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1.
Biophys J ; 118(8): 1820-1829, 2020 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-32191861

RESUMEN

We report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.


Asunto(s)
Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Luz , Microscopía Fluorescente
2.
Phytother Res ; 30(5): 790-6, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26806653

RESUMEN

Hydroxy-safflower yellow A (HSYA) is the major active component of safflower, a traditional Asia herbal medicine well known for its cardiovascular protective activities. The purpose of this study was to investigate the effect of HSYA on TNF-α-induced inflammatory responses in arterial endothelial cells (AECs) and to explore the mechanisms involved. The results showed that HSYA suppressed the up-regulation of ICAM-1 expression in TNF-α-stimulated AECs in a dose-dependent manner. High concentration (120 µM) HSYA significantly inhibited the TNF-α-induced adhesion of RAW264.7 cells to AECs. HSYA blocked the TNFR1-mediated phosphorylation and degradation of IκBα and also prevented the nuclear translocation of NF-κB p65. Moreover, HSYA reduced the cell surface level of TNFR1 and increased the content of sTNFR1 in the culture media. TNF-α processing inhibitor-0 (TAPI-0) prevented the HSYA inhibition of TNFR1-induced IκBα degradation, implying the occurrence of TNFR1 shedding. Furthermore, HSYA induced phosphorylation of TNF-α converting enzyme (TACE) at threonine 735, which is thought to be required for its activation. Conclusively, HSYA suppressed TNF-α-induced inflammatory responses in AECs, at least in part by inhibiting the TNFR1-mediated classical NF-κB pathway. TACE-mediated TNFR1 shedding can be involved in this effect. Our study provides new evidence for the antiinflammatory and anti-atherosclerotic effects of HSYA. Copyright © 2016 John Wiley & Sons, Ltd.


Asunto(s)
Chalcona/análogos & derivados , Medicina de Hierbas/métodos , FN-kappa B/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Chalcona/química , Humanos
3.
J Cell Physiol ; 230(3): 578-86, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25160801

RESUMEN

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.


Asunto(s)
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/metabolismo
4.
Am J Physiol Endocrinol Metab ; 306(8): E937-44, 2014 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-24619882

RESUMEN

Mechanical loading is an important regulator in skeletal growth, maintenance, and aging. Estrogen receptors have a regulatory role in mechanically induced bone adaptation. Estrogen receptor-α (ERα) is known to enhance load-induced bone formation, whereas ERß negatively regulates this process. We hypothesized that ERß regulates mechanical signaling in osteoblasts. We tested this hypothesis by subjecting primary calvarial cells isolated from wild-type and ERß-knockout mice (BERKO) to oscillatory fluid flow in the absence or presence of estradiol (E2). We found that the known responses to fluid shear stress, i.e., phosphorylation of the mitogen-activated protein kinase ERK and upregulation of COX-2 expression, were inhibited in BERKO cells in the absence of E2. Flow-induced increase in prostaglandin E2 (PGE2) release was not altered in BERKO cells in the absence of E2, but was increased when E2 was present. Additionally, immunofluorescence analysis and estrogen response element luciferase assays revealed increased ERα expression and flow- and ligand-induced nuclear translocation as well as transcriptional activity in BERKO cells in both the presence and absence of E2. Taken together, these data suggest that ERß plays both ligand-dependent and ligand-independent roles in mechanical signaling in osteoblasts. Furthermore, our data suggest that one mechanism by which ERß regulates mechanotransduction in osteoblasts may result from its inhibitory effect on ERα expression and function. Targeting estrogen receptors (e.g., inhibiting ERß) may represent an effective approach for prevention and treatment of age-related bone loss.


Asunto(s)
Receptor beta de Estrógeno/fisiología , Mecanotransducción Celular/genética , Osteoblastos/metabolismo , Animales , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Estradiol/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mecanotransducción Celular/efectos de los fármacos , Ratones , Ratones Noqueados , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Cráneo/citología
5.
J Cell Physiol ; 227(5): 1873-82, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-21732358

RESUMEN

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism.


Asunto(s)
Células de la Médula Ósea/fisiología , Proteínas Asociadas a Matriz Nuclear/genética , Osteoblastos/fisiología , Células del Estroma/fisiología , Telomerasa/metabolismo , Factores de Transcripción/genética , Adipocitos/citología , Adipocitos/fisiología , Animales , Células de la Médula Ósea/citología , Proteína Morfogenética Ósea 2/farmacología , Línea Celular , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Matriz Nuclear/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Hormona Paratiroidea/farmacología , Fenotipo , Células del Estroma/citología , Telomerasa/genética , Factores de Transcripción/metabolismo
6.
Crit Rev Eukaryot Gene Expr ; 22(3): 205-18, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23140162

RESUMEN

Chronic degenerative diseases are increasing with the aging U.S. population. One consequence of this phenomenon is the need for long-term osteoporosis therapies. Parathyroid hormone (PTH), the only FDA-approved treatment that adds bone to the aged skeleton, loses its potency within two years of initial treatment but the mechanism regulating its limited "anabolic window" is unknown. We have discovered that disabling the nucleocytoplasmic shuttling transcription factor nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ) in mice extends the PTH bone-forming capacity. Nmp4 was discovered during our search for nuclear matrix transcription factors that couple this hormone's impact on osteoblast cytoskeletal and nuclear organization with its anabolic capacity. CIZ was independently discovered as a protein that associates with the focal adhesion-associated mechanosensor p130Cas. The Nmp4/CIZ-knockout (KO) skeletal phenotype exhibits a modestly enhanced bone mineral density but manifests an exaggerated response to both PTH and to BMP2 and is resistant to disuse-induced bone loss. The cellular basis of the global Nmp4/CIZ-KO skeletal phenotype remains to be elucidated but may involve an expansion of the bone marrow osteoprogenitor population along with modestly enhanced osteoblast and osteoclast activities supporting anabolic bone turnover. As a shuttling Cys(2)His(2) zinc finger protein, Nmp4/CIZ acts as a repressive transcription factor perhaps associated with epigenetic remodeling complexes, but the functional significance of its interaction with p130Cas is not known. Despite numerous remaining questions, Nmp4/CIZ provides insights into how the anabolic window is regulated, and itself may provide an adjuvant therapy target for the treatment of osteoporosis by extending PTH anabolic efficacy.


Asunto(s)
Huesos/fisiología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Hormona Paratiroidea/fisiología , Factores de Transcripción/metabolismo , Animales , Densidad Ósea , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Adhesión Celular , Proteína Sustrato Asociada a CrK/genética , Proteína Sustrato Asociada a CrK/metabolismo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Ratones , Proteínas Asociadas a Matriz Nuclear/genética , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Hormona Paratiroidea/farmacología , Fenotipo , Factores de Transcripción/genética , Dedos de Zinc/genética
7.
Int J Mol Sci ; 13(11): 14385-400, 2012 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-23203070

RESUMEN

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing "FRET standard" fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.


Asunto(s)
Técnicas Biosensibles , Rastreo Celular/métodos , Microscopía Fluorescente/métodos , Animales , Humanos
8.
J Cell Physiol ; 226(4): 1044-51, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20857415

RESUMEN

Fluid shear stress protects cells from TNF-α-induced apoptosis. Oscillatory fluid shear stress (OFSS) is generally perceived as physiologically relevant biophysical signal for bone cells. Here we identify several cellular mechanisms responsible for mediating the protective effects of OFSS against TNF-α-induced apoptosis in vitro. We found that exposure of MC3T3-E1 osteoblast-like cells to as little as 5 min of OFSS suppressed TNF-α-induced activation of caspase-3, cleavage of PARP and phosphorylation of histone. In contrast, H(2)O(2)-induced apoptosis was not inhibited by OFSS suggesting that OFSS might not be protecting cells from TNF-α-induced apoptosis via stimulation of global pro-survival signaling pathways. In support of this speculation, OFSS inhibition of TNF-α-induced apoptosis was unaffected by inhibitors of several pro-survival signaling pathways including pI3-kinase (LY294002), MAPK/ERK kinase (PD98059 or U0126), intracellular Ca2+ release (U73122), NO production (L-NAME), or protein synthesis (cycloheximide) that were applied to cells during exposure to OFSS and during TNF-α treatment. However, TNF-α-induced phosphorylation and degradation of IκBα was blocked by pre-exposure of cells to OFSS suggesting a more specific effect of OFSS on TNF-α signaling. We therefore focused on the mechanism of OFSS regulation of TNF-receptor 1 (TNFR1) signaling and found that OFSS (1) reduced the amount of receptor on the cell surface, (2) prevented the association of ubiquitinated RIP in TNFR1 complexes with TRADD and TRAF2, and (3) reduced TNF-α-induced IL-8 promoter activity in the nucleus. We conclude that the anti-apoptotic effect of OFSS is not mediated by activation of universal pro-survival signaling pathways. Rather, OFSS inhibits TNF-α-induced pro-apoptotic signaling which can be explained by the down-regulation of TNFR1 on the cell surface and blockade of TNFR1 downstream signaling by OFSS.


Asunto(s)
Osteoblastos/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Reología , Transducción de Señal , Estrés Mecánico , Animales , Apoptosis/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Endocitosis/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Peróxido de Hidrógeno/farmacología , Proteínas I-kappa B/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Inhibidor NF-kappaB alfa , Óxido Nítrico/biosíntesis , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Reología/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitinación/efectos de los fármacos
9.
PLoS One ; 16(12): e0261660, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34941939

RESUMEN

Earlier, we proposed the "mechanosome" concept as a testable model for understanding how mechanical stimuli detected by cell surface adhesion molecules are transmitted to modulate gene expression inside cells. Here, for the first time we document a putative mechanosome involving Src, Pyk2 and MBD2 in MLO-Y4 osteocytes with high spatial resolution using FRET-FLIM. Src-Pyk2 complexes were concentrated at the periphery of focal adhesions and the peri-nuclear region. Pyk2-MBD2 complexes were located primarily in the nucleus and peri-nuclear region. Lifetime measurements indicated that Src and MBD2 did not interact directly. Finally, mechanical stimulation by fluid flow induced apparent accumulation of Src-Pyk2 protein complexes in the peri-nuclear/nuclear region, consistent with the proposed behavior of a mechanosome in response to a mechanical stimulus.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Osteocitos/metabolismo , Familia-src Quinasas/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/análisis , Transferencia Resonante de Energía de Fluorescencia , Quinasa 2 de Adhesión Focal/análisis , Adhesiones Focales/metabolismo , Mecanotransducción Celular , Ratones , Osteocitos/citología , Familia-src Quinasas/análisis
10.
J Cell Physiol ; 223(2): 435-41, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20112285

RESUMEN

Cellular mechanotransduction, the process of converting mechanical signals into biochemical responses within cells, is a critical aspect of bone health. While the effects of mechanical loading on bone are well recognized, elucidating the specific molecular pathways involved in the processing of mechanical signals by bone cells represents a challenge and an opportunity to identify therapeutic strategies to combat bone loss. In this study we have for the first time examined the relationship between the nucleocytoplasmic shuttling transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ) and beta-catenin signaling in response to a physiologic mechanical stimulation (oscillatory fluid shear stress, OFSS) in osteoblasts. Using calvaria-derived osteoblasts from Nmp4-deficient and wild-type mice, we found that the normal translocation of beta-catenin to the nucleus in osteoblasts that is induced by OFSS is enhanced when Nmp4/CIZ is absent. Furthermore, we found that other aspects of OFSS-induced mechanotransduction generally associated with the beta-catenin signaling pathway, including ERK, Akt, and GSK3beta activity, as well as expression of the beta-catenin-responsive protein cyclin D1 are also enhanced in cells lacking Nmp4/CIZ. Finally, we found that in the absence of Nmp4/CIZ, OFSS-induced cytoskeletal reorganization and the formation of focal adhesions between osteoblasts and the extracellular substrate is qualitatively enhanced, suggesting that Nmp4/CIZ may reduce the sensitivity of bone cells to mechanical stimuli. Together these results provide experimental support for the concept that Nmp4/CIZ plays an inhibitory role in the response of bone cells to mechanical stimulation induced by OFSS.


Asunto(s)
Mecanotransducción Celular/fisiología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Osteoblastos/metabolismo , Osteogénesis/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Animales , Resorción Ósea/metabolismo , Resorción Ósea/fisiopatología , Resorción Ósea/prevención & control , Adhesión Celular/fisiología , Células Cultivadas , Ciclina D1/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Adhesiones Focales/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Ratones , Ratones Noqueados , Proteínas Asociadas a Matriz Nuclear/genética , Osteoblastos/citología , Estimulación Física , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estrés Mecánico , Factores de Transcripción/genética , beta Catenina/genética
11.
Clin Rev Bone Miner Metab ; 8(4): 213-223, 2010 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-21479153

RESUMEN

We introduced the mechanosome hypothesis in 2003 as a heuristic model for investigating mechanotransduction in bone (Pavalko et al., J Cell Biochem, 2003, 88(1):104-112). This model suggested specific approaches for investigating how mechanical information is conveyed from the membrane of the sensor bone cell to the target genes and how this transmitted information from the membrane is converted into changes in transcription. The key concepts underlying the mechanosome hypothesis are that load-induced deformation of bone deforms the sensor cell membrane; embedded in the membrane are the focal adhesion and cadherin-catenin complexes, which in turn are physically connected to the chromatin via a solid-state scaffold. The physical stimulation of the membrane launches multiprotein complexes (mechanosomes) from the adhesion platforms while concomitantly tugging target genes into position for contact with the incoming mechanosomes, the carriers of the mechanical information to the nucleus. The mechanosome is comprised of an adhesion-associated protein and a nucleocytoplasmic shuttling transcription factor. Upon arrival at the target gene, mechanosomes alter DNA conformation and thus influence the interactions between trans-acting proteins along the gene, changing gene activity. Here, we update significant progress related to the mechanosome concept since publication of our original hypothesis. The launching of adhesion- and cytoskeletal-associated proteins into the nucleus toward target genes appears to be a common mechanism for regulating cell response to changes in its mechanical microenvironment.

12.
J Bone Miner Res ; 34(10): 1964-1975, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31173667

RESUMEN

Mechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of ß-catenin-an intracellular signaling node in the canonical Wnt pathway-in disuse mechanotransduction is not defined. Using the ß-catenin exon 3 flox (constitutively active [CA]) mouse model, in conjunction with a tamoxifen-inducible, osteocyte-selective Cre driver, we evaluated the effects of degradation-resistant ß-catenin on bone properties during disuse. We hypothesized that if ß-catenin plays an important role in Wnt-mediated osteoprotection, then artificial stabilization of ß-catenin in osteocytes would protect the limbs from disuse-induced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinum-toxin (botox)-induced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dual-energy X-ray absorptiometry (DXA), micro-computed tomography (µCT), and histomorphometry. Activation of the ßcatCA allele in tail-suspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botox-treated mice when the ßcatCA was activated. RNAseq analysis of altered gene regulation in tail-suspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tail-suspended ß-catenin stabilized mice and tail-suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of ß-catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease. © 2019 American Society for Bone and Mineral Research.


Asunto(s)
Mecanotransducción Celular , Osteocitos/metabolismo , Osteogénesis , Tibia/metabolismo , beta Catenina/metabolismo , Animales , Densidad Ósea , Ratones , Ratones Transgénicos , Osteocitos/patología , Tibia/diagnóstico por imagen , Tibia/patología , Microtomografía por Rayos X , beta Catenina/genética
13.
iScience ; 20: 205-215, 2019 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-31585407

RESUMEN

Wnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4KI), based on a published mutation in patients with high bone mass (HBM). Lrp4KI mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4KI mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4KI mice. In a disuse-induced bone wasting model, Lrp4KI mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse.

14.
Tissue Eng ; 13(11): 2653-66, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17883325

RESUMEN

The development of a completely tissue-engineered small-caliber prosthesis suitable for incorporation into an in vivo vascular network is fraught with many challenges, including overcoming resistance to endothelialization and susceptibility to thrombogenesis. In this work, recombinant human fibronectin-derived low-molecular-weight peptide fragments were studied for their ability to promote cell type-specific alpha(4) integrin-mediated adhesion. Two populations of primary human endothelial cells were examined and found to express alpha(4) integrin receptors on their surfaces; on the contrary, human platelets were not found to be expressers of alpha(4) integrins. A peptide fragment isolated from the variably spliced human fibronectin type III connecting segment-1 (CS-1) domain was determined to mediate statistically significant endothelial cell alpha(4) integrin-mediated adhesion. In contrast, the fibronectin type III CS-1 fragment did not support human platelet adhesion under physiological fluid shear conditions, although fully intact human fibronectin molecules supported shear-induced platelet adhesion. This suggests that platelets bind to fibronectin in regions not encompassing the CS-1 domain. In conclusion, this work has demonstrated that the low-molecular-weight peptide CS-1 could serve as a cell-selective adhesion mediator in the engineering of a more-compatible small-caliber vascular graft lumen interface.


Asunto(s)
Células Endoteliales/efectos de los fármacos , Fibronectinas/química , Fragmentos de Péptidos/farmacología , Adhesividad Plaquetaria/fisiología , Ingeniería de Tejidos/métodos , Secuencia de Aminoácidos , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Vasos Coronarios/citología , Medios de Cultivo , Células Endoteliales/citología , Células Endoteliales/fisiología , Endotelio Vascular/citología , Humanos , Integrina alfa4/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Estrés Mecánico , Venas Umbilicales/citología
15.
Mol Endocrinol ; 29(9): 1269-85, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26244796

RESUMEN

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.


Asunto(s)
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/cirugía
16.
Bone ; 33(3): 399-410, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-13678782

RESUMEN

Bone formation in response to exogenous mechanical loading is dependent on prostaglandin synthesis by the inducible isoform of cyclooxygenase, COX-2. While several transcription factors target the COX-2 gene, we examined the role of nuclear factor kappa B (NFkappaB) on COX-2 upregulation in osteoblasts in response to fluid shear due to its involvement in immune and inflammatory responses in other cell types. Application of 12 dyn/cm2 laminar flow to MC3T3-E1 osteoblast-like cells resulted in translocation of NFkappaB to the nucleus within 1 h of the onset of shear, with NFkappaB returning to the cytoplasm after 2 h of continuous flow. NFkappaB translocation in response to shear was inhibited by the protease inhibitor, Nalpha-p-tosyl-L-lysine chloromethylketone hydrochloride (TLCK), or a cell-permeant peptide that blocks the nuclear localization sequence (NLS) on NFkappaB. Block of NFkappaB translocation with these inhibitors blocked the shear-induced upregulation of COX-2. We found that disruption of the actin cytoskeleton with cytochalasin D or microtubules with nocodozol did not alter NFkappaB translocation in response to shear. However, addition of the intracellular Ca2+ chelator BAPTA completely blocked NFkappaB translocation. While block of Ca2+ entry with channel blockers failed to inhibit NFkappaB translocation, inhibition of phospholipase C (PLC)-induced intracellular Ca2+ release with the PLC inhibitor U73122 completely abrogated the NFkappaB response to shear. These data indicate that NFkappaB translocation to the nucleus is essential for the fluid shear-induced increase in COX-2. Further, these studies suggest that intracellular Ca2+ release, but not the cytoskeletal architecture, is important to NFkappaB translocation.


Asunto(s)
Calcio/metabolismo , Mecanotransducción Celular/fisiología , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Animales , Línea Celular , Ciclooxigenasa 2 , Citoesqueleto/metabolismo , Isoenzimas/metabolismo , Ratones , Osteoblastos/citología , Perfusión , Prostaglandina-Endoperóxido Sintasas/metabolismo , Estrés Mecánico , Fosfolipasas de Tipo C/metabolismo
17.
J Appl Physiol (1985) ; 97(1): 135-42, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15004000

RESUMEN

Mechanical loading of bone is important for the structural integrity of the skeleton and the maintenance of bone mass. Mechanically loading bone generates fluid shear stress (FSS) across the surface of bone cells resulting in the induction of cyclooxygenase-2 (COX-2) and release of prostaglandins, both of which are necessary for mechanically induced bone formation. However, the mechanisms by which cells transduce FSS-induced signals across the membrane and into the cell remain poorly understood. Focal adhesions, which are specialized sites of attachment between cells and the extracellular matrix, play a role in signal transduction and have been proposed to function as mechanosensors. To directly test whether focal adhesions mediate mechanotransduction in bone cells, we inhibited the formation of focal adhesions by 1). culturing MC3T3-E1 osteoblasts on bovine serum albumin (BSA), which does not contain integrin binding sites or by 2). treating cells cultured on fibronectin with soluble Arg-Gly-Asp-Ser (RGDS) peptide to specifically block integrin-fibronectin interactions. We then subjected the cells to FSS and measured COX-2 induction and PGE(2) release. Both COX-2 induction and PGE(2) release in response to FSS were significantly decreased when osteoblasts were treated with soluble RGDS peptide compared with controls. However, RGDS peptide treatment did not affect FSS-induced ERK phosphorylation. Interestingly, osteoblasts cultured on BSA to suppress focal adhesion formation secreted fibronectin and increased focal adhesion formation over time, which correlated with the induction of COX-2 in response to FSS. Together, these results suggest that fibronectin-induced formation of focal adhesions promotes FSS-induced PGE(2) release and upregulation of COX-2 protein.


Asunto(s)
Dinoprostona/metabolismo , Fibronectinas/fisiología , Adhesiones Focales/fisiología , Isoenzimas/biosíntesis , Osteoblastos/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Células 3T3 , Animales , Línea Celular , Medios de Cultivo , Ciclooxigenasa 2 , Inducción Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Immunoblotting , Ratones , Microscopía Fluorescente , Oligopéptidos/farmacología , Fosforilación , Albúmina Sérica Bovina , Resistencia al Corte , Regulación hacia Arriba/fisiología
18.
J Appl Physiol (1985) ; 96(3): 957-66, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-14617531

RESUMEN

Cultured osteoblasts express three major types of cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. The cytoskeletal network is thought to play an important role in the transmission and conversion of a mechanical stimulus into a biochemical response. To examine a role for the three different cytoskeletal networks in fluid shear stress-induced signaling in osteoblasts, we individually disrupted actin microfilaments, micro-tubules, and intermediate filaments in MC3T3-E1 osteoblasts with multiple pharmacological agents. We subjected these cells to 90 min of laminar fluid shear stress (10 dyn/cm(2)) and compared the PGE(2) and PGI(2) release and induction of cyclooxygenase-2 protein to control cells with intact cytoskeletons. Disruption of actin microfilaments, microtubules, or intermediate filaments in MC3T3-E1 cells did not prevent a significant fluid shear stress-induced release of PGE(2) or PGI(2). Furthermore, disruption of actin microfilaments or microtubules did not prevent a significant fluid shear stress-induced increase in cyclooxygenase-2 protein levels. Disruption of intermediate filaments with acrylamide did prevent the fluid shear stress-induced increase in cyclooxygenase-2 but also prevented a PGE(2)-induced increase in cyclooxygenase-2. Thus none of the three major cytoskeletal networks are required for fluid shear stress-induced prostaglandin release. Furthermore, although neither actin microfilaments nor microtubules are required for fluid shear stress-induced increase in cyclooxygenase-2 levels, the role of intermediate filaments in regulation of cyclooxygenase-2 expression is less clear.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Isoenzimas/biosíntesis , Microtúbulos/metabolismo , Osteoblastos/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandinas/metabolismo , Animales , Células Cultivadas , Ciclooxigenasa 2 , Isoenzimas/genética , Ratones , Osteoblastos/citología , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandinas/biosíntesis , Prostaglandinas/genética , Resistencia al Corte
19.
PLoS One ; 9(5): e97942, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24841674

RESUMEN

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/metabolismo
20.
J Bone Miner Res ; 28(6): 1434-45, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23362087

RESUMEN

Preclinical and clinical evidence from megakaryocyte (MK)-related diseases suggests that MKs play a significant role in maintaining bone homeostasis. Findings from our laboratories reveal that MKs significantly increase osteoblast (OB) number through direct MK-OB contact and the activation of integrins. We, therefore, examined the role of Pyk2, a tyrosine kinase known to be regulated downstream of integrins, in the MK-mediated enhancement of OBs. When OBs were co-cultured with MKs, total Pyk2 levels in OBs were significantly enhanced primarily because of increased Pyk2 gene transcription. Additionally, p53 and Mdm2 were both decreased in OBs upon MK stimulation, which would be permissive of cell cycle entry. We then demonstrated that OB number was markedly reduced when Pyk2-/- OBs, as opposed to wild-type (WT) OBs, were co-cultured with MKs. We also determined that MKs inhibit OB differentiation in the presence and absence of Pyk2 expression. Finally, given that MK-replete spleen cells from GATA-1-deficient mice can robustly stimulate OB proliferation and bone formation in WT mice, we adoptively transferred spleen cells from these mice into Pyk2-/- recipient mice. Importantly, GATA-1-deficient spleen cells failed to stimulate an increase in bone formation in Pyk2-/- mice, suggesting in vivo the important role of Pyk2 in the MK-induced increase in bone volume. Further understanding of the signaling pathways involved in the MK-mediated enhancement of OB number and bone formation will facilitate the development of novel anabolic therapies to treat bone loss diseases.


Asunto(s)
Diferenciación Celular/fisiología , Quinasa 2 de Adhesión Focal/metabolismo , Megacariocitos/enzimología , Osteoblastos/enzimología , Osteogénesis/fisiología , Animales , Células Cultivadas , Técnicas de Cocultivo , Quinasa 2 de Adhesión Focal/genética , Megacariocitos/citología , Ratones , Ratones Noqueados , Osteoblastos/citología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
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