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1.
J Bone Miner Metab ; 40(6): 900-913, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-35947191

RESUMEN

INTRODUCTION: This study was undertaken to gain mechanistic information about bone repair using the bone repletion model in aged Balb/cBy mice. MATERIALS AND METHODS: one month-old (young) mice were fed a calcium-deficient diet for 2 weeks and 8 month-old (adult) and 21-25 month-old (aged) female mice for 4 weeks during depletion, which was followed by feeding a calcium-sufficient diet for 16 days during repletion. To determine if prolonged repletion would improve bone repair, an additional group of aged mice were repleted for 4 additional weeks. Control mice were fed calcium-sufficient diet throughout. In vivo bone repletion response was assessed by bone mineral density gain and histomorphometry. In vitro response was monitored by osteoblastic proliferation, differentiation, and senescence. RESULTS:  There was no significant bone repletion in aged mice even with an extended repletion period, indicating an impaired bone repletion. This was not due to an increase in bone cell senescence or reduction in osteoblast proliferation, but to dysfunctional osteoblastic differentiation in aged bone cells. Osteoblasts of aged mice had elevated levels of cytosolic and ER calcium, which were associated with increased Cav1.2 and CaSR (extracellular calcium channels) expression but reduced expression of Orai1 and Stim1, key components of Stored Operated Ca2+ Entry (SOCE). Activation of Cav1.2 and CaSR leads to increased osteoblastic proliferation, but activation of SOCE is associated with osteoblastic differentiation. CONCLUSION: The bone repletion mechanism in aged Balb/cBy mice is defective that is caused by an impaired osteoblast differentiation through reducedactivation of SOCE.


Asunto(s)
Regeneración Ósea , Osteoblastos , Animales , Femenino , Ratones , Huesos/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Calcio de la Dieta/metabolismo , Osteoblastos/citología , Diferenciación Celular
2.
Am J Physiol Cell Physiol ; 322(5): C977-C990, 2022 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-35385325

RESUMEN

A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, store-operated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3-E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-κΒ ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.


Asunto(s)
Resorción Ósea , Osteogénesis , Animales , Resorción Ósea/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Diferenciación Celular , Ratones , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
3.
J Bone Miner Res ; 37(4): 660-674, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-34989027

RESUMEN

This study took advantage of the recent discovery that the EphA4 signaling has anti-catabolic effects on osteoclasts/macrophages/synoviocytes but pro-anabolic effects on articular chondrocytes and sought to develop an EphA4 signaling-based therapeutic strategy for osteoarthritis (OA) using a mouse model of OA/posttraumatic OA (PTOA). The injured joint of C57BL/6J mice received biweekly intraarticular injections of a soluble EphA4-binding ligand (EfnA4-fc) at 1 day after the tibial plateau injury or at 5 weeks post-injury. The animals were euthanized 5 weeks later. The injured right and contralateral uninjured left joints were analyzed for hallmarks of OA by histology. Relative severity was determined by a modified Mankin OA scoring system and serum COMP and CTX-II levels. Tibial plateau injury caused more severe OA in Epha4 null mice than in wild-type (WT) littermates, suggesting a protective role of EphA4 signaling in OA. A prototype strategy of an EphA4 signaling-based strategy involving biweekly injections of EfnA4-fc into injured joints was developed and was shown to be highly effective in preventing OA/PTOA when it was administered at 1 day post-injury and in treating OA/PTOA when it was applied after OA has been established. The efficacy of this prototype was dose- and time-dependent. The effects were not caused by the Fc moiety of EfnA4-fc. Other soluble EfnA ligands of EphA4, ie, EfnA1-fc and EfnA2-fc, were also effective. A prototype of a novel EphA4 signaling-based therapy was developed for OA/PTOA that not only reduces the progressive destruction of articular cartilage but may also promote regeneration of the damaged cartilage. © 2022 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.


Asunto(s)
Cartílago Articular , Osteoartritis , Sinoviocitos , Animales , Cartílago Articular/patología , Condrocitos/patología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Osteoartritis/patología , Sinoviocitos/patología
4.
Calcif Tissue Int ; 107(6): 576-592, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32816052

RESUMEN

The expression and activation of EphA4 in the various cell types in a knee joint was upregulated upon an intraarticular injury. To determine if EphA4 signaling plays a role in osteoarthritis, we determined whether deficient EphA4 expression (in EphA4 knockout mice) or upregulation of the EphA4 signaling (with the EfnA4-fc treatment) would alter cellular functions of synoviocytes and articular chondrocytes. In synoviocytes, deficient EphA4 expression enhanced, whereas activation of the EphA4 signaling reduced, expression and secretion of key inflammatory cytokines and matrix metalloproteases. Conversely, in articular chondrocytes, activation of the EphA4 signaling upregulated, while deficient EphA4 expression reduced, expression levels of chondrogenic genes (e.g., aggrecan, lubricin, type-2 collagen, and Sox9). EfnA4-fc treatment in wildtype, but not EphA4-deficient, articular chondrocytes promoted the formation and activity of acidic proteoglycan-producing colonies. Activation of the EphA4 signaling in articular chondrocytes upregulated Rac1/2 and downregulated RhoA via enhancing Vav1 and reducing Ephexin1 activation, respectively. However, activation of the EphA4 signaling in synoviocytes suppressed the Vav/Rac signaling while upregulated the Ephexin/Rho signaling. In summary, the EphA4 signaling in synoviocytes is largely of anti-catabolic nature through suppression of the expression of inflammatory cytokines and matrix proteases, but in articular chondrocytes the signaling is pro-anabolic in that it promotes the biosynthesis of articular cartilage. The contrasting action of the EphA4 signaling in synoviocytes as opposing to articular chondrocytes may in part be mediated through the opposite differential effects of the EphA4 signaling on the Vav/Rac signaling and Ephexin/Rho signaling in the two skeletal cell types.


Asunto(s)
Cartílago Articular , Condrocitos/metabolismo , Receptor EphA4/metabolismo , Sinoviocitos/metabolismo , Animales , Células Cultivadas , Colágeno Tipo II , Ratones , Ratones Noqueados , Transducción de Señal
5.
Calcif Tissue Int ; 106(2): 158-171, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31559470

RESUMEN

This study sought to develop a noninvasive, reliable, clinically relevant, and easy-to-implement mouse model that can be used for investigation of the pathophysiology of PTOA and for preclinical testing of new therapies of PTOA. Accordingly, we have established a closed intraarticular tibial plateau compression loading-induced injury model of PTOA in C57BL/6J mice. In this model, a single application of a defined loading force was applied with an indenter to the tibial plateau of the right knee to create injuries to the synovium, menisci, ligaments, and articular cartilage. The limiting loading force was set at 55 N with the loading speed of 60 N/s. This loading regimen limits the distance that the indenter would travel into the joint, but still yields substantial compression loading energy to cause significant injuries to the synovium, meniscus, and articular cartilage. The joint injury induced by this loading protocol consistently yielded evidence for key histological hallmarks of PTOA at 5-11 weeks post-injury, including loss of articular cartilage, disorganization of chondrocytes, meniscal hyperplasia and mineralization, osteophyte formation, and degenerative remodeling of subchondral bone. These arthritic changes were highly reproducible and of a progressive nature. Because 50% of patients with meniscal and/or ligament injuries without intraarticular fractures developed PTOA over time, this intraarticular tibial plateau compression loading-induced injury model is clinically relevant. In summary, we have developed a noninvasive intraarticular tibial plateau compression loading-induced injury model in the mouse that can be used to investigate the pathophysiology of PTOA and for preclinical testing for new therapies.


Asunto(s)
Osteoartritis/patología , Estrés Mecánico , Tibia , Fracturas de la Tibia/patología , Animales , Cartílago Articular/patología , Cartílago Articular/fisiología , Fuerza Compresiva/fisiología , Modelos Animales de Enfermedad , Femenino , Traumatismos de la Rodilla/complicaciones , Traumatismos de la Rodilla/patología , Articulación de la Rodilla/patología , Articulación de la Rodilla/fisiología , Traumatismos de la Pierna/complicaciones , Traumatismos de la Pierna/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoartritis/etiología , Tibia/patología , Tibia/fisiología , Fracturas de la Tibia/complicaciones , Soporte de Peso/fisiología
6.
Nitric Oxide ; 79: 57-67, 2018 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-30059767

RESUMEN

Dinitrosyl iron complexes (DNICs) are important intermediates in the metabolism of nitric oxide (NO). They have been considered to be NO storage adducts able to release NO, scavengers of excess NO during inflammatory hypotensive shock, and mediators of apoptosis in cancer cells, among many other functions. Currently, all studies of DNICs in biological matrices use electron paramagnetic resonance (EPR) for both detection and quantification. EPR is limited, however, by its ability to detect only paramagnetic mononuclear DNICs even though EPR-silent binuclear are likely to be prevalent. Furthermore, physiological concentrations of mononuclear DNICs are usually lower than the EPR detection limit (1 µM). We have thus developed a chemiluminescence-based method for the selective detection of both DNIC forms at physiological, pathophysiological, and pharmacologic conditions. We have also demonstrated the use of the new method in detecting DNIC formation in the presence of nitrite and nitrosothiols within biological fluids and tissue. This new method, which can be used alone or in tandem with EPR, has the potential to offer insight about the involvement of DNICs in many NO-dependent pathways.


Asunto(s)
Hierro/análisis , Luminiscencia , Óxidos de Nitrógeno/análisis , Ozono/química , Animales , Ovinos
7.
Arch Biochem Biophys ; 650: 30-38, 2018 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-29763590

RESUMEN

Information about the molecular mechanisms leading to the activation of the osteoclast is relatively limited. While there is compelling evidence that the signaling mechanisms of Src and integrin ß3 are essential for osteoclast activation, the regulation of these two signaling mechanisms is not fully understood. In this review, evidence supporting a novel regulatory axis of osteoclast activation that plays an upstream regulatory role in both the Src and integrin ß3 signaling during osteoclast activation is discussed. This regulatory axis contains three unique components: a structurally unique transmembrane protein-tyrosine phosphatase, PTP-oc, EphA4, and miR17. In the first component, PTP-oc activates the Src signaling through dephosphorylation of the inhibitory tyr-527 of Src. This in turn activates the integrin ß3 signaling, enhances the JNK2/NFκB signaling, promotes the ITAM/Syk signaling, and suppresses the ITIM/Shp1 signaling; the consequence of which is activation of the osteoclast. In the second component, EphA4 inhibits osteoclast activity by suppressing the integrin ß3 signaling. PTP-oc relieves the suppressive actions of EphA4 by directly dephosphorylating EphA4. In the third component, PTP-oc expression is negatively regulated by miR17. Accordingly, suppression of miR17 during osteoclast activation upregulates the PTP-oc signaling and suppresses the EphA4 signaling, resulting in the activation of the osteoclast. This regulatory axis is unique, in that each of the three components acts to exert suppressive action on their respective immediate downstream inhibitory step. Because the final downstream event is the EphA4-mediated inhibition of osteoclast activation, the overall effect of this mechanism is the stimulation of osteoclast activity.


Asunto(s)
Resorción Ósea/metabolismo , MicroARNs/metabolismo , Osteoclastos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptor EphA4/metabolismo , Transducción de Señal , Animales , Resorción Ósea/genética , Resorción Ósea/patología , Regulación de la Expresión Génica , Humanos , MicroARNs/genética , Osteoclastos/citología , Osteoclastos/patología , Proteínas Tirosina Fosfatasas/genética , Receptor EphA4/genética
8.
JBMR Plus ; 1(2): 73-85, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-29082358

RESUMEN

This study sought to understand the regulation of an osteoclastic protein-tyrosine phosphatase (PTP-oc), a positive regulator of osteoclast activaty. Our past studies suggested that PTP-oc is regulated post-transcriptionally. The 3'-UTR of PTP-oc mRNA contains a target site for miR17. During osteoclastic differentiation, there was an inverse relationship between the cellular levels of miR17 (expressed as one of the six cluster genes of miR17~92) and PTP-oc mRNA. Overexpression of pre-miR17~92 in mouse osteoclast precursors reduced PTP-oc mRNA level and the size of the derived osteoclasts; whereas deletion of miR17~92 or inhibition of miR17 resulted in the formation of larger osteoclasts containing more nuclei that expressed higher PTP-oc mRNA levels and created larger resorption pits. Thus, PTP-oc-mediated osteoclast activation is modulated in part by miR17~92, particularly miR17. The miR17~92 osteoclast conditional knockout (cKO) mutants, generated by breeding miR17~92loxp/loxp mice with Ctsk-Cre mice, had lower Tb.BV/TV, Tb.BMD, Tb.Conn-Dens, Tb.N, and Tb.Th, but larger Tb.Sp, and greater bone resorption without a change in bone formation compared to littermate controls. The cKO marrow-derived osteoclasts were twice as large, contained twice as many nuclei, and produced twice as large resorption pits as osteoclasts of littermate controls. The expression of genes associated with osteoclast activation was increased in cKO osteoclasts, suggesting that deletion of miR17~92 in osteoclasts promotes osteoclast activation. The cKO osteoblasts did not show differences in cellular miR17 level, alkaline phosphatase activity, and bone nodule formation ability. In conclusion, miR17-92 negatively regulates the osteoclast activity, in part via the miR17-mediated suppression of PTP-oc in osteoclasts.

9.
Endocrinology ; 158(4): 714-729, 2017 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-28324039

RESUMEN

The present study was undertaken to determine the mechanism whereby calcitropic hormones and mesenchymal stem cell progeny changes are involved in bone repletion, a regenerative bone process that restores the bone lost to calcium deficiency. To initiate depletion, weanling mice with a mixed C57BL/6 (75%) and CD1 (25%) genetic background were fed a calcium-deficient diet (0.01%) for 14 days. For repletion, the mice were fed a control diet containing 1.2% calcium for 14 days. Depletion decreased plasma calcium and increased plasma parathyroid hormone, 1,25(OH)2D (calcitriol), and C-terminal telopeptide of type I collagen. These plasma parameters quickly returned toward normal on repletion. The trabecular bone volume and connectivity decreased drastically during depletion but were completely restored by the end of repletion. This bone repletion process largely resulted from the development of new bone formation. When bromodeoxyuridine (BrdU) was administered in the middle of depletion for 3 days and examined by fluorescence-activated cell sorting at 7 days into repletion, substantial increases in BrdU incorporation were seen in several CD105 subsets of cells of osteoblastic lineage. When BrdU was administered on days 1 to 3 of repletion and examined 11 days later, no increases in BrdU were seen in these subsets. Additionally, osteocytes that stained positively for BrdU were increased during depletion. In conclusion, the results of the present study have established a unique regenerative mechanism to initiate bone repair during the bone insult. Calcium homeostatic mechanisms and the bone repletion mechanism are opposing functions but are simultaneously orchestrated such that both endpoints are optimized. These results have potential clinical relevance for disease entities such as type 2 osteoporosis.


Asunto(s)
Huesos/fisiología , Calcio de la Dieta , Calcio/deficiencia , Osteogénesis/fisiología , Hormona Paratiroidea/sangre , Regeneración/fisiología , Animales , Calcitriol/sangre , Calcio/sangre , Colágeno Tipo I/sangre , Citometría de Flujo , Masculino , Ratones , Péptidos/sangre
10.
Bone ; 92: 18-28, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27519969

RESUMEN

This study evaluated the effects of deficient IGF-I expression in osteocytes on fracture healing. Transgenic mice with conditional knockout (cKO) of Igf1 in osteocytes were generated by crossing Dmp1-Cre mice with Igf1 flox mice. Fractures were created on the mid-shaft of tibia of 12-week-old male cKO mice and wild-type (WT) littermates by three-point bending. At 21 and 28days post-fracture healing, the increases in cortical bone mineral density, mineral content, bone area, and thickness, as well as sub-cortical bone mineral content at the fracture site were each greater in cKO calluses than in WT calluses. There were 85% decrease in the cartilage area and >2-fold increase in the number of osteoclasts in cKO calluses at 14days post-fracture, suggesting a more rapid remodeling of endochondral bone. The upregulation of mRNA levels of osteoblast marker genes (cbfa1, alp, Opn, and Ocn) was greater in cKO calluses than in WT calluses. µ-CT analysis suggested an accelerated bony union of the fracture gap in cKO mice. The Sost mRNA level was reduced by 50% and the Bmp2 mRNA level was increased 3-fold in cKO fractures at 14days post-fracture, but the levels of these two mRNAs in WT fractures were unchanged, suggesting that the accelerated fracture repair may in part act through the Wnt and/or BMP signaling. In conclusion, conditional deletion of Igf1 in osteocytes not only did not impair, but unexpectedly enhanced, bony union of the fracture gap. The accelerated bony union was due in part to upregulation of the Wnt and BMP2 signaling in response to deficient osteocyte-derived IGF-I expression, which in turn favors intramembranous over endochondral bone repair.


Asunto(s)
Curación de Fractura/fisiología , Eliminación de Gen , Factor I del Crecimiento Similar a la Insulina/deficiencia , Osteocitos/fisiología , Fracturas de la Tibia/diagnóstico por imagen , Fracturas de la Tibia/metabolismo , Animales , Factor I del Crecimiento Similar a la Insulina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fracturas de la Tibia/genética
11.
J Cell Biochem ; 116(8): 1785-96, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25676701

RESUMEN

We have previously shown that PTP-oc is an enhancer of the functional activity of osteoclasts and that EphA4 is a suppressor. Here, we provide evidence that PTP-oc enhances osteoclast activity in part through inactivation of EphA4 by dephosphorylating key phosphotyrosine (pY) residues of EphA4. We show that EphA4 was pulled down by the PTP-oc trapping mutant but not by the wild-type (WT) PTP-oc and that transgenic overexpression of PTP-oc in osteoclasts drastically decreased pY602 and pY779 residues of EphA4. Consistent with the previous findings that EphA4 deficiency increased pY173-Vav3 level (Rac-GTP exchange factor [GEF]) and enhanced bone resorption activity of osteoclasts, reintroduction of WT-Epha4 in Epha4 null osteoclasts led to ∼50% reduction in the pY173-Vav3 level and ∼2-fold increase in bone resorption activity. Overexpression of Y779F-Epha4 mutant in WT osteoclasts markedly increased in pY173-Vav3 and reduced bone resorption activity, but overexpression of Y602F-Epha4 mutant had no effect, suggesting that pY779 residue plays an important role in the EphA4-mediated suppression of osteoclast activity. Deficient EphA4 in osteoclasts has been shown to up-regulate Rac-GTPase and down-regulate Rho-GTPase. PTP-oc overexpression in osteoclasts also increased the GTP-Rac level to 300% of controls, but decreased the GTP-Rho level to ∼50% of controls. PTP-oc overexpression or deficient Epha4 each also reduced pY87-Ephexin level, which is a Rho GEF. Thus, PTP-oc may differentially regulate Rac signaling versus Rho signaling through dephosphorylation of EphA4, which has shown to have opposing effects on Rac-GTPase versus Rho-GTPase through differential regulation of Vav3 versus Ephexin.


Asunto(s)
Resorción Ósea/metabolismo , Osteoclastos/fisiología , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Receptor EphA4/metabolismo , Tirosina/metabolismo , Animales , Resorción Ósea/genética , Línea Celular , Masculino , Ratones , Ratones Transgénicos , Mutación , Fosforilación , Proteínas Tirosina Fosfatasas no Receptoras/química , Receptor EphA4/genética , Transducción de Señal
12.
PLoS One ; 10(1): e0115897, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25635763

RESUMEN

The present study sought to evaluate the functional role of osteocyte-derived IGF-I in the bone repletion process by determining whether deficient expression of Igf1 in osteocytes would impair the bone repletion response to one week of dietary calcium repletion after two weeks of dietary calcium deprivation. As expected, the two-week dietary calcium depletion led to hypocalcemia, secondary hyperparathyroidism, and increases in bone resorption and bone loss in both Igf1 osteocyte conditional knockout (cKO) mutants and WT control mice. Thus, conditional disruption of Igf1 in osteocytes did not impair the calcium depletion-induced bone resorption. After one week of calcium repletion, both cKO mutants and WT littermates showed an increase in endosteal bone formation attended by the reduction in osteoclast number, indicating that deficient Igf1 expression in osteocytes also did not have deleterious effects on the bone repletion response. The lack of an effect of deficient osteocyte-derived IGF-I expression on bone repletion is unexpected since previous studies show that these Igf1 osteocyte cKO mice exhibited impaired developmental growth and displayed complete resistance to bone anabolic effects of loading. These studies suggest that there is a dichotomy between the mechanisms necessary for anabolic responses to mechanical loading and the regulatory hormonal and anabolic skeletal repletion following low dietary calcium challenge. In conclusion, to our knowledge this study has demonstrated for the first time that osteocyte-derived IGF-I, which is essential for anabolic bone response to mechanical loading, is not a key regulatory factor for bone repletion after a low calcium challenge.


Asunto(s)
Desarrollo Óseo/genética , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Osteogénesis , Animales , Resorción Ósea/dietoterapia , Resorción Ósea/genética , Resorción Ósea/fisiopatología , Calcio de la Dieta/administración & dosificación , Hipocalcemia/metabolismo , Hipocalcemia/patología , Factor I del Crecimiento Similar a la Insulina/genética , Ratones , Osteoclastos/metabolismo , Osteocitos/metabolismo
13.
J Bone Metab ; 21(1): 41-54, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24707466

RESUMEN

The osteocyte has long been considered to be the primary mechanosensory cell in the bone. Recent evidence has emerged that the osteocyte is also a key regulator of various bone and mineral metabolism and that its regulatory effects are in part mediated through locally produced osteocyte-derived factors, such as sclerostin, receptor activator of nuclear factor-kappa B ligand (RANKL), and fibroblast growth factor (FGF)-23. Osteocytes secrete large amounts of insulin-like growth factor (IGF)-I in bone. Although IGF-I produced locally by other bone cells, such as osteoblasts and chondrocytes, has been shown to play important regulatory roles in bone turnover and developmental bone growth, the functional role of osteocyte-derived IGF-I in the bone and mineral metabolism has not been investigated and remains unclear. However, results of recent studies in osteocyte Igf1 conditional knockout transgenic mice have suggested potential regulatory roles of osteocyte-derived IGF-I in various aspects of bone and mineral metabolism. In this review, evidence supporting a regulatory role for osteocyte-derived IGF-I in the osteogenic response to mechanical loading, the developmental bone growth, the bone response to dietary calcium depletion and repletion, and in fracture repair is discussed. A potential coordinated regulatory relationship between the effect of osteocyte-derived IGF-I on bone size and the internal organ size is also proposed.

14.
J Bone Miner Res ; 29(4): 804-19, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23983218

RESUMEN

Of the ephrin (Eph) receptors, mature osteoclasts express predominantly EphA4. This study sought to determine if EphA4 has a regulatory role in osteoclasts. Treatment of RAW/C4 cells with Epha4 small interfering RNAs (siRNAs) increased average size, Ctsk mRNA expression level, and bone resorption activity of the derived osteoclast-like cells. Activation of the EphA4 signaling in osteoclast precursors with EfnA4-fc chimeric protein reduced cell size and resorption activity of the derived osteoclasts. Homozygous Epha4 null mice had substantially less trabecular bone in femur and vertebra compared to wild-type controls. The bone loss was due to a decrease in trabecular number and an increase in trabecular spacing, but not to an increase in osteoclast-lined bone surface or an increase in the number of osteoclasts on bone surface. Dynamic histomorphometry and serum biomarker analyses indicate that bone formation in Epha4 null mice was reduced slightly but not significantly. Osteoclasts of Epha4 null mice were also larger, expressed higher levels of Mmp3 and Mmp9 mRNAs, and exhibited greater bone resorption activity than wild-type osteoclasts in vitro. Deficient Epha4 expression had no effects on the total number of osteoclast formed in response to receptor activator of NF-κB ligand nor on apoptosis of osteoclasts in vitro. It also did not affect the protein-tyrosine phosphorylation status of its ligands, EfnB2, EfnA2, and EfnA4, in osteoclasts. Deficient Epha4 expression in Epha4 null osteoclasts activated the ß3 -integrin signaling through reduced phosphorylation of the tyr-747 residue, which led to increased binding of the stimulatory talin and reduced binding of the inhibitory Dok1 to ß3 -integrin. This in turn activated Vav3 and the bone resorption activity of osteoclasts. In conclusion, we demonstrate for the first time that EphA4 is a potent negative regulator of osteoclastic activity, mediated in part through increased Dok1 binding to ß3 -integrin via an increase in EphA4-dependent tyr-747 phosphorylation.


Asunto(s)
Osteoclastos/citología , Receptor EphA4/fisiología , Animales , Remodelación Ósea , Línea Celular , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Receptor EphA4/metabolismo , Transducción de Señal , Microtomografía por Rayos X
15.
Am J Physiol Endocrinol Metab ; 305(2): E271-81, 2013 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-23715728

RESUMEN

This study sought to determine whether deficient Igf1 expression in osteocytes would affect loading-induced osteogenic response. Tibias of osteocyte Igf1 conditional knockout (KO) mice (generated by cross-breeding Igf1 floxed mice with Dmp1-Cre transgenic mice) and wild-type (WT) littermates were subjected to four-point bending for 2 wk. Microcomputed tomography confirmed that the size of tibias of conditional mutants was smaller. Loading with an equivalent loading strain increased periosteal woven bone and endosteal lamellar bone formation in WT mice but not in conditional KO mice. Consistent with the lack of an osteogenic response, the loading failed to upregulate expression of early mechanoresponsive genes (Igf1, Cox-2, c-fos) or osteogenic genes (Cbfa-1, and osteocalcin) in conditional KO bones. The lack of osteogenic response was not due to reduced osteocyte density or insufficient loading strain. Deficient osteocyte Igf1 expression reduced the loading-induced upregulation of expression of canonical Wnt signaling genes (Wnt10b, Lrp5, Dkk1, sFrp2). The loading also reduced (by 40%) Sost expression in WT mice, but the loading not only did not reduce but upregulated (~1.5-fold) Sost expression in conditional KO mice. Conditional disruption of Igf1 in osteocytes also abolished the loading-induced increase in the bone ß-catenin protein level. These findings suggest an impaired response in the loading-induced upregulation of the Wnt signaling in conditional KO mice. In summary, conditional disruption of Igf1 in osteocytes abolished the loading-induced activation of the Wnt signaling and the corresponding osteogenic response. In conclusion, osteocyte-derived IGF-I plays a key determining role in bone mechanosensitivity.


Asunto(s)
Huesos/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Mecanotransducción Celular/fisiología , Osteocitos/fisiología , Animales , Fenómenos Biomecánicos , Western Blotting , Desarrollo Óseo/genética , Desarrollo Óseo/fisiología , Huesos/anatomía & histología , Huesos/diagnóstico por imagen , ADN/genética , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Genotipo , Factor I del Crecimiento Similar a la Insulina/genética , Mecanotransducción Celular/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosfatidilinositoles/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Tibia/fisiología , Tomografía Computarizada por Rayos X , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo
16.
Bone ; 52(1): 133-44, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23032105

RESUMEN

This study evaluated the role of osteocyte-derived insulin-like growth factor 1 (IGF-1) in developmental bone growth by assessing the bone phenotype of osteocyte Igf1 conditional knockout (KO) mice, generated by crossing the Dmp1-driven Cre-expressing transgenic mice with Igf1 floxed mice containing loxP sites that flank exon 4 of the Igf1 gene. The periosteal diameter of femurs of homozygous conditional KO mutants was 8-12% smaller than wild-type (WT) littermates. The conditional mutants had 14-20%, 10-21%, and 15-31% reduction in total, trabecular, and cortical bone mineral contents, respectively. However, there were no differences in the total, trabecular, or cortical bone mineral densities, or in trabecular bone volume, thickness, number, and separation at secondary spongiosa between the mutants and WT littermates. The conditional KO mutants showed reduction in dynamic bone formation parameters at both periosteal and endosteal surfaces at the mid-diaphysis and in trabecular bone formation rate and resorption parameters at secondary spongiosa. The lower plasma levels of PINP and CTx in conditional KO mice support a regulatory role of osteocyte-derived IGF-1 in the bone turnover. The femur length of conditional KO mutants was 4-7% shorter due to significant reduction in the length of growth plate and hypertropic zone. The effect on periosteal expansion appeared to be bigger than that on longitudinal bone growth. The conditional KO mice had 14% thinner calvaria than WT littermates, suggesting that deficient osteocyte IGF-1 production also impairs developmental growth of intramembraneous bone. Conditional disruption of Igf1 in osteocytes did not alter plasma levels of IGF-1, calcium, or phosphorus. In summary, this study shows for the first time that osteocyte-derived IGF-1 plays an essential role in regulating bone turnover during developmental bone growth.


Asunto(s)
Desarrollo Óseo/genética , Factor I del Crecimiento Similar a la Insulina/genética , Osteocitos/metabolismo , Animales , Secuencia de Bases , Resorción Ósea , Diferenciación Celular , Cartilla de ADN , Placa de Crecimiento/crecimiento & desarrollo , Ratones , Ratones Noqueados , Osteocitos/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Tomografía Computarizada por Rayos X
17.
PLoS One ; 7(4): e35280, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22536365

RESUMEN

This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ∼2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by µ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption.


Asunto(s)
Resorción Ósea/metabolismo , Proteínas del Ojo/biosíntesis , Expresión Génica , Glicoproteínas de Membrana/biosíntesis , Osteoclastos/metabolismo , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Factores de Edad , Animales , Densidad Ósea , Resorción Ósea/sangre , Resorción Ósea/genética , Huesos/diagnóstico por imagen , Huesos/metabolismo , Huesos/patología , Tamaño del Núcleo Celular , Tamaño de la Célula , Proteínas del Ojo/genética , Femenino , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoblastos/metabolismo , Osteocalcina/sangre , Fragmentos de Péptidos/sangre , Fenotipo , Procolágeno/sangre , Regiones Promotoras Genéticas , Fosfatasa Ácida Tartratorresistente , Microtomografía por Rayos X , Familia-src Quinasas/metabolismo
18.
J Biol Chem ; 284(17): 11531-45, 2009 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-19244239

RESUMEN

This study evaluated whether transgenic expression of PTP-oc (osteoclastic transmembrane protein-tyrosine phosphatase) in cells of the osteoclast lineage would affect bone resorption and bone density in young adult mice. Transgenic mice were generated with a transgenic construct using a tartrate-resistant acid phosphatase exon 1C promoter to drive expression of rabbit PTP-oc in osteoclastic cells. pQCT evaluation of femurs of young adult male progeny of three lines showed that transgenic mice had reduced bone volume and area, cortical and trabecular bone mineral content, and density. Histomorphometric analyses at secondary spongiosa of the femur and at metaphysis of the L4 vertebra confirmed that male transgenic mice had decreased trabecular surface, reduced percentage of trabecular area, decreased trabecular number, increased trabecular separation, and increased osteoclast number per bone surface length. Consistent with an increase in bone resorption, the serum C-telopeptide level was 25% higher in transgenic mice than in wild-type littermates. However, the bone phenotype was not readily observed in female young adult transgenic mice. This could in part be due to potential interactions between estrogen and PTP-oc signaling, since the bone loss phenotype was seen in young adult ovariectomized transgenic mice by microcomputed tomography analysis. In vitro, the average pit area per resorption pit created by marrow-derived transgenic osteoclasts was approximately 50% greater than that created by wild-type osteoclasts. Transgenic osteoclasts showed a lower c-Src phosphotyrosine 527 level, greater c-Src kinase activity, and increased tyrosine phosphorylation of paxillin. In summary, this study provides compelling in vivo evidence that PTP-oc is a positive regulator of osteoclasts.


Asunto(s)
Resorción Ósea , Osteoclastos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/fisiología , Animales , Membrana Celular/metabolismo , Colágeno Tipo I/metabolismo , Fémur/metabolismo , Masculino , Ratones , Ratones Transgénicos , Péptidos/metabolismo , Regiones Promotoras Genéticas , Conejos , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Transducción de Señal , Familia-src Quinasas/metabolismo
19.
Bone ; 43(5): 880-8, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18708175

RESUMEN

This study sought to determine the role of the pro-apoptotic gene, Bax, in fracture healing by comparing femoral fracture healing in Bax knockout (KO) and wild-type C57BL/6J (background strain) mice. Bax KO fractures were larger, had more bone mineral content, had approximately 2-fold larger cartilage area per callus area in the first and second weeks of fracture healing, and showed an increased osteoclast surface area in the third and fourth weeks of fracture healing compared to C57BL/6J fractures. The increased cartilage area in the Bax KO fracture callus was due to increases in number of both pre-hypertropic and hypertropic chondrocytes. TUNEL analysis showed no significant differences in the number of either chondrocyte or non-chondrocyte apoptotic cells between Bax KO and C57BL/6J fractures at 7 or 14 days post-fracture, indicating that the increased number of chondrocytes in Bax KO fractures was not due to reduced apoptosis. Analysis of expression of apoptotic genes revealed that although the expression levels of Bcl-2 and Bcl-xL were not different between the Bax KO and C57BL/6J mice at 7 or 14 days post-fracture, the expression of BH3-domain only Bak and "Bik-like" pro-apoptotic gene increased approximately 1.5-fold and approximately 2-fold, respectively, in Bax KO fractures at 7 and 14 days post-fracture, compared to C57BL/6J fractures, suggesting that up-regulation of the Bak and Bik-like pro-apoptotic genes in Bax KO mice might compensate for the lack of Bax functions in the context of apoptosis. Analysis by in vivo incorporation of bromodeoxyuridine into chondrocytes within the fracture tissues indicated a highly significant increase in chondrocyte proliferation in Bax KO fractures compared to C57BL/6J fractures at day 7. The increased expression of collagen 2alpha1 and 9alpha1 gene in Bax KO fractures during early healing was consistent with an increased chondrocyte proliferation. In conclusion, this study demonstrates for the first time that Bax has an important role in the early stage of fracture healing, and that the increased callus size and cartilage area in Bax KO fractures was due to increased chondrocyte proliferation and not to reduced apoptosis or increased chondrocyte hypertrophy. The unexpected effect of Bax deficiency on chondrocyte proliferation implicates a novel regulatory function for Bax on chondrocyte proliferation during fracture repair.


Asunto(s)
Apoptosis/fisiología , Cartílago/fisiología , Condrocitos/fisiología , Curación de Fractura/fisiología , Proteína X Asociada a bcl-2/metabolismo , Animales , Callo Óseo/citología , Callo Óseo/metabolismo , Cartílago/citología , Proliferación Celular , Femenino , Fémur/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína X Asociada a bcl-2/genética
20.
FEBS Lett ; 582(10): 1451-8, 2008 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-18381073

RESUMEN

This study presents gene expression, protein expression, and in situ immunohistochemical evidence that osteoclasts express high levels of osteoactivin (OA), which had previously been reported to be an osteoblast-specific protein in bone. OA expression in osteoclasts was up-regulated upon receptor activator of NFkappaB ligand-induced differentiation. Suppression of functional activity of OA with neutralizing antibody reduced cell size, number of nuclei, fusion, and bone resorption activity of osteoclasts. OA was co-immunoprecipitated with integrin beta3 and beta1, indicating that OA co-localizes with integrin beta3 and/or beta1 in a hetero-polymeric complex in osteoclasts. These findings indicate that OA is a novel osteoclastic protein and plays a role in osteoclast differentiation and/or activity.


Asunto(s)
Diferenciación Celular , Proteínas del Ojo/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoclastos/citología , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Animales , Anticuerpos/farmacología , Diferenciación Celular/genética , Proteínas del Ojo/genética , Expresión Génica , Inmunoprecipitación , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Glicoproteínas de Membrana/genética , Ratones , FN-kappa B/metabolismo , Osteoclastos/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , Fosfatasa Ácida Tartratorresistente
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