RESUMEN
An estimated 100,000 patients each year in the United States suffer severe disability from bone defects that fail to heal, a condition where bone-regenerative therapies could provide substantial clinical benefits. Although recombinant human bone morphogenetic protein-2 (rhBMP2) is an osteogenic growth factor that is clinically approved for this purpose, it is only effective when used at exceedingly high doses that incur substantial costs, induce severe inflammation, produce adverse side effects, and form morphologically abnormal bone. Using a validated rat femoral segmental defect model, we show that bone formed in response to clinically relevant doses of rhBMP2 is accompanied by elevated expression of interleukin-1 (IL-1). Local delivery of cDNA encoding the IL-1 receptor antagonist (IL-1Ra) achieved bridging of segmental, critical size defects in bone with a 90% lower dose of rhBMP2. Unlike use of high-dose rhBMP2, bone formation in the presence of IL-1Ra occurred via the native process of endochondral ossification, resulting in improved quality without sacrificing the mechanical properties of the regenerated bone. Our results demonstrate that local immunomodulation may permit effective use of growth factors at lower doses to recapitulate more precisely the native biology of healing, leading to higher-quality tissue regeneration.
Asunto(s)
Proteína Antagonista del Receptor de Interleucina 1 , Osteogénesis , Humanos , Ratas , Animales , Osteogénesis/genética , Proteína Antagonista del Receptor de Interleucina 1/genética , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Factor de Crecimiento Transformador beta/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Regeneración Ósea/genética , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/farmacologíaRESUMEN
OBJECTIVE: To support the preclinical evaluation of therapeutics that target chondrogenesis, our goal was to generate a rat strain that can noninvasively report endogenous chondrogenic activity. DESIGN: A transgene was constructed in which the dual expression of bioluminescent (firefly luciferase) and fluorescent (mCherry) reporters is controlled by regulatory sequences from rat Col2a1. Candidate lines were established on a Lewis background and characterized by serial bioluminescence imaging as well as ex vivo measurement of molecular reporter levels in several tissues. The sensitivity and specificity of the reporter strain were assessed in models of orthotopic and ectopic chondrogenesis. RESULTS: Substantial bioluminescence signal was detected from cartilaginous regions, including the appendicular synovial joints, spine, sternum, nose, and pinnae. Bioluminescent radiance was intense at 1 month of age, rapidly declined with continued development, yet remained detectable in 2-year-old animals. Explant imaging and immunohistochemistry confirmed that both molecular reporters were localized to cartilage. Implantation of wild-type bone marrow stromal cells into osteochondral defects made in both young adult and aged reporter rats led to a time-dependent elevation of intra-articular reporter activity concurrent with cartilaginous tissue repair. To stimulate ectopic, endochondral bone formation, bone morphogenetic protein 2 was overexpressed in the gastrocnemius muscle, which led to bioluminescent signal that closely preceded heterotopic ossification. CONCLUSIONS: This strain can help develop strategies to stimulate cartilage repair and endochondral bone formation or to inhibit chondrogenesis associated with heterotopic ossification.
Asunto(s)
Condrogénesis , Ingeniería de Tejidos , Animales , Condrogénesis/genética , Osteogénesis , Ratas , Ratas Endogámicas Lew , Ratas Transgénicas , Ingeniería de Tejidos/métodosRESUMEN
In aging mice, osteoclast number increases in cortical bone but declines in trabecular bone, suggesting that different mechanisms underlie age-associated bone loss in these 2 compartments. Osteocytes produce the osteoclastogenic cytokine RANKL, encoded by Tnfsf11. Tnfsf11 mRNA increases in cortical bone of aged mice, suggesting a mechanism underlying the bone loss. To address this possibility, we aged mice lacking RANKL in osteocytes. Whereas control mice lost cortical bone between 8 and 24 months of age, mice lacking RANKL in osteocytes gained cortical bone during this period. Mice of both genotypes lost trabecular bone with age. Osteoclasts increased with age in cortical bone of control mice but not in RANKL conditional knockout mice. Induction of cellular senescence increased RANKL production in murine and human cell culture models, suggesting an explanation for elevated RANKL levels with age. Overexpression of the senescence-associated transcription factor Gata4 stimulated Tnfsf11 expression in cultured murine osteoblastic cells. Finally, elimination of senescent cells from aged mice using senolytic compounds reduced Tnfsf11 mRNA in cortical bone. Our results demonstrate the requirement of osteocyte-derived RANKL for age-associated cortical bone loss and suggest that increased Tnfsf11 expression with age results from accumulation of senescent cells in cortical bone.
Asunto(s)
Envejecimiento/patología , Resorción Ósea/patología , Senescencia Celular , Hueso Cortical/patología , Osteocitos/patología , Ligando RANK/fisiología , Envejecimiento/metabolismo , Animales , Resorción Ósea/etiología , Resorción Ósea/metabolismo , Hueso Cortical/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteocitos/metabolismoRESUMEN
IMPACT STATEMENT: The promoter characterized in this study has been made accessible as a resource for the skeletal tissue engineering and regenerative medicine community. When combined with suitable reporter vectors, the resulting tools can be used for noninvasive and/or high-throughput screening of test conditions for stimulating chondrogenesis by candidate stem/progenitor cells. As demonstrated in this study, they can also be used with small animal imaging platforms to monitor the chondrogenic activity of implanted progenitors within orthotopic models of bone and cartilage repair.
Asunto(s)
Huesos/citología , Condrocitos/citología , Condrogénesis , Articulaciones/fisiología , Células Madre Mesenquimatosas/citología , Osteogénesis , Ingeniería de Tejidos/métodos , Anciano , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Humanos , Articulaciones/lesiones , Masculino , Células Madre Mesenquimatosas/fisiología , Ratas , Ratas Endogámicas F344RESUMEN
Because muscle contains osteoprogenitor cells and has a propensity to form bone, we have explored its utility in healing large osseous defects. Healing is achieved by the insertion of muscle fragments transduced with adenovirus encoding BMP-2 (Ad.BMP-2). However, it is not known whether the genetically modified muscle contributes osteoprogenitor cells to healing defects or merely serves as a local source of BMP-2. This question is part of the larger debate on the fate of progenitor cells introduced into sites of tissue damage to promote regeneration. To address this issue, we harvested fragments of muscle from rats constitutively expressing GFP, transduced them with Ad.BMP-2, and implanted them into femoral defects in wild-type rats under various conditions. GFP+ cells persisted within defects for the entire 8 weeks of the experiments. In the absence of bone formation, these cells presented as fibroblasts. When bone was formed, GFP+ cells were present as osteoblasts and osteocytes and also among the lining cells of new blood vessels. The genetically modified muscle thus contributed progenitor cells as well as BMP-2 to the healing defect, a property of great significance in light of the extensive damage to soft tissue and consequent loss of endogenous progenitors in problematic fractures.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Osteoblastos/metabolismo , Osteogénesis , Absorciometría de Fotón , Animales , Biopsia , Regeneración Ósea , Expresión Génica , Genes Reporteros , Inmunohistoquímica , Masculino , Músculo Esquelético/metabolismo , ARN Mensajero/genética , Ratas , Cicatrización de Heridas , Microtomografía por Rayos XRESUMEN
MED26 is a subunit of Mediator, a large complex central to the regulation of gene transcription by RNA Polymerase II. MED26 plays a role in the switch between the initiation and elongation phases of RNA Polymerase II-mediated transcription process. Regulation of these steps requires successive binding of MED26 N-terminal domain (NTD) to TATA-binding protein-associated factor 7 (TAF7) and Eleven-nineteen lysine-rich in leukemia-Associated Factor 1 (EAF1). In order to investigate the mechanism of regulation by MED26, MED26-NTD structure was solved by NMR, revealing a 4-helix bundle. EAF1 (239-268) and TAF7 (205-235) peptide interactions were both mapped to the same groove formed by H3 and H4 helices of MED26-NTD. Both interactions are characterized by dissociation constants in the 10-µM range. Further experiments revealed a folding-upon-binding mechanism that leads to the formation of EAF1 (N247-S260) and TAF7 (L214-S227) helices. Chemical shift perturbations and nuclear Overhauser enhancement contacts support the involvement of residues I222/F223 in anchoring TAF7 helix to a hydrophobic pocket of MED26-NTD, including residues L48, W80 and I84. In addition, Ala mutations of charged residues located in the C-terminal disordered part of TAF7 and EAF1 peptides affected the binding, with a loss of affinity characterized by a 10-time increase of dissociation constants. A structural model of MED26-NTD/TAF7 complex shows bi-partite components, combining ordered and disordered segments, as well as hydrophobic and electrostatic contributions to the binding. This study provides molecular detail that will help to decipher the mechanistic basis for the initiation to elongation switch-function mediated by MED26-NTD.
Asunto(s)
Complejo Mediador/química , Complejo Mediador/metabolismo , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/metabolismo , Factores de Transcripción/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Conformación Proteica , Mapeo de Interacción de ProteínasRESUMEN
Orthopedic surgeons sometimes combine recombinant, human BMP-2 with autograft bone when dealing with problematic osseous fractures. Although some case reports indicate success with this off-label strategy, there have been no randomized controlled trials. Moreover, a literature search revealed only one pre-clinical study and this was in a cranial defect model. The present project examined the consequences of combining BMP-2 with particles of living bone in a rat femoral defect model. Human bone particles were recovered with a reamer-irrigator-aspirator (RIA). To allow acceptance of the xenograft as surrogate autograft, rats were administered an immunosuppressive cocktail that does not interfere with bone healing. Implantation of 200 µg living bone particles generated a small amount of new bone and defects did not heal. Graded amounts of BMP-2 that alone provoked no healing (1.1 µg), borderline healing (5.5 µg), or full healing (11 µg) were added to this amount of bone particles. Addition of BMP-2 (1.1 µg) increased osteogenesis, and produced bridging in 2 of 7 defects. The combination of BMP-2 (5.5 µg) and bone particles made healing more reliable and advanced the maturation of the regenerate. Bone formation with BMP-2 (11 µg) and bone particles showed improved maturation. Thus, the combination of autograft and BMP-2 may be helpful clinically under conditions where the healing response is suboptimal. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2137-2145, 2016. Clinical significance These data support the clinical use of recombinant, human BMP-2 with autograft bone when treating large segmental osseous defects. The combination leads to greater bone formation and accelerates the maturation of the regenerate.
Asunto(s)
Proteína Morfogenética Ósea 2/administración & dosificación , Trasplante Óseo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Evaluación Preclínica de Medicamentos , Fémur , Humanos , Masculino , Persona de Mediana Edad , Distribución Aleatoria , Ratas Endogámicas F344RESUMEN
MED26 is a subunit of the Mediator, a very large complex involved in regulation of gene transcription by RNA Polymerase II. MED26 regulates the switch between initiation and elongation phases of the transcription. This function requires interaction of its N-terminal domain (NTD) with several protein partners implicated in transcriptional regulation. Molecular details of the structure and interaction mode of MED26 NTD would improve understanding of this complex regulation. As a first step towards structural characterization, sequence specific (1)H, (13)C and (15)N assignments for MED26 NTD was performed based on Nuclear Magnetic Resonance spectroscopy. TALOS+ analysis of the chemical shifts data revealed a domain solely composed of helices. Assignments will be further used to solve NMR structure and dynamics of MED26 NTD and investigate the molecular details of its interaction with protein partners.
Asunto(s)
Complejo Mediador/química , Resonancia Magnética Nuclear Biomolecular , Subunidades de Proteína/química , Secuencia de Aminoácidos , Complejo Mediador/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Subunidades de Proteína/metabolismoRESUMEN
The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38-68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM-MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator-transactivator interactions.
Asunto(s)
Proteínas de Unión al ADN/química , Complejo Mediador/química , Transactivadores/química , Factores de Transcripción/química , Proteínas de Unión al ADN/metabolismo , Complejo Mediador/genética , Complejo Mediador/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transactivadores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Bone marrow contains mesenchymal stem cells (MSCs) that can differentiate along multiple mesenchymal lineages. In this capacity they are thought to be important in the intrinsic turnover and repair of connective tissues while also serving as a basis for tissue engineering and regenerative medicine. However, little is known of the biological responses of human MSCs to inflammatory conditions. When cultured with IL-1ß, marrow-derived MSCs from 8 of 10 human subjects deposited copious hydroxyapatite, in which authenticity was confirmed by Fourier transform infrared spectroscopy. Transmission electron microscopy revealed the production of fine needles of hydroxyapatite in conjunction with matrix vesicles. Alkaline phosphatase activity did not increase in response to inflammatory mediators, but PPi production fell, reflecting lower ectonucleotide pyrophosphatase activity in cells and matrix vesicles. Because PPi is the major physiological inhibitor of mineralization, its decline generated permissive conditions for hydroxyapatite formation. This is in contrast to MSCs treated with dexamethasone, where PPi levels did not fall and mineralization was fuelled by a large and rapid increase in alkaline phosphatase activity. Bone sialoprotein was the only osteoblast marker strongly induced by IL-1ß; thus these cells do not become osteoblasts despite depositing abundant mineral. RT-PCR did not detect transcripts indicative of alternative mesenchymal lineages, including chondrocytes, myoblasts, adipocytes, ligament, tendon, or vascular smooth muscle cells. IL-1ß phosphorylated multiple MAPKs and activated nuclear factor-κB (NF-κB). Certain inhibitors of MAPK and PI3K, but not NF-κB, prevented mineralization. The findings are of importance to soft tissue mineralization, tissue engineering, and regenerative medicine.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Citocinas/farmacología , Durapatita/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/metabolismo , Western Blotting , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/ultraestructura , Calcio/metabolismo , Células Cultivadas , Difosfatos/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Sialoproteína de Unión a Integrina/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/ultraestructura , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Fenotipo , Hidrolasas Diéster Fosfóricas/metabolismo , Fosforilación/efectos de los fármacos , Pirofosfatasas/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
PEA3, ERM and ER81 belong to the PEA3 subfamily of Ets transcription factors and play important roles in a number of tissue-specific processes. Transcriptional activation by PEA3 subfamily factors requires their characteristic amino-terminal acidic transactivation domain (TAD). However, the cellular targets of this domain remain largely unknown. Using ERM as a prototype, we show that the minimal N-terminal TAD activates transcription by contacting the activator interacting domain (ACID)/Prostate tumor overexpressed protein 1 (PTOV) domain of the Mediator complex subunit MED25. We further show that depletion of MED25 disrupts the association of ERM with the Mediator in vitro. Small interfering RNA-mediated knockdown of MED25 as well as the overexpression of MED25-ACID and MED25-VWA domains efficiently inhibit the transcriptional activity of ERM. Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM. Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment. In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Complejo Mediador/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Línea Celular , Proteínas de Unión al ADN/química , Humanos , Complejo Mediador/química , Complejo Mediador/genética , Mutación , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/químicaRESUMEN
The PEA3 (polyoma enhancer activator 3) group members [ERM (ETS-related molecule), ER81 (ETS-related 81) and PEA3] of the Ets transcription factor family are involved in migration and dissemination processes during organogenesis and cancer development. In the present study, we report that the hnRNP (heterogeneous nuclear ribonucleoprotein)-like protein CoAA (Coactivator activator) interacts with the PEA3 group members and modulates their transcriptional activity. We also demonstrate that the CoAA YQ domain, containing tyrosine/glutamine-rich hexapeptide repeats, is necessary for the interaction, whereas the two N-terminal RRMs (RNA recognition motifs) of CoAA are required to enhance transcriptional activity. Finally, we show that CoAA is involved in the migration-enhancing action of PEA3 on MCF7 human cancer cells, suggesting that CoAA might be an important regulator of PEA3 group member activity during metastasis.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/fisiología , Factores de Transcripción/biosíntesis , Activación Transcripcional/fisiología , Animales , Movimiento Celular/genética , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Conejos , Factores de Transcripción/genéticaRESUMEN
MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed ß-barrel with seven strands (Β1-Β7) and three α-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the ß-barrel, defined by strands B4-B7-B6.
Asunto(s)
Complejo Mediador/química , Resonancia Magnética Nuclear Biomolecular/métodos , Secuencia de Aminoácidos , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Bone marrow stromal cells (BMSCs) are the subject of intense research because of their biological properties and potential use for the repair of damaged tissues. Success of BMSC-based therapies, however, relies on a number of methodological improvements, including the establishment of a vascular network providing nutrients and oxygen to the transplanted cells and ensuring their immediate survival and long-term functionality. We described a method to enhance the autocrine expression of angiogenic factors by BMSCs. For this purpose, human BMSCs were treated with desferrioxamine (DFX). No PDGF-BB, VEGF-R1 or -R2 mRNA expression was detected under any of the conditions tested. mRNA and protein expression levels of TGFbeta1 were similar in BMSCs, whether they were exposed to DFX (50 microM) or to control conditions under normoxia for 48 h. In comparison with the results obtained with control conditions under normoxia, exposure of BMSCs to DFX for 48 h resulted in upregulation of bFGF at the protein (26-fold) but not at the mRNA levels and VEGF at both the mRNA (1.5-fold) and protein levels (4.5-fold). In comparison with the results obtained with control conditions under hypoxia, DFX induced a 50% increase in VEGF secretion but led to the same level of hypoxia inducible factor-1alpha protein expression (a transduction factor involved in angiogenic factor expression and known to be activated by DFX). Exposure of BMSCs to DFX resulted in oversecretion of angiogenic factors, suggesting that DFX-treated BMSCs could be used to supply angiogenic factors.
Asunto(s)
Inductores de la Angiogénesis/metabolismo , Células de la Médula Ósea/metabolismo , Deferoxamina/farmacología , Células del Estroma/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Secuencia de Bases , Células de la Médula Ósea/citología , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Humanos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/citologíaRESUMEN
Mesenchymal stem cells (MSCs) have been proposed for the repair of damaged tissue including bone, cartilage, and heart tissue. Upon in vivo transplantation, the MSCs encounter an ischemic microenvironment characterized by reduced oxygen (O2) tension and nutrient deprivation that may jeopardize viability of the tissue construct. The aim of this study was to assess the effects of serum deprivation and hypoxia on the MSC survival rates in vitro. As expanded MSCs are transferred from plastic to a scaffold in most tissue engineering approaches, possibly inducing loss of survival signals from matrix attachments, the effects of a scaffold shift on the MSC survival rates were also assessed. Human MSCs were exposed for 48 hours to (i) a scaffold substrate shift, (ii) serum deprivation, and (iii) O2 deprivation. MSCs were also exposed to prolonged (up to 120 hours) hypoxia associated with serum deprivation. Cell death was assessed by Live/Dead staining and image analysis. The MSC death rates were not affected by the shift to scaffold or 48-hour hypoxia, but increased with fetal bovine serum (FBS) starvation, suggesting that between the two components of ischemia, nutrient deprivation is the stronger factor. Long-term hypoxia combined with serum deprivation resulted in the complete death of MSCs (99 +/- 1%), but this rate was reduced by half when MSCs were exposed to hypoxia in the presence of 10% FBS (51 +/- 31%). These results show that MSCs are sensitive to the concurrent serum and O2 deprivation to which they are exposed when transplanted in vivo, and call for the development of new transplantation methods.
Asunto(s)
Apoptosis/fisiología , Técnicas de Cultivo de Célula/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Oxígeno/metabolismo , Ingeniería de Tejidos/métodos , Anciano , Hipoxia de la Célula/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Mesenchymal stromal cells (MSCs) seeded onto biocompatible scaffolds have been proposed for repairing bone defects. When transplanted in vivo, MSCs (expanded in vitro in 21% O(2)) undergo temporary oxygen deprivation due to the lack of pre-existing blood vessels within these scaffolds. In the present study, the effects of temporary (48 h) exposure to hypoxia (Asunto(s)
Proteínas Angiogénicas/metabolismo
, Hipoxia de la Célula/fisiología
, Células Madre Mesenquimatosas/citología
, Células Madre Mesenquimatosas/metabolismo
, Células Madre Multipotentes/citología
, Células Madre Multipotentes/metabolismo
, Osteogénesis/fisiología
, Células del Estroma/citología
, Células del Estroma/metabolismo
, Proteínas Angiogénicas/genética
, Secuencia de Bases
, Diferenciación Celular
, Células Cultivadas
, Citocinas/genética
, Citocinas/metabolismo
, Cartilla de ADN/genética
, Humanos
, ARN Mensajero/genética
, ARN Mensajero/metabolismo
RESUMEN
In the present study, we show that E2Fs (E2 promoter-binding factors) regulate the expression of ASK-1 (apoptosis signal-regulating kinase 1), which encodes a mitogen-activated protein kinase kinase kinase, also known as MAP3K5. Its mRNA expression is cell-cycle-regulated in human T98G cells released from serum starvation. Moreover, overexpression and RNA interference experiments support the requirement of endogenous E2F/DP (E2F dimerization partner) activity for ASK-1 expression. Characterization of the human ASK-1 promoter demonstrates that the -95/+11 region is critical for E2F-mediated up-regulation. Chromatin immunoprecipitation assays show that E2F1-E2F4 are bound in vivo to the ASK-1 promoter in cycling cells, probably through a non-consensus E2F-binding site located 12 bp upstream of the transcription start site. Mutation of this site completely abolishes the ASK-1 promoter response to E2Fs as well as the E2F1 binding in electrophoretic mobility-shift experiments. Our results indicate that E2Fs modulate the expression of ASK-1 and suggest that some of the cellular functions of ASK-1 may be under the control of E2F transcription factors. Moreover, the up-regulation of ASK-1 may also favour the p53-independent E2F1 apoptotic activity.
Asunto(s)
Factores de Transcripción E2F/fisiología , MAP Quinasa Quinasa Quinasa 5/biosíntesis , Sitios de Unión/genética , Línea Celular , Inmunoprecipitación de Cromatina , Factor de Transcripción E2F1/fisiología , Ensayo de Cambio de Movilidad Electroforética , Femenino , Humanos , Elementos de Nucleótido Esparcido Largo , MAP Quinasa Quinasa Quinasa 5/genética , Regiones Promotoras Genéticas/fisiología , Estructura Terciaria de Proteína , Interferencia de ARN , Factor de Transcripción DP1/fisiología , Regulación hacia ArribaRESUMEN
CONTEXTO: Fibromialgia é uma síndrome reumática caracterizada por dores músculo-esqueléticas difusas e crônicas e sítios dolorosos específicos à palpação, chamados de tender points, freqüentemente associados a fadiga, distúrbios do sono, rigidez matinal e, em alguns casos, dispnéia e ansiedade. Devido ao seu caráter crônico, a síndrome geralmente causa impacto negativo na qualidade de vida dos fibromiálgicos. OBJETIVO: Comparar a eficácia de instrumentos que avaliam a qualidade de vida de fibromiálgicos mensurada pelo Fibromyalgia Impact Questionnaire(FIQ) e pelo Medical Outcomes Study 36-item Short-Form Healthy Survey (SF-36), e a ansiedade avaliada pelo Inventário de Ansiedade Traço-estado (IDATE). TIPO DE ESTUDO: Transversal. LOCAL: Ambulatório de Reumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC/FMUSP). MÉTODOS: Participaram do estudo 80 sujeitos: 40 com fibromialgia (grupo teste) e 40 saudáveis (grupo controle). Três questionários (dois para avaliação de qualidade de vida - FIQ e SF-36 û; e um para ansiedade - IDATE) foram aplicados aos indivíduos dos dois grupos em uma única entrevista. Toda a análise estatística foi realizada utilizando-se o Teste "t" Student e o teste de Correlação de Pearson (r), com significância p < 0,05. Além disso, o teste estatístico Qui-quadrado de Pearson, para homogeneidade, foi usado para comparar o grau de escolaridade entre os grupos teste e controle. RESULTADOS: Os resultados obtidos mostram que houve diferença estatisticamente significante entre os grupos (p = 0,00), indicando que os fibromiálgicos têm pior qualidade de vida e níveis mais altos de ansiedade. A correlação entre os três questionários foi alta (r = 0,90). DISCUSSAO: O impacto negativo na qualidade de vida decorrente da Fibromialgia tem sido relatado em muitos estudos, nos quais os protocolos de avaliação são os principais istrumentos de medida. O FIQ é um instrumento utilizado em vários estudos clínicos para avaliar a função física. Este estudo comprovou a eficiência do FIQ para avaliar o impacto da fibromialgia sobre a qualidade de vida. O SF-36 é menos específico que o FIQ, mas também se mostrou eficiente para a avaliação da qualidade de vida de fibromiálgicos, uma vez que os discrimina dos indivíduos saudáveis. A ansiedade é considerada um ...
Asunto(s)
Humanos , Femenino , Ansiedad/psicología , Fibromialgia/psicología , Inventario de Personalidad/normas , Psicometría/normas , Calidad de Vida , Encuestas y Cuestionarios/normas , Métodos Epidemiológicos , Dimensión del DolorRESUMEN
CONTEXT: Fibromyalgia is a syndrome characterized by chronic, diffuse musculoskeletal pain, and by a low pain threshold at specific anatomical points. The syndrome is associated with other symptoms such as fatigue, sleep disturbance, morning stiffness and anxiety. Because of its chronic nature, it often has a negative impact on patients' quality of life. OBJECTIVE: To assess the quality of life and anxiety level of patients with fibromyalgia. TYPE OF STUDY: Cross-sectional. SETTING: Rheumatology outpatient service of Hospital das Clínicas (Medical School, Universidade de São Paulo). METHODS: This study evaluated 80 individuals, divided between test and control groups. The test group included 40 women with a confirmed diagnosis of fibromyalgia. The control group was composed of 40 healthy women. Three questionnaires were used: two to assess quality of life (FIQ and SF-36) and one to assess anxiety (STAI). They were applied to the individuals in both groups in a single face-to-face interview. The statistical analysis used Student's t test and Pearson's correlation test (r), with a significance level of 95%. Also, the Pearson chi-squared statistics test for homogeneity, with Yates correction, was used for comparing schooling between test and control groups. RESULTS: There was a statistically significant difference between the groups (p = 0.000), thus indicating that fibromyalgia patients have a worse quality of life and higher levels of anxiety. The correlations between the three questionnaires were high (r = 0.9). DISCUSSION: This study has confirmed the efficacy of FIQ for evaluating the impact of fibromyalgia on the quality of life. SF-36 is less specific than FIQ, although statistically significant values were obtained when analyzed separately, STAI showed lower efficacy for discriminating the test group from the control group. The test group showed worse quality of life than did the control group, which was demonstrated by both FIQ and SF-36. Even though STAI was a less efficient instrument, it presented significant results, showing that fibromyalgia patients presented higher levels of anxiety, both on the state and trait scales. Thus, patients with fibromyalgia had higher levels of tension, nervousness, preoccupation and apprehension, and higher propensity towards anxiety. CONCLUSION: The three instruments utilized showed efficiency in evaluating fibromyalgia patients. FIQ was found to be the most efficient instrument for discriminating and assessing the impact of fibromyalgia on their quality of life. It can be concluded that such patients have a worse quality of life and higher levels of anxiety.
Asunto(s)
Ansiedad/psicología , Fibromialgia/psicología , Calidad de Vida , Métodos Epidemiológicos , Femenino , Humanos , Persona de Mediana Edad , Dimensión del Dolor , Inventario de Personalidad/normas , Psicometría/normas , Encuestas y Cuestionarios/normasRESUMEN
Ações para se vencer uma doença dependem, fundamentalmente, do quanto se sabe acerca da mesma. Daí justificam-se os procedimentos adotados ao longo da história contra a tuberculose, outrora bizarros, hoje quimioterapêuticos e profiláticos. A era pré-quimioterápica da tuberculose foi marcadamente sombria, devido às altas taxas de morbidade e de mortalidade. Felizmente, com o advento dos quimioterápicos o quadro epidemiológico da doença mudou, alcançou-se o declínio por volta da década de 50 e a estabilidade até a década de 80. Mas, a tuberculose está de volta, atingindo números crescentes, em função de diversos fatores, entre eles, o descaso público e a falta de vontade política governamental, aumento da migração para e de áreas endêmicas da doença, surgimento de cepas multi-resistentes aos fármacos de uso corrente e, sobretudo da epidemia de infecção pelo HIV...