RESUMEN
A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.
Asunto(s)
Citomegalovirus/fisiología , Células Epiteliales/metabolismo , Complejos Multiproteicos/metabolismo , Transducción de Señal , Proteínas Virales/metabolismo , Tropismo Viral/fisiología , Células A549 , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Epiteliales/patología , Células Epiteliales/virología , Células HEK293 , Células HeLa , Humanos , Complejos Multiproteicos/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Virales/genéticaRESUMEN
Streptococcus pneumoniae commonly resides asymptomatically in the nasopharyngeal (NP) cavity of healthy individuals but can cause life-threatening pulmonary and systemic infections, particularly in the elderly. NP colonization results in a robust immune response that protects against invasive infections. However, the duration, mechanism, and cellular component of such responses are poorly understood. In this study, we found that repeated NP exposure of mice to S. pneumoniae TIGR4 strain results in pneumococcal-specific Ab responses that protect against lethal lung challenge. Abs were necessary and sufficient for protection because Ab-deficient µMT mice did not develop postexposure protection, only becoming resistant to lung infection after transfer of immune sera from NP-exposed mice. T cells contributed to immunity at the time of NP exposure, but neither CD4+ nor CD8+ T cells were required. The protective activity was detectable 20 wk after exposure and was maintained in irradiated mice, suggesting involvement of long-lived Ab-secreting cells (ASC), which are radioresistant and secrete Abs for extended periods of time in the absence of T cells or persistent Ag. CD138+ bone marrow cells, likely corresponding to long-lived ASC, were sufficient to confer protection. NP exposure of aged mice failed to protect against subsequent lung infection despite eliciting a robust Ab response. Furthermore, transfer of CD138+ bone marrow cells or sera from NP-exposed old mice failed to protect naive young mice. These findings suggest that NP exposure elicits extended protection against pneumococcal lung infection by generating long-lived CD138+ ASC and that the protective efficacy of these responses declines with age.
RESUMEN
Alcoholic liver disease results from a combination of immune and metabolic pathogenic events. In addition to liver injury, chronic alcohol consumption also causes adipose tissue inflammation. The specific immune mechanisms that drive this process are unknown. Here, we sought to determine the role of the innate immune receptor Toll-like receptor 4 (TLR4) in alcohol-induced adipose tissue inflammation. Using a model of chronic, multiple-binge alcohol exposure, we showed that alcohol-mediated accumulation of proinflammatory adipose tissue macrophages was absent in global TLR4 knockout mice. Proinflammatory macrophage accumulation did not depend on macrophage TLR4 expression; LysMCre-driven deletion of Tlr4 from myeloid cells did not affect circulating endotoxin or the accumulation of M1 macrophages in adipose tissue following alcohol exposure. Proinflammatory cytokine/chemokine production in the adipose stromal vascular fraction also occurred independently of TLR4. Finally, the levels of other adipose immune cells, such as dendritic cells, neutrophils, B cells, and T cells, were modulated by chronic, multiple-binge alcohol and the presence of TLR4. Together, these data indicate that TLR4 expression on cells, other than myeloid cells, is important for the alcohol-induced increase in proinflammatory adipose tissue macrophages.
Asunto(s)
Adipocitos/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Etanol/farmacología , Macrófagos/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Macrófagos/metabolismo , Ratones Transgénicos , Obesidad/tratamiento farmacológico , Obesidad/metabolismoRESUMEN
The germinal center (GC) reaction produces high-affinity Abs for a robust adaptive immune response. When dysregulated, the same processes cause GC B cells to become susceptible to lymphomagenesis. It is important to understand how the GC reaction is regulated. In this study, we show that transcription factor YY1 is required to maintain a robust GC reaction in mice. Selective ablation of YY1 significantly decreased in the frequency and number of GC B cells during the GC reaction. This decrease of GC B cells was accompanied by increased apoptosis in these cells. Furthermore, we found that loss of YY1 disrupted the balance between dark zones and light zones, leading to a preferential decrease in dark zone cells. Collectively, these results indicate that YY1 plays an important role in regulating the balance between dark zone and light zone cells in GCs and between survival and death of GC B cells.
Asunto(s)
Apoptosis , Linfocitos B/inmunología , Linfocitos B/fisiología , Regulación de la Expresión Génica , Centro Germinal/inmunología , Factor de Transcripción YY1/fisiología , Animales , Centro Germinal/citología , Ratones , Factor de Transcripción YY1/deficiencia , Factor de Transcripción YY1/genéticaRESUMEN
Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (D(H)) and joining (J(H)) gene segments were methylated, DJ(H) junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eµ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination.
Asunto(s)
Metilación de ADN , Reordenamiento Génico de Cadena Pesada de Linfocito B , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Recombinación V(D)J , Animales , Linfocitos B/inmunología , Islas de CpG/genética , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I , Elementos de Facilitación Genéticos , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Histonas/genética , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Secuencias Reguladoras de Ácidos Nucleicos , Linfocitos T/inmunologíaRESUMEN
Foxn1 is essential for thymic organogenesis and T lymphopoiesis. Whereas reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with early T cell progenitors (ETP), derived from multipotent progenitors (MPP) in the bone marrow (BM). A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1 transgenic (Tg); whether the effect is initiated in the BM with MPP is not known. In this study, we report that Foxn1 is expressed in wild-type BM and overexpressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of hematopoietic stem cells. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, common lymphoid progenitors and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared with wild type. We identified a novel population of lineage(neg/low), CD45(pos) EpCAM(pos), SCA1(pos), CD117(neg), CD138(neg), MHCII(neg) cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, and the Foxn1 BM niche may function in skewing MPP development toward T lineage progenitors.
Asunto(s)
Envejecimiento/inmunología , Linfocitos B/inmunología , Factores de Transcripción Forkhead/metabolismo , Células Progenitoras Linfoides/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD/metabolismo , Médula Ósea/inmunología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Inmunofenotipificación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Recuento de Linfocitos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nicho de Células Madre/inmunología , Transgenes/genéticaRESUMEN
B cell development involves rapid cellular proliferation, gene rearrangements, selection, and differentiation, and it provides a powerful model to study DNA repair processes in vivo. Analysis of the contribution of the base excision repair pathway in lymphocyte development has been lacking primarily owing to the essential nature of this repair pathway. However, mice deficient for the base excision repair enzyme, apurinic/apyrimidinic endonuclease 2 (APE2) protein develop relatively normally, but they display defects in lymphopoiesis. In this study, we present an extensive analysis of bone marrow hematopoiesis in mice nullizygous for APE2 and find an inhibition of the pro-B to pre-B cell transition. We find that APE2 is not required for V(D)J recombination and that the turnover rate of APE2-deficient progenitor B cells is nearly normal. However, the production rate of pro- and pre-B cells is reduced due to a p53-dependent DNA damage response. FACS-purified progenitors from APE2-deficient mice differentiate normally in response to IL-7 in in vitro stromal cell cocultures, but pro-B cells show defective expansion. Interestingly, APE2-deficient mice show a delay in recovery of B lymphocyte progenitors following bone marrow depletion by 5-fluorouracil, with the pro-B and pre-B cell pools still markedly decreased 2 wk after a single treatment. Our data demonstrate that APE2 has an important role in providing protection from DNA damage during lymphoid development, which is independent from its ubiquitous and essential homolog APE1.
Asunto(s)
Subgrupos de Linfocitos B/enzimología , Subgrupos de Linfocitos B/inmunología , Endonucleasas/fisiología , Fluorouracilo/administración & dosificación , Células Madre Hematopoyéticas/enzimología , Subgrupos Linfocitarios/enzimología , Linfopoyesis/inmunología , Animales , Subgrupos de Linfocitos B/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Daño del ADN/inmunología , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Reparación del ADN/inmunología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/fisiología , Endonucleasas/deficiencia , Endonucleasas/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Depleción Linfocítica , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Linfopoyesis/efectos de los fármacos , Linfopoyesis/genética , Ratones , Ratones Noqueados , Enzimas Multifuncionales , Mielopoyesis/efectos de los fármacos , Mielopoyesis/genética , Mielopoyesis/inmunología , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Endemic Burkitt lymphoma (BL) is a childhood cancer in sub-Saharan Africa characterized by Epstein-Barr virus and malaria-associated aberrant B-cell activation and MYC chromosomal translocation. Survival rates hover at 50% after conventional chemotherapies; therefore, clinically relevant models are necessary to test additional therapies. Hence, we established five patient-derived BL tumor cell lines and corresponding NSG-BL avatar mouse models. Transcriptomics confirmed that our BL lines maintained fidelity from patient tumors to NSG-BL tumors. However, we found significant variation in tumor growth and survival among NSG-BL avatars and in Epstein-Barr virus protein expression patterns. We tested rituximab responsiveness and found one NSG-BL model exhibiting direct sensitivity, characterized by apoptotic gene expression counterbalanced by unfolded protein response and mTOR pro-survival pathways. In rituximab-unresponsive tumors, we observed an IFN-α signature confirmed by the expression of IRF7 and ISG15. Our results demonstrate significant inter-patient tumor variation and heterogeneity, and that contemporary patient-derived BL cell lines and NSG-BL avatars are feasible tools to guide new therapeutic strategies and improve outcomes for these children.
Asunto(s)
Linfoma de Burkitt , Infecciones por Virus de Epstein-Barr , Animales , Ratones , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/genética , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/genética , Rituximab/farmacología , Rituximab/uso terapéutico , Herpesvirus Humano 4/genética , Línea Celular Tumoral , Modelos Animales de EnfermedadRESUMEN
Signaling from the BCR and B cell activating factor receptor (BAFF-R or BR3) differentially regulates apoptosis within early transitional (T1) and late transitional (T2; CD21(int)-T2) B cells during selection processes to generate mature B lymphocytes. However, molecular mechanisms underlying the differential sensitivity of transitional B cells to apoptosis remain unclear. In this study, we demonstrate that BCR signaling induced more long-term c-Rel activation in T2 and mature than in T1 B cells leading to increased expression of anti-apoptotic genes as well as prosurvival BAFF-R and its downstream substrate p100 (NF-kappaB2). Sustained c-Rel activation required de novo c-Rel gene transcription and translation via Btk-dependent mechanisms. Like T1 cells, mature B cells from Btk- and c-Rel-deficient mice also failed to activate these genes. These findings suggest that the gain of survival potential within transitional B cells is dependent on the ability to produce a long-term c-Rel response, which plays a critical role in T2 B cell survival and differentiation in vivo by inducing anti-apoptotic genes, BAFF-R and NF-kappaB2, an essential component for BAFF-R survival signaling. Thus, acquisition of resistance to apoptosis during transitional B cell maturation is achieved by integration of BCR and BAFF-R signals.
Asunto(s)
Receptor del Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Subunidad p52 de NF-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Linfocitos B/citología , Supervivencia Celular , Células Cultivadas , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-rel/genética , Transducción de Señal/inmunología , Especificidad por Sustrato , Factores de TiempoRESUMEN
In B lineage progenitors, V(D)J recombination occurs only during distinct stages of development and is restricted to immunoglobulin loci. This process is thought to be controlled by both regulated expression of the V(D)J recombinase and by limited accessibility of target loci to the recombinase complex. However, it is unknown whether these two processes occur concomitantly in developing B lineage progenitors or whether these events are temporally distinct and, therefore, potentially independently regulated. To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors. We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable. Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.
Asunto(s)
Linfocitos B/inmunología , Células Madre Hematopoyéticas/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , VDJ Recombinasas/genética , Animales , Linfocitos B/enzimología , Secuencia de Bases , Cartilla de ADN , Citometría de Flujo , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Recombinación GenéticaRESUMEN
During aging, adaptive immunity is severely compromised, due in part to decreased production of B lymphocytes and loss of immunoglobulin (Ig) diversity. However, the molecular mechanisms that underlie age-associated diminished B cell production remain unclear. Using in vivo labeling, we find that this reduction in marrow pre-B cells reflects increased attrition during passage from the pro-B to pre-B cell pool. Analyses of reciprocal bone marrow chimeras reveal that the magnitude and production rates of pre-B cells are controlled primarily by microenvironmental factors, rather than intrinsic events. To understand changes in pro-B cells that could diminish production of pre-B cells, we evaluated rag2 expression and V(D)J recombinase activity in pro-B cells at the single cell level. The percentage of pro-B cells that express rag2 is reduced in aged mice and is correlated with both a loss of V(D)J recombinase activity in pro-B cells and reduced numbers of pre-B cells. Reciprocal bone marrow chimeras revealed that the aged microenvironment also determines rag2 expression and recombinase activity in pro-B cells. Together, these observations suggest that extrinsic factors in the bone marrow that decline with age are largely responsible for less efficient V(D)J recombination in pro-B cells and diminished progression to the pre-B cell stage.
Asunto(s)
Envejecimiento/inmunología , Linfocitos B/inmunología , Médula Ósea/inmunología , Proteínas de Unión al ADN/metabolismo , Inmunidad Celular/inmunología , VDJ Recombinasas/metabolismo , Animales , Bromodesoxiuridina , Citometría de Flujo , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos , Factores de TiempoRESUMEN
Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in approximately 5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.
Asunto(s)
Linfocitos B/inmunología , Células Madre Hematopoyéticas/inmunología , Linfopoyesis/inmunología , VDJ Recombinasas/metabolismo , Animales , Linfocitos B/enzimología , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas/enzimología , Proteínas de Homeodominio/genética , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , VDJ Recombinasas/genéticaRESUMEN
Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor, but it is not known if these two genetic lesions act together to transform cells. To answer this question, we infected PTEN-/- neural precursor cells with a retrovirus encoding EGFRvIII, which is a constitutively activated receptor. EGFRvIII PTEN-/- cells formed highly mitotic tumors with nuclear pleomorphism, necrotic areas, and glioblastoma markers. The transformed cells showed increased cell proliferation, centrosome amplification, colony formation in soft agar, self-renewal, expression of the stem cell marker CD133, and resistance to oxidative stress and ionizing radiation. The RAS/mitogen-activated protein kinase (ERK) and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathways were activated, and checkpoint kinase 1 (Chk1), the DNA damage regulator, was phosphorylated at S280 by Akt, suppressing Chk1 phosphorylation at S345 in response to ionizing irradiation. The PTEN-/- cells showed low levels of DNA damage in the absence of irradiation, which was increased by EGFRvIII expression. Finally, secondary changes occurred during tumor growth in mice. Cells from these tumors showed decreased tumor latencies and additional chromosomal aberrations. Most of these tumor lines showed translocations of mouse chromosome 15. Intracranial injections of one of these lines led to invasive, glial fibrillary acidic protein-positive, nestin-positive tumors. These results provide a molecular basis for the occurrence of these two genetic lesions in brain tumors and point to a role in induction of genomic instability.
Asunto(s)
Neoplasias Encefálicas/genética , Inestabilidad Cromosómica , Receptores ErbB/metabolismo , Glioma/genética , Fosfohidrolasa PTEN/fisiología , Animales , Western Blotting , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular/efectos de la radiación , Transformación Celular Neoplásica/efectos de la radiación , Células Cultivadas , Centrosoma/metabolismo , Receptores ErbB/genética , Femenino , Glioma/metabolismo , Humanos , Integrasas/metabolismo , Proteínas de Filamentos Intermediarios/fisiología , Cariotipificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Microscopía Fluorescente , Células 3T3 NIH , Proteínas del Tejido Nervioso/fisiología , Nestina , Fosforilación/efectos de la radiación , Radiación Ionizante , Retroviridae/genéticaRESUMEN
PURPOSE: Approximately 50% of patients with diffuse large B-cell lymphoma (DLBCL) enter long-term remission after standard chemotherapy. Patients with DLBCL who do not respond to chemotherapy have few treatment options. There remains a critical need to identify effective and targeted therapeutics for DLBCL. EXPERIMENTAL DESIGN: Recent studies have highlighted the incidence of increased c-MYC protein in DLBCL and the correlation between high levels of c-MYC protein and poor survival prognosis of patients with DLBCL, suggesting that c-MYC is a compelling target for DLBCL therapy. The small molecule JQ1 suppresses c-MYC expression through inhibition of the bromodomain and extra-terminal (BET) family of bromodomain proteins. We investigated whether JQ1 can inhibit proliferation of DLBCL cells in culture and xenograft models in vivo. RESULTS: We show that JQ1 at nanomolar concentrations efficiently inhibited proliferation of human DLBCL cells in a dose-dependent manner regardless of their molecular subtypes, suggesting a broad effect of JQ1 in DLBCL. The initial G1 arrest induced by JQ1 treatment in DLBCL cells was followed by either apoptosis or senescence. The expression of c-MYC was suppressed as a result of JQ1 treatment from the natural, chromosomally translocated, or amplified loci. Furthermore, JQ1 treatment significantly suppressed growth of DLBCL cells engrafted in mice and improved survival of engrafted mice. CONCLUSION: Our results demonstrate that inhibition of the BET family of bromodomain proteins by JQ1 has potential clinical use in the treatment of DLBCL.
Asunto(s)
Azepinas/administración & dosificación , Proliferación Celular/efectos de los fármacos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Triazoles/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de Señal/efectos de los fármacos , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The age-dependent decline in the self-renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age-dependent decline of stem cell self-renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2-deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2-deficient mice had a significantly better repopulating capacity than aged wild-type HSCs, suggesting that decline in HSC function with age is Smurf2 dependent. Furthermore, Smurf2-deficient HSCs exhibited elevated long-term self-renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self-renewal and aging.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Proliferación Celular/fisiología , Senescencia Celular/fisiología , Femenino , Trasplante de Células Madre Hematopoyéticas , Masculino , Ratones , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Hematopoietic stem cells (HSCs) have the ability to self-renew and replenish the blood and immune system for the life span of an individual. An age-associated decline in HSC function is responsible for the decreased immune function and increased incidence of myeloid diseases and anemia in the elderly. The changes in HSC function are thought to occur as the result of an intrinsic defect in the self-renewal potential of HSCs as they age. In this chapter, we describe a bone marrow serial transplantation protocol designed to test the self-renewal capacity of HSCs in vivo.
Asunto(s)
Trasplante de Médula Ósea/métodos , Diferenciación Celular , Proliferación Celular , Células Madre Hematopoyéticas/citología , Animales , Células Madre Hematopoyéticas/fisiología , Humanos , RatonesRESUMEN
About half of patients with diffuse large B-cell lymphoma (DLBCL) do not respond to or relapse soon after the standard chemotherapy, indicating a critical need to better understand the specific pathways perturbed in DLBCL for developing effective therapeutic approaches. Mice deficient in the E3 ubiquitin ligase Smurf2 spontaneously develop B-cell lymphomas that resemble human DLBCL with molecular features of germinal centre or post-germinal centre B cells. Here we show that Smurf2 mediates ubiquitination and degradation of YY1, a key germinal centre transcription factor. Smurf2 deficiency enhances YY1-mediated transactivation of c-Myc and B-cell proliferation. Furthermore, Smurf2 expression is significantly decreased in primary human DLBCL samples, and low levels of Smurf2 expression correlate with inferior survival in DLBCL patients. The Smurf2-YY1-c-Myc regulatory axis represents a novel pathway perturbed in DLBCL that suppresses B-cell proliferation and lymphomagenesis, suggesting pharmaceutical targeting of Smurf2 as a new therapeutic paradigm for DLBCL.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Proteínas Proto-Oncogénicas c-myc/genética , Ubiquitina-Proteína Ligasas/genética , Factor de Transcripción YY1/genética , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular Tumoral , Proliferación Celular , Centro Germinal/metabolismo , Centro Germinal/patología , Humanos , Activación de Linfocitos , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Transcripción Genética , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Factor de Transcripción YY1/metabolismoRESUMEN
Definitive hematopoiesis requires the master hematopoietic transcription factor Runx1, which is a frequent target of leukemia-related chromosomal translocations. Several of the translocation-generated fusion proteins retain the DNA binding activity of Runx1, but lose subnuclear targeting and associated transactivation potential. Complete loss of these functions in vivo resembles Runx1 ablation, which causes embryonic lethality. We developed a knock-in mouse that expresses full-length Runx1 with a mutation in the subnuclear targeting cofactor interaction domain, Runx1(HTY350-352AAA). Mutant mice survive to adulthood, and hematopoietic stem cell emergence appears to be unaltered. However, defects are observed in multiple differentiated hematopoietic lineages at stages where Runx1 is known to play key roles. Thus, a germline mutation in Runx1 reveals uncoupling of its functions during developmental hematopoiesis from subsequent differentiation across multiple hematopoietic lineages in the adult. These findings indicate that subnuclear targeting and cofactor interactions with Runx1 are important in many compartments throughout hematopoietic differentiation.