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1.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-38473716

RESUMEN

Despite the well-known relevance of polyamines to many forms of life, little is known about how polyamines regulate osteogenesis and skeletal homeostasis. Here, we report a series of in vitro studies conducted with human-bone-marrow-derived pluripotent stromal cells (MSCs). First, we show that during osteogenic differentiation, mRNA levels of most polyamine-associated enzymes are relatively constant, except for the catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), which is strongly increased at both mRNA and protein levels. As a result, the intracellular spermidine to spermine ratio is significantly reduced during the early stages of osteoblastogenesis. Supplementation of cells with exogenous spermidine or spermine decreases matrix mineralization in a dose-dependent manner. Employing N-cyclohexyl-1,3-propanediamine (CDAP) to chemically inhibit spermine synthase (SMS), the enzyme catalyzing conversion of spermidine into spermine, also suppresses mineralization. Intriguingly, this reduced mineralization is rescued with DFMO, an inhibitor of the upstream polyamine enzyme ornithine decarboxylase (ODC1). Similarly, high concentrations of CDAP cause cytoplasmic vacuolization and alter mitochondrial function, which are also reversible with the addition of DFMO. Altogether, these studies suggest that excess polyamines, especially spermidine, negatively affect hydroxyapatite synthesis of primary MSCs, whereas inhibition of polyamine synthesis with DFMO rescues most, but not all of these defects. These findings are relevant for patients with Snyder-Robinson syndrome (SRS), as the presenting skeletal defects-associated with SMS deficiency-could potentially be ameliorated by treatment with DFMO.


Asunto(s)
Células Madre Mesenquimatosas , Espermidina , Humanos , Espermidina/metabolismo , Espermina/metabolismo , Espermina Sintasa/genética , Ornitina Descarboxilasa/metabolismo , Osteogénesis , Poliaminas/metabolismo , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero
2.
Curr Osteoporos Rep ; 17(1): 26-35, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30725321

RESUMEN

PURPOSE OF REVIEW: We reviewed recent literature on oxygen sensing in osteogenic cells and its contribution to development of a skeletal phenotype, the coupling of osteogenesis with angiogenesis and integration of hypoxia into canonical Wnt signaling, and opportunities to manipulate oxygen sensing to promote skeletal repair. RECENT FINDINGS: Oxygen sensing in osteocytes can confer a high bone mass phenotype in murine models; common and unique targets of HIF-1α and HIF-2α and lineage-specific deletion of oxygen sensing machinery suggest differentia utilization and requirement of HIF-α proteins in the differentiation from mesenchymal stem cell to osteoblast to osteocyte; oxygen-dependent but HIF-α-independent signaling may contribute to observed skeletal phenotypes. Manipulating oxygen sensing machinery in osteogenic cells influences skeletal phenotype through angiogenesis-dependent and angiogenesis-independent pathways and involves HIF-1α, HIF-2α, or both proteins. Clinically, an FDA-approved iron chelator promotes angiogenesis and osteogenesis, thereby enhancing the rate of fracture repair.


Asunto(s)
Huesos/metabolismo , Hipoxia/metabolismo , Osteocitos/metabolismo , Osteogénesis/fisiología , Oxígeno/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Densidad Ósea/fisiología , Regeneración Ósea/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Neovascularización Fisiológica/fisiología , Vía de Señalización Wnt/fisiología
3.
Proc Natl Acad Sci U S A ; 109(35): 14092-7, 2012 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-22886088

RESUMEN

The Wnt antagonist Sost has emerged as a key regulator of bone homeostasis through the modulation of Lrp4/5/6 Wnt coreceptors. In humans, lack of Sclerostin causes sclerosteosis and van Buchem (VB) disease, two generalized skeletal hyperostosis disorders that result from hyperactive Wnt signaling. Unlike sclerosteosis, VB patients lack SOST coding mutations but carry a homozygous 52 kb noncoding deletion that is essential for the transcriptional activation of SOST in bone. We recently identified a putative bone enhancer, ECR5, in the VB deletion region, and showed that the transcriptional activity of ECR5 is controlled by Mef2C transcription factor in vitro. Here we report that mice lacking ECR5 or Mef2C through Col1-Cre osteoblast/osteocyte-specific ablation result in high bone mass (HBM) due to elevated bone formation rates. We conclude that the absence of the Sost-specific long-range regulatory element ECR5 causes VB disease in rodents, and that Mef2C is the main transcription factor responsible for ECR5-dependent Sost transcriptional activation in the adult skeleton.


Asunto(s)
Remodelación Ósea/genética , Elementos de Facilitación Genéticos/genética , Glicoproteínas/genética , Hiperostosis/genética , Factores Reguladores Miogénicos/genética , Osteocitos/fisiología , Sindactilia/genética , Proteínas Adaptadoras Transductoras de Señales , Factores de Edad , Animales , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/metabolismo , Femenino , Fémur/citología , Fémur/fisiología , Eliminación de Gen , Glicoproteínas/metabolismo , Hiperostosis/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Operón Lac , Factores de Transcripción MEF2 , Masculino , Mandíbula/anomalías , Mandíbula/metabolismo , Ratones , Ratones Transgénicos , Factores Reguladores Miogénicos/metabolismo , Osteocondrodisplasias , Osteosclerosis/genética , Osteosclerosis/metabolismo , Transducción de Señal/genética , Cráneo/anomalías , Cráneo/metabolismo , Sindactilia/metabolismo , Activación Transcripcional/genética
4.
JBMR Plus ; 7(4): e10733, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37065626

RESUMEN

Hypoxia-inducible factors (HIFs) are oxygen-dependent heterodimeric transcription factors that mediate molecular responses to reductions in cellular oxygen (hypoxia). HIF signaling involves stable HIF-ß subunits and labile, oxygen-sensitive HIF-α subunits. Under hypoxic conditions, the HIF-α subunit is stabilized, complexes with nucleus-confined HIF-ß subunit, and transcriptionally regulates hypoxia-adaptive genes. Transcriptional responses to hypoxia include altered energy metabolism, angiogenesis, erythropoiesis, and cell fate. Three isoforms of HIF-α-HIF-1α, HIF-2α, and HIF-3α-are found in diverse cell types. HIF-1α and HIF-2α serve as transcriptional activators, whereas HIF-3α restricts HIF-1α and HIF-2α. The structure and isoform-specific functions of HIF-1α in mediating molecular responses to hypoxia are well established across a wide range of cell and tissue types. The contributions of HIF-2α to hypoxic adaptation are often unconsidered if not outrightly attributed to HIF-1α. This review establishes what is currently known about the diverse roles of HIF-2α in mediating the hypoxic response in skeletal tissues, with specific focus on development and maintenance of skeletal fitness. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

5.
JBMR Plus ; 7(4): e10724, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37065633

RESUMEN

Molecular oxygen levels vary during development and disease. Adaptations to decreased oxygen bioavailability (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are composed of an oxygen-dependent α subunit (HIF-α), of which there are two transcriptionally active isoforms (HIF-1α and HIF-2α), and a constitutively expressed ß subunit (HIFß). Under normoxic conditions, HIF-α is hydroxylated via prolyl hydroxylase domain (PHD) proteins and targeted for degradation via Von Hippel-Lindau (VHL). Under hypoxic conditions, hydroxylation via PHD is inhibited, allowing for HIF-α stabilization and induction of target transcriptional changes. Our previous studies showed that Vhl deletion in osteocytes (Dmp1-cre; Vhl f/f ) resulted in HIF-α stabilization and generation of a high bone mass (HBM) phenotype. The skeletal impact of HIF-1α accumulation has been well characterized; however, the unique skeletal impacts of HIF-2α remain understudied. Because osteocytes orchestrate skeletal development and homeostasis, we investigated the role of osteocytic HIF-α isoforms in driving HBM phenotypes via osteocyte-specific loss-of-function and gain-of-function HIF-1α and HIF-2α mutations in C57BL/6 female mice. Deletion of Hif1a or Hif2a in osteocytes showed no effect on skeletal microarchitecture. Constitutively stable, degradation-resistant HIF-2α (HIF-2α cDR), but not HIF-1α cDR, generated dramatic increases in bone mass, enhanced osteoclast activity, and expansion of metaphyseal marrow stromal tissue at the expense of hematopoietic tissue. Our studies reveal a novel influence of osteocytic HIF-2α in driving HBM phenotypes that can potentially be harnessed pharmacologically to improve bone mass and reduce fracture risk. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

6.
Endocr Relat Cancer ; 30(9)2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-37226936

RESUMEN

Prostate cancer (PCa) is an increasingly prevalent health problem in the developed world. Effective treatment options exist for localized PCa, but metastatic PCa has fewer treatment options and shorter patient survival. PCa and bone health are strongly entwined, as PCa commonly metastasizes to the skeleton. Since androgen receptor signaling drives PCa growth, androgen-deprivation therapy whose sequelae reduce bone strength constitutes the foundation of advanced PCa treatment. The homeostatic process of bone remodeling - produced by concerted actions of bone-building osteoblasts, bone-resorbing osteoclasts, and regulatory osteocytes - may also be subverted by PCa to promote metastatic growth. Mechanisms driving skeletal development and homeostasis, such as regional hypoxia or matrix-embedded growth factors, may be subjugated by bone metastatic PCa. In this way, the biology that sustains bone is integrated into adaptive mechanisms for the growth and survival of PCa in bone. Skeletally metastatic PCa is difficult to investigate due to the entwined nature of bone biology and cancer biology. Herein, we survey PCa from origin, presentation, and clinical treatment to bone composition and structure and molecular mediators of PCa metastasis to bone. Our intent is to quickly yet effectively reduce barriers to team science across multiple disciplines that focuses on PCa and metastatic bone disease. We also introduce concepts of tissue engineering as a novel perspective to model, capture, and study complex cancer-microenvironment interactions.


Asunto(s)
Neoplasias Óseas , Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata/patología , Neoplasias Óseas/secundario , Antagonistas de Andrógenos/uso terapéutico , Huesos/metabolismo , Resultado del Tratamiento , Microambiente Tumoral
7.
Exp Cell Res ; 317(6): 737-44, 2011 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-21237152

RESUMEN

Fluid shear stress regulates gene expression in osteoblasts, in part by activation of the transcription factor NF-κB. We examined whether this process was under the control of purinoceptor activation. MC3T3-E1 osteoblasts under static conditions expressed the NF-κB inhibitory protein IκBα and exhibited cytosolic localization of NF-κB. Under fluid shear stress, IκBα levels decreased, and concomitant nuclear localization of NF-κB was observed. Cells exposed to fluid shear stress in ATP-depleted medium exhibited no significant reduction in IκBα, and NF-κB remained within the cytosol. Similar results were found using oxidized ATP or Brilliant Blue G, P2X(7) receptor antagonists, indicating that the P2X(7) receptor is responsible for fluid shear-stress-induced IκBα degradation and nuclear accumulation of NF-κB. Pharmacologic blockage of the P2Y6 receptor also prevented shear-induced IκBα degradation. These phenomena involved neither ERK1/2 signaling nor autocrine activation by P2X(7)-generated lysophosphatidic acid. Our results suggest that fluid shear stress regulates NF-κB activity through the P2Y(6) and P2X(7) receptor.


Asunto(s)
FN-kappa B/metabolismo , Osteoblastos/metabolismo , Transducción de Señal , Estrés Mecánico , Animales , Western Blotting , Células Cultivadas , Inmunohistoquímica , Ratones , Transporte de Proteínas , Receptores Purinérgicos P2X7/metabolismo
8.
Dev Biol ; 342(2): 169-79, 2010 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20359476

RESUMEN

SOST is a negative regulator of bone formation, and mutations in human SOST are responsible for sclerosteosis. In addition to high bone mass, sclerosteosis patients occasionally display hand defects, suggesting that SOST may function embryonically. Here we report that overexpression of SOST leads to loss of posterior structures of the zeugopod and autopod by perturbing anterior-posterior and proximal-distal signaling centers in the developing limb. Mutant mice that overexpress SOST in combination with Grem1 and Lrp6 mutations display more severe limb defects than single mutants alone, while Sost(-/-) significantly rescues the Lrp6(-/-) skeletal phenotype, signifying that SOST gain-of-function impairs limb patterning by inhibiting the WNT signaling through LRP5/6.


Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Extremidades/embriología , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Marcadores Genéticos , Humanos , Ratones , Ratones Transgénicos
9.
J Cell Biochem ; 112(11): 3354-63, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21751239

RESUMEN

Tenascins regulate cell interaction with the surrounding pericellular matrix. Within bone, tenascins C and W influence osteoblast adhesion and differentiation, although little is known about the regulation of tenascin expression. In this study we examined the effect of osteogenic differentiation, bone morphogenetic protein (BMP) and Wnt growth factors, and mechanical loading on tenascin expression in osteogenic cells. Osteogenic differentiation increased tenascin C (TnC), and decreased tenascin W (TnW), expression. Both growth factors and mechanical loading increased both TnC and TnW expression, albeit via distinct signaling mechanisms. Both BMP-2 and Wnt5a induction of tenascin expression were mediated by MAP kinases. These data establish a role for BMP, Wnts, and mechanical loading in the regulation of tenascin expression in osteoblasts.


Asunto(s)
Huesos/metabolismo , Tenascina/metabolismo , Animales , Western Blotting , Proteínas Morfogenéticas Óseas/fisiología , Huesos/citología , Diferenciación Celular , Línea Celular , Mecanotransducción Celular , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Transcripción NFATC/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Transducción de Señal , Tenascina/genética , Fosfolipasas de Tipo C/metabolismo , Proteínas Wnt/fisiología
10.
Biotechnol Bioeng ; 108(11): 2727-35, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21656707

RESUMEN

Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to non-mineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration.


Asunto(s)
Apatitas/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Células Madre Mesenquimatosas/fisiología , Osteogénesis , Fosfatasa Alcalina/metabolismo , Calcio/metabolismo , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Perfilación de la Expresión Génica , Humanos , Sialoproteína de Unión a Integrina/biosíntesis , Células Madre Mesenquimatosas/efectos de los fármacos , Osteonectina/biosíntesis , Factor de Transcripción Sp7 , Factores de Transcripción/biosíntesis
11.
Cell Biol Int ; 35(10): 981-9, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21574962

RESUMEN

A variety of pathologies such as skeletal fracture, neoplasia and inflammation compromise tissue perfusion and thereby decrease tissue oxygen tension. We and others have demonstrated that hypoxia is a potent stimulant for MSC (mesenchymal stem cell) recruitment and differentiation, yet to date little research has focused on the effects of oxygen tension on MSC migration. In the present study, we examined the effects of hypoxia and the potential role of the GTPase RhoA and HIF-1α (hypoxia-inducible factor 1α) on MSC migration. Our results demonstrate that hypoxia decreases MSC migration through an HIF-1α and RhoA-mediated pathway. The active GTP-bound form of RhoA was reduced in 1% oxygen, whereas activation of RhoA under hypoxic conditions rescued migration. Furthermore, stabilization of HIF-1α under normoxic conditions attenuated cell migration similar to that of hypoxia. These results suggest that hypoxia negatively affects MSC migration by regulating activation of GTPases. These results highlight the importance of oxygen in regulating the recruitment of progenitor cells to areas of ischaemic tissue damage.


Asunto(s)
Hipoxia de la Célula , Movimiento Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Madre Mesenquimatosas/citología , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Adolescente , Células de la Médula Ósea/citología , Humanos , Masculino , Transducción de Señal
12.
JBMR Plus ; 5(5): e10462, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33977198

RESUMEN

Sclerostin antibody (romosozumab) was recently approved for clinical use in the United States to treat osteoporosis. We and others have explored Wnt-based combination therapy to disproportionately improve the anabolic effects of sclerostin inhibition, including cotreatment with sclerostin antibody (Scl-mAb) and Dkk1 antibody (Dkk1-mAb). To determine the optimal ratio of Scl-mAb and Dkk1-mAb for producing maximal anabolic action, the proportion of Scl-mAb and Dkk1-mAb were systematically varied while holding the total antibody dose constant. A 3:1 mixture of Scl-mAb to Dkk1-mAb produced two to three times as much cancellous bone mass as an equivalent dose of Scl-mAb alone. Further, a 75% reduction in the dose of the 3:1 mixture was equally efficacious to a full dose of Scl-mAb in the distal femur metaphysis. The Scl-mAb/Dkk1-mAb combination approach was highly efficacious in the cancellous bone mass, but the cortical compartment was much more subtly affected. The osteoanabolic effects of Wnt pathway targeting can be made more efficient if multiple antagonists are simultaneously targeted. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

13.
J Cell Biochem ; 110(1): 87-96, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20213746

RESUMEN

Skeletal defects commonly suffer from poor oxygen microenvironments resulting from compromised vascularization associated with injury or disease. Adipose stem cells (ASCs) represent a promising cell population for stimulating skeletal repair by differentiating toward the osteogenic lineage or by secreting trophic factors. However, the osteogenic or trophic response of ASCs to reduced oxygen microenvironments is poorly understood. Moreover, a direct comparison between 2D and 3D response of ASCs to hypoxia is lacking. Thus, we characterized the osteogenic and angiogenic potential of human ASCs under hypoxic (1%), normoxic (5%), and atmospheric (21%) oxygen tensions in both 2D and 3D over 4 weeks in culture. We detected greatest alkaline phosphatase activity and extracellular calcium deposition in cells cultured in both 2D and 3D under 21% oxygen, and reductions in enzyme activity corresponded to reductions in oxygen tension. ASCs cultured in 1% oxygen secreted more vascular endothelial growth factor (VEGF) over the 4-week period than cells cultured in other conditions, with cells cultured in 2D secreting VEGF in a more sustained manner than those in 3D. Expression of osteogenic markers revealed temporal changes under different oxygen conditions with peak expression occurring earlier in 3D. In addition, the increase of most osteogenic markers was significantly higher in 2D compared to 3D cultures at 1% and 5% oxygen. These results suggest that oxygen, in conjunction with dimensionality, affects the timing of the differentiation program in ASCs. These findings offer new insights for the use of ASCs in bone repair while emphasizing the importance of the culture microenvironment.


Asunto(s)
Tejido Adiposo/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Oxígeno/farmacología , Células Madre/citología , Células Madre/efectos de los fármacos , Adulto , Fosfatasa Alcalina/metabolismo , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Neovascularización Fisiológica/efectos de los fármacos , Osteogénesis/genética , Células Madre/enzimología , Células Madre/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
14.
J Cell Biochem ; 110(2): 457-67, 2010 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-20336693

RESUMEN

Mutations in sclerostin function or expression cause sclerosing bone dysplasias, involving decreased antagonism of Wnt/Lrp5 signaling. Conversely, deletion of the VHL tumor suppressor in osteoblasts, which stabilize HIF-alpha isoforms and thereby enables HIF-alpha/beta-driven gene transcription, increases bone mineral content and cross-sectional area compared to wild-type controls. We examined the influence of cellular hypoxia (1% oxygen) upon sclerostin expression and canonical Wnt signaling. Osteoblasts and osteocytes cultured under hypoxia revealed decreased sclerostin transcript and protein, and increased expression and nuclear localization of activated beta-catenin. Similarly, both hypoxia and the hypoxia mimetic DFO increased beta-catenin gene reporter activity. Hypoxia and its mimetics increased expression of the BMP antagonists gremlin and noggin and decreased Smad-1/5/8 phosphorylation. As a partial explanation for the mechanism of regulation of sclerostin by oxygen, MEF2 reporter assays revealed decreased activity. Modulation of VEGF signaling under normoxia or hypoxia revealed no influence upon Sost transcription. These data suggest that hypoxia inhibits sclerostin expression, through enhanced antagonism of BMP signaling independent of VEGF.


Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Hipoxia de la Célula , Osteoblastos/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Western Blotting , Línea Celular , Marcadores Genéticos , Humanos , Transcripción Genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , beta Catenina/metabolismo
15.
Cell Tissue Res ; 340(1): 81-9, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20165885

RESUMEN

Cellular signaling via epidermal growth factor (EGF) and EGF-like ligands can determine cell fate and behavior. Osteoblasts, which are responsible for forming and mineralizing osteoid, express EGF receptors and alter rates of proliferation and differentiation in response to EGF receptor activation. Transgenic mice over-expressing the EGF-like ligand betacellulin (BTC) exhibit increased cortical bone deposition; however, because the transgene is ubiquitously expressed in these mice, the identity of cells affected by BTC and responsible for increased cortical bone thickness remains unknown. We have therefore examined the influence of BTC upon mesenchymal stem cell (MSC) and pre-osteoblast differentiation and proliferation. BTC decreases the expression of osteogenic markers in both MSCs and pre-osteoblasts; interestingly, increases in proliferation require hypoxia-inducible factor-alpha (HIF-alpha), as an HIF antagonist prevents BTC-driven proliferation. Both MSCs and pre-osteoblasts express EGF receptors ErbB1, ErbB2, and ErbB3, with no change in expression under osteogenic differentiation. These are the first data that demonstrate an influence of BTC upon MSCs and the first to implicate HIF-alpha in BTC-mediated proliferation.


Asunto(s)
Diferenciación Celular/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Osteogénesis/genética , Adolescente , Betacelulina , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Adulto Joven
16.
Cells Tissues Organs ; 191(3): 175-84, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-19690399

RESUMEN

The use of mesenchymal stem cells in tissue engineering to augment the repair of a variety of tissues including bone is a rapidly growing and exciting field. Although oxygen tension is a powerful stimulus for cells both in vitro and in vivo, the oxygen environment in which such cells would undergo differentiation is commonly overlooked. We examined the effect of long-term (21-days) low oxygen tension (1, 2 and 5%) on the osteogenic differentiation and matrix metalloproteinase (MMP)/tissue inhibitor of MMP (TIMP) expression of human mesenchymal stem cells (MSCs). Our data suggest that MSCs undergo osteoblastic differentiation most rapidly under 21% oxygen while oxygen tensions below 5% have an inhibitory effect. Interestingly, there was not a statistically significant difference in osteogenic markers between 5 and 21% oxygen. In addition, our data suggest that oxygen tension affects the expression of individual MMP and TIMPs differently. Low oxygen tension has an inhibitory effect on MMP-13 and TIMP-1 expression, which are involved in extracellular matrix remodeling and potentially vascular invasion. In contrast, MMP-2, a metalloproteinase involved in cell migration was not affected by oxygen tension. This data suggests that 21% oxygen may be beneficial for rapid osteogenic differentiation as would be required for the production of individual patient ex vivo constructs. In addition, this has important in vivo implications relating to the importance of early vascularization of sites of orthopedic injury. By augmenting the neovascularization process, it may be possible to facilitate more rapid differentiation of progenitors and thus the repair process.


Asunto(s)
Diferenciación Celular , Metaloproteinasas de la Matriz/biosíntesis , Células Madre Mesenquimatosas/citología , Osteogénesis , Oxígeno/metabolismo , Inhibidores Tisulares de Metaloproteinasas/biosíntesis , Fosfatasa Alcalina/metabolismo , Calcio/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Metaloproteinasa 13 de la Matriz/biosíntesis , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Células Madre Mesenquimatosas/fisiología , Osteocalcina/biosíntesis , Osteopontina/biosíntesis , Oxígeno/química , Factores de Tiempo , Ingeniería de Tejidos
17.
J Bone Miner Metab ; 28(1): 8-16, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-19471853

RESUMEN

Conditions such as fracture and unloading have been shown to be associated with tissue and cellular hypoxia in bone. The effects of hypoxia on bone cell physiology and ultimately its impact on bone tissue repair and remodeling are not well understood. In this study, we investigated the role of hypoxia on prostaglandin release from osteoblastic cells cultured in 2% (hypoxia), 5% (potentially cellular normoxia), and 21% (normoxia for standard cell culture conditions) oxygen for up to 24 h. We quantified the effects of reduced oxygen tension on the release of prostaglandin (PG)E(2), PGF(2alpha), PGD(2), and PGI(2). The mechanism by which hypoxia increases PG production was investigated by examining the various regulatory components of the PG biosynthetic pathway. Our data show that PGE(2) levels alone are significantly elevated under hypoxic conditions. Also, we show that cyclooxygenase (COX)-1 and COX-2 play an important role in hypoxia-induced PGE(2) production, possibly via a mechanism involving changes in their respective activity levels under low oxygen conditions. The effect of hypoxia on PGE(2) levels was mimicked by dimethyloxaloglycine, a known activator of the HIF pathway. In addition, we confirmed that HIF-1alpha was stabilized in osteoblastic cells under hypoxia. Taken together these data suggest a role for the HIF pathway in regulation of PGE(2) levels under hypoxic conditions. Previous studies have detected release of prostaglandins from areas of damaged bone, such as a fracture site, and our data may contribute to an understanding of how this release is regulated.


Asunto(s)
Hipoxia de la Célula , Dinoprostona/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Osteoblastos/fisiología , Oxígeno/fisiología , Animales , Remodelación Ósea/fisiología , Línea Celular , Medios de Cultivo Condicionados/química , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprost/análisis , Dinoprost/metabolismo , Dinoprostona/análisis , Epoprostenol/análisis , Epoprostenol/metabolismo , Hidroxiprostaglandina Deshidrogenasas/genética , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/agonistas , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Osteoblastos/metabolismo , Fosfolipasas A2/genética , Fosfolipasas A2/metabolismo , Reacción en Cadena de la Polimerasa , Prostaglandina D2/análisis , Prostaglandina D2/metabolismo , ARN Mensajero , Factores de Tiempo
18.
Am J Vet Res ; 71(10): 1237-45, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20919913

RESUMEN

OBJECTIVE: To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. ANIMALS: 10 adult Quarter Horses and 8 newborn Thoroughbred foals. PROCEDURES: eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. RESULTS: BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP activity and calcium deposition. There was no significant difference in BM-eMSC yield from the tuber coxae or sternum, but yield was significantly greater from the first 60-mL aspirate than from subsequent aspirates. The BM-eMSC expansion rate was significantly higher when cells were cultured in fetal bovine serum instead of autologous serum (AS). CONCLUSIONS AND CLINICAL RELEVANCE: eMSCs from BM possessed the highest in vitro osteogenic potential; eMSCs from adipose tissue also had robust osteogenic potential. The tuber coxae and the sternum were viable sources of BM-eMSCs in yearlings, and 60 mL of BM aspirate was sufficient for culture and expansion. Expanding BM-eMSCs in AS to avoid potential immunologic reactions decreased the total yield because BM-eMSCs grew significantly slower in AS than in fetal bovine serum. Additional studies are needed to determine optimal ex vivo eMSC culture and expansion conditions, including the timing and use of growth factor­supplemented AS.


Asunto(s)
Tejido Adiposo/citología , Células de la Médula Ósea/fisiología , Sangre Fetal/citología , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Cordón Umbilical/citología , Animales , Diferenciación Celular , Caballos , Células Madre Mesenquimatosas/fisiología
19.
Artículo en Inglés | MEDLINE | ID: mdl-33071963

RESUMEN

The skeleton is well-innervated, but only recently have the functions of this complex network in bone started to become known. Although our knowledge of skeletal sensory and sympathetic innervation is incomplete, including the specific locations and subtypes of nerves in bone, we are now able to reconcile early studies utilizing denervation models with recent work dissecting the molecular signaling between bone and nerve. In total, sensory innervation functions in bone much as it does elsewhere in the body-to sense and respond to stimuli, including mechanical loading. Similarly, sympathetic nerves regulate autonomic functions related to bone, including homeostatic remodeling and vascular tone. However, more study is required to translate our current knowledge of bone-nerve crosstalk to novel therapeutic strategies that can be effectively utilized to combat skeletal diseases, disorders of low bone mass, and age-related decreases in bone quality.


Asunto(s)
Adaptación Fisiológica , Envejecimiento/fisiología , Desarrollo Óseo , Huesos/inervación , Huesos/fisiología , Animales , Sistema Nervioso Autónomo/fisiología , Humanos
20.
J Cell Biochem ; 107(2): 233-9, 2009 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-19277991

RESUMEN

Changes in regional oxygen tension that occur during skeletal development and fracture stimulate local bone cell activity to regulate bone formation, maintenance, and repair. The adaptive responses of bone cells to hypoxia are only beginning to be understood. The transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) is activated under hypoxia and promotes expression of genes required for adaptation and cell survival, and also regulates both bone development and fracture repair. We have previously demonstrated that hypoxic osteoblasts increase PGE(2) release and expression of the PGE(2) receptor EP1. In the present studies, we investigated the impact of altered HIF-1alpha activity and expression on EP1 expression in osteoblasts. HIF-1alpha stabilization was induced in cells cultured in 21% oxygen by treatment with dimethyloxaloglycine (DMOG) or siRNA targeted against PHD2. To implicate HIF-1alpha in hypoxia-induced EP1 expression, osteoblastic cells were treated with siRNA targeted against HIF-1alpha prior to exposure to hypoxia. EP1 expression was significantly increased in cells cultured in 21% oxygen with DMOG or PHD2 siRNA treatment compared to controls. Hypoxia responsive element (HRE) activation in hypoxia was attenuated in cells treated with HIF-1alpha siRNA compared to controls, indicating HIF-1alpha as the functional HIF-alpha isoform in this system. Furthermore, hypoxic cells treated with HIF-1alpha siRNA demonstrated reduced EP1 expression in hypoxia compared to controls. Inhibition of SAPK/JNK activity significantly reduced hypoxia-induced EP1 expression but had no impact on HIF-1alpha expression or activity. These data strongly implicate a role for HIF-1alpha in hypoxia-induced EP1 expression and may provide important insight into the mechanisms by which HIF-1alpha regulates bone development and fracture repair.


Asunto(s)
Hipoxia de la Célula/fisiología , Regulación de la Expresión Génica/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Osteoblastos/metabolismo , Receptores de Prostaglandina E/biosíntesis , Transducción de Señal/fisiología , Animales , Western Blotting , Línea Celular , Silenciador del Gen , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia , MAP Quinasa Quinasa 4/metabolismo , Ratones , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/genética , ARN Interferente Pequeño , Subtipo EP1 de Receptores de Prostaglandina E , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección
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