RESUMEN
Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.
Asunto(s)
Afinidad de Anticuerpos , Linfocitos B , Centro Germinal , Anticuerpos Anti-VIH , VIH-1 , Centro Germinal/inmunología , Animales , Ratones , Humanos , Linfocitos B/inmunología , VIH-1/inmunología , Anticuerpos Anti-VIH/inmunología , Afinidad de Anticuerpos/inmunología , Anticuerpos Neutralizantes/inmunología , Infecciones por VIH/inmunología , Vacunas contra el SIDA/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Técnicas de Sustitución del Gen , Ratones Transgénicos , Anticuerpos ampliamente neutralizantes/inmunología , Ratones Endogámicos C57BLRESUMEN
Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.
RESUMEN
Skin sun exposure induces two protection programs: stress responses and pigmentation, the former within minutes and the latter only hours afterward. Although serving the same physiological purpose, it is not known whether and how these programs are coordinated. Here, we report that UVB exposure every other day induces significantly more skin pigmentation than the higher frequency of daily exposure, without an associated increase in stress responses. Using mathematical modeling and empirical studies, we show that the melanocyte master regulator, MITF, serves to synchronize stress responses and pigmentation and, furthermore, functions as a UV-protection timer via damped oscillatory dynamics, thereby conferring a trade-off between the two programs. MITF oscillations are controlled by multiple negative regulatory loops, one at the transcriptional level involving HIF1α and another post-transcriptional loop involving microRNA-148a. These findings support trait linkage between the two skin protection programs, which, we speculate, arose during furless skin evolution to minimize skin damage.
Asunto(s)
Factor de Transcripción Asociado a Microftalmía/metabolismo , Piel/metabolismo , Piel/efectos de la radiación , Animales , Línea Celular , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Masculino , Melanocitos/fisiología , Melanocitos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , MicroARNs/fisiología , Factor de Transcripción Asociado a Microftalmía/efectos de la radiación , Cultivo Primario de Células , Pigmentación de la Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversosRESUMEN
Metastases are the major cause of cancer-related death, yet, molecular weaknesses that could be exploited to prevent tumor cells spreading are poorly known. Here, we found that perturbing hydrolase transport to lysosomes by blocking either the expression of IGF2R, the main receptor responsible for their trafficking, or GNPT, a transferase involved in the addition of the specific tag recognized by IGF2R, reduces melanoma invasiveness potential. Mechanistically, we demonstrate that the perturbation of this traffic, leads to a compensatory lysosome neo-biogenesis devoided of degradative enzymes. This regulatory loop relies on the stimulation of TFEB transcription factor expression. Interestingly, the inhibition of this transcription factor playing a key role of lysosome production, restores melanomas' invasive potential in the absence of hydrolase transport. These data implicate that targeting hydrolase transport in melanoma could serve to develop new therapies aiming to prevent metastasis by triggering a physiological response stimulating TFEB expression in melanoma.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Hidrolasas , Lisosomas , Melanoma , Humanos , Melanoma/genética , Melanoma/patología , Melanoma/metabolismo , Lisosomas/metabolismo , Hidrolasas/metabolismo , Hidrolasas/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Línea Celular Tumoral , Receptor IGF Tipo 2/metabolismo , Receptor IGF Tipo 2/genética , Metástasis de la Neoplasia , Transporte de Proteínas , Regulación Neoplásica de la Expresión GénicaRESUMEN
Introduction: The first cells affected by UVB exposure are epidermal keratinocytes, and p53, the genome guardian, is activated in these cells when skin is exposed to UVB. UVB exposure induces appetite, but it remains unclear whether p53 in epidermal keratinocytes plays a role in this appetite stimulation. Results: Here we found that food intake was increased following chronic daily UVB exposure in a manner that depends on p53 expression in epidermal keratinocytes. p53 conditional knockout in epidermal keratinocytes reduced food intake in mice upon UVB exposure. Methods: To investigate the effects of p53 activation following UVB exposure, mice behavior was assessed using the staircase, open-field, elevated-plus maze, and conditioned-place preference tests. In addition to effects on appetite, loss of p53 resulted in anxiety-related behaviors with no effect on activity level. Discussion: Since skin p53 induces production of ß-endorphin, our data suggest that UVB-mediated activation of p53 results in an increase in ß-endorphin levels which in turn influences appetite. Our study positions UVB as a central environmental factor in systemic behavior and has implications for the treatment of eating and anxiety-related disorders.
RESUMEN
Anti-Müllerian hormone (AMH) is produced exclusively by granulosa cells of ovarian follicles and is an indicator of ovarian reserve which declines with age. Seasonality in AMH levels have been reported to be correlated with variations in Vitamin D levels, which is dependent on sunlight exposure. However, the effects of age and its association with solar radiation intensity with respect to AMH was never studied before. In this study, we investigated the relationship between AMH levels with season and with solar radiation intensity in a cohort of 2235 women aged 19-40 years undergoing hormonal work-up over a four-year period. Our findings revealed that among women aged 20-29 years, there was no significant association between AMH levels and either season or solar radiation intensity. However, for women aged 30-40 years, a seasonal pattern was observed, with higher AMH levels during spring and autumn months characterized by moderate solar radiation intensity. Women in their declining ovarian reserve age were found to be more sensitive to the effects of moderate solar radiation. Moderate solar radiation exposure positively impacted AMH levels, whereas low and high intensity exposure had a negative effect. Our findings indicate that age and solar radiation intensity must be considered when assessing AMH levels and provide valuable insights into the intricate relationship between AMH, seasonality, and UVB exposure in the context of reproductive health.
Asunto(s)
Hormona Antimülleriana , Folículo Ovárico , Femenino , Humanos , Estaciones del Año , Células de la GranulosaRESUMEN
Skin pigmentation is paused after sun exposure; however, the mechanism behind this pausing is unknown. In this study, we found that the UVB-induced DNA repair system, led by the ataxia telangiectasia mutated (ATM) protein kinase, represses MITF transcriptional activity of pigmentation genes while placing MITF in DNA repair mode, thus directly inhibiting pigment production. Phosphoproteomics analysis revealed ATM to be the most significantly enriched pathway among all UVB-induced DNA repair systems. ATM inhibition in mouse or human skin, either genetically or chemically, induces pigmentation. Upon UVB exposure, MITF transcriptional activation is blocked owing to ATM-dependent phosphorylation of MITF on S414, which modifies MITF activity and interactome toward DNA repair, including binding to TRIM28 and RBBP4. Accordingly, MITF genome occupancy is enriched in sites of high DNA damage that are likely repaired. This suggests that ATM harnesses the pigmentation key activator for the necessary rapid, efficient DNA repair, thus optimizing the chances of the cell surviving. Data are available from ProteomeXchange with the identifier PXD041121.
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Ataxia Telangiectasia , Humanos , Animales , Ratones , Pigmentación de la Piel/genética , Reparación del ADN , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Transducción de Señal , Daño del ADN , Fosforilación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismoRESUMEN
Normal growth and development in mammals are tightly controlled by numerous genetic factors and metabolic conditions. The growth hormone (GH)-insulin-like growth factor-1 (IGF1) hormonal axis is a key player in the regulation of these processes. Dysregulation of the GH-IGF1 endocrine system is linked to a number of pathologies, ranging from growth deficits to cancer. Laron syndrome (LS) is a type of dwarfism that results from mutation of the GH receptor (GHR) gene, leading to GH resistance and short stature as well as a number of metabolic abnormalities. Of major clinical relevance, epidemiological studies have shown that LS patients do not develop cancer. While the mechanisms associated with cancer protection in LS have not yet been elucidated, genomic analyses have identified a series of metabolic genes that are over-represented in LS patients. We hypothesized that these genes might constitute novel targets for IGF1 action. With a fold-change of 11.09, UDP-glucuronosyltransferase 2B15 (UGT2B15) was the top up-regulated gene in LS. The UGT2B15 gene codes for an enzyme that converts xenobiotic substances into lipophilic compounds and thereby facilitates their clearance from the body. We investigated the regulation of UGT2B15 gene expression by IGF1 and insulin. Both hormones inhibited UGT2B15 mRNA levels in endometrial and breast cancer cell lines. Regulation of UGT2B15 protein levels by IGF1/insulin, however, was more complex and not always correlated with mRNA levels. Furthermore, UGT2B15 expression was dependent on p53 status. Thus, UGT2B15 mRNA levels were higher in cell lines expressing a wild-type p53 compared to cells containing a mutated p53. Animal studies confirmed an inverse correlation between UGT2B15 and p53 levels. In summary, increased UGT2B15 levels in LS might confer upon patient's protection from genotoxic damage.
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Glucuronosiltransferasa/metabolismo , Síndrome de Laron , Neoplasias , Animales , Glucuronosiltransferasa/genética , Glicosiltransferasas/metabolismo , Hormona del Crecimiento/metabolismo , Humanos , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Síndrome de Laron/genética , Síndrome de Laron/metabolismo , Mamíferos/metabolismo , Neoplasias/metabolismo , ARN Mensajero/genética , Proteína p53 Supresora de Tumor/genética , Uridina DifosfatoRESUMEN
Exercise prevents cancer incidence and recurrence, yet the underlying mechanism behind this relationship remains mostly unknown. Here we report that exercise induces the metabolic reprogramming of internal organs that increases nutrient demand and protects against metastatic colonization by limiting nutrient availability to the tumor, generating an exercise-induced metabolic shield. Proteomic and ex vivo metabolic capacity analyses of murine internal organs revealed that exercise induces catabolic processes, glucose uptake, mitochondrial activity, and GLUT expression. Proteomic analysis of routinely active human subject plasma demonstrated increased carbohydrate utilization following exercise. Epidemiologic data from a 20-year prospective study of a large human cohort of initially cancer-free participants revealed that exercise prior to cancer initiation had a modest impact on cancer incidence in low metastatic stages but significantly reduced the likelihood of highly metastatic cancer. In three models of melanoma in mice, exercise prior to cancer injection significantly protected against metastases in distant organs. The protective effects of exercise were dependent on mTOR activity, and inhibition of the mTOR pathway with rapamycin treatment ex vivo reversed the exercise-induced metabolic shield. Under limited glucose conditions, active stroma consumed significantly more glucose at the expense of the tumor. Collectively, these data suggest a clash between the metabolic plasticity of cancer and exercise-induced metabolic reprogramming of the stroma, raising an opportunity to block metastasis by challenging the metabolic needs of the tumor. SIGNIFICANCE: Exercise protects against cancer progression and metastasis by inducing a high nutrient demand in internal organs, indicating that reducing nutrient availability to tumor cells represents a potential strategy to prevent metastasis. See related commentary by Zerhouni and Piskounova, p. 4124.
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Ejercicio Físico , Melanoma , Nutrientes , Proteómica , Animales , Humanos , Ratones , Glucosa/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Estudios Prospectivos , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Ejercicio Físico/fisiología , Nutrientes/genética , Nutrientes/metabolismoRESUMEN
Sexual dimorphisms are responsible for profound metabolic differences in health and behavior. Whether males and females react differently to environmental cues, such as solar ultraviolet (UV) exposure, is unknown. Here we show that solar exposure induces food-seeking behavior, food intake, and food-seeking behavior and food intake in men, but not in women, through epidemiological evidence of approximately 3,000 individuals throughout the year. In mice, UVB exposure leads to increased food-seeking behavior, food intake and weight gain, with a sexual dimorphism towards males. In both mice and human males, increased appetite is correlated with elevated levels of circulating ghrelin. Specifically, UVB irradiation leads to p53 transcriptional activation of ghrelin in skin adipocytes, while a conditional p53-knockout in mice abolishes UVB-induced ghrelin expression and food-seeking behavior. In females, estrogen interferes with the p53-chromatin interaction on the ghrelin promoter, thus blocking ghrelin and food-seeking behavior in response to UVB exposure. These results identify the skin as a major mediator of energy homeostasis and may lead to therapeutic opportunities for sex-based treatments of endocrine-related diseases.
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Ghrelina , Proteína p53 Supresora de Tumor , Animales , Apetito , Femenino , Ghrelina/farmacología , Humanos , Masculino , Ratones , Proteína p53 Supresora de Tumor/genética , Rayos Ultravioleta , Aumento de PesoRESUMEN
Almost half of the human microRNAs (miRNAs) are encoded in clusters. Although transcribed as a single unit, the levels of individual mature miRNAs often differ. The mechanisms underlying differential biogenesis of clustered miRNAs and the resulting physiological implications are mostly unknown. In this study, we report that the melanoma master transcription regulator MITF regulates the differential expression of the 99a/let-7c/125b-2 cluster by altering the distribution of RNA polymerase II along the cluster. We discovered that MITF interacts with TRIM28, a known inhibitor of RNA polymerase II transcription elongation, at the mIR-let-7c region, resulting in the pausing of RNA polymerase II activity and causing an elevation in mIR-let-7c expression; low levels of RNA polymerase II occupation over miR-99a and miR-125b-2 regions decreases their biogenesis. Furthermore, we showed that this differential expression affects the phenotypic state of melanoma cells. RNA-sequencing analysis of proliferative melanoma cells that express miR-99a and miR-125b mimics revealed a transcriptomic shift toward an invasive phenotype. Conversely, expression of a mIR-let-7c mimic in invasive melanoma cells induced a shift to a more proliferative state. We confirmed direct target genes of these miRNAs, including FGFR3, BAP1, Bcl2, TGFBR1, and CDKN1A. Our study demonstrates an MITF-governed biogenesis mechanism that results in differential expression of clustered 99a/let-7c/125b-2 miRNAs that control melanoma progression.
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Adaptación Fisiológica/fisiología , Melanoma/genética , MicroARNs/genética , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Melanoma/fisiopatología , Ratones , Factor de Transcripción Asociado a Microftalmía/fisiología , Transcripción Genética , Proteína 28 que Contiene Motivos Tripartito/fisiologíaRESUMEN
Ultraviolet (UV) light affects endocrinological and behavioral aspects of sexuality via an unknown mechanism. Here we discover that ultraviolet B (UVB) exposure enhances the levels of sex-steroid hormones and sexual behavior, which are mediated by the skin. In female mice, UVB exposure increases hypothalamus-pituitary-gonadal axis hormone levels, resulting in larger ovaries; extends estrus days; and increases anti-Mullerian hormone (AMH) expression. UVB exposure also enhances the sexual responsiveness and attractiveness of females and male-female interactions. Conditional knockout of p53 specifically in skin keratinocytes abolishes the effects of UVB. Thus, UVB triggers a skin-brain-gonadal axis through skin p53 activation. In humans, solar exposure enhances romantic passion in both genders and aggressiveness in men, as seen in analysis of individual questionaries, and positively correlates with testosterone level. Our findings suggest opportunities for treatment of sex-steroid-related dysfunctions.
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Hormona Antimülleriana/biosíntesis , Sistema Hipotálamo-Hipofisario/metabolismo , Ovario/metabolismo , Conducta Sexual/efectos de la radiación , Piel/metabolismo , Testosterona/biosíntesis , Rayos Ultravioleta , Animales , Estro/metabolismo , Femenino , Técnicas de Inactivación de Genes , Queratinocitos/metabolismo , Masculino , RatonesRESUMEN
MicroRNA (miRNA) biogenesis initiates co-transcriptionally, but how the Microprocessor machinery pinpoints the locations of short precursor miRNA sequences within long flanking regions of the transcript is not known. Here we show that miRNA biogenesis depends on DNA methylation. When the regions flanking the miRNA coding sequence are highly methylated, the miRNAs are more highly expressed, have greater sequence conservation, and are more likely to drive cancer-related phenotypes than miRNAs encoded by unmethylated loci. We show that the removal of DNA methylation from miRNA loci leads to their downregulation. Further, we found that MeCP2 binding to methylated miRNA loci halts RNA polymerase II elongation, leading to enhanced processing of the primary miRNA by Drosha. Taken together, our data reveal that DNA methylation directly affects miRNA biogenesis.
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MicroARNs/genética , Animales , Línea Celular , Metilación de ADN , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , MicroARNs/metabolismo , Sistemas de Lectura Abierta , Procesamiento Postranscripcional del ARNRESUMEN
Melanoma, a melanocyte origin neoplasm, is the most lethal type of skin cancer, and incidence is increasing. Several familial and somatic mutations have been identified in the gene encoding the melanocyte lineage master regulator, MITF; however, the neoplastic mechanisms of these mutant MITF variants are mostly unknown. Here, by performing unbiased analysis of the transcriptomes in cells expressing mutant MITF, we identified calcium-binding protein S100A4 as a downstream target of MITF-E87R. By using wild-type and mutant MITF melanoma lines, we found that both endogenous wild-type and MITF-E87R variants occupy the S100A4 promoter. Remarkably, whereas wild-type MITF represses S100A4 expression, MITF-E87R activates its transcription. The opposite effects of wild-type and mutant MITF result in opposing cellular phenotypes, because MITF-E87R via S100A4 enhanced invasion and reduced adhesion in contrast to wild-type MITF activity. Finally, we found that melanoma patients with altered S100A4 expression have poor prognosis. These data show that a change in MITF transcriptional activity from repression to activation of S100A4 that results from a point mutation in MITF alters melanoma invasive ability. These data suggest new opportunities for diagnosis and treatment of metastatic melanoma.
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ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Mutación , Proteína de Unión al Calcio S100A4/genética , Neoplasias Cutáneas/genética , Análisis Mutacional de ADN , Progresión de la Enfermedad , Humanos , Immunoblotting , Melanoma/metabolismo , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteína de Unión al Calcio S100A4/biosíntesis , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
Ectopic expression of lineage master regulators induces transdifferentiation. Whether cell fate transitions can be induced during various developmental stages has not been systemically examined. Here we discover that amongst different developmental stages, mouse embryonic stem cells (mESCs) are resistant to cell fate conversion induced by the melanocyte lineage master regulator MITF. By generating a transgenic system we exhibit that in mESCs, the pluripotency master regulator Oct4, counteracts pro-differentiation induced by Mitf by physical interference with MITF transcriptional activity. We further demonstrate that mESCs must be released from Oct4-maintained pluripotency prior to ectopically induced differentiation. Moreover, Oct4 induction in various differentiated cells represses their lineage identity in vivo. Alongside, chromatin architecture combined with ChIP-seq analysis suggest that Oct4 competes with various lineage master regulators for binding promoters and enhancers. Our analysis reveals pluripotency and transdifferentiation regulatory principles and could open new opportunities in the field of regenerative medicine.