RESUMEN
Acute kidney injury (AKI) is a common condition associated with significant morbidity, mortality, and cost. Injured kidney tissue can regenerate after many forms of AKI. However, there are no treatments in routine clinical practice to encourage recovery. In part, this shortcoming is due to an incomplete understanding of the genetic mechanisms that orchestrate kidney recovery. The advent of high-throughput sequencing technologies and genetic mouse models has opened an unprecedented window into the transcriptional dynamics that accompany both successful and maladaptive repair. AKI recovery shares similar cell-state transformations with kidney development, which can suggest common mechanisms of gene regulation. Several powerful bioinformatic strategies have been developed to infer the activity of gene regulatory networks by combining multiple forms of sequencing data at single-cell resolution. These studies highlight not only shared stress responses but also key changes in gene regulatory networks controlling metabolism. Furthermore, chromatin immunoprecipitation studies in injured kidneys have revealed dynamic epigenetic modifications at enhancer elements near target genes. This review will highlight how these studies have enhanced our understanding of gene regulation in injury response and regeneration.
RESUMEN
Chronic kidney disease is increasing at an alarming rate and correlates with the increase in diabetes, obesity, and hypertension that disproportionately impact socioeconomically disadvantaged communities. Iron plays essential roles in many biological processes including oxygen transport, mitochondrial function, cell proliferation, and regeneration. However, excess iron induces the generation and propagation of reactive oxygen species, which lead to oxidative stress, cellular damage, and ferroptosis. Iron homeostasis is regulated in part by the kidney through iron resorption from the glomerular filtrate and exports into the plasma by ferroportin (FPN). Yet, the impact of iron overload in the kidney has not been addressed. To test more directly whether excess iron accumulation is toxic to kidneys, we generated a kidney proximal tubule-specific knockout of FPN. Despite significant intracellular iron accumulation in FPN mutant tubules, basal kidney function was not measurably different from wild type kidneys. However, upon induction of acute kidney injury (AKI), FPN mutant kidneys exhibited significantly more damage and failed recovery, evidence for ferroptosis, and increased fibrosis. Thus, disruption of iron export in proximal tubules, leading to iron overload, can significantly impair recovery from AKI and can contribute to progressive renal damage indicative of chronic kidney disease. Understanding the mechanisms that regulate iron homeostasis in the kidney may provide new therapeutic strategies for progressive kidney disease and other ferroptosis-associated disorders.NEW & NOTEWORTHY Physiological iron homeostasis depends in part on renal resorption and export into the plasma. We show that specific deletion of iron exporters in the proximal tubules sensitizes cells to injury and inhibits recovery. This can promote a chronic kidney disease phenotype. Our paper demonstrates the need for iron balance in the proximal tubules to maintain and promote healthy recovery after acute kidney injury.
Asunto(s)
Lesión Renal Aguda , Proteínas de Transporte de Catión , Sobrecarga de Hierro , Insuficiencia Renal Crónica , Humanos , Riñón/metabolismo , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Hierro/metabolismo , Sobrecarga de Hierro/metabolismo , Homeostasis/fisiología , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismoRESUMEN
Acute kidney injury (AKI) is a common condition that lacks effective treatments. In part, this shortcoming is due to an incomplete understanding of the genetic mechanisms that control pathogenesis and recovery. Identifying the molecular and genetic regulators unique to nephron segments that dictate vulnerability to injury and regenerative potential could lead to new therapeutic targets to treat ischemic kidney injury. Pax2 and Pax8 are homologous transcription factors with overlapping functions that are critical for kidney development and are re-activated in AKI. Here, we examined the role of Pax2 and Pax8 in recovery from ischemic AKI and found them upregulated after severe AKI and correlated with chronic injury. Surprisingly, proximal-tubule-selective deletion of Pax2 and Pax8 resulted in a less severe chronic injury phenotype. This effect was mediated by protection against the acute insult, similar to pre-conditioning. Prior to injury, Pax2 and Pax8 mutant mice develop a unique subpopulation of proximal tubule cells in the S3 segment that displayed features usually seen only in acute or chronic injury. The expression signature of these cells was strongly enriched with genes associated with other mechanisms of protection against ischemic AKI including caloric restriction, hypoxic pre-conditioning, and female sex. Thus, our results identified a novel role for Pax2 and Pax8 in mature proximal tubules that regulates critical genes and pathways involved in both the injury response and protection from ischemic AKI.
Asunto(s)
Lesión Renal Aguda , Túbulos Renales Proximales , Factor de Transcripción PAX2 , Factor de Transcripción PAX8 , Insuficiencia Renal Crónica , Animales , Femenino , Ratones , Lesión Renal Aguda/complicaciones , Lesión Renal Aguda/genética , Isquemia/complicaciones , Túbulos Renales Proximales/patología , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/genética , Daño por Reperfusión/genética , Factor de Transcripción PAX8/genética , Factor de Transcripción PAX8/metabolismo , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismoRESUMEN
Acute kidney injury (AKI) is a common condition that lacks effective treatments. In part this shortcoming is due to an incomplete understanding of the genetic mechanisms that control pathogenesis and recovery. Pax2 and Pax8 are homologous transcription factors with overlapping functions that are critical for kidney development and are re-activated in AKI. In this report, we examined the role of Pax2 and Pax8 in recovery from ischemic AKI. We found that Pax2 and Pax8 are upregulated after severe AKI and correlate with chronic injury. Surprisingly, we then discovered that proximal-tubule-selective deletion of Pax2 and Pax8 resulted in a less severe chronic injury phenotype. This effect was mediated by protection against the acute insult, similar to preconditioning. Prior to injury, Pax2 and Pax8 mutant mice develop a unique subpopulation of S3 proximal tubule cells that display features usually seen only in acute or chronic injury. The expression signature of these cells was strongly enriched with genes associated with other mechanisms of protection against ischemic AKI including caloric restriction, hypoxic preconditioning, and female sex. Taken together, our results identify a novel role for Pax2 and Pax8 in mature proximal tubules that regulates critical genes and pathways involved in both injury response and protection from ischemic AKI. TRANSLATIONAL STATEMENT: Identifying the molecular and genetic regulators unique to the nephron that dictate vulnerability to injury and regenerative potential could lead to new therapeutic targets to treat ischemic kidney injury. Pax2 and Pax8 are two homologous nephron-specific transcription factors that are critical for kidney development and physiology. Here we report that proximal-tubule-selective depletion of Pax2 and Pax8 protects against both acute and chronic injury and induces an expression profile in the S3 proximal tubule with common features shared among diverse conditions that protect against ischemia. These findings highlight a new role for Pax proteins as potential therapeutic targets to treat AKI.
RESUMEN
Recovery from acute kidney injury can vary widely in patients and in animal models. Immunofluorescence staining can provide spatial information about heterogeneous injury responses, but often only a fraction of stained tissue is analyzed. Deep learning can expand analysis to larger areas and sample numbers by substituting for time-intensive manual or semi-automated quantification techniques. Here we report one approach to leverage deep learning tools to quantify heterogenous responses to kidney injury that can be deployed without specialized equipment or programming expertise. We first demonstrated that deep learning models generated from small training sets accurately identified a range of stains and structures with performance similar to that of trained human observers. We then showed this approach accurately tracks the evolution of folic acid induced kidney injury in mice and highlights spatially clustered tubules that fail to repair. We then demonstrated that this approach captures the variation in recovery across a robust sample of kidneys after ischemic injury. Finally, we showed markers of failed repair after ischemic injury were correlated both spatially within and between animals and that failed repair was inversely correlated with peritubular capillary density. Combined, we demonstrate the utility and versatility of our approach to capture spatially heterogenous responses to kidney injury.
Asunto(s)
Lesión Renal Aguda , Aprendizaje Profundo , Humanos , Ratones , Animales , Riñón/irrigación sanguínea , Modelos Animales , Ácido FólicoRESUMEN
The Pax family of developmental control genes are frequently deregulated in human disease. In the kidney, Pax2 is expressed in developing nephrons but not in adult proximal and distal tubules, whereas polycystic kidney epithelia or renal cell carcinoma continues to express high levels. Pax2 reduction in mice or cell culture can slow proliferation of cystic epithelial cells or renal cancer cells. Thus, inhibition of Pax activity may be a viable, cell-type-specific therapy. We designed an unbiased, cell-based, high-throughput screen that identified triazolo pyrimidine derivatives that attenuate Pax transactivation ability. We show that BG-1 inhibits Pax2-positive cancer cell growth and target gene expression but has little effect on Pax2-negative cells. Chromatin immunoprecipitation suggests that these inhibitors prevent Pax protein interactions with the histone H3K4 methylation complex at Pax target genes in renal cells. Thus, these compounds may provide structural scaffolds for kidney-specific inhibitors with therapeutic potential.
Asunto(s)
Antineoplásicos , Neoplasias , Factor de Transcripción PAX2 , Animales , Antineoplásicos/farmacología , Proliferación Celular , Expresión Génica , Riñón/metabolismo , Metilación , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Factor de Transcripción PAX2/antagonistas & inhibidores , Factor de Transcripción PAX2/genéticaRESUMEN
The vertebrate eye anlage grows out of the brain and folds into bilayered optic cups. The eye is patterned along multiple axes, precisely controlled by genetic programs, to delineate neural retina, pigment epithelium, and optic stalk tissues. Pax genes encode developmental regulators of key morphogenetic events, with Pax2 being essential for interpreting inductive signals, including in the eye. PAX2 mutations cause ocular coloboma, when the ventral optic fissure fails to close. Previous studies established that Pax2 is necessary for fissure closure and to maintain the neural retina -- glial optic stalk boundary. Using a Pax2GFP/+ knock-in allele we discovered that the mutant optic nerve head (ONH) lacks molecular boundaries with the retina and RPE, rendering the ONH larger than normal. This was preceded by ventronasal cup mispatterning, a burst of overproliferation and followed by optic cup apoptosis. Our findings support the hypothesis that ONH cells are tripotential, requiring Pax2 to remain committed to glial fates. This work extends current models of ocular development, contributes to broader understanding of tissue boundary formation and informs the underlying mechanisms of human coloboma.
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Ojo/embriología , Ojo/metabolismo , Disco Óptico/embriología , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Proliferación Celular/genética , Coloboma/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Disco Óptico/anomalías , Disco Óptico/citología , Retina/embriología , Células Madre/metabolismoRESUMEN
BACKGROUND: As the glomerular filtrate passes through the nephron and into the renal medulla, electrolytes, water, and urea are reabsorbed through the concerted actions of solute carrier channels and aquaporins at various positions along the nephron and in the outer and inner medulla. Proliferating stem cells expressing the nuclear transcription factor Pax2 give rise to renal epithelial cells. Pax2 expression ends once the epithelial cells differentiate into mature proximal and distal tubules, whereas expression of the related Pax8 protein continues. The collecting tubules and renal medulla are derived from Pax2-positive ureteric bud epithelia that continue to express Pax2 and Pax8 in adult kidneys. Despite the crucial role of Pax2 in renal development, functions for Pax2 or Pax8 in adult renal epithelia have not been established. METHODS: To examine the roles of Pax2 and Pax8 in the adult mouse kidney, we deleted either Pax2, Pax8, or both genes in adult mice and examined the resulting phenotypes and changes in gene expression patterns. We also explored the mechanism of Pax8-mediated activation of potential target genes in inner medullary collecting duct cells. RESULTS: Mice with induced deletions of both Pax2 and Pax8 exhibit severe polyuria that can be attributed to significant changes in the expression of solute carriers, such as the urea transporters encoded by Slc14a2, as well as aquaporins within the inner and outer medulla. Furthermore, Pax8 expression is induced by high-salt levels in collecting duct cells and activates the Slc14a2 gene by recruiting a histone methyltransferase complex to the promoter. CONCLUSIONS: These data reveal novel functions for Pax proteins in adult renal epithelia that are essential for retaining water and concentrating urine.
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Acuaporinas/fisiología , Capacidad de Concentración Renal/fisiología , Riñón/fisiología , Proteínas de Transporte de Membrana/fisiología , Factor de Transcripción PAX2/fisiología , Factor de Transcripción PAX8/fisiología , Animales , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Osmorregulación , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX8/genética , Transportadores de UreaRESUMEN
A terminally differentiated cellular phenotype is thought to be maintained, at least in part, by both active and repressive histone marks. However, it is unclear whether regenerating cells after injury need to replicate such epigenetic marks to recover. To test whether renal epithelial cell regeneration is dependent on histone H3K4 methylation, we generated a mouse model that deleted the Paxip1 gene in mature renal proximal tubules. Paxip1 encodes PTIP, an essential protein in the Mll3/4 histone H3K4 methyltransferase complex. Mice with PTIP deletions in the adult kidney proximal tubules were viable and fertile. Upon acute kidney injury, such mice failed to regenerate damaged tubules, leading to scarring and interstitial fibrosis. The inability to repair damage was likely due to a failure to reenter mitosis and reactivate regulatory genes such as Sox9. PTIP deletion reduced histone H3K4 methylation in uninjured adult kidneys but did not significantly affect function or the expression of epithelial specific markers. Strikingly, cell lineage tracing revealed that surviving PTIP mutant cells could alter their phenotype and lose epithelial markers. These data demonstrate that PTIP and associated MLL3/4-mediated histone methylation are needed for regenerating proximal tubules and to maintain or reestablish the cellular epithelial phenotype.
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Lesión Renal Aguda/fisiopatología , Proteínas de Unión al ADN/fisiología , Epigénesis Genética/fisiología , Regeneración Hepática , Lesión Renal Aguda/patología , Animales , Biomarcadores/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/genética , Eliminación de Gen , Histonas/metabolismo , Metilación , RatonesRESUMEN
Bone morphogenetic protein (BMP) signaling is critical in renal development and disease. In animal models of chronic kidney disease (CKD), re-activation of BMP signaling is reported to be protective by promoting renal repair and regeneration. Clinical use of recombinant BMPs, however, requires harmful doses to achieve efficacy and is costly because of BMPs' complex synthesis. Therefore, alternative strategies are needed to harness the beneficial effects of BMP signaling in CKD. Key aspects of the BMP signaling pathway can be regulated by both extracellular and intracellular molecules. In particular, secreted proteins like noggin and chordin inhibit BMP activity, whereas kielin/chordin-like proteins (KCP) enhance it and attenuate kidney fibrosis or CKD. Clinical development of KCP, however, is precluded by its size and complexity. Therefore, we propose an alternative strategy to enhance BMP signaling by using small molecules, which are simpler to synthesize and more cost-effective. To address our objective, here we developed a small-molecule high-throughput screen (HTS) with human renal cells having an integrated luciferase construct highly responsive to BMPs. We demonstrate the activity of a potent benzoxazole compound, sb4, that rapidly stimulated BMP signaling in these cells. Activation of BMP signaling by sb4 increased the phosphorylation of key second messengers (SMAD-1/5/9) and also increased expression of direct target genes (inhibitors of DNA binding, Id1 and Id3) in canonical BMP signaling. Our results underscore the feasibility of utilizing HTS to identify compounds that mimic key downstream events of BMP signaling in renal cells and have yielded a lead BMP agonist.
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Benzoxazoles/farmacología , Proteínas Morfogenéticas Óseas/agonistas , Proteínas Morfogenéticas Óseas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Portadoras/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Fosfoproteínas/metabolismo , Proteínas Smad/metabolismoRESUMEN
BACKGROUND: Renal interstitial fibrosis results from activation and proliferation of fibroblasts to myofibroblasts, secretion and accumulation of extracellular matrix, and displacement of normal renal tubules. In contrast to chronic renal disease, acute injury may be repaired, a process that includes a decrease in the number of myofibroblasts in the interstitium and degradation of the accumulated extracellular matrix, leaving little evidence of prior injury. METHODS: To investigate whether activated fibroblasts demonstrate changes in gene expression that correspond with regression after acute injury but are not observed in chronic models of fibrosis, we used microarrays to analyze gene expression patterns among fibroblast populations at different stages of injury or repair. We then mined the data for signaling pathways in fibroblasts corresponding to the acute proliferative, regression, and chronic phases of renal injury. RESULTS: We identified multiple gene clusters with changes that correlate with the three phases of renal injury, including changes in levels of receptors for the antifibrotic factor PGE2. In adult renal fibroblast cultures, PGE2 was able to upregulate many genes that are suppressed by the profibrotic cytokine TGF-ß, whereas many PGE2-downregulated genes were activated by TGF-ß. High levels of TGF-ß suppressed expression of a subset of PG receptors in fibroblast cultures, making these cells resistant to any effects of PGE2. CONCLUSIONS: Inherent gene expression changes in activated fibroblasts accompany the transition from AKI to repair and regeneration. In chronic models, however, activated fibroblasts are resistant to the antifibrotic effects of PGE2 due to suppression of a subset of PGE receptors.
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Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Dinoprostona/farmacología , Regulación de la Expresión Génica , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Animales , Diferenciación Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibrosis/genética , Fibrosis/patología , Perfilación de la Expresión Génica , Inmunohistoquímica , Ratones , Miofibroblastos/citología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transducción de Señal/genéticaRESUMEN
Aberrant expression of HOXA9 is a prominent feature of acute leukemia driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid and B progenitor cells leads to significant enhancer reorganizations with prominent emergence of leukemia-specific de novo enhancers. Alterations in the enhancer landscape lead to activation of an ectopic embryonic gene program. We show that HOXA9 functions as a pioneer factor at de novo enhancers and recruits CEBPα and the MLL3/MLL4 complex. Genetic deletion of MLL3/MLL4 blocks histone H3K4 methylation at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis in vivo. These results suggest that therapeutic targeting of HOXA9-dependent enhancer reorganization can be an effective therapeutic strategy in acute leukemia with HOXA9 overexpression.
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Proteínas de Homeodominio/genética , Leucemia Mieloide Aguda/genética , Animales , Transformación Celular Neoplásica , Elementos de Facilitación Genéticos/genética , Humanos , Metilación , Regiones Promotoras Genéticas/genéticaRESUMEN
The in vivo niche and basic cellular properties of nephron progenitors are poorly described. Here we studied the cellular organization and function of the MAPK/ERK pathway in nephron progenitors. Live-imaging of ERK activity by a Förster resonance energy transfer biosensor revealed a dynamic activation pattern in progenitors, whereas differentiating precursors exhibited sustained activity. Genetic experiments demonstrate that MAPK/ERK activity controls the thickness, coherence, and integrity of the nephron progenitor niche. Molecularly, MAPK/ERK activity regulates niche organization and communication with extracellular matrix through PAX2 and ITGA8, and is needed for CITED1 expression denoting undifferentiated status. MAPK/ERK activation in nephron precursors propels differentiation by priming cells for distal and proximal fates induced by the Wnt and Notch pathways. Thus, our results demonstrate a mechanism through which MAPK/ERK activity controls both progenitor maintenance and differentiation by regulating a distinct set of targets, which maintain the biomechanical milieu of tissue-residing progenitors and prime precursors for nephrogenesis.
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Diferenciación Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Nefronas/citología , Nicho de Células Madre , Células Madre/citología , Animales , Apoptosis , Técnicas Biosensibles , Tipificación del Cuerpo , Proliferación Celular , Autorrenovación de las Células , Activación Enzimática , Regulación del Desarrollo de la Expresión Génica , Cadenas alfa de Integrinas/metabolismo , Ratones , Organogénesis , Factor de Transcripción PAX2/metabolismo , Células Madre/metabolismoRESUMEN
Pax genes encode developmental regulators that are expressed in a variety of tissues and control critical events in morphogenesis. In the kidney, Pax2 and Pax8 are expressed in embryonic development and in specific renal diseases associated with aberrant epithelial cell proliferation. Prior genetic and cell biological studies suggest that reducing the activity of Pax proteins in renal cancer or in polycystic kidney disease can slow the progression of these conditions. The Pax proteins may be critical for providing tissue and locus specificity to recruit epigenetic modifiers that control gene expression and chromatin structure. Although they are nuclear, targeting Pax proteins to inhibit function may be feasible with small molecules. Such inhibition of Pax protein function may provide novel therapies for subsets of renal disorders that are tissue- and cell type-specific and avoid systemic effects on non-Pax-expressing cells and tissues. Given the paucity of effective treatments for renal cancer and cystic disease, the Pax family of proteins represents new pharmaceutical targets that merit exploration and further development.
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Antineoplásicos/farmacología , Neoplasias Renales/tratamiento farmacológico , Factor de Transcripción PAX2/antagonistas & inhibidores , Factor de Transcripción PAX8/antagonistas & inhibidores , Enfermedades Renales Poliquísticas/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Progresión de la Enfermedad , Epigénesis Genética/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Riñón/citología , Riñón/crecimiento & desarrollo , Riñón/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/patología , Terapia Molecular Dirigida/métodos , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Factor de Transcripción PAX8/genética , Factor de Transcripción PAX8/metabolismo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Dominios Proteicos/efectos de los fármacos , Urotelio/citología , Urotelio/efectos de los fármacos , Urotelio/metabolismo , Urotelio/patologíaRESUMEN
Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.
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Oído Interno/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células Ciliadas Auditivas/metabolismo , Ratones , Organoides/metabolismo , Factor de Transcripción PAX2/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Oído Interno/citología , Oído Interno/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Organogénesis/genética , Organoides/citología , Factor de Transcripción PAX2/genéticaRESUMEN
Obesity and its associated complications such as insulin resistance and non-alcoholic fatty liver disease are reaching epidemic proportions. In mice, the TGF-ß superfamily is implicated in the regulation of white and brown adipose tissue differentiation. The kielin/chordin-like protein (KCP) is a secreted regulator of the TGF-ß superfamily pathways that can inhibit both TGF-ß and activin signals while enhancing bone morphogenetic protein (BMP) signaling. However, KCP's effects on metabolism and obesity have not been studied in animal models. Therefore, we examined the effects of KCP loss or gain of function in mice that were maintained on either a regular or a high-fat diet. KCP loss sensitized the mice to obesity and associated complications such as glucose intolerance and adipose tissue inflammation and fibrosis. In contrast, transgenic mice that expressed KCP in the kidney, liver, and adipose tissues were resistant to developing high-fat diet-induced obesity and had significantly reduced white adipose tissue. Moreover, KCP overexpression shifted the pattern of SMAD signaling in vivo, increasing the levels of phospho (P)-SMAD1 and decreasing P-SMAD3. Adipocytes in culture showed a cell-autonomous effect in response to added TGF-ß1 or BMP7. Metabolic profiling indicated increased energy expenditure in KCP-overexpressing mice and reduced expenditure in the KCP mutants with no effect on food intake or activity. These findings demonstrate that shifting the TGF-ß superfamily signaling with a secreted protein can alter the physiology and thermogenic properties of adipose tissue to reduce obesity even when mice are fed a high-fat diet.
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Adipocitos/metabolismo , Proteínas Portadoras/metabolismo , Grasas de la Dieta/efectos adversos , Síndrome Metabólico/metabolismo , Obesidad/metabolismo , Transducción de Señal , Adipocitos/patología , Animales , Proteína Morfogenética Ósea 7/genética , Proteína Morfogenética Ósea 7/metabolismo , Proteínas Portadoras/genética , Grasas de la Dieta/farmacología , Síndrome Metabólico/inducido químicamente , Síndrome Metabólico/genética , Síndrome Metabólico/patología , Ratones , Ratones Noqueados , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/patología , Especificidad de Órganos/genética , Proteína smad3/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The Pax gene family encodes DNA binding transcription factors that control critical steps in embryonic development and differentiation of specific cell lineages. Often, Pax proteins are re-expressed or ectopically expressed in cancer and other diseases of abnormal proliferation, making them attractive targets for tissue specific inhibition by small molecules. In this report, we used a homology model of the Pax2 paired domain and a virtual screen to identify small molecules that can inhibit binding of the paired domain to DNA and Pax2 mediated transcription activation. Candidates from the virtual screen were then confirmed in a cell based Pax2 transactivation assay. Subsequently, we tested analogs of these hits to identify a single compound that effectively blocked Pax2 activity and DNA binding with a Kd of 1.35-1.5 µM. The compound, termed EG1, was used to inhibit embryonic kidney development, a process directly dependent on Pax2 activity. Furthermore, we show that EG1 can inhibit proliferation of Pax2 positive renal and ovarian cancer cell lines but has little effect on Pax2 negative cancer cells. These data confirm that small molecules targeting the DNA binding paired domain can be identified and may be good lead compounds for developing tissue and cell-type specific anticancer therapies.
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ADN/metabolismo , Factor de Transcripción PAX2/metabolismo , Bibliotecas de Moléculas Pequeñas , Activación Transcripcional , Animales , Línea Celular Tumoral , Simulación por Computador , Células HEK293 , Humanos , Riñón/crecimiento & desarrollo , Riñón/metabolismo , Factor de Transcripción PAX2/antagonistas & inhibidoresRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease and is increasing with the rising rate of obesity in the developed world. Signaling pathways known to influence the rate of lipid deposition in liver, known as hepatic steatosis, include the transforming growth factor (TGF) superfamily, which function through the SMAD second messengers. The kielin/chordin-like protein (KCP) is a large secreted protein that can enhance bone morphogenetic protein signaling while suppressing TGF-ß signaling in cells and in genetically modified mice. In this report, we show that aging KCP mutant (Kcp-/-) mice are increasingly susceptible to developing hepatic steatosis and liver fibrosis. When young mice are put on a high-fat diet, Kcp-/- mice are also more susceptible to developing liver pathology, compared with their wild-type littermates. Furthermore, mice that express a Pepck-KCP transgene (KcpTg) in the liver are resistant to developing liver pathology even when fed a high-fat diet. Analyses of liver tissues reveal a significant reduction of P-Smad3, consistent with a role for KCP in suppressing TGF-ß signaling. Transcriptome analyses show that livers from Kcp-/- mice fed a normal diet are more like wild-type livers from mice fed a high-fat diet. However, the KCP transgene can suppress many of the changes in liver gene expression that are due to a high-fat diet. These data demonstrate that shifting the TGF-ß signaling paradigm with the secreted regulatory protein KCP can significantly alter the liver pathology in aging mice and in diet-induced NAFLD.
Asunto(s)
Proteínas Portadoras/metabolismo , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Transducción de Señal/fisiología , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Proteínas Portadoras/genética , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Hígado/patología , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/genética , Fosforilación , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Pax genes encode developmental regulatory proteins that specify cell lineages and tissues in metazoans. Upon binding to DNA through the conserved paired domain, Pax proteins can recruit both activating and repressing complexes that imprint distinct patterns of histone methylation associated with either gene activation or silencing. How the switch from Pax-mediated activation to repression is regulated remains poorly understood. In this report, we identify the phosphatase PPM1B as an essential component of the Groucho4 repressor complex that is recruited by Pax2 to chromatin. PPM1B can dephosphorylate the Pax2 activation domain and displace the adaptor protein PTIP, thus inhibiting H3K4 methylation and gene activation. Loss of PPM1B prevents Groucho-mediated gene repression. Thus, PPM1B helps switch Pax2 from a transcriptional activator to a repressor protein. This can have profound implications for developmental regulation by Pax proteins and suggests a model for imprinting specific epigenetic marks depending on the availability of co-factors.
Asunto(s)
Proteínas Portadoras/metabolismo , Silenciador del Gen , Proteínas Nucleares/metabolismo , Factor de Transcripción PAX2/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Represoras/metabolismo , Activación Transcripcional , Proteínas Portadoras/análisis , Cromatina/metabolismo , Proteínas de Unión al ADN , Células HEK293 , Humanos , Proteínas Nucleares/análisis , Factor de Transcripción PAX2/análisis , Fosfoproteínas Fosfatasas/análisis , Mapas de Interacción de Proteínas , Proteína Fosfatasa 2C , Proteínas Represoras/análisisRESUMEN
Activation of the Pax2 gene marks the intermediate mesoderm shortly after gastrulation, as the mesoderm becomes compartmentalized into paraxial, intermediate, and lateral plate. Using an EGFP knock-in allele of Pax2 to identify and sort cells of the intermediate mesodermal lineage, we compared gene expression patterns in EGFP positive cells that were heterozygous or homozygous null for Pax2. Thus, we identified critical regulators of intermediate mesoderm and kidney development whose expression depended on Pax2 function. In cell culture models, Pax2 is thought to recruit epigenetic modifying complex to imprint activating histone methylation marks through interactions with the adaptor protein PTIP. In kidney organ culture, conditional PTIP deletion showed that many Pax2 target genes, which were activated early in renal progenitor cells, remained on once activated, whereas Pax2 target genes expressed later in kidney development were unable to be fully activated without PTIP. In Pax2 mutants, we also identified a set of genes whose expression was up-regulated in EGFP positive cells and whose expression was consistent with a cell fate transformation to paraxial mesoderm and its derivatives. These data provide evidence that Pax2 specifies the intermediate mesoderm and renal epithelial cells through epigenetic mechanisms and in part by repressing paraxial mesodermal fate.