RESUMEN
Preclinical human-relevant modeling of organ-specific vasculature offers a unique opportunity to recreate pathophysiological intercellular, tissue-tissue, and cell-matrix interactions for a broad range of applications. Here, we present a reliable, and simply reproducible process for constructing user-controlled long rounded extracellular matrix (ECM)-embedded vascular microlumens on-chip for endothelization and co-culture with stromal cells obtained from human lung. We demonstrate the critical impact of microchannel cross-sectional geometry and length on uniform distribution and magnitude of vascular wall shear stress, which is key when emulating in vivo-observed blood flow biomechanics in health and disease. In addition, we provide an optimization protocol for multicellular culture and functional validation of the system. Moreover, we show the ability to finely tune rheology of the three-dimensional natural matrix surrounding the vascular microchannel to match pathophysiological stiffness. In summary, we provide the scientific community with a matrix-embedded microvasculature on-chip populated with all-primary human-derived pulmonary endothelial cells and fibroblasts to recapitulate and interrogate lung parenchymal biology, physiological responses, vascular biomechanics, and disease biogenesis in vitro. Such a mix-and-match synthetic platform can be feasibly adapted to study blood vessels, matrix, and ECM-embedded cells in other organs and be cellularized with additional stromal cells.
RESUMEN
Tissue injury induces metabolic changes in stem cells, which likely modulate regeneration. Using a model of organ regeneration called wound-induced hair follicle neogenesis (WIHN), we identified skin-resident bacteria as key modulators of keratinocyte metabolism, demonstrating a positive correlation between bacterial load, glutamine metabolism, and regeneration. Specifically, through comprehensive multiomic analysis and single-cell RNA sequencing in murine skin, we show that bacterially induced hypoxia drives increased glutamine metabolism in keratinocytes with attendant enhancement of skin and hair follicle regeneration. In human skin wounds, topical broad-spectrum antibiotics inhibit glutamine production and are partially responsible for reduced healing. These findings reveal a conserved and coherent physiologic context in which bacterially induced metabolic changes improve the tolerance of stem cells to damage and enhance regenerative capacity. This unexpected proregenerative modulation of metabolism by the skin microbiome in both mice and humans suggests important methods for enhancing regeneration after injury.
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Glutamina , Folículo Piloso , Animales , Humanos , Ratones , Glutamina/metabolismo , Queratinocitos , Regeneración , Piel/metabolismo , Cicatrización de Heridas , MicrobiotaRESUMEN
Uniquely among mammalian organs, skin is capable of marked size change in adults, yet the mechanisms underlying this notable capacity are unclear. Here, we use a system of controlled tissue expansion in mice to uncover cellular and molecular determinants of skin growth. Through machine learning-guided three-dimensional tissue reconstruction, we capture morphometric changes in growing skin. We find that most growth is driven by the proliferation of the epidermis in response to mechanical tension, with more limited changes in dermal and subdermal compartments. Epidermal growth is achieved through preferential activation and differentiation of Lgr6+ stem cells of the epidermis, driven in part by the Hippo pathway. By single-cell RNA sequencing, we uncover further changes in mechanosensitive and metabolic pathways underlying growth control in the skin. These studies point to therapeutic strategies to enhance skin growth and establish a platform for understanding organ size dynamics in adult mammals.
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Células Epidérmicas , Receptores Acoplados a Proteínas G , Piel , Células Madre , Animales , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Epidermis/crecimiento & desarrollo , Epidermis/metabolismo , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Piel/crecimiento & desarrollo , Piel/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Chronic and low-grade inflammation associated with persistent bacterial infections has been linked to colon tumor development; however, the impact of transient and self-limited infections in bacterially driven colon tumorigenesis has remained enigmatic. Here we report that UshA is a novel genotoxin in attaching/effacing (A/E) pathogens, which include the human pathogens enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and their murine equivalent Citrobacter rodentium (CR). UshA harbors direct DNA digestion activity with a catalytic histidine-aspartic acid dyad. Injected via the type III secretion system (T3SS) into host cells, UshA triggers DNA damage and initiates tumorigenic transformation during infections in vitro and in vivo. Moreover, UshA plays an indispensable role in CR infection-accelerated colon tumorigenesis in genetically susceptible Apc MinΔ716/+ mice. Collectively, our results reveal that UshA, functioning as a bacterial T3SS-dependent genotoxin, plays a critical role in prompting transient and noninvasive bacterial infection-accelerated colon tumorigenesis in mice. SIGNIFICANCE: We identified UshA, a novel T3SS-dependent genotoxin in A/E pathogens that possesses direct DNA digestion activity and confers bacterially accelerated colon tumorigenesis in mice. Our results demonstrate that acute and noninvasive infection with A/E pathogens harbors a far-reaching impact on the development of colon cancer.This article is highlighted in the In This Issue feature, p. 1.
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Transformación Celular Neoplásica/patología , Citrobacter rodentium/patogenicidad , Neoplasias Colorrectales/patología , Escherichia coli Enteropatógena/patogenicidad , Mutágenos/farmacología , Animales , Línea Celular Tumoral/efectos de los fármacos , Neoplasias Colorrectales/microbiología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound-induced hair follicle neogenesis (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound-associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single-cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3-/- mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder regeneration.
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Trampas Extracelulares , Neutrófilos/inmunología , Neutrófilos/metabolismo , Regeneración , Animales , Biomarcadores , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Ratones , Ratones Noqueados , Infiltración Neutrófila , Análisis de la Célula Individual/métodos , Piel/metabolismo , Cicatrización de Heridas/genética , Cicatrización de Heridas/inmunologíaRESUMEN
Environmental factors that enhance regeneration are largely unknown. The immune system and microbiome are attributed roles in repairing and regenerating structure but their precise interplay is unclear. Here, we assessed the function of skin bacteria in wound healing and wound-induced hair follicle neogenesis (WIHN), a rare adult organogenesis model. WIHN levels and stem cell markers correlate with bacterial counts, being lowest in germ-free (GF), intermediate in conventional specific pathogen-free (SPF), and highest in wild-type mice, even those infected with pathogenic Staphylococcus aureus. Reducing skin microbiota via cage changes or topical antibiotics decreased WIHN. Inflammatory cytokine IL-1ß and keratinocyte-dependent IL-1R-MyD88 signaling are necessary and sufficient for bacteria to promote regeneration. Finally, in a small trial, a topical broad-spectrum antibiotic also slowed skin wound healing in adult volunteers. These results demonstrate a role for IL-1ß to control morphogenesis and support the need to reconsider routine applications of topical prophylactic antibiotics.
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Interleucina-1beta/metabolismo , Piel/microbiología , Piel/fisiopatología , Heridas y Lesiones/microbiología , Heridas y Lesiones/fisiopatología , Adolescente , Adulto , Animales , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Femenino , Humanos , Interleucina-1beta/genética , Queratinocitos/metabolismo , Queratinocitos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Microbiota , Persona de Mediana Edad , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Regeneración , Transducción de Señal , Piel/metabolismo , Cicatrización de Heridas , Heridas y Lesiones/genética , Heridas y Lesiones/metabolismo , Adulto JovenRESUMEN
IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL36 cytokines in human atopic dermatitis skin and in IL36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL36 responses represent a key mechanism and potential therapeutic target against allergic diseases.
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Dermatitis Atópica/inmunología , Inmunoglobulina E/inmunología , Interleucina-1/inmunología , Queratinocitos/inmunología , Células Plasmáticas/inmunología , Staphylococcus aureus/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Dermatitis Atópica/genética , Dermatitis Atópica/microbiología , Humanos , Cambio de Clase de Inmunoglobulina , Inmunoglobulina E/genética , Interleucina-1/genética , Interleucina-4/genética , Interleucina-4/inmunología , Queratinocitos/microbiología , Ratones , Ratones Noqueados , Células Plasmáticas/patologíaRESUMEN
Wound-induced hair follicle neogenesis (WIHN) is a phenomenon that occurs in adult mammalian skin, where fully functional hair follicles are regenerated in the center of large full-thickness excisional wounds. Although originally discovered over 50 years ago in mice and rabbits, within the last decade it has received renewed interest, as the molecular mechanism has begun to be defined. This de novo regeneration of hair follicles largely recapitulates embryonic hair development, requiring canonical Wnt signaling in the epidermis, however, important differences between the two are beginning to come to light. TLR3 mediated double stranded RNA sensing is critical for the regeneration, activating retinoic acid signaling following wounding. Inflammatory cells, including Fgf9-producing γ-δ T cells and macrophages, are also emerging as important mediators of WIHN. Additionally, while dispensable in embryonic hair follicle development, Shh signaling plays a major role in WIHN and may be able to redirect cells fated to scarring wounds into a regenerative phenotype. The cellular basis of WIHN is also becoming clearer, with increasing evidence suggesting an incredible level of cellular plasticity. Multiple stem cell populations, along with lineage switching of differentiated cells all contribute towards the regeneration present in WIHN. Further study of WIHN will uncover key steps in mammalian development and regeneration, potentially leading to new clinical treatments for hair-related disorders or fibrotic scarring.
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Folículo Piloso/crecimiento & desarrollo , Regeneración , Piel/metabolismo , Cicatrización de Heridas , Animales , Folículo Piloso/metabolismo , Humanos , Piel/crecimiento & desarrolloRESUMEN
How developmental programs reactivate in regeneration is a fundamental question in biology. We addressed this question through the study of Wound Induced Hair follicle Neogenesis (WIHN), an adult organogenesis model where stem cells regenerate de novo hair follicles following deep wounding. The exact mechanism is uncertain. Here we show that self-noncoding dsRNA activates the anti-viral receptor toll like receptor 3 (TLR3) to induce intrinsic retinoic acid (RA) synthesis in a pattern that predicts new hair follicle formation after wounding in mice. Additionally, in humans, rejuvenation lasers induce gene expression signatures for dsRNA and RA, with measurable increases in intrinsic RA synthesis. These results demonstrate a potent stimulus for RA synthesis by non-coding dsRNA, relevant to their broad functions in development and immunity.
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Folículo Piloso/fisiología , ARN Bicatenario/fisiología , Regeneración/fisiología , Receptor Toll-Like 3/metabolismo , Tretinoina/metabolismo , Animales , Benzoatos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Cabello/crecimiento & desarrollo , Humanos , Interleucina-6/administración & dosificación , Interleucina-6/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Interferencia de ARN , ARN Interferente Pequeño , Estilbenos/farmacología , Cicatrización de HeridasRESUMEN
Previously we reported that Src-associated-substrate-during-mitosis-of-68kDa (Sam68/KHDRBS1) is pivotal for DNA damage-stimulated NF-κB transactivation of anti-apoptotic genes (Fu et al., 2016). Here we show that Sam68 is critical for genotoxic stress-induced NF-κB activation in the γ-irradiated colon and animal and that Sam68-dependent NF-κB activation provides radioprotection to colon epithelium in vivo. Sam68 deletion diminishes γ-irradiation-triggered PAR synthesis and NF-κB activation in colon epithelial cells (CECs), thus hampering the expression of anti-apoptotic molecules in situ and facilitating CECs to undergo apoptosis in mice post whole-body γ-irradiation (WBIR). Sam68 knockout mice suffer more severe damage in the colon and succumb more rapidly from acute radiotoxicity than the control mice following WBIR. Our results underscore the critical role of Sam68 in orchestrating genotoxic stress-initiated NF-κB activation signaling in the colon tissue and whole animal and reveal the pathophysiological relevance of Sam68-dependent NF-κB activation in colonic cell survival and recovery from extrinsic DNA damage.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Colon/efectos de la radiación , Rayos gamma , Mucosa Intestinal/efectos de la radiación , Subunidad p50 de NF-kappa B/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Ratones NoqueadosRESUMEN
The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/fisiología , Adenosina Difosfato/metabolismo , Animales , Línea Celular Tumoral , Activación Enzimática , Humanos , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Traumatismos Experimentales por Radiación/enzimología , Transducción de Señal , Timo/enzimología , Timo/efectos de la radiaciónRESUMEN
Animal models of colon cancer are widely used to understand the molecular mechanisms and pathogenesis of the disease. These animal models require a substantial investment of time and traditionally necessitate the killing of the animal to measure the tumor progression. Several in vivo imaging techniques are being used in both human clinics and preclinical studies, albeit at high cost and requiring particular expertise. Here, we report that the progression of splenomegaly coincides with and positively correlates to colon tumor development in Apc(min716/+) mice expressing a mutant gene encoding an adenomatous polyposis coli protein truncated at amino acid 716. Ultrasound image-based spleen size measurement precisely mirrors splenomegaly development in vivo in the tumor-laden Apc(min716/+) mice. Moreover, the spleen dimensions extracted from the ultrasound sonograms are positively correlated with normalized spleen weight and the number and area of colon tumors. Hence, we propose measuring the spleen size in vivo by ultrasound imaging as a novel approach to estimate splenomegaly development and to indirectly monitor colon tumor development in Apc(min716/+) mice. The widespread use of ultrasound machines in the laboratory setting, coupled with the fact that it is a noninvasive method, make it a straightforward and useful tool for monitoring the experimental progress of colon cancer in mice and determining end points without killing animals strictly for diagnostics purposes.
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Proteína de la Poliposis Adenomatosa del Colon/genética , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/genética , Predisposición Genética a la Enfermedad , Mutación , Esplenomegalia/diagnóstico por imagen , Animales , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Transgénicos , UltrasonografíaRESUMEN
Nuclear factor kappa B (NF-κB)-mediated transcription is an important mediator for cellular responses to DNA damage. Genotoxic agents trigger a 'nuclear-to-cytoplasmic' NF-κB activation signaling pathway; however, the early nuclear signaling cascade linking DNA damage and NF-κB activation is poorly understood. Here we report that Src-associated-substrate-during-mitosis-of-68kDa/KH domain containing, RNA binding, signal transduction associated 1 (Sam68/KHDRBS1) is a key NF-κB regulator in genotoxic stress-initiated signaling pathway. Sam68 deficiency abolishes DNA damage-stimulated polymers of ADP-ribose (PAR) production and the PAR-dependent NF-κB transactivation of anti-apoptotic genes. Sam68 deleted cells are hypersensitive to genotoxicity caused by DNA damaging agents. Upregulated Sam68 coincides with elevated PAR production and NF-κB-mediated anti-apoptotic transcription in human and mouse colon cancer. Knockdown of Sam68 sensitizes human colon cancer cells to genotoxic stress-induced apoptosis and genetic deletion of Sam68 dampens colon tumor burden in mice. Together our data reveal a novel function of Sam68 in the genotoxic stress-initiated nuclear signaling, which is crucial for colon tumorigenesis.
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Proteínas Adaptadoras Transductoras de Señales/genética , Adenoma/genética , Carcinogénesis/genética , Neoplasias del Colon/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , FN-kappa B/genética , Proteínas de Unión al ARN/genética , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenoma/metabolismo , Adenoma/patología , Adenosina Difosfato Ribosa/metabolismo , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Colon/metabolismo , Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Daño del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Trasplante de Neoplasias , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Transducción de SeñalRESUMEN
Caspase-3-mediated p65 cleavage is believed to suppress nuclear factor-kappa B (NF-κB)-mediated anti-apoptotic transactivation in cells undergoing apoptosis. However, only a small percentage of p65 is cleaved during apoptosis, not in proportion to the dramatic reduction in NF-κB transactivation. Here we show that the p65(1-97) fragment generated by Caspase-3 cleavage interferes with ribosomal protein S3 (RPS3), an NF-κB "specifier" subunit, and selectively retards the nuclear translocation of RPS3, thus dampening the RPS3/NF-κB-dependent anti-apoptotic gene expression. Our findings reveal a novel cell fate determination mechanism to ensure cells undergo programed cell death through interfering with RPS3/NF-κB-conferred anti-apoptotic transcription by the fragment from partial p65 cleavage by activated Caspase-3.
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Apoptosis , Caspasa 3/metabolismo , Fragmentos de Péptidos/metabolismo , Proteolisis , Proteínas Ribosómicas/metabolismo , Factor de Transcripción ReIA/metabolismo , Transcripción Genética , Animales , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Fragmentos de Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Factor de Transcripción ReIA/químicaRESUMEN
Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹â»³8 fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹â»³8 fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.
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Proteínas Bacterianas/inmunología , Infecciones por Enterobacteriaceae/inmunología , Interacciones Huésped-Parásitos/fisiología , Metaloproteasas/inmunología , FN-kappa B/inmunología , Transducción de Señal/inmunología , Animales , Proteínas Bacterianas/metabolismo , Citrobacter rodentium , Infecciones por Enterobacteriaceae/metabolismo , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunoprecipitación , Inflamación/inmunología , Inflamación/metabolismo , Metaloproteasas/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Ribosómicas/inmunología , Proteínas Ribosómicas/metabolismo , Factor de Transcripción ReIA/inmunología , Factor de Transcripción ReIA/metabolismo , TransfecciónRESUMEN
CD25, the alpha chain of the interleukin-2 receptor, is expressed in activated T cells and has a significant role in autoimmune disease and tumorigenesis; however, the mechanisms regulating transcription of CD25 remain elusive. Here we identify the Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel non-Rel component in the nuclear factor-kappaB (NF-κB) complex that confers CD25 transcription. Our results demonstrate that Sam68 has an essential role in the induction and maintenance of CD25 in T cells. T-cell receptor engagement triggers translocation of the inhibitor of NF-κB kinase alpha (IKKα) from the cytoplasm to the nucleus, where it phosphorylates Sam68, causing complex formation with NF-κB in the nucleus. These findings reveal the important roles of KH domain-containing components and their spatial interactions with IKKs in determining the binding targets of NF-κB complexes, thus shedding novel insights into the regulatory specificity of NF-κB.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos/inmunología , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas de Unión al ARN/metabolismo , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Quinasa I-kappa B/metabolismo , Subunidad alfa del Receptor de Interleucina-2/genética , Células Jurkat , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Ribosómicas/metabolismo , Transducción de Señal/genética , Factor de Transcripción ReIA/metabolismo , Transcripción GenéticaRESUMEN
NF-κB is a pleiotrophic transcription factor that plays a prominent regulatory role in various cellular processes. Although previous efforts have focused on its activation, how NF-κB selects specific target genes in response to discrete signals remains puzzling. In addition to the well defined Rel protein components of NF-κB, the ribosomal protein S3 (RPS3) was identified to be an essential component of specific NF-κB complexes. RPS3 synergistically interacts with the NF-κB p65 subunit to achieve optimal binding and transactivation of a subset of NF-κB target genes, thus providing regulatory specificity. Emerging evidence suggests an important role for the RPS3-p65 interaction in context-specific NF-κB gene transcription. The food-borne pathogen Escherichia coli O157:H7 impacts the transcription of a subset of NF-κB target genes encoding proinflammatory cytokines and chemokines in host cells by preventing the nuclear translocation of RPS3, but not p65. The N terminus of p65 is crucial for RPS3 binding. Although several p65 N-terminal fragments are generated by either protease cleavage or alternative mRNA splicing under certain pathophysiological conditions, the role of these fragments in modulating NF-κB signaling, in particular RPS3-dependent selective gene transcription, has not been fully characterized. Here we report that an N-terminal fragment of p65 (amino acids 21-186) can selectively modulate NF-κB gene transcription by competing for RPS3 binding to p65. This 21-186 fragment preferentially localizes in the cytoplasm where it delays stimuli-induced RPS3 nuclear translocation, without affecting the nuclear translocation of p65. Our findings thus uncover a new cytoplasmic function for the N-terminal domain of p65 and provide a novel strategy for selective inhibition of NF-κB gene transcription.
Asunto(s)
Regulación de la Expresión Génica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Ribosómicas/metabolismo , Factor de Transcripción ReIA/química , Factor de Transcripción ReIA/metabolismo , Animales , Caspasa 3/metabolismo , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Células Jurkat , Activación de Linfocitos/genética , Ratones , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Subunidades de Proteína/genética , Transporte de Proteínas , Transducción de Señal/genética , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/inmunología , Factor de Transcripción ReIA/genética , Transcripción GenéticaRESUMEN
BACKGROUND: The developing limb has served as an excellent model for studying pattern formation and signal transduction in mammalians. Many of the crucial genes that regulate growth and patterning of the limb following limb bud formation are now well known. However, details regarding the control of limb initiation and early stages of outgrowth remain to be defined. This report is focused on genetic events that pave the way for the establishment of a hindlimb bud. RESULTS: Fgf10 and Tbx are crucial for early phases of limb bud initiation. Here we show that in the absence of Isl1 or of Ldb1/2, there is no hindlimb bud development. Fgf10 expression in the bud mesenchyme is dependent on Isl1 and its Ldb co-regulators. CONCLUSIONS: Thus, Isl1 and the Ldb co-regulators of transcription are essential early determinants of mouse limb development. Isl1/Ldb complexes regulate Fgf10 to orchestrate the earliest stages of hindlimb formation.