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1.
Exp Cell Res ; 350(1): 284-297, 2017 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-27964926

RESUMEN

Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic ß-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/ß-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice.


Asunto(s)
Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Huesos/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Ácido Retinoico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Tretinoina/farmacología
2.
Oncogene ; 35(40): 5282-5294, 2016 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-27041566

RESUMEN

Osteosarcoma (OS) is the most common cancer of bone and the 5th leading cause of cancer-related death in young adults. Currently, 5-year survival rates have plateaued at ~70% for patients with localized disease. Those with disseminated disease have an ~20% 5-year survival. An improved understanding of the molecular genetics of OS may yield new approaches to improve outcomes for OS patients. To this end, we applied murine models that replicate human OS to identify and understand dysregulated microRNAs (miRNAs) in OS. miRNA expression patterns were profiled in murine primary osteoblasts, osteoblast cultures and primary OS cell cultures (from primary and paired metastatic locations) isolated from two genetically engineered murine models of OS. The differentially expressed miRNA were further assessed by a cross-species comparison with human osteoblasts and OS cultures. We identified miR-155-5p and miR-148a-3p as deregulated in OS. miR-155-5p suppression or miR-148a-3p overexpression potently reduced proliferation and induced apoptosis in OS cells, yet strikingly, did not impact normal osteoblasts. To define how these miRNAs regulated OS cell fate, we used an integrated computational approach to identify putative candidate targets and then correlated these with the cell biological impact. Although we could not resolve the mechanism through which miR-148a-3p impacts OS, we identified that miR-155-5p overexpression suppressed its target Ripk1 (receptor (TNFRSF)-interacting serine-threonine kinase 1) expression, and miR-155-5p inhibition elevated Ripk1 levels. Ripk1 is directly involved in apoptosis/necroptosis. In OS cells, small interfering RNA against Ripk1 prevented cell death induced by the sequestration of miR-155-5p. Collectively, we show that miR-148a-3p and miR-155-5p are species-conserved deregulated miRNA in OS. Modulation of these miRNAs was specifically toxic to tumor cells but not normal osteoblasts, raising the possibility that these may be tractable targets for miRNA-based therapies for OS.


Asunto(s)
MicroARNs/biosíntesis , Osteosarcoma/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Animales , Apoptosis/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , MicroARNs/genética , Osteosarcoma/patología
3.
Leukemia ; 27(6): 1369-80, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23228968

RESUMEN

All-trans retinoic acid (ATRA) is used successfully in the treatment of acute promyelocytic leukemia (APL). ATRA enhances hematopoietic stem cell self-renewal through retinoic acid receptor (RAR)γ activation while promoting differentiation of committed myeloid progenitors through RARα activation. Its lack of success in the treatment of non-APL acute myeloid leukemia (AML) may be related to ATRA's non-selectivity for the RARα and RARγ isotypes, and specific RARα activation may be more beneficial in promoting myeloid differentiation. To investigate this hypothesis, the effects of ATRA and the specific RARα agonist NRX195183 was assessed in AML1-ETO (AE)-expressing murine bone marrow (BM) progenitors. ATRA potentiated the in vitro clonogenicity of these cells while NRX195183 had the opposite effect. Morphological and flow cytometric analysis confirmed a predominantly immature myeloid population in the ATRA-treated AE cells while the NRX195183-treated cells demonstrated an increase in the mature myeloid population. Similarly, NRX195183 treatment promoted myeloid differentiation in an AE9a in vivo murine model. In the ATRA-treated AE cells, gene expression analyses revealed functional networks involving SERPINE1 and bone morphogenetic protein 2; AKT phosphorylation was upregulated. Collectively, these findings confirm the contrasting roles of specific RARα and RARγ activation in the clonogenicity and differentiation of AE cells with potential significant implications in the treatment of non-APL AML using a specific RARα agonist.


Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas de Fusión Oncogénica/genética , Receptores de Ácido Retinoico/agonistas , Tretinoina/farmacología , Animales , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Proteína 1 Compañera de Translocación de RUNX1 , Receptor alfa de Ácido Retinoico
4.
Blood Cancer J ; 2: e91, 2012 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-22983434

RESUMEN

In the adult mammal, normal haematopoiesis occurs predominantly in the bone marrow, where primitive haematopoietic stem cells (HSC) and their progeny reside in specialised microenvironments. The bone marrow microenvironment contains specific anatomical areas (termed niches) that are highly specialised for the development of certain blood cell types, for example HSCs. The HSC niche provides important cell-cell interactions and signalling molecules that regulate HSC self-renewal and differentiation processes. These same signals and interactions are also important in the progression of haematological malignancies, such as multiple myeloma (MM). This review provides an overview of the bone marrow microenvironment and its involvement in normal, physiological HSC maintenance and plasma cell growth throughout MM disease progression.

6.
Blood ; 94(2): 483-95, 1999 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-10397716

RESUMEN

All-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. In this study, we investigated the effect of ATRA on highly enriched murine hematopoietic precursor cells (lin-c-kit+Sca-1(+)) grown in liquid suspension culture for 28 days. ATRA initially slowed the growth of these hematopoietic precursors but prolonged and markedly enhanced their colony-forming cell production compared with the hematopoietic precursors cultured in its absence. At 7 and 14 days of culture, a substantially greater percentage of cells cultured with ATRA did not express lineage-associated antigens (55.4% at day 7 and 68.6% at day 14) and retained expression of Sca-1 (44.7% at day 7 and 79.9% at day 14) compared with cells grown in its absence (lin- cells: 31.5% at day 7 and 4% at day 14; Sca-1(+): 10.4% at day 7 and 0.7% at day 14). Moreover, a marked inhibition of granulocyte production was observed in cultures continuously incubated with ATRA. Significantly, ATRA markedly prolonged and enhanced the production of transplantable colony-forming unit-spleen (CFU-S) during 14 days of liquid suspension culture. In contrast with its effects on primitive lin-c-kit+Sca-1(+) hematopoietic precursors, ATRA did not exert the same effects on the more committed lin-c-kit+Sca-1(-) progenitor cells. Moreover, the late addition of ATRA (7 days post-culture initiation) to cultures of primitive hematopoietic precursors resulted in a marked decrease in colony-forming cell production in these cultures, which was associated with enhanced granulocyte differentiation. These observations indicate that ATRA has different effects on hematopoietic cells depending on their maturational state, preventing and/or delaying the differentiation of primitive hematopoietic precursors while enhancing the terminal differentiation of committed granulocyte/monocyte progenitors.


Asunto(s)
Granulocitos/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Monocitos/citología , Tretinoina/farmacología , Animales , Antígenos Ly/análisis , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Femenino , Células Madre Hematopoyéticas/citología , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-kit/análisis , Organismos Libres de Patógenos Específicos , Factores de Tiempo
7.
Blood ; 87(5): 1802-8, 1996 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-8634426

RESUMEN

Single-cell suspensions of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) cultured in alpha minimal essential medium (alphaMEM) containing 10% fetal bovine serum formed multicellular aggregates within 24 hours. In six separate experiments, formation of aggregates appeared to be dependent on cell density per surface area, so that 5.8 +/- 1.3 aggregates formed per 1 x 10(5) cells when G-PBMC were cultured at densities greater than or equal to 1 x 10(5) cells/cm2. The frequency of aggregate formation was less than 1 per 10(5) cells when G-PBMC were cultured at densities less than 1 x 10(5) cells/cm2. Once formed, aggregates became adherent within 72 hours, and then, over the course of 21 days, released CD3/CD4/CD25-positive cells into the supernatant. This T-cell production peaked between days 7 and 14, reaching a total of 1,269 +/- 125.9 cells released per aggregate by day 21. Between days 14 and 21, the aggregates also generated macroscopic clusters of adherent mononuclear and giant multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). At 4 weeks, the macroscopic foci coalesced into monolayers. Multinucleated TRAP-positive cells were distinguished from macrophage polykaryons by the absence of CD14 expression and the presence of osteoclast-specific membrane receptors for calcitonin and alphavbeta3-vitronectin. The osteoclast nature of these cells was further demonstrated by their ability to form resorption lacunae on dentine slices. Comparable osteoclast formation was not detected in cultures of normal marrow or normal nonmobilized peripheral blood.


Asunto(s)
Factor Estimulante de Colonias de Granulocitos/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Osteoclastos/citología , Receptores de Calcitonina/biosíntesis , Fosfatasa Ácida/análisis , Animales , Biomarcadores , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Bovinos , Agregación Celular , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colorantes , Dentina/metabolismo , Humanos , Isoenzimas/análisis , Especificidad de Órganos , Receptores de Vitronectina/biosíntesis , Fosfatasa Ácida Tartratorresistente , Resorción Dentaria
8.
Blood ; 95(2): 470-7, 2000 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-10627451

RESUMEN

The retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. We previously reported that pharmacologic levels (1 micromol) of ATRA enhanced the generation of colony-forming cell (CFC) and colony-forming unit-spleen (CFU-S) in liquid suspension cultures of lin- c-kit+ Sca-1+ murine hematopoietic precursors. In this study, we further investigated the effects of ATRA as well as an RAR antagonist, AGN 193109, on the generation of transplantable cells, including pre-CFU-S, short-term repopulating stem cells (STRCs), and long-term repopulating stem cells (LTRCs). ATRA enhanced the ex vivo maintenance and production of competitive repopulating STRCs and LTRCs from lin- c-kit+ Sca-1+ cells cultured in liquid suspension for 14 days. In addition, ATRA prevented the differentiation of these primitive stem cells into more mature pre-CFU-S during the 14 days of culture. In marked contrast, lin- c-kit+ Sca-1+ cells cultured with AGN 193109 for 7 days had virtually no short- or long-term repopulating ability, but displayed an approximately 6-fold increase in the pre-CFU-S population. The data suggest that the RAR agonist ATRA enhances the maintenance and self-renewal of short- and long-term repopulating stem cells. In contrast, the RAR antagonist AGN 193109 abrogates reconstituting ability, most likely by promoting the differentiation of the primitive stem cells. These results imply an important and unexpected role of retinoids in regulating hematopoietic stem cell differentiation. (Blood. 2000;95:470-477)


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Tretinoina/farmacología , Animales , Antígenos Ly/análisis , Trasplante de Médula Ósea/fisiología , División Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Femenino , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Naftalenos/farmacología , Proteínas Proto-Oncogénicas c-kit/análisis , Receptores de Ácido Retinoico/antagonistas & inhibidores
9.
Blood ; 98(8): 2382-8, 2001 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-11588034

RESUMEN

The transcription factor C/EBPalpha is an important mediator of granulocyte differentiation and regulates the expression of multiple granulocyte-specific genes including the granulocyte-colony-stimulating factor (G-CSF) receptor, neutrophil elastase, and myeloperoxidase. Indeed C/EBPalpha knockout mice display a profound block in granulocyte differentiation. To study this block in granulocytic differentiation in more detail, retroviral vector-mediated transduction of a dominant-negative retinoic acid receptor was used to establish hematopoietic growth factor-dependent, lympho-myeloid progenitor cell lines from the fetal livers of both the C/EBPalpha knockout animals (C/EBPalpha(-/-)) and their heterozygous littermates (C/EBPalpha(+/-)). Surprisingly, the C/EBPalpha(-/-) cell lines displayed significant spontaneous granulocytic differentiation, and this differentiation was markedly enhanced when the cells were stimulated with granulocyte macrophage (GM)-CSF. This GM-CSF-mediated differentiation was associated with the up-regulation of G-CSF receptor mRNA, and the combination of GM-CSF and G-CSF generated more than 95% mature neutrophils in the C/EBPalpha(-/-) cultures. The addition of all-trans retinoic acid also enhanced this granulocytic differentiation of the cultured C/EBPalpha(-/-) cells, indicating that the activated retinoic acid receptors can enhance granulocytic differentiation through a molecular pathway that is independent of C/EBPalpha. These studies clearly indicate that terminal granulocytic differentiation associated with the up-regulation of C/EBPalpha-responsive genes can occur in the absence of C/EBPalpha, and they indicate the existence of multiple independent molecular pathways potentially used by primitive hematopoietic precursors that can lead to the development of mature granulocytes.


Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Diferenciación Celular/fisiología , Factor Estimulante de Colonias de Granulocitos/sangre , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Granulocitos/citología , Animales , Northern Blotting , Proteína alfa Potenciadora de Unión a CCAAT/deficiencia , Proteína alfa Potenciadora de Unión a CCAAT/genética , Línea Celular , Genotipo , Hígado/citología , Hígado/embriología , Ratones , Ratones Noqueados , ARN/sangre , ARN/genética , ARN/aislamiento & purificación , Valores de Referencia , Retroviridae/genética , Regulación hacia Arriba , Integración Viral
10.
J Hematother Stem Cell Res ; 10(6): 815-25, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11798508

RESUMEN

A major limiting factor in achieving high levels of gene transfer into hematopoietic stem cells is the ability to retain significant repopulating activity of the stem cells during the ex vivo exposure to oncoretroviral vectors. Recently, we reported that pharmacological levels (1 microM) of all-trans retinoic acid (ATRA) enhanced the maintenance of in vivo repopulating hematopoietic stem cells during liquid suspension culture. Therefore, we investigated the use of ATRA to improve transduction of hematopoietic repopulating cells. Hematopoietic precursors cultured and transduced with a GFP-containing oncoretroviral vector with or without ATRA were transplanted immediately post-transduction (day 3 post-culture initiation) or following extended culture without further transduction (day 7 post-culture initiation). Mice transplanted with 3-day ATRA-treated cells had four-fold more donor cells than the untreated cells. In contrast, there were more GFP-expressing donor cells in recipients of cells cultured without ATRA (31.31 +/- 8.47% no ATRA vs. 16.52 +/- 9.35% ATRA). After 7 days of culture, however, the repopulating ability of the hematopoietic precursors was the same for both treatment groups, but the ATRA-treated cells had significantly more green fluorescence protein (GFP)-expressing donor cells (5.57 +/- O.53% no ATRA vs. 13.67 +/- 2.14% ATRA). Secondary recipients of marrow from recipients of the 3 day cultured cells had similar donor cell levels, but the percentage of GFP-expressing cells within the donor cell population was higher in the recipients of ATRA-treated cells (3.25 +/- 0.70% no ATRA vs. 7.97 +/- 2.71% ATRA). Our data show that the addition of ATRA to cultures of hematopoietic precursors resulted in increased gene transfer into murine hematopoietic repopulating cells. These data suggest that ATRA may be useful in clinical gene therapy protocols using oncoretroviral vectors.


Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Transducción Genética/métodos , Tretinoina/farmacología , Animales , Técnicas de Cultivo de Célula , Femenino , Vectores Genéticos , Supervivencia de Injerto , Proteínas Fluorescentes Verdes , Cinética , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Retroviridae/genética , Trasplante de Células Madre/métodos
11.
Blood ; 91(11): 4084-91, 1998 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-9596653

RESUMEN

We examined the expression of two members of the Notch family, Notch-1 and Notch-2, and one Notch ligand, Jagged-1, in hematopoietic cells. Both Notch-1 and Notch-2 were detected in murine marrow precursors (Lin-Sca-1+c-kit+). The Notch ligand, Jagged-1, was not detected in whole marrow or in precursors. However, Jagged-1 was seen in cultured primary murine fetal liver stroma, cultured primary murine bone marrow stroma, and in stromal cell lines. These results indicate a potential role for Notch-Notch ligand interactions in hematopoiesis. To further test this possibility, the effect of Jagged-1 on murine marrow precursor cells was assessed by coculturing sorted precursor cells (Lin-Sca-1+c-kit+) with a 3T3 cell layer that expressed human Jagged-1 or by incubating sorted precursors with beads coated with the purified extracellular domain of human Jagged-1 (Jagged-1(ext)). We found that Jagged-1, presented both on the cell surface and on beads, promoted a twofold to threefold increase in the formation of primitive precursor cell populations. These results suggest a potential use for Notch ligands in expanding precursor cell populations in vitro.


Asunto(s)
Células Madre Hematopoyéticas/citología , Proteínas de la Membrana/biosíntesis , Factores de Transcripción , Células 3T3 , Animales , Proteínas de Unión al Calcio , Diferenciación Celular , Fibroblastos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-1 , Ligandos , Proteínas de la Membrana/metabolismo , Ratones , Unión Proteica , Receptor Notch1 , Receptor Notch2 , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/metabolismo , Proteínas Serrate-Jagged , Transfección
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