RESUMO
Not simply an attribute of the adaptive immune system, immunological memory can be viewed on multiple levels. Accordingly, the molecular basis of memory comprises multiple mechanisms. The advent of new sequencing technologies has greatly enhanced the understanding of gene regulation and lymphocyte specification, and improved measurement of chromatin states affords new insights into the epigenomic and transcriptomic programs that underlie memory. Beyond canonical genes, the involvement of long noncoding RNAs (lncRNAs) is becoming increasingly apparent, and it appears that there are more than two to three times as many lncRNAs as protein-coding genes. lncRNAs can directly interact with DNA, RNA, and proteins, and a single lncRNA can contain multiple modular domains and thus interact with different classes of molecules. Yet, most lncRNAs have not been tested for function, and even fewer knockout mice have been generated. It is therefore timely to consider new potential mechanisms that may contribute to immune memory.
Assuntos
RNA Longo não Codificante , Animais , Cromatina , Epigenômica , Regulação da Expressão Gênica , Linfócitos , Camundongos , RNA Longo não Codificante/metabolismoRESUMO
Induced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and eventually, treat patients. However, these cell sources produce progeny that retain embryonic and/or fetal characteristics. The failure to mature to definitive, adult-type cells is a major barrier for iPSC-based disease modeling and drug discovery. To directly address these concerns, we have developed a chemically defined, serum and feeder-free-directed differentiation platform to generate hematopoietic stem-progenitor cells (HSPCs) and resultant adult-type progeny from iPSCs. This system allows for strict control of signaling pathways over time through growth factor and/or small molecule modulation. Through direct comparison with our previously described protocol for the production of primitive wave hematopoietic cells, we demonstrate that induced HSPCs are enhanced for erythroid and myeloid colony forming potential, and strikingly, resultant erythroid-lineage cells display enhanced expression of adult ß globin indicating definitive pathway patterning. Using this system, we demonstrate the stage-specific roles of two key signaling pathways, Notch and the aryl hydrocarbon receptor (AHR), in the derivation of definitive hematopoietic cells. We illustrate the stage-specific necessity of Notch signaling in the emergence of hematopoietic progenitors and downstream definitive, adult-type erythroblasts. We also show that genetic or small molecule inhibition of the AHR results in the increased production of CD34+ CD45+ HSPCs while conversely, activation of the same receptor results in a block of hematopoietic cell emergence. Results presented here should have broad implications for hematopoietic stem cell transplantation and future clinical translation of iPSC-derived blood cells. Stem Cells 2018;36:1004-1019.
Assuntos
Hematopoese/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Notch/genética , Diferenciação Celular , Humanos , Transdução de SinaisRESUMO
The HBS1L-MYB intergenic region (chr6q23) regulates erythroid cell proliferation, maturation, and fetal hemoglobin (HbF) expression. An enhancer element within this locus, highlighted by a 3-bp deletion polymorphism (rs66650371), is known to interact with the promoter of the neighboring gene, MYB, to increase its expression, thereby regulating HbF production. RNA polymerase II binding and a 50-bp transcript from this enhancer region reported in ENCODE datasets suggested the presence of a long noncoding RNA (lncRNA). We characterized a novel 1283bp transcript (HMI-LNCRNA; chr6:135,096,362-135,097,644; hg38) that was transcribed from the enhancer region of MYB. Within erythroid cells, HMI-LNCRNA was almost exclusively present in nucleus, and was much less abundant than the mRNA for MYB. HMI-LNCRNA expression was significantly higher in erythroblasts derived from cultured adult peripheral blood CD34+ cells which expressed more HBB, compared to erythroblasts from cultured cord blood CD34+ cells which expressed much more HBG. Down-regulation of HMI-LNCRNA in HUDEP-2 cells, which expressed mostly HBB, significantly upregulated HBG expression both at the mRNA (200-fold) and protein levels, and promoted erythroid maturation. No change was found in the expression of BCL11A and other key transcription factors known to modulate HBG expression. HMI-LNCRNA plays an important role in regulating HBG expression, and its downregulation can result in a significant increase in HbF. HMI-LNCRNA might be a potential therapeutic target for HbF induction treatment in sickle cell disease and ß-thalassemia.
Assuntos
Cromossomos Humanos Par 6 , DNA Intergênico/genética , Hemoglobina Fetal/genética , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Genes myb , RNA Longo não Codificante , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Eritroblastos/metabolismo , Células Eritroides/metabolismo , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/metabolismo , Humanos , Locos de Características QuantitativasRESUMO
Despite high expression levels, the role of Tsc1 in cardiovascular tissue is ill defined. We launched this study to examine the role of Tsc1 in cardiac physiology and pathology. Mice in which Tsc1 was deleted in cardiac tissue and vascular smooth muscle (Tsc1c/cSM22cre(+/-)), developed progressive cardiomegaly and hypertension and died early. Hearts of Tsc1c/cSM22cre(+/-) mice displayed a progressive increase in cardiomyocyte number, and to a lesser extent, size between the ages of 1 and 6 weeks. In addition, compared to control hearts, proliferation markers (phospho-histone 3 and PCNA) were elevated in Tsc1c/cSM22cre(+/-) cardiomyocytes at 0-4 weeks, suggesting that cardiomyocyte proliferation was the predominant mechanism underlying cardiomegaly in Tsc1c/cSM22cre(+/-) mice. To examine the contribution of Tsc1 deletion in peripheral vascular smooth muscle to the cardiac phenotype, Tsc1c/cSM22cre(+/-) mice were treated with the antihypertensive, hydralazine. Prevention of hypertension had no effect on survival, cardiac size, or cardiomyocyte number in these mice. We furthermore generated mice in which Tsc1 was deleted only in vascular smooth muscle but not in cardiac tissue (Tsc1c/cSMAcre-ER(T2+/-)). The Tsc1c/cSMAcre-ER(T2+/-) mice also developed hypertension. However, their survival was normal and no cardiac abnormalities were observed. Our results suggest that loss of Tsc1 in the heart causes cardiomegaly, which is driven by increased cardiomyocyte proliferation that also appears to confer relative resistance to afterload reduction. These findings support a critical role for the Tsc1 gene as gatekeeper in the protection against uncontrolled cardiac growth.
Assuntos
Cardiomegalia/metabolismo , Proliferação de Células/genética , Miócitos Cardíacos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Modelos Animais de Doenças , Hemodinâmica/fisiologia , Hiperplasia/genética , Hiperplasia/metabolismo , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Multiplex , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos Cardíacos/patologia , Reação em Cadeia da Polimerase , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)-2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)-2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM.
Assuntos
Estradiol/análogos & derivados , Estradiol/efeitos adversos , Matriz Extracelular/efeitos dos fármacos , Neoplasias Pulmonares/secundário , Linfangioleiomiomatose/patologia , Remodelação das Vias Aéreas , Animais , Antineoplásicos/farmacologia , Colágeno Tipo IV/metabolismo , Avaliação Pré-Clínica de Medicamentos , Estradiol/farmacologia , Matriz Extracelular/metabolismo , Feminino , Fulvestranto , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Linfangioleiomiomatose/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos SCID , Ratos , Receptores de Estradiol/antagonistas & inibidores , Análise de Sobrevida , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome characterized by benign tumors in multiple organs, including the brain and kidney. TSC-associated tumors exhibit hyperactivation of mammalian target of rapamycin complex 1 (mTORC1), a direct inhibitor of autophagy. Autophagy can either promote or inhibit tumorigenesis, depending on the cellular context. The role of autophagy in the pathogenesis and treatment of the multisystem manifestations of TSC is unknown. We found that the combination of mTORC1 and autophagy inhibition was more effective than either treatment alone in inhibiting the survival of tuberin (TSC2)-null cells, growth of TSC2-null xenograft tumors, and development of spontaneous renal tumors in Tsc2(+/-) mice. Down-regulation of Atg5 induced extensive central necrosis in TSC2-null xenograft tumors, and loss of one allele of Beclin1 almost completely blocked macroscopic renal tumor formation in Tsc2(+/-) mice. Surprisingly, given the finding that lowering autophagy blocks TSC tumorigenesis, genetic down-regulation of p62/sequestosome 1 (SQSTM1), the autophagy substrate that accumulates in TSC tumors as a consequence of low autophagy levels, strongly inhibited the growth of TSC2-null xenograft tumors. These data demonstrate that autophagy is a critical component of TSC tumorigenesis, suggest that mTORC1 inhibitors may have autophagy-dependent prosurvival effects in TSC, and reveal two distinct therapeutic targets for TSC: autophagy and the autophagy target p62/SQSTM1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Choque Térmico/metabolismo , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Autofagia/fisiologia , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Genes p53 , Proteínas de Choque Térmico/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Complexos Multiproteicos , Proteínas/genética , Proteínas/metabolismo , Proteína Sequestossoma-1 , Serina-Treonina Quinases TOR , Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
Lymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking female predominance of LAM are unknown. We report here that 17-beta-estradiol (E(2)) causes a 3- to 5-fold increase in pulmonary metastases in male and female mice, respectively, and a striking increase in circulating tumor cells in mice bearing tuberin-null xenograft tumors. E(2)-induced metastasis is associated with activation of p42/44 MAPK and is completely inhibited by treatment with the MEK1/2 inhibitor, CI-1040. In vitro, E(2) inhibits anoikis of tuberin-null cells. Finally, using a bioluminescence approach, we found that E(2) enhances the survival and lung colonization of intravenously injected tuberin-null cells by 3-fold, which is blocked by treatment with CI-1040. Taken together these results reveal a new model for LAM pathogenesis in which activation of MEK-dependent pathways by E(2) leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells.