RESUMO
A classic model for identification of novel differentiation mechanisms and pathways is the eye lens that consists of a monolayer of quiescent epithelial cells that are the progenitors of a core of mature fully differentiated fiber cells. The differentiation of lens epithelial cells into fiber cells follows a coordinated program involving cell cycle exit, expression of key structural proteins and the hallmark elimination of organelles to achieve transparency. Although multiple mechanisms and pathways have been identified to play key roles in lens differentiation, the entirety of mechanisms governing lens differentiation remain to be discovered. A previous study established that specific chromatin accessibility changes were directly associated with the expression of essential lens fiber cell genes, suggesting that the activity of transcription factors needed for expression of these genes could be regulated through binding access to the identified chromatin regions. Sequence analysis of the identified chromatin accessible regions revealed enhanced representation of the binding sequence for the transcription factor FOXO4 suggesting a direct role for FOXO4 in expression of these genes. FOXO4 is known to regulate a variety of cellular processes including cellular response to metabolic and oxidative stress, cell cycle withdrawal, and homeostasis, suggesting a previously unidentified role for FOXO4 in the regulation of lens cell differentiation. To further evaluate the role of FOXO4 we employed a multiomics approach to analyze the relationship between genome-wide FOXO4 binding, the differentiation-specific expression of key genes, and chromatin accessibility. To better identify active promoters and enhancers we also examined histone modification through analysis of H3K27ac. Specific methods included CUT&RUN (FOXO4 binding and H3K27ac modification), RNA-seq (differentiation state specific gene expression), and ATAC-seq (chromatin accessibility). CUT&RUN identified 20,966 FOXO4 binding sites and 33,921 H3K27ac marked regions across the lens fiber cell genome. RNA-seq identified 956 genes with significantly greater expression levels in fiber cells compared to epithelial cells (log2FC > 0.7, q < 0.05) and 2548 genes with significantly lower expression levels (log2FC < -0.7, q < 0.05). Integrated analysis identified 1727 differentiation-state specific genes that were nearest neighbors to at least one FOXO4 binding site, including genes encoding lens gap junctions (GJA1, GJA3), lens structural proteins (BFSP1, CRYBB1, ASL1), and genes required for lens transparency (HSF4, NRCAM). Multiomics analysis comparing the identified FOXO4 binding sites in published ATAC-seq data revealed that chromatin accessibility was associated with FOXO4-dependent gene expression during lens differentiation. The results provide evidence for an important requirement for FOXO4 in the regulated expression of key genes required for lens differentiation and link epigenetic regulation of chromatin accessibility and H3K27ac histone modification with the function of FOXO4 in controlling lens gene expression during lens fiber cell differentiation.
Assuntos
Epigênese Genética , Cristalino , Multiômica , Regulação da Expressão Gênica , Diferenciação Celular/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Cristalino/metabolismoRESUMO
The potential of adoptive cell therapy with regulatory T cells (Tregs) to promote transplant tolerance is under active exploration. However, the impact of specific transplant settings and protocols on Treg manufacturing is not well-delineated. Here, we compared the use of peripheral blood mononuclear cells (PBMCs) from patients before or after liver transplantation to the use of healthy control PBMCs to determine their suitability for Treg manufacture using ex vivo costimulatory blockade with belatacept. Despite liver failure or immunosuppressive therapy, the capacity for Treg expansion during the manufacturing process was preserved. These experiments did not identify performance or quality issues that disqualified the use of posttransplant PBMCs-the currently favored protocol design. However, as Treg input correlated with output, significant CD4-lymphopenia in both pre- and posttransplant patients limited Treg yield. We therefore turned to leukapheresis posttransplant to improve absolute yield. To make deceased donor use feasible, we also developed protocols to substitute splenocytes for PBMCs as allostimulators. In addition to demonstrating that this Treg expansion strategy works in a liver transplant context, this preclinical study illustrates how characterizing cellular input populations and their performance can both inform and respond to clinical trial design and Treg manufacturing requirements.
Assuntos
Transplante de Fígado , Linfócitos T Reguladores , Abatacepte/farmacologia , Humanos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Leucócitos Mononucleares , Transplantados , Tolerância ao TransplanteRESUMO
BACKGROUND: During eye lens development the embryonic vasculature regresses leaving the lens without a direct oxygen source. Both embryonically and throughout adult life, the lens contains a decreasing oxygen gradient from the surface to the core that parallels the natural differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia and the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1 (HIF1), in the regulation of genes required for lens fiber cell differentiation, structure and transparency. Here, we employed a multiomics approach combining CUT&RUN, RNA-seq and ATACseq analysis to establish the genomic complement of lens HIF1α binding sites, genes activated or repressed by HIF1α and the chromatin states of HIF1α-regulated genes. RESULTS: CUT&RUN analysis revealed 8375 HIF1α-DNA binding complexes in the chick lens genome. One thousand one hundred ninety HIF1α-DNA binding complexes were significantly clustered within chromatin accessible regions (χ2 test p < 1 × 10- 55) identified by ATACseq. Formation of the identified HIF1α-DNA complexes paralleled the activation or repression of 526 genes, 116 of which contained HIF1α binding sites within 10kB of the transcription start sites. Some of the identified HIF1α genes have previously established lens functions while others have novel functions never before examined in the lens. GO and pathway analysis of these genes implicate HIF1α in the control of a wide-variety of cellular pathways potentially critical for lens fiber cell formation, structure and function including glycolysis, cell cycle regulation, chromatin remodeling, Notch and Wnt signaling, differentiation, development, and transparency. CONCLUSIONS: These data establish the first functional map of genomic HIF1α-DNA complexes in the eye lens. They identify HIF1α as an important regulator of a wide-variety of genes previously shown to be critical for lens formation and function and they reveal a requirement for HIF1α in the regulation of a wide-variety of genes not yet examined for lens function. They support a requirement for HIF1α in lens fiber cell formation, structure and function and they provide a basis for understanding the potential roles and requirements for HIF1α in the development, structure and function of more complex tissues.
Assuntos
Cristalino , Diferenciação Celular , Cromatina , DNA , Genômica , Subunidade alfa do Fator 1 Induzível por HipóxiaRESUMO
A hallmark feature of lens development and differentiation is the complete elimination of organelles from the center of the eye lens. A long unanswered question in lens biology is what are the mechanisms that control the elimination of organelles during the terminal remodeling program to form mature lens fiber cells? Recent advances have expanded our understanding of these mechanisms including newly discovered signaling pathways, proteasomal regulators, autophagy proteins, transcription factors and the hypoxic environment of the lens itself. These recent discoveries suggest that distinct mechanisms coordinate the elimination of the nucleus, mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell differentiation. Since regulation of organelle number and distribution is also a feature of the terminal remodeling programs of more complex cell-types and tissues, these advances are likely to impact a wide-variety of fields.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Cristalino/crescimento & desenvolvimento , Animais , Autofagia , Diferenciação Celular , Humanos , Cristalino/citologia , Mitocôndrias/metabolismo , Modelos AnimaisRESUMO
Changes in chromatin accessibility regulate the expression of multiple genes by controlling transcription factor access to key gene regulatory sequences. Here, we sought to establish a potential function for altered chromatin accessibility in control of key gene expression events during lens cell differentiation by establishing genome-wide chromatin accessibility maps specific for four distinct stages of lens cell differentiation and correlating specific changes in chromatin accessibility with genome-wide changes in gene expression. ATAC sequencing was employed to generate chromatin accessibility profiles that were correlated with the expression profiles of over 10,000 lens genes obtained by high-throughput RNA sequencing at the same stages of lens cell differentiation. Approximately 90,000 regions of the lens genome exhibited distinct changes in chromatin accessibility at one or more stages of lens differentiation. Over 1000 genes exhibited high Pearson correlation coefficients (r â> â0.7) between altered expression levels at specific stages of lens cell differentiation and changes in chromatin accessibility in potential promoter (-7.5kbp/+2.5kbp of the transcriptional start site) and/or other potential cis-regulatory regions ( ±10 âkb of the gene body). Analysis of these regions identified consensus binding sequences for multiple transcription factors including members of the TEAD, FOX, and NFAT families of transcription factors as well as HIF1a, RBPJ and IRF1. Functional mapping of genes with high correlations between altered chromatin accessibility and differentiation state-specific gene expression changes identified multiple families of proteins whose expression could be regulated through changes in chromatin accessibility including those governing lens structure (BFSP1,BFSP2), gene expression (Pax-6, Sox 2), translation (TDRD7), cell-cell communication (GJA1), autophagy (FYCO1), signal transduction (SMAD3, EPHA2), and lens transparency (CRYBB1, CRYBA4). These data provide a novel relationship between altered chromatin accessibility and lens differentiation and they identify a wide-variety of lens genes and functions that could be regulated through altered chromatin accessibility. The data also point to a large number of potential DNA regulatory sequences and transcription factors whose functional analysis is likely to provide insight into novel regulatory mechanisms governing the lens differentiation program.
Assuntos
Diferenciação Celular/genética , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cristalino/citologia , Animais , Sequência de Bases , Sítios de Ligação , Biomarcadores/metabolismo , Galinhas/genética , Sequência Consenso/genética , DNA/metabolismo , Genoma , Cristalino/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Formation of the eye lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To attain their mature structure and transparent function, nascent lens fiber cells must complete a precise cellular remodeling program hallmarked by the complete elimination of organelles to form the core lens organelle-free zone (OFZ). Lacking a blood supply, the lens resides in a hypoxic environment that results in a decreasing oxygen concentration from the lens surface to the lens core. This oxygen gradient results in a hypoxic microenvironment in the region of the lens where immature lens fiber cells initiate loss of organelles to form the core OFZ. These features of the lens suggest a potential role for low lens oxygen levels in the regulation of organelle degradation and other events critical for mature lens fiber cell formation. Hypoxia activates the master regulator of the hypoxic response, hypoxia-inducible factor 1a (HIF1a) that regulates hypoxia-responsive genes. To identify a potential role for hypoxia and HIF1a in the elimination of organelles during lens fiber cell maturation, we tested the requirement for hypoxia in the degradation of non-nuclear organelles in ex vivo cultured embryonic chick lenses by monitoring the degradation of mitochondria (MT), Golgi apparatus (GA) and endoplasmic reticulum (ER) under conditions of low (1% O2) and high (21% O2) oxygen. We also examined the requirement for HIF1a activation for elimination of these organelles under the same conditions using a specific HIF1a activator (DMOG) and a specific HIF1a inhibitor (chetomin) and examined the requirements for hypoxia and HIF1a for regulating transcription of BNIP3L that we previously showed to be required for elimination of non-nuclear lens organelles. We used ChIP-qPCR to confirm direct binding of HIF1a to the 5' untranslated region of the BNIP3L gene. Finally, we examined the effects of expressing an oxygen insensitive mutant form of HIF1a (P402A/P565A) and BNIP3L on non-nuclear organelle degradation. Our data demonstrate that hypoxia and HIF1a are required for the degradation of non-nuclear organelles during lens fiber cell formation and that they regulate this process by governing BNIP3L transcription. Our results also provide evidence that hypoxia and HIF1a are essential for achieving mature lens structure.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/genética , Cristalino/metabolismo , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Animais , Diferenciação Celular , Embrião de Galinha , Modelos Animais de Doenças , Hipóxia/embriologia , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Cristalino/embriologia , Proteínas de Membrana/metabolismo , Técnicas de Cultura de Órgãos , Organelas/metabolismo , Organelas/patologia , Proteínas Proto-Oncogênicas/metabolismo , RNA/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Ocular lens morphogenesis is a model for investigating mechanisms of cellular differentiation, spatial and temporal gene expression control, and chromatin regulation. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of distinct ATP-dependent chromatin remodeling complexes implicated in transcriptional regulation. Previous studies have shown that Brg1 regulates both lens fiber cell differentiation and organized degradation of their nuclei (denucleation). Here, we employed a conditional Snf2h(flox) mouse model to probe the cellular and molecular mechanisms of lens formation. Depletion of Snf2h induces premature and expanded differentiation of lens precursor cells forming the lens vesicle, implicating Snf2h as a key regulator of lens vesicle polarity through spatial control of Prox1, Jag1, p27(Kip1) (Cdkn1b) and p57(Kip2) (Cdkn1c) gene expression. The abnormal Snf2h(-/-) fiber cells also retain their nuclei. RNA profiling of Snf2h(-/) (-) and Brg1(-/-) eyes revealed differences in multiple transcripts, including prominent downregulation of those encoding Hsf4 and DNase IIß, which are implicated in the denucleation process. In summary, our data suggest that Snf2h is essential for the establishment of lens vesicle polarity, partitioning of prospective lens epithelial and fiber cell compartments, lens fiber cell differentiation, and lens fiber cell nuclear degradation.
Assuntos
Adenosina Trifosfatases/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos/metabolismo , Cristalino/citologia , Cristalino/embriologia , Animais , Autofagia , Compartimento Celular , Ciclo Celular , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição de Choque Térmico , Camundongos Knockout , Mitofagia , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX6/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
The formation and life-long growth of the ocular lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To achieve their mature structure and transparent function, newly formed lens fiber cells undergo a series of cellular remodeling events including the complete elimination of cellular organelles to form the lens organelle-free zone (OFZ). To date, the mechanisms and requirements for organelle elimination by lens fiber cells remain to be fully elucidated. In previous studies, we detected the presence of mitochondria contained within autophagolysosomes throughout human and chick lenses suggesting that proteins targeting mitochondria for degradation by mitophagy could be required for the elimination of mitochondria during OFZ formation. Consistently, high-throughput RNA sequencing of microdissected embryonic chick lenses revealed that expression of a protein that targets mitochondria for elimination during erythrocyte formation, called BCL2 interacting protein 3-like (BNIP3L/NIX), peaks in the region of lens where organelle elimination occurs. To examine the potential role for BNIP3L in the elimination of mitochondria during lens fiber cell remodeling, we analyzed the expression pattern of BNIP3L in newborn mouse lenses, the effect of its deletion on organelle elimination and its co-localization with lens organelles. We demonstrate that the expression pattern of BNIP3L in the mouse lens is consistent with it playing an important role in the elimination of mitochondria during lens fiber cell organelle elimination. Importantly, we demonstrate that deletion of BNIP3L results in retention of mitochondria during lens fiber cell remodeling, and, surprisingly, that deletion of BNIP3L also results in the retention of endoplasmic reticulum and Golgi apparatus but not nuclei. Finally, we show that BNIP3L localizes to the endoplasmic reticulum and Golgi apparatus of wild-type newborn mouse lenses and is contained within mitochondria, endoplasmic reticulum and Golgi apparatus isolated from adult mouse liver. These data identify BNIP3L as a novel requirement for the elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell remodeling and they suggest a novel function for BNIP3L in the regulation of endoplasmic reticulum and Golgi apparatus populations in the lens and non-lens tissues.
Assuntos
Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Cristalino/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Animais , Western Blotting , Perfilação da Expressão Gênica , Cristalino/embriologia , Fígado/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Age-related cataract is associated with oxidative stress and death of lens epithelial cells (LECs) whose survival is dependent on functional mitochondrial populations. Oxidative stress-induced depolarization/damage of LEC mitochondria results in increased reactive oxygen species (ROS) levels and cell death suggesting the need for a LEC mechanism to remove mitochondria depolarized/damaged upon oxidative stress exposure to prevent ROS release and LEC death. To date, a mechanism(s) for removal of depolarized/damaged LEC mitochondria has yet to be identified and the importance of eliminating oxidative stress-damaged mitochondria to prevent LEC ROS release and death has not been established. Here, we demonstrate that Parkin levels increase in LECs exposed to H2O2-oxidative stress. We establish that Parkin translocates to LEC mitochondria depolarized upon oxidative stress exposure and that Parkin recruits p62/SQSTM1 to depolarized LEC mitochondria. We demonstrate that translocation of Parkin results in the elimination of depolarized/damaged mitochondria and that Parkin clearance of LEC mitochondria is dependent on its ubiquitin ligase activity. Importantly, we demonstrate that Parkin elimination of damaged LEC mitochondria results in reduced ROS levels and increased survival upon oxidative stress exposure. These results establish that Parkin functions to eliminate LEC mitochondria depolarized/damaged upon oxidative stress exposure and that elimination of damaged mitochondria by Parkin is important for LEC homeostasis and survival. The data also suggest that mitochondrial quality control by Parkin could play a role in lens transparency.
Assuntos
Células Epiteliais/citologia , Cristalino/citologia , Mitocôndrias/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Galinhas , Células Epiteliais/metabolismo , Humanos , Cristalino/metabolismo , Transporte ProteicoRESUMO
Chronic heart failure (HF) is a disease with significantly higher prevalence in the elderly or patients older than 65 years old. Typically, older patients have more risk factors for HF, more comorbidities, and are more likely to have recurrent admissions for acute decompensations. With HF burden on health care systems primarily related to hospital and nursing home costs, it is critical that elderly patients are approached with a clear understanding of certain unique clinical, laboratory, imaging, and pharmacokinetic differences that can alter their management and outcomes. Psychosocial factors have major implications on adherence to therapy as well as decisions on advanced care for elderly HF patients. In this article, we highlight ten peculiar management considerations when approaching older patients with HF. We discuss issues related to epidemiology, diagnostic challenges, pharmacotherapy, and palliative care; all of which can impact this unique population and, more importantly, the disease burden as a whole.
Assuntos
Gerenciamento Clínico , Avaliação Geriátrica , Insuficiência Cardíaca/epidemiologia , Fatores Etários , Idoso , Comorbidade , Saúde Global , Insuficiência Cardíaca/terapia , Humanos , Polimedicação , Prevalência , Fatores de RiscoRESUMO
Accumulation of apoptotic material is toxic and associated with cataract and other disease states. Identification of mechanisms that prevent accumulation of apoptotic debris is important for establishing the etiology of these diseases. The ocular lens is routinely assaulted by UV light that causes lens cell apoptosis and is associated with cataract formation. To date, no molecular mechanism for removal of toxic apoptotic debris has been identified in the lens. Vesicular debris within lens cells exposed to UV light has been observed raising speculation that lens cells themselves could act as phagocytes to remove toxic apoptotic debris. However, phagocytosis has not been confirmed as a function of the intact eye lens, and no mechanism for lens phagocytosis has been established. Here, we demonstrate that the eye lens is capable of phagocytizing extracellular lens cell debris. Using high throughput RNA sequencing and bioinformatics analysis, we establish that lens epithelial cells express members of the integrin αVß5-mediated phagocytosis pathway and that internalized cell debris co-localizes with αVß5 and with RAB7 and Rab-interacting lysosomal protein that are required for phagosome maturation and fusion with lysosomes. We demonstrate that the αVß5 receptor is required for lens epithelial cell phagocytosis and that UV light treatment of lens epithelial cells results in damage to the αVß5 receptor with concomitant loss of phagocytosis. These data suggest that loss of αVß5-mediated phagocytosis by the eye lens could result in accumulation of toxic cell debris that could contribute to UV light-induced cataract formation.
Assuntos
Apoptose/efeitos da radiação , Proteínas Aviárias/metabolismo , Células Epiteliais/metabolismo , Proteínas do Olho/metabolismo , Cristalino/metabolismo , Receptores de Vitronectina/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Humanos , Lisossomos/metabolismo , Fagocitose/efeitos da radiação , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
BACKGROUND: αB-crystallin/sHSP protects cells against oxidative stress damage. Here, we mechanistically examined its ability to preserve mitochondrial function in lens and retinal cells and protect cytochrome c under oxidative stress conditions. METHODS: αB-crystallin/sHSP was localized in human lens (HLE-B3) and retinal (ARPE-19) cells. αB-crystallin/sHSP was stably over-expressed and its ability to preserve mitochondrial membrane potential under oxidative stress conditions was monitored. Interactions between αB-crystallin/sHSP and cytochrome c were examined by fluorescent resonance energy transfer (FRET) and by co-immune precipitation. The ability of αB-crystallin/sHSP to protect cytochrome c against methionine-80 oxidation was monitored. RESULTS: αB-crystallin/sHSP is present in the mitochondria of lens and retinal cells and is translocated to the mitochondria under oxidative conditions. αB-crystallin/sHSP specifically interacts with cytochrome c in vitro and in vivo and its overexpression preserves mitochondrial membrane potential under oxidative stress conditions. αB-crystallin/sHSP directly protects cytochrome c against oxidation. GENERAL SIGNIFICANCE: These data demonstrate that αB-crystallin/sHSP maintains lens and retinal cells under oxidative stress conditions at least in part by preserving mitochondrial function and by protecting cytochrome c against oxidation. Since oxidative stress and loss of mitochondrial function are associated with eye lens cataract and age-related macular degeneration, loss of these αB-crystallin/sHSP functions likely plays a key role in the development of these diseases. αB-crystallin/sHSP is expressed throughout the body and its ability to maintain mitochondrial function is likely important for the prevention of multiple degenerative diseases.
Assuntos
Citocromos c/metabolismo , Cristalino/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Western Blotting , Células Cultivadas , Humanos , Imunoprecipitação , Cristalino/citologia , Potencial da Membrana Mitocondrial , Oxirredução , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/efeitos dos fármacosRESUMO
αB-crystallin is a small heat shock protein that exhibits chaperone activity and can protect multiple cell types against oxidative stress damage. Altered levels and specific mutations of αB-crystallin are associated with multiple degenerative diseases. We previously found that αB-crystallin translocates to lens and retinal cell mitochondria upon oxidative stress exposure where it provides protection against oxidative stress damage. To date, the role of the chaperone function of αB-crystallin in mitochondrial translocation and protection has not been established. Here, we sought to determine the relationship between the chaperone activity of αB-crystallin and its ability to translocate to and protect retinal cell mitochondria against oxidative stress damage. Our data provide evidence that three forms of αB-crystallin exhibiting different chaperone activity levels including wild-type, R120G (decreased chaperone activity) and M68A (increased chaperone activity) provide comparable levels of mitochondrial translocation and protection to retinal cells exposed to oxidative stress. The results provide evidence that mitochondrial translocation and protection by αB-crystallin is independent of its chaperone activity and that other functions of αB-crystallin may also be independent of its chaperone activity.
Assuntos
Mitocôndrias/metabolismo , Chaperonas Moleculares/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Cadeia B de alfa-Cristalina/fisiologia , Western Blotting , Células Cultivadas , Clonagem Molecular , Citoproteção , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Humanos , Peróxido de Hidrogênio/toxicidade , Potencial da Membrana Mitocondrial , Mutagênese Sítio-Dirigida , Estresse Oxidativo , Mutação Puntual , Transporte Proteico , TransfecçãoRESUMO
The eye lens consists of a layer of epithelial cells that overlay a series of differentiating fiber cells that upon maturation lose their mitochondria, nuclei and other organelles. Lens transparency relies on the metabolic function of mitochondria contained in the lens epithelial cells and in the immature fiber cells and the programmed degradation of mitochondria and other organelles occurring upon lens fiber cell maturation. Loss of lens mitochondrial function in the epithelium or failure to degrade mitochondria and other organelles in lens fiber cells results in lens cataract formation. To date, the mechanisms that govern the maintenance of mitochondria in the lens and the degradation of mitochondria during programmed lens fiber cell maturation have not been fully elucidated. Here, we demonstrate using electron microscopy and dual-label confocal imaging the presence of autophagic vesicles containing mitochondria in lens epithelial cells, immature lens fiber cells and during early stages of lens fiber cell differentiation. We also show that mitophagy is induced in primary lens epithelial cells upon serum starvation. These data provide evidence that autophagy occurs throughout the lens and that mitophagy functions in the lens to remove damaged mitochondria from the lens epithelium and to degrade mitochondria in the differentiating lens fiber cells for lens development. The results provide a novel mechanism for how mitochondria are maintained to preserve lens metabolic function and how mitochondria are degraded upon lens fiber cell maturation.
Assuntos
Autofagia , Catarata/patologia , Cristalino/ultraestrutura , Mitofagia , Organelas/ultraestrutura , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Catarata/metabolismo , Proliferação de Células , Embrião de Galinha , Humanos , Cristalino/metabolismo , Microscopia Eletrônica , Pessoa de Meia-Idade , Adulto JovemRESUMO
Chemotherapy dosing in hematopoietic cell therapy (HCT) conditioning regimens is based on patient weight. We hypothesized that potential underdosing or overdosing of patients with significant deviation of weight from normal might alter HCT outcomes, such as early mortality, overall or organ-specific toxicity, and/or relapse. We therefore conducted a retrospective analysis of 400 children between the ages of 2 and 18 years who underwent HCT for malignant or nonmalignant disease at Boston Children's Hospital over a 10-year period. Using the Centers for Disease Control and Prevention standard weight classification schema, we found no evidence to suggest a difference in survival or in time to engraftment or in relapse in patients with malignant disease. In the subgroups of patients either receiving autologous HCT or with underlying malignancy, combined overweight and obese patients had a higher rate of any organ, but not organ-specific, Grade 3-5 toxicity compared with the normal weight group. The study was not powered to detect a difference between underweight and normal weight patients. These data suggest that multiple outcome measures over the first year after HCT are unaffected by weight.
Assuntos
Transplante de Células-Tronco Hematopoéticas/mortalidade , Obesidade/mortalidade , Adolescente , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Neoplasias/mortalidade , Neoplasias/terapia , Estudos Retrospectivos , Taxa de Sobrevida , Transplante Autólogo , Transplante HomólogoRESUMO
Recent advances in next-generation sequencing and data analysis have provided new gateways for identification of novel genome-wide genetic determinants governing tissue development and disease. These advances have revolutionized our understanding of cellular differentiation, homeostasis, and specialized function in multiple tissues. Bioinformatic and functional analysis of these genetic determinants and the pathways they regulate have provided a novel basis for the design of functional experiments to answer a wide range of long-sought biological questions. A well-characterized model for the application of these emerging technologies is the development and differentiation of the ocular lens and how individual pathways regulate lens morphogenesis, gene expression, transparency, and refraction. Recent applications of next-generation sequencing analysis on well-characterized chicken and mouse lens differentiation models using a variety of omics techniques including RNA-seq, ATAC-seq, whole-genome bisulfite sequencing (WGBS), chip-seq, and CUT&RUN have revealed a wide range of essential biological pathways and chromatin features governing lens structure and function. Multiomics integration of these data has established new gene functions and cellular processes essential for lens formation, homeostasis, and transparency including the identification of novel transcription control pathways, autophagy remodeling pathways, and signal transduction pathways, among others. This review summarizes recent omics technologies applied to the lens, methods for integrating multiomics data, and how these recent technologies have advanced our understanding ocular biology and function. The approach and analysis are relevant to identifying the features and functional requirements of more complex tissues and disease states.
Assuntos
Cristalino , Multiômica , Animais , Camundongos , Cristalino/metabolismo , Regulação da Expressão Gênica , Cromatina/metabolismo , GenomaRESUMO
Recent evidence points to autophagy as an essential cellular requirement for achieving the mature structure, homeostasis, and transparency of the lens. Collective evidence from multiple laboratories using chick, mouse, primate, and human model systems provides evidence that classic autophagy structures, ranging from double-membrane autophagosomes to single-membrane autolysosomes, are found throughout the lens in both undifferentiated lens epithelial cells and maturing lens fiber cells. Recently, key autophagy signaling pathways have been identified to initiate critical steps in the lens differentiation program, including the elimination of organelles to form the core lens organelle-free zone. Other recent studies using ex vivo lens culture demonstrate that the low oxygen environment of the lens drives HIF1a-induced autophagy via upregulation of essential mitophagy components to direct the specific elimination of the mitochondria, endoplasmic reticulum, and Golgi apparatus during lens fiber cell differentiation. Pioneering studies on the structural requirements for the elimination of nuclei during lens differentiation reveal the presence of an entirely novel structure associated with degrading lens nuclei termed the nuclear excisosome. Considerable evidence also indicates that autophagy is a requirement for lens homeostasis, differentiation, and transparency, since the mutation of key autophagy proteins results in human cataract formation.
Assuntos
Catarata , Cristalino , Camundongos , Humanos , Animais , Cristalino/metabolismo , Autofagia , Núcleo Celular/metabolismo , Catarata/metabolismo , Diferenciação CelularRESUMO
Purpose: During lens fiber cell differentiation, organelles are removed in an ordered manner to ensure lens clarity. A critical step in this process is removal of the cell nucleus, but the mechanisms by which this occurs are unclear. In this study, we investigate the role of a cyclin-dependent kinase 1 (CDK1) regulatory loop in controlling lens fiber cell denucleation (LFCD). Methods: We examined lens differentiation histologically in two different vertebrate models. An embryonic chick lens culture system was used to test the role of CDK1, cell division cycle 25 (CDC25), WEE1, and PP2A in LFCD. Additionally, we used three mouse models that express high levels of the CDK inhibitor p27 to test whether increased p27 levels affect LFCD. Results: Using chick lens organ cultures, small-molecule inhibitors of CDK1 and CDC25 inhibit LFCD, while inhibiting the CDK1 inhibitory kinase WEE1 potentiates LFCD. Additionally, treatment with an inhibitor of PP2A, which indirectly inhibits CDK1 activity, also increased LFCD. Three different mouse models that express increased levels of p27 through different mechanisms show impaired LFCD. Conclusions: Here we define a conserved nonmitotic role for CDK1 and its upstream regulators in controlling LFCD. We find that CDK1 functionally interacts with WEE1, a nuclear kinase that inhibits CDK1 activity, and CDC25 activating phosphatases in cells where CDK1 activity must be exquisitely regulated to allow for LFCD. We also provide genetic evidence in multiple in vivo models that p27, a CDK1 inhibitor, inhibits lens growth and LFCD.
Assuntos
Proteína Quinase CDC2 , Mitose , Camundongos , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Fosforilação , Proteínas de Ciclo Celular/genética , Diferenciação CelularRESUMO
PURPOSE: Mutation of the autophagy gene FYVE (named after the four cysteine-rich proteins: Fab 1 [yeast orthologue of PIKfyve], YOTB, Vac 1 [vesicle transport protein], and EEA1) and coiled coil containing 1 (fyco1) causes human cataract suggesting a role for autophagy in lens function. Here, we analyzed the range and spatial expression patterns of lens autophagy genes and we evaluated whether autophagy could be induced in lens cells exposed to stress. METHODS: Autophagy gene expression levels and their spatial distribution patterns were evaluated between microdissected human lens epithelium and fibers at the mRNA and protein levels by microarray data analysis, real-time PCR and western blot analysis. Selected autophagy protein spatial expression patterns were also examined in newborn mouse lenses by immunohistochemistry. The autophagosomal content of cultured human lens epithelial cells was determined by counting the number of microtubule-associated protein 1 light chain 3B (LC3B)-positive puncta in cells cultured in the presence or absence of serum. RESULTS: A total of 42 autophagy genes were detected as being expressed by human lens epithelium and fibers. The autophagosomal markers LC3B and FYCO1 were detected throughout the newborn mouse lens. Consistently, the autophagy active form of LC3B (LC3B II) was detected in microdissected human lens fibers. An increased number of LC3B-positive puncta was detected in cultured lens cells upon serum starvation suggesting induction of autophagy in lens cells under stress conditions. CONCLUSIONS: The data provide evidence that autophagy is an important component for the function of lens epithelial and fiber cells. The data are consistent with the notion that disruption of lens autophagy through mutation or inactivation of specific autophagy proteins could lead to loss of lens resistance to stress and/or loss of lens differentiation resulting in cataract formation.
Assuntos
Autofagia/genética , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Fibroblastos/metabolismo , Expressão Gênica , Cristalino/metabolismo , Idoso , Animais , Animais Recém-Nascidos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Proteínas do Olho/metabolismo , Fibroblastos/citologia , Perfilação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Cristalino/citologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Soro/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
INTRODUCTION: The purpose of this study is to examine student perceptions of accomplishment among 6 subdomains of Center for Advancement of Pharmacy Education (CAPE) Domain 3 "Approach to Practice and Care" outcomes in Advanced Pharmacy Practice Experiences (APPE) across distinct geographical regions. METHODS: An 18-item electronic survey was distributed to 88 student pharmacists at a private university completing APPEs in 5 distinct regions and 2 concentrated learning experiences during their penultimate rotation. The survey assessed whether students had at least 1 opportunity to achieve Domain 3 outcomes. Students were prompted to report a percentage of perceived successful accomplishment of outcomes if they stated they had at least 1 opportunity for achievement. RESULTS: Survey response rate was 52% (n = 46). Respondents reported a median accomplishment of at least 85% for each question. For 2 questions, respondents reported a median accomplishment of 99%. Students perceived successful accomplishment for most of the questions related to communication outcomes, while the lowest completion percentages were noted in outcomes related to patient advocacy (85%) and problem solving (88%). Student perceptions of accomplishment among the 6 subdomains were similar across regions and concentrated learning experiences. CONCLUSIONS: Students felt confident in accomplishing the outcomes associated with CAPE Domain 3. Regional assignments did not impact student perceptions of outcome accomplishment. Preceptors may play a pivotal role in providing students with opportunities to further polish their skills and increase confidence, specifically in the areas of patient advocacy and problem solving.