RESUMEN
The transcription factor Nrf2 is the master regulator of cellular stress response, facilitating the expression of cytoprotective genes, including those responsible for drug detoxification, immunomodulation, and iron metabolism. FDA-approved Nrf2 activators, Tecfidera and Skyclarys for patients with multiple sclerosis and Friedreich's ataxia, respectively, are non-specific alkylating agents exerting side effects. Nrf2 is under feedback regulation through its target gene, transcriptional repressor Bach1. Specifically, in Parkinson's disease and other neurodegenerative diseases with Bach1 dysregulation, excessive Bach1 accumulation interferes with Nrf2 activation. Bach1 is a heme sensor protein, which, upon heme binding, is targeted for proteasomal degradation, relieving the repression of Nrf2 target genes. Ideally, a combination of Nrf2 stabilization and Bach1 inhibition is necessary to achieve the full therapeutic benefits of Nrf2 activation. Here, we discuss recent advances and future perspectives in developing small molecule inhibitors of Bach1, highlighting the significance of the Bach1/Nrf2 signaling pathway as a promising neurotherapeutic strategy.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Factor 2 Relacionado con NF-E2 , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Regulación de la Expresión Génica , HemoRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of nigrostriatal dopaminergic neurons. Mounting evidence suggests that Nrf2 is a promising target for neuroprotective interventions in PD. However, electrophilic chemical properties of the canonical Nrf2-based drugs cause irreversible alkylation of cysteine residues on cellular proteins resulting in side effects. Bach1 is a known transcriptional repressor of the Nrf2 pathway. We report that Bach1 levels are up-regulated in PD postmortem brains and preclinical models. Bach1 knockout (KO) mice were protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity and associated oxidative damage and neuroinflammation. Functional genomic analysis demonstrated that the neuroprotective effects in Bach1 KO mice was due to up-regulation of Bach1-targeted pathways that are associated with both Nrf2-dependent antioxidant response element (ARE) and Nrf2-independent non-ARE genes. Using a proprietary translational technology platform, a drug library screen identified a substituted benzimidazole as a Bach1 inhibitor that was validated as a nonelectrophile. Oral administration of the Bach1 inhibitor attenuated MPTP neurotoxicity in pre- and posttreatment paradigms. Bach1 inhibitor-induced neuroprotection was associated with the up-regulation of Bach1-targeted pathways in concurrence with the results from Bach1 KO mice. Our results suggest that genetic deletion as well as pharmacologic inhibition of Bach1 by a nonelectrophilic inhibitor is a promising therapeutic approach for PD.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Neuroprotección , Enfermedad de Parkinson/terapia , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Anciano , Anciano de 80 o más Años , Animales , Elementos de Respuesta Antioxidante , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Enfermedad de Parkinson/metabolismo , RatasRESUMEN
The Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of Receptor Activator of NFkB Ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C-terminal end. Here we show that the Mitf(ce) allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitf(ce/ce) mice. The loss of the leucine zipper domain in Mitf(ce/ce) mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitf(ce). This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site-directed mutations in the docking site interfered with the interaction between MITF and its co-factors FUS and BRG1. MITF-ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/p38 MAPK interactions in osteoclast differentiation.
Asunto(s)
Diferenciación Celular/genética , Sistema de Señalización de MAP Quinasas , Factor de Transcripción Asociado a Microftalmía/metabolismo , Microftalmía/genética , Osteoclastos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Sistema de Señalización de MAP Quinasas/genética , Ratones , Mutación/genética , Osteoclastos/citología , Fosforilación , Ligando RANK/metabolismoRESUMEN
The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a signaling effector engaged by macrophage colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). MITF exerts its regulatory functions through its association with cofactors. Discovering the identity of its various partners will provide insights into the mechanisms governing gene expression during osteoclastogenesis. Here, we demonstrate that the proto-oncogene fused in sarcoma (FUS), the chromatin remodeling ATPase BRG1, and MITF form a trimeric complex that is regulated by phosphorylation of MITF at Ser-307 by p38 MAPK during osteoclast differentiation. FUS was recruited to MITF target gene promoters Acp5 and Ctsk during osteoclast differentiation, and FUS knockdown abolished efficient transcription of Acp5 and Ctsk. Furthermore, sumoylation of MITF at Lys-316, known to negatively regulate MITF transcriptional activity, inhibited MITF interactions with FUS and BRG1 in a p38 MAPK phosphorylation-dependent manner. These results demonstrate that FUS is a coregulator of MITF activity and provide new insights into how the RANKL/p38 MAPK signaling nexus controls gene expression in osteoclasts.
Asunto(s)
ADN Helicasas/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Osteoclastos/metabolismo , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Fosfatasa Ácida/biosíntesis , Fosfatasa Ácida/genética , Animales , Células COS , Catepsina K/biosíntesis , Catepsina K/genética , Chlorocebus aethiops , ADN Helicasas/genética , Regulación de la Expresión Génica/fisiología , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Osteoclastos/citología , Fosforilación/fisiología , Proto-Oncogenes Mas , Ligando RANK/genética , Ligando RANK/metabolismo , Proteína FUS de Unión a ARN , Fosfatasa Ácida Tartratorresistente , Factores de Transcripción/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Fibroblastos/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Fosfohidrolasa PTEN/metabolismo , Células del Estroma/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Matriz Extracelular/metabolismo , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunidad Innata , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Proteína Proto-Oncogénica c-ets-2/deficiencia , Proteína Proto-Oncogénica c-ets-2/metabolismoRESUMEN
Neurodegenerative diseases represent a pressing global health challenge, and the identification of novel mechanisms underlying their pathogenesis is of utmost importance. Ferroptosis, a non-apoptotic form of regulated cell death characterized by iron-dependent lipid peroxidation, has emerged as a pivotal player in the pathogenesis of neurodegenerative diseases. This review delves into the discovery of ferroptosis, the critical players involved, and their intricate role in the underlying mechanisms of neurodegeneration, with an emphasis on Alzheimer's and Parkinson's diseases. We critically appraise unsolved mechanistic links involved in the initiation and propagation of ferroptosis, such as a signaling cascade resulting in the de-repression of lipoxygenase translation and the role played by mitochondrial voltage-dependent anionic channels in iron homeostasis. Particular attention is given to the dual role of heme oxygenase in ferroptosis, which may be linked to the non-specific activity of P450 reductase in the endoplasmic reticulum. Despite the limited knowledge of ferroptosis initiation and progression in neurodegeneration, Nrf2/Bach1 target genes have emerged as crucial defenders in anti-ferroptotic pathways. The activation of Nrf2 and the inhibition of Bach1 can counteract ferroptosis and present a promising avenue for future therapeutic interventions targeting ferroptosis in neurodegenerative diseases.
RESUMEN
Parkinson's disease (PD) is the second most common neurodegenerative movement disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Although a complex interplay of multiple environmental and genetic factors has been implicated, the etiology of neuronal death in PD remains unresolved. Various mechanisms of neuronal degeneration in PD have been proposed, including oxidative stress, mitochondrial dysfunction, neuroinflammation, α-synuclein proteostasis, disruption of calcium homeostasis, and other cell death pathways. While many drugs individually targeting these pathways have shown promise in preclinical PD models, this promise has not yet translated into neuroprotective therapies in human PD. This has consequently spurred efforts to identify alternative targets with multipronged therapeutic approaches. A promising therapeutic target that could modulate multiple etiological pathways involves drug-induced activation of a coordinated genetic program regulated by the transcription factor, nuclear factor E2-related factor 2 (Nrf2). Nrf2 regulates the transcription of over 250 genes, creating a multifaceted network that integrates cellular activities by expressing cytoprotective genes, promoting the resolution of inflammation, restoring redox and protein homeostasis, stimulating energy metabolism, and facilitating repair. However, FDA-approved electrophilic Nrf2 activators cause irreversible alkylation of cysteine residues in various cellular proteins resulting in side effects. We propose that the transcriptional repressor of BTB and CNC homology 1 (Bach1), which antagonizes Nrf2, could serve as a promising complementary target for the activation of both Nrf2-dependent and Nrf2-independent neuroprotective pathways. This review presents the current knowledge on the Nrf2/Bach1 signaling pathway, its role in various cellular processes, and the benefits of simultaneously inhibiting Bach1 and stabilizing Nrf2 using non-electrophilic small molecules as a novel therapeutic approach for PD.
RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is associated with an incredibly dense stroma, which contributes to its recalcitrance to therapy. Cancer-associated fibroblasts (CAFs) are one of the most abundant cell types within the PDAC stroma and have context-dependent regulation of tumor progression in the tumor microenvironment (TME). Therefore, understanding tumor-promoting pathways in CAFs is essential for developing better stromal targeting therapies. Here, we show that disruption of the STAT3 signaling axis via genetic ablation of Stat3 in stromal fibroblasts in a Kras G12D PDAC mouse model not only slows tumor progression and increases survival, but re-shapes the characteristic immune-suppressive TME by decreasing M2 macrophages (F480+CD206+) and increasing CD8+ T cells. Mechanistically, we show that loss of the tumor suppressor PTEN in pancreatic CAFs leads to an increase in STAT3 phosphorylation. In addition, increased STAT3 phosphorylation in pancreatic CAFs promotes secretion of CXCL1. Inhibition of CXCL1 signaling inhibits M2 polarization in vitro. The results provide a potential mechanism by which CAFs promote an immune-suppressive TME and promote tumor progression in a spontaneous model of PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Linfocitos T CD8-positivos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Fibroblastos/metabolismo , Ratones , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Osteoporosis results from an imbalance in skeletal remodeling that favors bone resorption over bone formation. Bone matrix is degraded by osteoclasts, which differentiate from myeloid precursors in response to the cytokine RANKL. To gain insight into the transcriptional regulation of bone resorption during growth and disease, we generated a conditional knockout of the transcription factor nuclear factor of activated T cells c1 (Nfatc1). Deletion of Nfatc1 in young mice resulted in osteopetrosis and inhibition of osteoclastogenesis in vivo and in vitro. Transcriptional profiling revealed NFATc1 as a master regulator of the osteoclast transcriptome, promoting the expression of numerous genes needed for bone resorption. In addition, NFATc1 directly repressed osteoclast progenitor expression of osteoprotegerin, a decoy receptor for RANKL previously thought to be an osteoblast-derived inhibitor of bone resorption. "Cherubism mice", which carry a gain-of-function mutation in SH3-domain binding protein 2 (Sh3bp2), develop osteoporosis and widespread inflammation dependent on the proinflammatory cytokine, TNF-alpha. Interestingly, deletion of Nfatc1 protected cherubism mice from systemic bone loss but did not inhibit inflammation. Taken together, our study demonstrates that NFATc1 is required for remodeling of the growing and adult skeleton and suggests that NFATc1 may be an effective therapeutic target for osteoporosis associated with inflammatory states.
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Enfermedades Óseas Metabólicas/patología , Querubismo/metabolismo , Inflamación/patología , Factores de Transcripción NFATC/metabolismo , Osteoclastos/fisiología , Osteoprotegerina/metabolismo , Animales , Querubismo/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Osteoclastos/metabolismo , Osteoprotegerina/genéticaRESUMEN
The ras/Raf/Mek/Erk pathway plays a central role in coordinating endothelial cell activities during angiogenesis. Transcription factors Ets1 and Ets2 are targets of ras/Erk signaling pathways that have been implicated in endothelial cell function in vitro, but their precise role in vascular formation and function in vivo remains ill-defined. In this work, mutation of both Ets1 and Ets2 resulted in embryonic lethality at midgestation, with striking defects in vascular branching having been observed. The action of these factors was endothelial cell autonomous as demonstrated using Cre/loxP technology. Analysis of Ets1/Ets2 target genes in isolated embryonic endothelial cells demonstrated down-regulation of Mmp9, Bcl-X(L), and cIAP2 in double mutants versus controls, and chromatin immunoprecipitation revealed that both Ets1 and Ets2 were loaded at target promoters. Consistent with these observations, endothelial cell apoptosis was significantly increased both in vivo and in vitro when both Ets1 and Ets2 were mutated. These results establish essential and overlapping functions for Ets1 and Ets2 in coordinating endothelial cell functions with survival during embryonic angiogenesis.
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Apoptosis/genética , Desarrollo Embrionario/genética , Células Endoteliales/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Neovascularización Fisiológica/genética , Proteína Proto-Oncogénica c-ets-1/fisiología , Proteína Proto-Oncogénica c-ets-2/fisiología , Animales , Vasos Sanguíneos/embriología , Vasos Sanguíneos/ultraestructura , Supervivencia Celular/genética , Quimera , Edema/embriología , Edema/genética , Transferencia de Embrión , Muerte Fetal/genética , Muerte Fetal/patología , Enfermedades Fetales/genética , Enfermedades Fetales/patología , Regulación del Desarrollo de la Expresión Génica/genética , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Hemorragia/embriología , Hemorragia/genética , Homocigoto , Ratones , Ratones Noqueados , Fenotipo , Proteína Proto-Oncogénica c-ets-1/deficiencia , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-2/deficiencia , Proteína Proto-Oncogénica c-ets-2/genéticaRESUMEN
The molecular events linking lipid accumulation in atherosclerotic plaques to complications such as aneurysm formation and plaque disruption are poorly understood. BALB/c-Apoe(-/-) mice bearing a null mutation in the Npc1 gene display prominent medial erosion and atherothrombosis, whereas their macrophages accumulate free cholesterol in late endosomes and show increased cathepsin K (Ctsk) expression. We now show increased cathepsin K immunostaining and increased cysteinyl proteinase activity using near infrared fluorescence imaging over proximal aortas of Apoe(-/-), Npc1(-/-) mice. In mechanistic studies, cholesterol loading of macrophage plasma membranes (cyclodextrin-cholesterol) or endosomal system (AcLDL+U18666A or Npc1 null mutation) activated Toll-like receptor (TLR) signaling, leading to sustained phosphorylation of p38 mitogen-activated protein kinase and induction of p38 targets, including Ctsk, S100a8, Mmp8, and Mmp14. Studies in macrophages from knockout mice showed major roles for TLR4, following plasma membrane cholesterol loading, and for TLR3, after late endosomal loading. TLR signaling via p38 led to phosphorylation and activation of the transcription factor Microphthalmia transcription factor, acting at E-box elements in the Ctsk promoter. These studies suggest that free cholesterol enrichment of either plasma or endosomal membranes in macrophages leads to activation of signaling via various TLRs, prolonged p38 mitogen-activated protein kinase activation, and induction of Mmps, Ctsk, and S100a8, potentially contributing to plaque complications.
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Catepsinas/biosíntesis , Membrana Celular/metabolismo , Colesterol/metabolismo , Endosomas/metabolismo , Macrófagos/metabolismo , Transducción de Señal , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Aorta/metabolismo , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Calgranulina A , Catepsina K , Membrana Celular/enzimología , Membrana Celular/inmunología , Células Cultivadas , Elementos E-Box , Endosomas/enzimología , Endosomas/inmunología , Inducción Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular , Macrófagos/enzimología , Macrófagos/inmunología , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteína Niemann-Pick C1 , Fosforilación , Regiones Promotoras Genéticas , Proteínas/genética , Proteínas/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Proteínas S100/metabolismo , Factores de Tiempo , Receptor Toll-Like 3/deficiencia , Receptor Toll-Like 3/genética , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Proteínas Quinasas p38 Activadas por Mitógenos/deficiencia , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Significance: Advancements in and access to health care have led to unprecedented improvements in the quality of life and increased lifespan of human beings in the past century. However, aging is a significant risk factor for neurodegenerative diseases (NDs). Hence, improved life expectancy has led to an increased incidence of NDs. Despite intense research, effective treatments for NDs remain elusive. The future of neurotherapeutics development depends on effective disease modification strategies centered on carefully scrutinized targets. Recent Advances: As a promising new direction, recent evidence has demonstrated that epigenetic processes modify diverse biochemical pathways, including those related to NDs. Small non-coding RNAs, known as microRNAs (miRNAs), are components of the epigenetic system that alter the expression of target genes at the post-transcriptional level. Critical Issues: miRNAs are expressed abundantly in the central nervous system and are critical for the normal functioning and survival of neurons. Here, we review recent advances in elucidating miRNAs' roles in NDs and discuss their potential as therapeutic targets. In particular, neuroinflammation is a major pathological hallmark of NDs and miR146a is a crucial regulator of inflammation. Future Directions: Finally, we explore the possibilities of developing miR146a as a potential biomarker and therapeutic target where additional research may help facilitate the detection and amelioration of neuroinflammation in NDs. Antioxid. Redox Signal. 35, 580-594.
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MicroARNs , Enfermedades Neurodegenerativas , Sistema Nervioso Central/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Calidad de VidaRESUMEN
The Keap1-Nrf2 signaling axis is a validated and promising target for cellular defense and survival pathways. This minireview discusses the potential off-target effects and their impact on future drug development originating from Keap1-targeting small molecules that function as displacement activators of the redox-sensitive transcription factor Nrf2. We argue that small-molecule displacement activators, similarly to electrophiles, will release both Nrf2 and other Keap1 client proteins from the ubiquitin ligase complex. This non-specificity is likely unavoidable and may result in off-target effects during Nrf2 activation by targeting Keap1. The small molecule displacement activators may also target Kelch domains in proteins other than Keap1, causing additional off-target effects unless designed to ensure specificity for the Kelch domain only in Keap1. A potentially promising and alternative therapeutic approach to overcome this non-specificity emerging from targeting Keap1 is to inhibit the Nrf2 repressor Bach1 for constitutive activation of the Nrf2 pathway and bypass the Keap1-Nrf2 complex.
RESUMEN
Transcription factors MITF and PU.1 collaborate to increase expression of target genes like cathepsin K (Ctsk) and acid phosphatase 5 (Acp5) during osteoclast differentiation. We show that these factors can also repress transcription of target genes in committed myeloid precursors capable of forming either macrophages or osteoclasts. The direct interaction of MITF and PU.1 with the zinc finger protein Eos, an Ikaros family member, was necessary for repression of Ctsk and Acp5. Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of corepressors CtBP (C-terminal binding protein) and Sin3A, but during osteoclast differentiation, Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited coactivators to these target genes, resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors disrupted osteoclast differentiation and selectively repressed transcription of MITF/PU.1 targets, while small interfering RNA knockdown of Eos resulted in increased basal expression of Ctsk and Acp5. This work provides a mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.
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Proteínas Portadoras/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción Asociado a Microftalmía/metabolismo , Células Progenitoras Mieloides/fisiología , Proteínas del Tejido Nervioso/metabolismo , Osteoclastos/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Animales , Proteínas Portadoras/genética , Catepsina K , Catepsinas/genética , Catepsinas/metabolismo , Diferenciación Celular/fisiología , Proteínas de Unión al ADN , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Células Progenitoras Mieloides/citología , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Ligando RANK/genética , Ligando RANK/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Fosfatasa Ácida Tartratorresistente , Transactivadores/genética , Dedos de ZincRESUMEN
The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.
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Diferenciación Celular , ADN Helicasas/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Osteopetrosis/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Fosfatasa Ácida/metabolismo , Factores de Edad , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Catepsina K , Catepsinas/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Regulación de la Expresión Génica , Isoenzimas/metabolismo , Ratones , Ratones Mutantes , Factor de Transcripción Asociado a Microftalmía/genética , FN-kappa B/metabolismo , Osteoclastos/enzimología , Osteogénesis/genética , Osteopetrosis/genética , Osteopetrosis/patología , Regiones Promotoras Genéticas , Ligando RANK/metabolismo , ARN Mensajero/metabolismo , Fosfatasa Ácida TartratorresistenteRESUMEN
The microphthalmia-associated transcription factor (MITF) is required for terminal osteoclast differentiation and is a target for signaling pathways engaged by colony stimulating factor (CSF)-1 and receptor-activator of nuclear factor-kappaB ligand (RANKL). Work presented here demonstrates that MITF can shuttle from cytoplasm to nucleus dependent upon RANKL/CSF-1 action. 14-3-3 was identified as a binding partner of MITF in osteoclast precursors, and overexpression of 14-3-3 in a transgenic model resulted in increased cytosolic localization of MITF and decreased expression of MITF target genes. MITF/14-3-3 interaction was phosphorylation dependent, and Ser173 residue, within the minimal interaction region of amino acid residues 141-191, was required. The Cdc25C-associated kinase (C-TAK)1 interacted with an overlapping region of MITF. C-TAK1 increased MITF/14-3-3 complex formation and thus promoted cytoplasmic localization of MITF. C-TAK1 interaction was disrupted by RANKL/CSF-1 treatment. The results indicate that 14-3-3 regulates MITF activity by promoting the cytosolic localization of MITF in the absence of signals required for osteoclast differentiation. This work identifies a mechanism that regulates MITF activity in monocytic precursors that are capable of undergoing different terminal differentiation programs, and it provides a mechanism that allows committed precursors to rapidly respond to signals in the bone microenvironment to promote specifically osteoclast differentiation.
Asunto(s)
Proteínas 14-3-3/metabolismo , Diferenciación Celular , Factor de Transcripción Asociado a Microftalmía/metabolismo , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Expresión Génica , Humanos , Ratones , Factor de Transcripción Asociado a Microftalmía/química , Modelos Biológicos , Osteoclastos/citología , Fosfoserina/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de ProteínasRESUMEN
Despite advances in diabetic wound care, the significant number of amputations that occur every year demands more effective therapeutics. Herein, we offer an aminated polyethersulfone nanofiber-expanded human umbilical cord blood-derived CD34+ cells (henceforth CD34+ cells) effective therapy, tested in cutaneous wounds developed in streptozotocin-induced diabetic NOD/SCID mice. We show that systemic administration of CD34+ cells homed to the wound site and significantly accelerated wound closure. Wound closure was associated with improved re-epithelialization and increased neovascularization; and with decreased sustained pro-inflammatory activity of NF-κB and its downstream effector molecules TNF-α, IL-1ß, and IL-6 at the wound bed. This finding was further supported by the observation of a decreased number of myeloperoxidase positive neutrophils, and concomitantly increased levels of IL-10. In addition, improved granulation tissue formation was observed along with higher collagen deposition and myofibroblasts and decreased expressions of MMP-1. Mechanistically, CD34+ cells reduced the level of MMP-1 expression by inhibiting recruitment of NF-κB to the MMP-1 promoter site in dermal fibroblasts. In summary, we provide evidence of a novel nanofiber-expanded CD34+ stem cell therapeutic development for treating diabetic wounds by defining their cellular and molecular mechanisms.
Asunto(s)
Antígenos CD34/metabolismo , Diabetes Mellitus Experimental/patología , Nanofibras/química , Piel/patología , Cicatrización de Heridas , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Dermis/patología , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Sangre Fetal/citología , Fibroblastos/efectos de los fármacos , Tejido de Granulación/patología , Humanos , Inflamación/patología , Metaloproteinasas de la Matriz/metabolismo , Ratones Endogámicos NOD , Ratones SCID , FN-kappa B/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Estreptozocina , Factor de Necrosis Tumoral alfa/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Bone-resorbing osteoclasts (OCs) are derived from myeloid precursors (MPs). Several transcription factors are implicated in OC differentiation and function; however, their hierarchical architecture and interplay are not well known. Analysis for enriched motifs in PU.1 and MITF chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) data from differentiating OCs identified eomesodermin (EOMES) as a potential novel binding partner of PU.1 and MITF at genes critical for OC differentiation and function. We were able to demonstrate using co-immunoprecipitation and sequential ChIP analysis that PU.1, MITF, and EOMES are in the same complex and present as a complex at OC genomic loci. Furthermore, EOMES knockdown in MPs led to osteopetrosis associated with decreased OC differentiation and function both in vitro and in vivo. Although EOMES is associated with embryonic development and other hematopoietic lineages, this is the first study demonstrating the requirement of EOMES in the myeloid compartment.
RESUMEN
Expression of the alpha(v)beta(3) integrin is required for normal osteoclast function. We previously showed that an evolutionary conserved NFATc1 binding site is required for RANKL induction and NFATc1 transactivation of the human beta(3) promoter. The mechanism conferring specificity for RANKL induction and NFATc1 transduction of the beta(3) gene in osteoclast differentiation is unclear since NFATc1 is expressed and activated in numerous cell types that do not express the beta(3) gene. PU.1 is an ETS family transcription factor in myeloid cells associated with expression of various osteoclast genes. The present study investigates the role of NFATc1 in concert with PU.1 in osteoclast-specific transcription of the mouse beta(3) integrin gene. The mouse beta(3) promoter was transactivated by NFATc1 in RAW264.7 cells and deletion or mutation of either of the conserved NFAT and PU.1 binding sites abrogated transactivation. NFATc1 transactivation of the mouse beta(3) promoter was specifically dependent on co-transfected PU.1 in HEK293 cells, to the exclusion of other ETS family members. Direct binding of NFATc1 and PU.1 to their cognate sequences was demonstrated by EMSA and NFATc1 and PU.1 occupy their cognate sites in RANKL-treated mouse marrow precursors in chromatin immuno-precipitation (ChIP) assays. TAT-mediated transduction with dominant-negative NFATc1 dose-dependently blocked endogenous expression of the mouse beta(3) integrin and the formation of TRAP positive multinucleated cells in RANKL-treated mouse macrophages. These data provide evidence that NFATc1, in concert with PU.1, are involved in regulation of beta(3) integrin expression during osteoclast differentiation and suggest that PU.1 confers specificity to the NFATc1 response to macrophage lineage cells.
Asunto(s)
Regulación de la Expresión Génica , Integrina beta3/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Línea Celular , Genes Dominantes , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Factores de Transcripción NFATC/genética , Osteoclastos/citología , Unión Proteica , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Activación Transcripcional/genética , TransfecciónRESUMEN
Genome-wide association studies (GWASs) have been instrumental in understanding complex phenotypic traits. However, they have rarely been used to understand lineage-specific pathways and functions that contribute to the trait. In this study, by integrating lineage-specific enhancers from mesenchymal and myeloid compartments with bone mineral density loci, we were able to segregate osteoblast- and osteoclast (OC)-specific functions. Specifically, in OCs, a PU.1-dependent transcription factor (TF) network was revealed. Deletion of PU.1 in OCs in mice resulted in severe osteopetrosis. Functional genomic analysis indicated PU.1 and MITF orchestrated a TF network essential for OC differentiation. Several of these TFs were regulated by cooperative binding of PU.1 with BRD4 to form superenhancers. Further, PU.1 is essential for conformational changes in the superenhancer region of Nfatc1. In summary, our study demonstrates that combining GWASs with genome-wide binding studies and model organisms could decipher lineage-specific pathways contributing to complex disease states.