohorts included global Nmp4-/- and Nmp4+/+ littermates and three conditional knockout models. Nmp4-floxed (Nmp4fl/fl) mice were crossed with mice harboring one of three Cre-drivers (i) Prx1Cre+ targeting MSPCs, (ii) BglapCre+ (mature osteocalcin-expressing osteoblasts), and (iii) Dmp1Cre+ (osteocytes). Female mice were treated with Scl-mAb or 0.9 % saline vehicle for 4 or 7 weeks from 10 weeks of age. Skeletal response was assessed using micro-computed tomography, dual-energy X-ray absorptiometry, bone histomorphometry, and serum analysis. Global Nmp4-/- mice exhibited enhanced Scl-mAb-induced increases in trabecular bone in the femur and spine and a heightened increase in whole body areal bone mineral density compared to global Nmp4+/+ controls. This improved Scl-mAb potency was primarily driven by enhanced increases in bone formation. Nmp4fl/fl;PrxCre+ mice showed an exaggerated Scl-mAb-induced increase in femoral bone but not in the spine since Prrx1 is not expressed in vertebra. The Nmp4fl/fl;BglapCre+ and Nmp4fl/fl;Dmp1Cre+ mice did not exhibit an improved Scl-mAb response. We conclude that Nmp4 expression in MSPCs interferes with the bone anabolic response to anti-sclerostin therapy.
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The ability to behaviorally regulate body conditions is critical for ectotherms, particularly in the face of global climate change when seeking stable refugia in a changing environment could facilitate survival. This is especially important for montane species that are limited to high elevations. In the Northern Gray-cheeked salamander (Plethodon montanus), studies have demonstrated that population demographics improve at higher elevations and physiological constraints may prevent them from moving into lower-elevation habitats. However, little is known about the species' ability to utilize microhabitats and behaviorally regulate by selecting preferable microclimates. Here, we used continuous position-sensing gradient chambers to examine the behavioral preference for temperature and relative humidity (RH) in P. montanus to better understand their microhabitat use and behavioral thermoregulation across an elevation gradient. We investigated the seasonal variation in both thermal and RH preference of P. montanus collected from different elevations. Our results suggest that most recently experienced environmental temperatures influence thermal preference in animals at high elevations but not those at lower elevations. Salamanders preferred the highest available RH conditions regardless of environmental conditions or elevation. Data on shuttling behavior (movement across the behavior arena) from the experiments suggest that while salamanders shuttled a similar number of times in both types of trials, they spent significantly less time exploring when exposed to the RH gradient compared to the thermal gradient. Together these results suggest that while thermal preference is influenced by acclimation, preference for moisture conditions is less elastic.
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Regulación de la Temperatura Corporal , Microclima , Animales , Temperatura , Ecosistema , UrodelosRESUMEN
The skeleton is a secretory organ, and the goal of some osteoporosis therapies is to maximize bone matrix output. Nmp4 encodes a novel transcription factor that regulates bone cell secretion as part of its functional repertoire. Loss of Nmp4 enhances bone response to osteoanabolic therapy, in part, by increasing the production and delivery of bone matrix. Nmp4 shares traits with scaling factors, which are transcription factors that influence the expression of hundreds of genes to govern proteome allocation for establishing secretory cell infrastructure and capacity. Nmp4 is expressed in all tissues and while global loss of this gene leads to no overt baseline phenotype, deletion of Nmp4 has broad tissue effects in mice challenged with certain stressors. In addition to an enhanced response to osteoporosis therapies, Nmp4-deficient mice are less sensitive to high fat diet-induced weight gain and insulin resistance, exhibit a reduced disease severity in response to influenza A virus (IAV) infection, and resist the development of some forms of rheumatoid arthritis. In this review, we present the current understanding of the mechanisms underlying Nmp4 regulation of the skeletal response to osteoanabolics, and we discuss how this unique gene contributes to the diverse phenotypes among different tissues and stresses. An emerging theme is that Nmp4 is important for the infrastructure and capacity of secretory cells that are critical for health and disease.
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Osteoporosis , Hormona Paratiroidea , Ratones , Animales , Hormona Paratiroidea/metabolismo , Ratones Noqueados , Factores de Transcripción/genética , Regulación de la Expresión Génica , Osteoporosis/tratamiento farmacológico , Osteoporosis/genéticaRESUMEN
Activation of bone anabolic pathways is a fruitful approach for treating severe osteoporosis, yet FDA-approved osteoanabolics, eg, parathyroid hormone (PTH), have limited efficacy. Improving their potency is a promising strategy for maximizing bone anabolic output. Nmp4 (Nuclear Matrix Protein 4) global knockout mice exhibit enhanced PTH-induced increases in trabecular bone but display no overt baseline skeletal phenotype. Nmp4 is expressed in all tissues; therefore, to determine which cell type is responsible for driving the beneficial effects of Nmp4 inhibition, we conditionally removed this gene from cells at distinct stages of osteogenic differentiation. Nmp4-floxed (Nmp4fl/fl ) mice were crossed with mice bearing one of three Cre drivers including (i) Prx1Cre+ to remove Nmp4 from mesenchymal stem/progenitor cells (MSPCs) in long bones; (ii) BglapCre+ targeting mature osteoblasts, and (iii) Dmp1Cre+ to disable Nmp4 in osteocytes. Virgin female Cre+ and Cre- mice (10 weeks of age) were sorted into cohorts by weight and genotype. Mice were administered daily injections of either human PTH 1-34 at 30 µg/kg or vehicle for 4 weeks or 7 weeks. Skeletal response was assessed using dual-energy X-ray absorptiometry, micro-computed tomography, bone histomorphometry, and serum analysis for remodeling markers. Nmp4fl/fl ;Prx1Cre+ mice virtually phenocopied the global Nmp4-/- skeleton in the femur, ie, a mild baseline phenotype but significantly enhanced PTH-induced increase in femur trabecular bone volume/total volume (BV/TV) compared with their Nmp4fl/fl ;Prx1Cre- controls. This was not observed in the spine, where Prrx1 is not expressed. Heightened response to PTH was coincident with enhanced bone formation. Conditional loss of Nmp4 from the mature osteoblasts (Nmp4fl/fl ;BglapCre+ ) failed to increase BV/TV or enhance PTH response. However, conditional disabling of Nmp4 in osteocytes (Nmp4fl/fl ;Dmp1Cre+ ) increased BV/TV without boosting response to hormone under our experimental regimen. We conclude that Nmp4-/- Prx1-expressing MSPCs drive the improved response to PTH therapy and that this gene has stage-specific effects on osteoanabolism. © 2022 American Society for Bone and Mineral Research (ASBMR).
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Células Madre Mesenquimatosas , Osteogénesis , Animales , Femenino , Humanos , Ratones , Huesos , Densidad Ósea , Proteínas de Homeodominio/genética , Ratones Noqueados , Hormona Paratiroidea/farmacología , Microtomografía por Rayos XRESUMEN
A bidirectional and complex relationship exists between bone and glycemia. Persons with type 2 diabetes (T2D) are at risk for bone loss and fracture, however, heightened osteoanabolism may ameliorate T2D-induced deficits in glycemia as bone-forming osteoblasts contribute to energy metabolism via increased glucose uptake and cellular glycolysis. Mice globally lacking nuclear matrix protein 4 (Nmp4), a transcription factor expressed in all tissues and conserved between humans and rodents, are healthy and exhibit enhanced bone formation in response to anabolic osteoporosis therapies. To test whether loss of Nmp4 similarly impacted bone deficits caused by diet-induced obesity, male wild-type and Nmp4-/- mice (8 weeks) were fed either low-fat diet or high-fat diet (HFD) for 12 weeks. Endpoint parameters included bone architecture, structural and estimated tissue-level mechanical properties, body weight/composition, glucose-stimulated insulin secretion, glucose tolerance, insulin tolerance, and metabolic cage analysis. HFD diminished bone architecture and ultimate force and stiffness equally in both genotypes. Unexpectedly, the Nmp4-/- mice exhibited deficits in pancreatic ß-cell function and were modestly glucose intolerant under normal diet conditions. Despite the ß-cell deficits, the Nmp4-/- mice were less sensitive to HFD-induced weight gain, increases in % fat mass, and decreases in glucose tolerance and insulin sensitivity. We conclude that Nmp4 supports pancreatic ß-cell function but suppresses peripheral glucose utilization, perhaps contributing to its suppression of induced skeletal anabolism. Selective disruption of Nmp4 in peripheral tissues may provide a strategy for improving both induced osteoanabolism and energy metabolism in comorbid patients.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Animales , Dieta Alta en Grasa/efectos adversos , Humanos , Insulina , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Matriz Nuclear/metabolismo , Hormona Paratiroidea , Factores de Transcripción/metabolismoRESUMEN
Severe influenza A virus infection typically triggers excessive and detrimental lung inflammation with massive cell infiltration and hyper-production of cytokines and chemokines. We identified a novel function for nuclear matrix protein 4 (NMP4), a zinc-finger-containing transcription factor playing roles in bone formation and spermatogenesis, in regulating antiviral immune response and immunopathology. Nmp4-deficient mice are protected from H1N1 influenza infection, losing only 5% body weight compared to a 20% weight loss in wild type mice. While having no effects on viral clearance or CD8/CD4 T cell or humoral responses, deficiency of Nmp4 in either lung structural cells or hematopoietic cells significantly reduces the recruitment of monocytes and neutrophils to the lungs. Consistent with fewer innate cells in the airways, influenza-infected Nmp4-deficient mice have significantly decreased expression of chemokine genes Ccl2, Ccl7 and Cxcl1 as well as pro-inflammatory cytokine genes Il1b and Il6. Furthermore, NMP4 binds to the promoters and/or conserved non-coding sequences of the chemokine genes and regulates their expression in mouse lung epithelial cells and macrophages. Our data suggest that NMP4 functions to promote monocyte- and neutrophil-attracting chemokine expression upon influenza A infection, resulting in exaggerated innate inflammation and lung tissue damage.
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Inmunidad Innata , Inmunomodulación , Virus de la Influenza A/inmunología , Proteínas Asociadas a Matriz Nuclear/genética , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/inmunología , Factores de Transcripción/genética , Inmunidad Adaptativa , Animales , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno/inmunología , Inmunomodulación/genética , Mediadores de Inflamación/metabolismo , Ratones , Ratones Noqueados , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/patología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Factores de Transcripción/metabolismoRESUMEN
Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43def ), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.
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Proteína HMGB1/metabolismo , Osteoclastos/citología , Osteocitos/metabolismo , Osteogénesis/fisiología , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Apoptosis/genética , Células de la Médula Ósea/metabolismo , Línea Celular , Conexina 43/genética , Femenino , Proteína HMGB1/antagonistas & inhibidores , Macrófagos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteocitos/citología , Osteogénesis/genética , Ligando RANK/metabolismo , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Receptor Toll-Like 4/antagonistas & inhibidoresRESUMEN
A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor nuclear matrix protein 4 (Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared with wild-type (WT) animals. Nmp4-/- mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyperanabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4-/- MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion: a key step in bone anabolism. Nmp4-/- cells showed elevated collagen translation and secretion. The expression of matrix genes that contribute to bone material-level mechanical properties was elevated in Nmp4-/- cells, an observation that was supported by biomechanical testing of bone samples from Nmp4-/- and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality.
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Matriz Ósea/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Osteoblastos/metabolismo , Osteogénesis/genética , Factores de Transcripción/genética , Animales , Calcificación Fisiológica/genética , Colágeno/genética , Colágeno/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glucólisis/genética , Ratones , Ratones Noqueados , Osteoblastos/citología , Osteoporosis/metabolismo , ARN Mensajero/metabolismoRESUMEN
The response of aquatic species to contaminants is often context dependent as illustrated by the influence that predation cues can have on the toxicity of some chemicals. We sought to gain additional insight into this interaction by examining how predation cues (alarm cue and fish kairomone) influence metabolic rate and the acute toxicity of sodium chloride and cadmium to fathead minnow larvae (Pimephales promelas) and sodium chloride to Daphnia pulex neonates. Consistent with a "flight or fight" response, the metabolic rate of fish larvae was elevated in the presence of alarm cue and growth of the minnows was also significantly reduced when exposed to alarm cue. The average 48-h LC50 for fathead minnows exposed to sodium chloride was significantly lower in the presence of alarm cue and kairomone combined as compared to tests with the salt alone. Analysis of the dose and survival response indicated alarm cue increased sensitivity of the fish to mid-range salt concentrations in particular. These results suggest an energetic cost of exposure to predation cues that resulted in enhanced toxicity of NaCl. Exposure to kairomone alone had no significant effect on salt toxicity to the minnows, which could be related to a lack of previous exposure to that cue. The acute toxicity of cadmium to the fish larvae was also not affected by the presence of predation cues which could be due to a metal-induced sensory system dysfunction or reduced bioavailability of the metal due to organic exudates from the predation cues. In contrast to the fathead minnow results, the metabolic rate of D. pulex and toxicity of NaCl to the daphnids were reduced in the presence of certain predator kairomones. This suggests an anti-predator response that enhanced tolerance to the salt. This study illustrates that the effect of predation cues on toxicity of aquatic contaminants can vary significantly based on the prey species, type of cue, and chemical stressor.
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Señales (Psicología) , Cyprinidae/fisiología , Conducta Predatoria/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Daphnia , Larva/efectos de los fármacos , FeromonasRESUMEN
Megakaryocytes (MKs) can induce proliferation of calvarial osteoblasts [Ciovacco et al., 2009], but this same phenomenon has not been reported for bone marrow stromal populations from long bones. Bone marrow contains several types of progenitor cells which can be induced to differentiate into multiple cell types. Herein, we examined mesenchymal stromal cell proliferation and osteoblastic differentiation when rabbit or mouse MK were cultured with i) rabbit bone marrow stromal cells, ii) rabbit dental pulp stromal cells, or iii) mouse bone marrow stromal cells. Our results demonstrated that rabbit and mouse stromal cells co-cultured with rabbit MK or mouse MK, have significant increases in proliferation on day 7 by 52%, 46%, and 24%, respectively, compared to cultures without MK. Conversely, alkaline phosphatase (ALP) activity was lower at various time points in these cells when cultures contain MK. Similarly, calcium deposition observed at day 14 rabbit bone marrow and dental pulp stromal cells and day 21 mouse bone marrow stromal cells was 63%, 69%, and 30% lower respectively, when co-cultured with MK. Gene expression studies reveal transcriptional changes broadly consistent with increased proliferation and decreased differentiation. Transcript levels of c-fos (associated with cell proliferation) trended higher after 3, 7, and 14 days in culture. Also, expression of alkaline phosphatase, osteonectin, osterix, and osteopontin, which are markers for osteoblast differentiation, showed MK-induced decreases in a cell type and time dependent manner. Taken together, these data suggest that MK play a role in stromal cell proliferation and differentiation, from multiple sites/locations in multiple species. This article is protected by copyright. All rights reserved.
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Combining anticatabolic agents with parathyroid hormone (PTH) to enhance bone mass has yielded mixed results in osteoporosis patients. Toward the goal of enhancing the efficacy of these regimens, we tested their utility in combination with loss of the transcription factor Nmp4 because disabling this gene amplifies PTH-induced increases in trabecular bone in mice by boosting osteoblast secretory activity. We addressed whether combining a sustained anabolic response with an anticatabolic results in superior bone acquisition compared with PTH monotherapy. Additionally, we inquired whether Nmp4 interferes with anticatabolic efficacy. Wild-type and Nmp4-/- mice were ovariectomized at 12 weeks of age, followed by therapy regimens, administered from 16 to 24 weeks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Anabolic therapeutic efficacy generally corresponded with PTH + RAL = PTH + ZOL > PTH + ALN = PTH > vehicle control. Loss of Nmp4 enhanced femoral trabecular bone increases under PTH + RAL and PTH + ZOL. RAL and ZOL promoted bone restoration, but unexpectedly, loss of Nmp4 boosted RAL-induced increases in femoral trabecular bone. The combination of PTH, RAL, and loss of Nmp4 significantly increased bone marrow osteoprogenitor number, but did not affect adipogenesis or osteoclastogenesis. RAL, but not ZOL, increased osteoprogenitors in both genotypes. Nmp4 status did not influence bone serum marker responses to treatments, but Nmp4-/- mice as a group showed elevated levels of the bone formation marker osteocalcin. We conclude that the heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, in part by increasing hyperanabolic osteoprogenitors. Nmp4 provides a promising target pathway for identifying barriers to pharmacologically induced bone formation.
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Huesos/efectos de los fármacos , Huesos/metabolismo , Difosfonatos/administración & dosificación , Imidazoles/administración & dosificación , Osteoporosis/tratamiento farmacológico , Hormona Paratiroidea/administración & dosificación , Clorhidrato de Raloxifeno/administración & dosificación , Animales , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/genética , Resorción Ósea/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Femenino , Ratones , Ratones Noqueados , Proteínas Asociadas a Matriz Nuclear/genética , Osteoporosis/genética , Osteoporosis/patología , Factores de Transcripción/genética , Ácido ZoledrónicoRESUMEN
The unfolded protein response (UPR) maintains protein homeostasis by governing the processing capacity of the endoplasmic reticulum (ER) to manage ER client loads; however, key regulators within the UPR remain to be identified. Activation of the UPR sensor PERK (EIFAK3/PEK) results in the phosphorylation of the α subunit of eIF2 (eIF2α-P), which represses translation initiation and reduces influx of newly synthesized proteins into the overloaded ER. As part of this adaptive response, eIF2α-P also induces a feedback mechanism through enhanced transcriptional and translational expression of Gadd34 (Ppp1r15A),which targets type 1 protein phosphatase for dephosphorylation of eIF2α-P to restore protein synthesis. Here we describe a novel mechanism by which Gadd34 expression is regulated through the activity of the zinc finger transcription factor NMP4 (ZNF384, CIZ). NMP4 functions to suppress bone anabolism, and we suggest that this occurs due to decreased protein synthesis of factors involved in bone formation through NMP4-mediated dampening of Gadd34 and c-Myc expression. Loss of Nmp4 resulted in an increase in c-Myc and Gadd34 expression that facilitated enhanced ribosome biogenesis and global protein synthesis. Importantly, protein synthesis was sustained during pharmacological induction of the UPR through a mechanism suggested to involve GADD34-mediated dephosphorylation of eIF2α-P. Sustained protein synthesis sensitized cells to pharmacological induction of the UPR, and the observed decrease in cell viability was restored upon inhibition of GADD34 activity. We conclude that NMP4 is a key regulator of ribosome biogenesis and the UPR, which together play a central role in determining cell viability during endoplasmic reticulum stress.
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Regulación de la Expresión Génica/fisiología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteína Fosfatasa 1/biosíntesis , Ribosomas/metabolismo , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Factor 1 Eucariótico de Iniciación/genética , Factor 1 Eucariótico de Iniciación/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Matriz Nuclear/genética , Fosforilación/fisiología , Proteína Fosfatasa 1/genética , Ribosomas/genética , Factores de Transcripción/genéticaRESUMEN
PTH is an osteoanabolic for treating osteoporosis but its potency wanes. Disabling the transcription factor nuclear matrix protein 4 (Nmp4) in healthy, ovary-intact mice enhances bone response to PTH and bone morphogenetic protein 2 and protects from unloading-induced osteopenia. These Nmp4(-/-) mice exhibit expanded bone marrow populations of osteoprogenitors and supporting CD8(+) T cells. To determine whether the Nmp4(-/-) phenotype persists in an osteoporosis model we compared PTH response in ovariectomized (ovx) wild-type (WT) and Nmp4(-/-) mice. To identify potential Nmp4 target genes, we performed bioinformatic/pathway profiling on Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 w) were ovx or sham operated 4 weeks before the initiation of PTH therapy. Skeletal phenotype analysis included microcomputed tomography, histomorphometry, serum profiles, fluorescence-activated cell sorting and the growth/mineralization of cultured WT and Nmp4(-/-) bone marrow mesenchymal stem progenitor cells (MSPCs). ChIP-seq data were derived using MC3T3-E1 preosteoblasts, murine embryonic stem cells, and 2 blood cell lines. Ovx Nmp4(-/-) mice exhibited an improved response to PTH coupled with elevated numbers of osteoprogenitors and CD8(+) T cells, but were not protected from ovx-induced bone loss. Cultured Nmp4(-/-) MSPCs displayed enhanced proliferation and accelerated mineralization. ChIP-seq/gene ontology analyses identified target genes likely under Nmp4 control as enriched for negative regulators of biosynthetic processes. Interrogation of mRNA transcripts in nondifferentiating and osteogenic differentiating WT and Nmp4(-/-) MSPCs was performed on 90 Nmp4 target genes and differentiation markers. These data suggest that Nmp4 suppresses bone anabolism, in part, by regulating IGF-binding protein expression. Changes in Nmp4 status may lead to improvements in osteoprogenitor response to therapeutic cues.
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Resorción Ósea/tratamiento farmacológico , Linfocitos T CD8-positivos/citología , Proteínas Asociadas a Matriz Nuclear/genética , Osteoporosis/tratamiento farmacológico , Hormona Paratiroidea/uso terapéutico , Factores de Transcripción/genética , Animales , Densidad Ósea/efectos de los fármacos , Enfermedades Óseas Metabólicas/prevención & control , Proteína Morfogenética Ósea 2/metabolismo , Resorción Ósea/genética , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Mapeo Cromosómico , Células Madre Embrionarias/citología , Femenino , Terapia Genética , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteogénesis/efectos de los fármacos , Osteoporosis/genética , Ovariectomía , Ovario/cirugíaRESUMEN
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.
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Diferenciación Celular/genética , Megacariocitos/citología , Osteoblastos/citología , Transducción de Señal/genética , Ciclo Celular/genética , Linaje de la Célula , Proliferación Celular/genética , Células Cultivadas , Humanos , Sistema de Señalización de MAP Quinasas/genética , Megacariocitos/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismoRESUMEN
Mechanical stimulation of the skeleton promotes bone gain and suppresses bone loss, ultimately resulting in improved bone strength and fracture resistance. The molecular mechanisms directing anabolic and/or anti-catabolic actions on the skeleton during loading are not fully understood. Identifying molecular mechanisms of mechanotransduction (MTD) signaling cascades could identify new therapeutic targets. Most research into MTD mechanisms is typically focused on understanding the signaling pathways that stimulate new bone formation in response to load. However, we investigated the structural, signaling and transcriptional molecules that suppress the stimulatory effects of loading. The high bone mass phenotype of mice with global deletion of either Pyk2 or Src suggests a role for these tyrosine kinases in repression of bone formation. We used fluid shear stress as a MTD stimulus to identify a novel Pyk2/Src-mediated MTD pathway that represses mechanically-induced bone formation. Our results suggest Pyk2 and Src function as molecular switches that inhibit MTD in our mechanically stimulated osteocyte culture experiments. Once activated by oscillatory fluid shear stress (OFSS), Pyk2 and Src translocate to and accumulate in the nucleus, where they associate with a protein involved in DNA methylation and the interpretation of DNA methylation patterns -methyl-CpG-binding domain protein 2 (MBD2). OFSS-induced Cox-2 and osteopontin expression was enhanced in Pyk2 KO osteoblasts, while inhibition of Src enhanced osteocalcin expression in response to OFSS. We found that Src kinase activity increased in the nucleus of osteocytes in response to OFSS and an interaction activated between Src (Y418) and Pyk2 (Y402) increased in response to OFSS. Thus, as a mechanism to prevent an over-reaction to physical stimulation, mechanical loading may induce the formation of a Src/Pyk2/MBD2 complex in the nucleus that functions to suppress anabolic gene expression.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Mecanotransducción Celular/fisiología , Complejos Multiproteicos/metabolismo , Osteocitos/fisiología , Estrés Mecánico , Animales , Antracenos , Western Blotting , Metilación de ADN/genética , Cartilla de ADN/genética , Proteínas de Unión al ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Técnica del Anticuerpo Fluorescente , Quinasa 2 de Adhesión Focal/metabolismo , Ratones , Complejos Multiproteicos/biosíntesis , Osteocitos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Resistencia al Corte , Familia-src Quinasas/metabolismoRESUMEN
The hydrogeomorphic approach (HGM) to wetland classification and functional assessment has been applied regionally throughout the United States, but the ability of HGM functional assessment models to reflect wetland condition has limited verification. Our objective was to determine how variability derived from anthropogenic effects and natural variability impacted site assessment variables within regional wetland subclasses in central Oklahoma. We collected data for nine potential assessment variables including vegetation physiognomy (e.g., tree basal area, herbaceous cover, canopy cover, etc.) and soil organic matter at wetlands of two HGM riverine subclasses (oxbow and riparian) in May and June, 2010. Using Akaike Information Criteria, we identified limited relationships between landscape disturbance metrics and assessment variables within subclasses. The high degree of natural variability from climatic and hydrologic factors within both subclasses may be masking the impact of landscape disturbance on the other measured assessment variables. Precipitation had significant effects on assessment variables within each of the subclasses. To reduce natural climatic variability, the reference domain may need to be further subdivided. The approach used in this study provides fairly rapid and quantitative methods for evaluating the effectiveness of using HGM assessment variables in assessing wetland condition regionally.
Asunto(s)
Modelos Teóricos , Humedales , Oklahoma , LluviaRESUMEN
The skeleton is a high-renewal organ that undergoes ongoing cycles of remodeling. The regenerative bone formation arm ultimately declines in the aging, postmenopausal skeleton, but current therapies do not adequately address this deficit. Bone marrow is the primary source of the skeletal anabolic response and the mesenchymal stem cells (MSCs), which give rise to bone matrix-producing osteoblasts. The identity of these stem cells is emerging, but it now appears that the term 'MSC' has often been misapplied to the bone marrow stromal cell (BMSC), a progeny of the MSC. Nevertheless, the changes in BMSC phenotype associated with age and estrogen depletion likely contribute to the attenuated regenerative competence of the marrow and may reflect alterations in MSC phenotype. Here we summarize current concepts in bone marrow MSC identity, and within this context, review recent observations on changes in bone marrow population dynamics associated with aging and menopause.
Asunto(s)
Envejecimiento/patología , Células de la Médula Ósea/patología , Regeneración Ósea/fisiología , Osteoblastos/patología , Osteogénesis , Osteoporosis/patología , Diferenciación Celular , Proliferación Celular , HumanosRESUMEN
Parathyroid hormone (PTH) anabolic osteoporosis therapy is intrinsically limited by unknown mechanisms. We previously showed that disabling the transcription factor Nmp4/CIZ in mice expanded this anabolic window while modestly elevating bone resorption. This enhanced bone formation requires a lag period to materialize. Wild-type (WT) and Nmp4-knockout (KO) mice exhibited equivalent PTH-induced increases in bone at 2 weeks of treatment, but by 7 weeks, the null mice showed more new bone. At 3-week treatment, serum osteocalcin, a bone formation marker, peaked in WT mice, but continued to increase in null mice. To determine if 3 weeks is the time when the addition of new bone diverges and to investigate its cellular basis, we treated 10-week-old null and WT animals with human PTH (1-34) (30 µg/kg/day) or vehicle before analyzing femoral trabecular architecture and bone marrow (BM) and peripheral blood phenotypic cell profiles. PTH-treated Nmp4-KO mice gained over 2-fold more femoral trabecular bone than WT by 3 weeks. There was no difference between genotypes in BM cellularity or profiles of several blood elements. However, the KO mice exhibited a significant elevation in CFU-F cells, CFU-F(Alk)(Phos+) cells (osteoprogenitors), and a higher percentage of CFU-F(Alk)(Phos+) cells/CFU-F cells consistent with an increase in CD45-/CD146+/CD105+/nestin+ mesenchymal stem cell frequency. Null BM exhibited a 2-fold enhancement in CD8+ T cells known to support osteoprogenitor differentiation and a 1.6-fold increase in CFU-GM colonies (osteoclast progenitors). We propose that Nmp4/CIZ limits the PTH anabolic window by restricting the number of BM stem, progenitor, and blood cells that support anabolic bone remodeling.