RESUMEN

The functional maturation status of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has a notable impact upon their use in pharmacological studies, disease modeling, and therapeutic applications. Non-cardiomyocytes (non-CMs) produced in the differentiation process have previously been identified as having an extrinsic influence upon hiPSC-CM development, yet the underlying mechanisms are not fully understood. Herein, we aimed to modulate electrophysiological properties of hiPSC-CMs within co-cultures containing varied proportions of non-CMs and investigate the nature of interactions between these different cell types. Therefore, we sorted cardiac differentiations on day 10 and subsequently replated the cells at ratios of 7:3, 1:1, 3:7, and 1:9 non-CMs to CMs. After a month of co-culture, we evaluated electrophysiological properties through the genetically encoded voltage indicator ArcLight. We ultimately identified that co-cultures with approximately 70%-90% CM purity demonstrated the highest action potential (AP) amplitude and maximum upstroke velocity by day 40 of differentiation, indicative of enhanced electrophysiological maturation, as well as more ventricular-like AP morphologies. Notably, these findings were distinct from those observed for co-cultures of hiPSC-CMs and dermal fibroblasts. We determined that the co-culture phenotypes could not be attributed to paracrine effects of non-CMs due to the inability of conditioned media to recapitulate the observed effects. This led to the further observation of a distinctive expression pattern of connexin 43 (Cx43) at cell-cell interfaces between both CMs and non-CMs. Depletion of Cx43 by short hairpin RNA (shRNA) specifically in the non-CM population within a co-culture environment was able to recapitulate electrophysiological phenotypes of a purer hiPSC-CM population. Collectively, our data demonstrate that abundant non-CM content exerts a significant negative influence upon the electrophysiological maturation of hiPSC-CMs through Cx43-mediated cell-cell-contacts, and thus should be considered regarding the future production of purpose-built hiPSC-CM systems.

Asunto(s)

Potenciales de Acción/fisiología , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Conexina 43/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Células Cultivadas , Conexina 43/genética , Medios de Cultivo Condicionados/farmacología , Fenómenos Electrofisiológicos/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Técnicas de Placa-Clamp

RESUMEN

Induced pluripotent stem cells (iPSCs) offer great promise in the areas of disease modeling, basic research, drug development, and regenerative medicine. Much of their value comes from the fact that they can be used to create otherwise inaccessible cell types, such as cardiomyocytes, which are genetically matched to a patient or any other individual of interest. A consistent issue plaguing the iPSC platform, however, involves excessive variability exhibited in the differentiated products. This includes discrepancies in genetic, epigenetic, and transcriptional features, cell signalling, the cell types produced from cardiac differentiation, and cardiomyocyte functionality. These properties can result from both the somatic source cells and environmental conditions related to the derivation and handling of these cells. Understanding the potential sources of variability, along with determining which factors are most relevant to a given application, are essential in advancing iPSC-based technologies.

Asunto(s)

Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Medicina Regenerativa , Diferenciación Celular , Humanos

RESUMEN

Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high-throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time-consuming, and incompatible with high-throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells' documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419-42-0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of <300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells. Stem Cells Translational Medicine 2017;6:1829-1839.

Asunto(s)

Ensayo de Unidades Formadoras de Colonias/métodos , Etopósido/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Inhibidores de Topoisomerasa/farmacología , Células Cultivadas , Ensayos Clínicos como Asunto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Transcriptoma

RESUMEN

We have previously demonstrated that TGFß Inducible Early Gene-1 (TIEG1), also known as KLF10, plays important roles in mediating skeletal development and homeostasis in mice. TIEG1 has also been identified in clinical studies as one of a handful of genes whose altered expression levels or allelic variations are associated with decreased bone mass and osteoporosis in humans. Here, we provide evidence for the first time that TIEG1 is involved in regulating the canonical Wnt signaling pathway in bone through multiple mechanisms of action. Decreased Wnt signaling in the absence of TIEG1 expression is shown to be in part due to impaired ß-catenin nuclear localization resulting from alterations in the activity of AKT and GSK-3ß. We also provide evidence that TIEG1 interacts with, and serves as a transcriptional co-activator for, Lef1 and ß-catenin. Changes in Wnt signaling in the setting of altered TIEG1 expression and/or activity may in part explain the observed osteopenic phenotype of TIEG1 KO mice as well as the known links between TIEG1 expression levels/allelic variations and patients with osteoporosis.

Asunto(s)

Huesos/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Huesos/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Ligandos , Cloruro de Litio/farmacología , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Cráneo/citología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética

RESUMEN

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability, the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.

Asunto(s)

Diferenciación Celular , ADN Mitocondrial/genética , Variación Genética , Células Madre Pluripotentes Inducidas/fisiología , Respiración de la Célula , ADN Mitocondrial/química , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Miocitos Cardíacos/fisiología , Fenotipo

RESUMEN

BACKGROUND: Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors may desynchronize natural developmental schedules. This study aims to evaluate the effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells. METHODS AND RESULTS: Targeted inclusion and exclusion of reprogramming transgenes (c-MYC, KLF4, OCT4, and SOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system. Pulsed transgene overexpression, before iPS cell differentiation, hindered cardiogenic outcomes. Delayed in counterparts with maintained integrated transgenes, transgene removal enabled proficient differentiation of iPS cells into functional cardiac tissue. Transgene-free iPS cells generated reproducible beating activity with robust expression of cardiac α-actinin, connexin 43, myosin light chain 2a, α/ß-myosin heavy chain, and troponin I. Although operational excitation-contraction coupling was demonstrable in the presence or absence of transgenes, factor-free derivatives exhibited an expedited maturing phenotype with canonical responsiveness to adrenergic stimulation. CONCLUSIONS: A disproportionate stemness load, caused by integrated transgenes, affects the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of nonintegrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics.

Asunto(s)

Células Madre Pluripotentes Inducidas/citología , Transgenes , Actinina/biosíntesis , Animales , Miosinas Cardíacas/biosíntesis , Diferenciación Celular , Separación Celular , Reprogramación Celular , Conexina 43/biosíntesis , Electrofisiología , Fibroblastos/metabolismo , Citometría de Flujo , Técnicas Genéticas , Factor 4 Similar a Kruppel , Ratones , Microscopía Electrónica , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Ligeras de Miosina/biosíntesis , Troponina I/biosíntesis

RESUMEN

Dilated cardiomyopathy (DCM) due to mutations in RBM20, a gene encoding an RNA-binding protein, is associated with high familial penetrance, risk of progressive heart failure and sudden death. Although genetic investigations and physiological models have established the linkage of RBM20 with early-onset DCM, the underlying basis of cellular and molecular dysfunction is undetermined. Modeling human genetics using a high-throughput pluripotent stem cell platform was herein designed to pinpoint the initial transcriptome dysfunction and mechanistic corruption in disease pathogenesis. Tnnt2-pGreenZeo pluripotent stem cells were engineered to knockdown Rbm20 (shRbm20) to determine the cardiac-pathogenic phenotype during cardiac differentiation. Intracellular Ca(2+) transients revealed Rbm20-dependent alteration in Ca(2+) handling, coinciding with known pathological splice variants of Titin and Camk2d genes by Day 24 of cardiogenesis. Ultrastructural analysis demonstrated elongated and thinner sarcomeres in the absence of Rbm20 that is consistent with human cardiac biopsy samples. Furthermore, Rbm20-depleted transcriptional profiling at Day 12 identified Rbm20-dependent dysregulation with 76% of differentially expressed genes linked to known cardiac pathology ranging from primordial Nkx2.5 to mature cardiac Tnnt2 as the initial molecular aberrations. Notably, downstream consequences of Rbm20-depletion at Day 24 of differentiation demonstrated significant dysregulation of extracellular matrix components such as the anomalous overexpression of the Vtn gene. By using the pluripotent stem cell platform to model human cardiac disease according to a stage-specific cardiogenic roadmap, we established a new paradigm of familial DCM pathogenesis as a developmental disorder that is patterned during early cardiogenesis and propagated with cellular mechanisms of pathological cardiac remodeling.

Asunto(s)

Calcio/metabolismo , Cardiomiopatía Dilatada/etiología , Corazón/crecimiento & desarrollo , Proteínas de Unión al ARN/metabolismo , Sarcómeros/patología , Animales , Cardiomiopatía Dilatada/patología , Diferenciación Celular , Línea Celular , Cuerpos Embrioides/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Modelos Biológicos , Células Madre Pluripotentes/metabolismo , Empalme del ARN , Sarcómeros/ultraestructura

RESUMEN

Activation of the Janus kinase family/signal transducer and activator of transcription (JAK/STAT) signaling pathway has been associated with the pathogenesis and progression of both solid and hematologic malignancies. We have detected constitutive activation of STAT5 in malignant B cells derived from patients with Waldenström's macroglobulinemia (WM). Although short hairpin RNA-mediated knockdown of the STAT5A and STAT5B isoforms did not affect cellular proliferation, loss of STAT5 significantly decreased immunoglobulin M (IgM) secretion. A similar dose-dependent inhibition of IgM secretion was observed when WM cell lines were treated with a small molecule inhibitor of STAT5. These data suggest that STAT5 is involved in regulating IgM production in WM and that inhibition of STAT5 may represent a novel therapeutic strategy for lowering IgM levels in WM patients.

Asunto(s)

Inmunoglobulina M/metabolismo , Factor de Transcripción STAT5/genética , Proteínas Supresoras de Tumor/genética , Macroglobulinemia de Waldenström/genética , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , ARN Interferente Pequeño/farmacología , Factor de Transcripción STAT5/metabolismo , Vías Secretoras/efectos de los fármacos , Vías Secretoras/genética , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología , Proteínas Supresoras de Tumor/metabolismo , Macroglobulinemia de Waldenström/inmunología , Macroglobulinemia de Waldenström/metabolismo

RESUMEN

B-cell activating factor-receptor (BAFF-R) is the primary BAFF receptor that is responsible for promoting B-cell development and survival. Malignant B-cells exploit the BAFF/BAFF-R system, and high serum BAFF levels or genetic alterations in BAFF receptors have been found in B-cell cancers. BAFF signaling impacts pro-survival pathways. However, other than nuclear factor-κB2 (NF-κB2), little is known about the specific pathways activated by individual BAFF receptors. Using a novel BAFF-R expression model we have demonstrated that activation of BAFF-R, independent of transmembrane activator and cytophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), can induce phosphorylation of Akt and glycogen synthase kinase 3ß (GSK3ß). Expression of an activated form of BAFF-R also enhanced a pro-survival gene expression pattern, including the novel BAFF-regulated gene Pin1, whose expression was phosphatidyl inositol 3-kinase (PI3K)-dependent. Additionally, we showed that TRAF6 is essential for mediating BAFF-R dependent activation of Akt. Together these data describe a novel role for TRAF6 in BAFF-R-specific activation of the PI3K pathway and provide evidence suggesting a new role for Pin1 in BAFF-R signaling.

Asunto(s)

Linfoma de Células B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Interleucina-4/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor Activador de Células B/farmacología , Línea Celular Tumoral , Activación Enzimática , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Interleucina-4/genética

RESUMEN

While the effect of TGF-ß on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-ß in tumor immunity in B-cell NHL are limited. We found that soluble TGF-ß, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-ß promoted regulatory T (T(reg)) cells by enhancing expression of Foxp3 in CD4(+) T cells and suppressed effector helper T (T(H)) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-ß on T cell differentiation. Furthermore, we found that membrane-bound TGF-ß is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-ß was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-ß receptor. We showed that pretreatment of lymphoma B cells with TGF-ß significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-ß are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.

Asunto(s)

Linfocitos B/citología , Diferenciación Celular , Membrana Celular/metabolismo , Linfoma no Hodgkin/patología , Linfocitos T/citología , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismo , Linfocitos B/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Interleucina-2/biosíntesis , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Solubilidad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología

RESUMEN

Genetic aberrations, including trisomies 3 and 18, and well-defined IGH translocations, have been described in marginal zone lymphomas (MZLs); however, these known genetic events are present in only a subset of cases. Here, we report the cloning of an IGH translocation partner on chromosome X, t(X;14)(p11.4;q32) that deregulates expression of an poorly characterized orphan G-protein-coupled receptor, GPR34. Elevated GPR34 gene expression was detected independent of the translocation in multiple subtypes of non-Hodgkin lymphoma and distinguished a unique molecular subtype of MZL. Increased expression of GPR34 was also detected in tissue from brain tumors and surface expression of GPR34 was detected on human MZL tumor cells and normal immune cells. Overexpression of GPR34 in lymphoma and HeLa cells resulted in phosphorylation of ERK, PKC, and CREB; induced CRE, AP1, and NF-κB-mediated gene transcription; and increased cell proliferation. In summary, these results are the first to identify a role for a GPR34 in lymphoma cell growth, provide insight into GPR34-mediated signaling, identify a genetically unique subset of MZLs that express high levels of GPR34, and suggest that MEK inhibitors may be useful for treatment of GPR34-expressing tumors.

Asunto(s)

Cromosomas Humanos Par 14 , Cromosomas Humanos X , Linfoma de Células B de la Zona Marginal/genética , Receptores Lisofosfolípidos/genética , Translocación Genética , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Puntos de Rotura del Cromosoma , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Orden Génico , Células HeLa , Humanos , Linfoma de Células B de la Zona Marginal/metabolismo , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Receptores Lisofosfolípidos/metabolismo , Factores de Transcripción SOXC/genética , Transducción de Señal

RESUMEN

Cytokines within the tumor microenvironment play an important role in supporting the growth and survival of B-cell malignancies. One such cytokine, IL-21, promotes the growth of myeloma and Hodgkin lymphoma cells while inducing apoptosis in chronic lymphocytic leukemia. However, the biologic significance of IL-21 has not been examined in Waldenstrom macroglobulinemia (WM), a B-cell lymphoma characterized by elevated serum IgM and a lymphoplasmacytic bone marrow infiltrate. We report here on the presence of IL-21 in the bone marrow of patients with WM and have identified activated T cells as the source of this cytokine. We readily detected the IL-21 receptor on malignant WM B cells and show that IL-21 significantly increases both IgM secretion and cellular proliferation of these cells with no effect on viability. IL-21 rapidly induces phosphorylation of STAT3 in WM cells, and treatment of the WM cell line MWCL-1 with a STAT3 inhibitor abolished the IL-21-mediated increases in cellular proliferation and IgM secretion. IL-21 also increased the expression of known STAT3 targets involved in B-cell differentiation, including BLIMP-1, XBP-1, IL-6, and IL-10. Overall, our data indicate that IL-21 in the bone marrow microenvironment significantly affects the biology of WM tumor cells through a STAT3-dependent mechanism.

Asunto(s)

Proliferación Celular , Inmunoglobulina M/biosíntesis , Interleucinas/metabolismo , Microambiente Tumoral/inmunología , Macroglobulinemia de Waldenström/metabolismo , Macroglobulinemia de Waldenström/patología , Médula Ósea/inmunología , Médula Ósea/metabolismo , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Immunoblotting , Inmunoglobulina M/inmunología , Inmunohistoquímica , Interleucinas/inmunología , Activación de Linfocitos/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Macroglobulinemia de Waldenström/inmunología

RESUMEN

Lymphoid enhancer-binding factor (Lef) 1 is a high mobility group protein best known as a Wnt-responsive transcription factor that associates with ß-catenin. Lef1ΔN is a short isoform of Lef1 that lacks the first 113 amino acids and a well characterized high affinity ß-catenin binding domain present in the full-length protein. Both Lef1 isoforms bind DNA and regulate gene expression. We previously reported that Lef1 is expressed in proliferating osteoblasts and blocks osteocalcin expression. In contrast, Lef1ΔN is only detectable in the later stages of osteoblast differentiation and promotes osteogenesis in vitro. Here, we show that Lef1ΔN retains the ability to interact physically and functionally with ß-catenin. Unlike what has been reported in T cells and colon cancer cell lines, Lef1ΔN activated gene transcription in the absence of exogenous ß-catenin and cooperated with constitutively active ß-catenin to stimulate gene transcription in mesenchymal and osteoblastic cells. Residues at the N terminus of Lef1ΔN were required for ß-catenin binding and the expression of osteoblast differentiation genes. To determine the role of Lef1ΔN on bone formation in vivo, a Lef1ΔN transgene was expressed in committed osteoblasts using the 2.3-kb fragment of the type 1 collagen promoter. The Lef1ΔN transgenic mice had higher trabecular bone volume in the proximal tibias and L5 vertebrae. Histological analyses of tibial sections revealed no differences in osteoblast, osteoid, or osteoclast surface areas. However, bone formation and mineral apposition rates as well as osteocalcin levels were increased in Lef1ΔN transgenic mice. Together, our data indicate that Lef1ΔN binds ß-catenin, stimulates Lef/Tcf reporter activity, and promotes terminal osteoblast differentiation.

Asunto(s)

Diferenciación Celular/fisiología , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Osteoblastos/metabolismo , Osteogénesis/fisiología , beta Catenina/metabolismo , Animales , Células COS , Chlorocebus aethiops , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Factor de Unión 1 al Potenciador Linfoide/genética , Ratones , Ratones Transgénicos , Tamaño de los Órganos/fisiología , Osteoblastos/citología , Osteocalcina/biosíntesis , Osteocalcina/genética , Regiones Promotoras Genéticas/fisiología , Unión Proteica/fisiología , Isoformas de Proteínas , Estructura Terciaria de Proteína , Columna Vertebral/citología , Columna Vertebral/metabolismo , Tibia/citología , Tibia/metabolismo , beta Catenina/genética

RESUMEN

Histone deacetylase (Hdac) inhibitors are used clinically to treat cancer and epilepsy. Although Hdac inhibition accelerates osteoblast maturation and suppresses osteoclast maturation in vitro, the effects of Hdac inhibitors on the skeleton are not understood. The purpose of this study was to determine how the pan-Hdac inhibitor, suberoylanilide hydroxamic acid (SAHA; a.k.a. vorinostat or Zolinza(TM)) affects bone mass and remodeling in vivo. Male C57BL/6J mice received daily SAHA (100mg/kg) or vehicle injections for 3 to 4weeks. SAHA decreased trabecular bone volume fraction and trabecular number in the distal femur. Cortical bone at the femoral midshaft was not affected. SAHA reduced serum levels of P1NP, a bone formation marker, and also suppressed tibial mRNA levels of type I collagen, osteocalcin and osteopontin, but did not alter Runx2 or osterix transcripts. SAHA decreased histological measures of osteoblast number but interestingly increased indices of osteoblast activity including mineral apposition rate and bone formation rate. Neither serum (TRAcP 5b) nor histological markers of bone resorption were affected by SAHA. P1NP levels returned to baseline in animals which were allowed to recover for 4weeks after 4weeks of daily SAHA injections, but bone density remained low. In vitro, SAHA suppressed osteogenic colony formation, decreased osteoblastic gene expression, induced cell cycle arrest, and caused DNA damage in bone marrow-derived adherent cells. Collectively, these data demonstrate that bone loss following treatment with SAHA is primarily due to a reduction in osteoblast number. Moreover, these decreases in osteoblast number can be attributed to the deleterious effects of SAHA on immature osteoblasts, even while mature osteoblasts are resistant to the harmful effects and demonstrate increased activity in vivo, indicating that the response of osteoblasts to SAHA is dependent upon their differentiation state. These studies suggest that clinical use of SAHA and other Hdac inhibitors to treat cancer, epilepsy or other conditions may potentially compromise skeletal structure and function.

Asunto(s)

Resorción Ósea/inducido químicamente , Diferenciación Celular/efectos de los fármacos , Ácidos Hidroxámicos/efectos adversos , Ácidos Hidroxámicos/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Acetilación/efectos de los fármacos , Animales , Biomarcadores/sangre , Peso Corporal/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Remodelación Ósea/efectos de los fármacos , Resorción Ósea/sangre , Recuento de Células , Ciclo Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Daño del ADN , Fémur/efectos de los fármacos , Fémur/metabolismo , Fémur/patología , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Procolágeno/metabolismo , Factores de Tiempo , Vorinostat

RESUMEN

Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.

Asunto(s)

Adipogénesis/fisiología , Densidad Ósea/fisiología , Células de la Médula Ósea/citología , Histona Desacetilasas/metabolismo , Osteogénesis/fisiología , Células Madre/citología , Células Madre/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis/genética , Animales , Western Blotting , Densidad Ósea/genética , Células de la Médula Ósea/metabolismo , Genotipo , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Histona Desacetilasas/genética , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteogénesis/genética , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp7 , Factores de Transcripción/genética , Microtomografía por Rayos X

RESUMEN

Lymphoid Enhancer Binding Factor (Lef) 1 is a transcriptional effector of the Wnt/Lrp5/beta-catenin signaling cascade, which regulates osteoblast differentiation, bone density, and skeletal strength. In this study, we describe the expression and function of an alternative Lef1 isoform in osseous cells. Lef1DeltaN is a naturally occurring isoform driven by a promoter (p2) within the intron between exons 3 and 4 of Lef1. Lef1DeltaN is induced during late osteoblast differentiation. This is converse to the expression pattern of the full-length Lef1 protein, which as we previously showed, decreases during differentiation. Agonists of osteoblast maturation differentially affected Lef1DeltaN expression. BMP2 stimulated Lef1DeltaN expression, whereas Wnt3a blocked basal and BMP2-induced expression of Lef1DeltaN transcripts during osteoblast differentiation. We determined that the Lef1DeltaN p2 promoter is active in osteoblasts and Runx2 regulates its activity. Stable overexpression of Lef1DeltaN in differentiating osteoblasts induced the expression of osteoblast differentiation genes, osteocalcin and type 1 collagen. Taken together, our results suggest Lef1DeltaN is a crucial regulator of terminal differentiation in osseous cells.

Asunto(s)

Empalme Alternativo , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Factor de Unión 1 al Potenciador Linfoide/genética , Osteoblastos/citología , Osteoblastos/metabolismo , Empalme Alternativo/genética , Animales , Northern Blotting , Diferenciación Celular/genética , Línea Celular , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Osteocalcina/genética , Osteocalcina/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3A

RESUMEN

Bone is one of the few tissues in the body with the capacity to regenerate and repair itself. Fractures usually are completely repaired in a relatively short time, but in a small percentage of cases, healing never occurs and nonunion is the result. Fracture repair and bone regeneration require the localized reactivation of signaling cascades that are crucial for skeletal development. The Wnt/beta-catenin signaling pathway is one such developmental pathway whose role in bone formation and regeneration recently has been appreciated. During the past decade, much has been learned about how Wnt pathways regulate bone mass. Small molecules and biologics aimed at this pathway are now being tested as potential new anabolic agents. This article reviews recent data demonstrating that Wnt pathways are active during fracture repair and that increasing the activities of Wnt pathway components accelerates bone regeneration.

Asunto(s)

Curación de Fractura/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/fisiología , Regeneración Ósea/fisiología , Fracturas Óseas/fisiopatología , Humanos , beta Catenina/fisiología

RESUMEN

BACKGROUND: There is a need to develop new bone anabolic agents because current bone regeneration regimens have limitations. The Wingless-type MMTV integration site (Wnt) pathway has emerged as a regulator of bone formation and regeneration. OBJECTIVE: To review the molecular basis for Wnt pathway modulation and discuss strategies that target it and improve bone mass. METHODS: Data in peer-reviewed reports and meeting abstracts are discussed. RESULTS/CONCLUSIONS: Neutralizing inhibitors of Wnt signaling have emerged as promising strategies. Small-molecule inhibitors of glycogen synthase kinase 3beta increase bone mass, lower adiposity and reduce fracture risk. Neutralizing antibodies to Dickkopf 1, secreted Frizzled-related protein 1 and sclerostin produce similar outcomes in animal models. These drugs are exciting breakthroughs but are not without risks. The challenges include tissue-specific targeting and consequently, long-term safety.

Asunto(s)

Enfermedades Óseas/tratamiento farmacológico , Osteoblastos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Animales , Enfermedades Óseas/fisiopatología , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/fisiología , Resorción Ósea/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Fracturas Óseas/tratamiento farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos/fisiología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Humanos , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/fisiología , Proteínas Relacionadas con Receptor de LDL/deficiencia , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/fisiología , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Especificidad de Órganos , Osteoblastos/fisiología , Osteogénesis/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Regeneración/fisiología , Proteínas Wnt/fisiología , beta Catenina/biosíntesis , beta Catenina/genética

RESUMEN

The actions of 17beta-estradiol (E2) and selective estrogen receptor modulators (SERMs) have been extensively investigated regarding their ability to act through estrogen receptor-alpha (ERalpha) to perturb estrogen receptor positive (ER+) breast cancer (BC) growth. However, many BCs also express ERbeta, along with multiple estrogen receptor (ER) splice variants such as ERbetacx, an ERbeta splice variant incapable of binding ligand. To gain a more comprehensive understanding of ER action in BC cells, we stably expressed ERalpha, ERbeta, or ERbetacx under doxycycline (Dox) control in Hs578T cells. Microarrays performed on E2 or 4OH-tamoxifen (4HT) treated Hs578T ERalpha and ERbeta cells revealed distinct ligand and receptor-dependent patterns of gene regulation, while the induction of ERbetacx did not alter gene expression patterns. E2 stimulation of Hs578T ERbeta cells resulted in a 27% decrease in cellular proliferation, however, no significant change in proliferation was observed following the exposure of Hs578T ERalpha or ERbeta cells to 4HT. Expression of ERbetacx in Hs578T cells did not effect cellular proliferation. Flow cytometry assays revealed a 50% decrease in E2-stimulated Hs578T ERbeta cells entering S-phase, along with a 17% increase in G0/G1 cell-cycle arrest. We demonstrate here that ERalpha and ERbeta regulate unique gene expression patterns in Hs578T cells, and such regulation likely is responsible for the observed isoform-specific changes in cell proliferation. Hs578T ER expressing cell-lines provide a unique BC model system, permitting the comparison of ERalpha, ERbeta, and ERbetacx actions in the same cell-line.

Asunto(s)

Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxiciclina/farmacología , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Relacionados con las Neoplasias , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología

RESUMEN

Estrogen (E2) is involved in mediating many important functions relevant to osteoblast biology through the actions of the estrogen receptors (ER) alpha and beta. To further understand the mechanisms of ER-specific regulation, we used microarray and reverse transcription-PCR analyses of E2-treated U2OS-ERalpha or -ERbeta cells and identified retinoblastoma-binding protein 1 (RBBP1) as a major E2-regulated gene. RBBP1 is a retinoblastoma cofactor involved in the control of osteoblastic proliferation. Although RBBP1 mRNA levels rapidly increased after 2 h of E2 treatment in both U2OS-ER-expressing lines, a sustained induction was only observed in U2OS-ERalpha cells. Examination of the RBBP1 genomic sequence revealed an ER response element and a Sp1 site located within the first intron. Chromatin immunoprecipitation analyses demonstrated that E2-dependent ERalpha binding to the intron 1 enhancer region was constitutive, whereas ERbeta binding was transient, consistent with the mRNA time course. Interestingly, transient transfection and receptor mutational studies revealed that RBBP1 induction by ERalpha only requires the Sp1 site, whereas ERbeta utilizes both the Sp1 and estrogen response elements binding sites for maximal E2-dependent activation. Stable U2OS transfectants containing a deletion of the ERalpha activation function 1 (AF1) resulted in a temporal mRNA induction profile similar to that of wild type ERbeta. Further, overexpression and chromatin immunoprecipitation analyses also demonstrated that E2-dependent RBBP1 induction is SRC2-dependent for both ER isoforms. These results describe an E2-dependent, ER isoform-specific transcriptional activation of the RBBP1 gene, which in part, is explained by the differential activity of ER AF1 and enhancer element binding.

Asunto(s)

Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Receptores de Estrógenos/química , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Receptor alfa de Estrógeno/metabolismo , Humanos , Datos de Secuencia Molecular , Osteoblastos/metabolismo , Isoformas de Proteínas , Estructura Terciaria de Proteína , Receptores de Estrógenos/metabolismo , Proteína 1 de Unión a Retinoblastoma