RESUMEN
Wnt ligands are considered classical morphogens, for which the strength of the cellular response is proportional to the concentration of the ligand. Herein, we show an emergent property of bistability arising from feedback among the Wnt destruction complex proteins that target the key transcriptional co-activator ß-catenin for degradation. Using biochemical reconstitution, we identified positive feedback between the scaffold protein Axin and the kinase glycogen synthase kinase 3 (GSK3). Theoretical modeling of this feedback between Axin and GSK3 suggested that the activity of the destruction complex exhibits bistable behavior. We experimentally confirmed these predictions by demonstrating that cellular cytoplasmic ß-catenin concentrations exhibit an "all-or-none" response with sustained memory (hysteresis) of the signaling input. This bistable behavior was transformed into a graded response and memory was lost through inhibition of GSK3. These findings provide a mechanism for establishing decisive, switch-like cellular response and memory upon Wnt pathway stimulation.
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Complejo de Señalización de la Axina , beta Catenina , Complejo de Señalización de la Axina/metabolismo , beta Catenina/metabolismo , Proteína Axina/genética , Proteína Axina/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Retroalimentación , Fosforilación , Vía de Señalización Wnt/fisiologíaRESUMEN
SMAD4 is a tumor suppressor mutated or silenced in multiple cancers, including oral cavity squamous cell carcinoma (OSCC). Human clinical samples and cell lines, mouse models and organoid culture were used to investigate the role that SMAD4 plays in progression from benign disease to invasive OSCC. Human OSCC lost detectable SMAD4 protein within tumor epithelium in 24% of cases, and this loss correlated with worse progression-free survival independent of other major clinical and pathological features. A mouse model engineered for KrasG12D expression in the adult oral epithelium induced benign papillomas, however the combination of KrasG12D with loss of epithelial Smad4 expression resulted in rapid development of invasive carcinoma with features of human OSCC. Examination of regulatory pathways in 3D organoid cultures of SMAD4+ and SMAD4- mouse tumors with Kras mutation found that either loss of SMAD4 or inhibition of TGFß signaling upregulated the WNT pathway and altered the extracellular matrix. The gene signature of the mouse tumor organoids lacking SMAD4 was highly similar to the gene signature of human head and neck squamous cell carcinoma. In summary, this work has uncovered novel mechanisms by which SMAD4 acts as a tumor suppressor in OSCC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Progresión de la Enfermedad , Neoplasias de la Boca , Proteína Smad4 , Vía de Señalización Wnt , Proteína Smad4/metabolismo , Proteína Smad4/genética , Humanos , Animales , Vía de Señalización Wnt/fisiología , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/genética , Ratones , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Línea Celular Tumoral , Mutación , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Masculino , Organoides/metabolismo , Organoides/patologíaRESUMEN
PURPOSE OF REVIEW: The objective of this manuscript is to examine the current literature on the epidemiology of metabolic dysfunction-associated steatotic liver disease (MASLD), its correlation with cardiovascular disease (CVD) outcomes, as well as to evaluate the update in nomenclature from non-alcoholic liver disease (NAFLD). RECENT FINDINGS: The update of diagnostic criteria from NAFLD to MASLD reduces the stigma associated with alcohol consumption and poor health choices. It also shines a light on the crucial role of cardiometabolic risk factors in disease pathophysiology. The incidence and prevalence of MASLD are projected to increase significantly in the future as the population burden of cardiometabolic risk factors rises. MASLD is also a potent risk factor for developing CVD that should be tackled by using a multi-disciplinary team with a holistic approach. As the new nomenclature for metabolic liver disease is adopted on a global scale, more research is needed to investigate the applicability of findings from previous trials focusing on NAFLD. It is anticipated that the epidemic of MASLD will continue to increase globally, hence the urgent need for therapeutic approaches to reverse this trend.
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Enfermedades Cardiovasculares , Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/epidemiología , Factores de Riesgo , Consumo de Bebidas Alcohólicas , Enfermedades Cardiovasculares/epidemiologíaRESUMEN
The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN-CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.
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Caseína Quinasa Ialfa , Compuestos de Pirvinio , Caseína Quinasa Ialfa/metabolismo , Compuestos de Pirvinio/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización WntRESUMEN
BACKGROUND: Recent studies suggest that tirzepatide, a dual glucose-dependent insulinotropic-peptide (GIP) and glucagon-like peptide-1 receptor agonist (GLP-1 RA), has significant weight loss effects. This systematic review and meta-analysis aims to assess the efficacy and safety of tirzepatide for weight loss in patients with overweight or obesity. METHODS: Medline, Embase and Cochrane CENTRAL were searched for randomized controlled trials (RCTs) on tirzepatide's weight loss efficacy for these patients. A single arm meta-analysis of proportions estimated primary outcomes, ≥5%, ≥10%, and ≥15% weight loss, and adverse events (AEs); while meta-analysis of means estimated secondary outcomes. Comparative meta-analysis was conducted between tirzepatide and control arms where mean differences and odds ratios were estimated for continuous and dichotomous outcomes respectively. RESULTS: RCTs included in this study revealed that among 5800 patients, 78.22% (95% CI: 72.15% to 83.73%), 55.60% (95% CI: 46.54% to 64.47%), 32.28% (95% CI: 23.17% to 42.12%) achieved ≥5%, ≥10%, and ≥15% weight loss, respectively. Tirzepatide 5 mg demonstrated weight loss superiority relative to placebo (MD: -12.47 kg, 95% CI: -13.94 kg to -11.00 kg) and semaglutide (n = 1409, MD: -1.90 kg, 95% CI: -2.97 kg to -0.83 kg) with dose-dependent increase for 10 mg and 15 mg doses. The comparison between tirzepatide and semaglutide was examined in the SURPASS-2 trial that was included in this systematic review. For AEs, there was increase odds of experiencing gastrointestinal AEs with tirzepatide compared to placebo, but no significant difference with semaglutide. CONCLUSION: Tirzepatide has significant potential as a weight loss drug in patients with overweight and obesity, with little increase in AEs compared to other weight loss drugs. With its ability to concurrently target multiple aspects of metabolic syndrome, it should be considered as the next helm of weight loss therapies.
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Fármacos Antiobesidad , Diabetes Mellitus Tipo 2 , Humanos , Sobrepeso/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Polipéptido Inhibidor Gástrico , Fármacos Antiobesidad/efectos adversos , Pérdida de Peso , Hipoglucemiantes , Receptor del Péptido 1 Similar al GlucagónRESUMEN
Chromatin structure plays a fundamental role in regulating gene expression, with histone modifiers shaping the structure of chromatin by adding or removing chemical changes to histone proteins. The p53 transcription factor controls gene expression, binds target genes, and regulates their activity. While p53 has been extensively studied in cancer research, specifically in relation to fundamental cellular processes, including gene transcription, apoptosis, and cell cycle progression, its association with histone modifiers has received limited attention. This review explores the interplay between histone modifiers and p53 in regulating gene expression. We discuss how histone modifications can influence how p53 binds to target genes and how this interplay can be disrupted in cancer cells. This review provides insights into the complex mechanisms underlying gene regulation and their implications for potential cancer therapy.
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Histonas , Proteína p53 Supresora de Tumor , Histonas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Cromatina , Regulación de la Expresión Génica , Expresión GénicaRESUMEN
Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive inflammatory lung disease without effective molecular markers of disease activity or treatment responses. Monocyte and interstitial macrophages that express the C-C motif CCR2 (chemokine receptor 2) are active in IPF and central to fibrosis.Objectives: To phenotype patients with IPF for potential targeted therapy, we developed 64Cu-DOTA-ECL1i, a radiotracer to noninvasively track CCR2+ monocytes and macrophages using positron emission tomography (PET).Methods: CCR2+ cells were investigated in mice with bleomycin- or radiation-induced fibrosis and in human subjects with IPF. The CCR2+ cell populations were localized relative to fibrotic regions in lung tissue and characterized using immunolocalization, single-cell mass cytometry, and Ccr2 RNA in situ hybridization and then correlated with parallel quantitation of lung uptake by 64Cu-DOTA-ECL1i PET.Measurements and Main Results: Mouse models established that increased 64Cu-DOTA-ECL1i PET uptake in the lung correlates with CCR2+ cell infiltration associated with fibrosis (n = 72). As therapeutic models, the inhibition of fibrosis by IL-1ß blockade (n = 19) or antifibrotic pirfenidone (n = 18) reduced CCR2+ macrophage accumulation and uptake of the radiotracer in mouse lungs. In lung tissues from patients with IPF, CCR2+ cells concentrated in perifibrotic regions and correlated with radiotracer localization (n = 21). Human imaging revealed little lung uptake in healthy volunteers (n = 7), whereas subjects with IPF (n = 4) exhibited intensive signals in fibrotic zones.Conclusions: These findings support a role for imaging CCR2+ cells within the fibrogenic niche in IPF to provide a molecular target for personalized therapy and monitoring.Clinical trial registered with www.clinicaltrials.gov (NCT03492762).
Asunto(s)
Biomarcadores/química , Fibrosis Pulmonar Idiopática/fisiopatología , Pulmón/diagnóstico por imagen , Pulmón/fisiopatología , Macrófagos/fisiología , Monocitos/fisiología , Receptores CCR2/química , Adulto , Anciano , Anciano de 80 o más Años , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Imagen Molecular , Tomografía de Emisión de PositronesRESUMEN
Rationale: Bronchopulmonary dysplasia (BPD) is a leading complication of preterm birth that affects infants born in the saccular stage of lung development at <32 weeks of gestation. Although the mechanisms driving BPD remain uncertain, exposure to hyperoxia is thought to contribute to disease pathogenesis.Objectives: To determine the effects of hyperoxia on epithelial-mesenchymal interactions and to define the mediators of activated Wnt/ß-catenin signaling after hyperoxia injury.Methods: Three hyperoxia models were used: A three-dimensional organotypic coculture using primary human lung cells, precision-cut lung slices (PCLS), and a murine in vivo hyperoxia model. Comparisons of normoxia- and hyperoxia-exposed samples were made by real-time quantitative PCR, RNA in situ hybridization, quantitative confocal microscopy, and lung morphometry.Measurements and Main Results: Examination of an array of Wnt ligands in the three-dimensional organotypic coculture revealed increased mesenchymal expression of WNT5A. Inhibition of Wnt5A abrogated the BPD transcriptomic phenotype induced by hyperoxia. In the PCLS model, Wnt5A inhibition improved alveolarization following hyperoxia exposure, and treatment with recombinant Wnt5a reproduced features of the BPD phenotype in PCLS cultured in normoxic conditions. Chemical inhibition of NF-κB with BAY11-7082 reduced Wnt5a expression in the PCLS hyperoxia model and in vivo mouse hyperoxia model, with improved alveolarization in the PCLS model.Conclusions: Increased mesenchymal Wnt5A during saccular-stage hyperoxia injury contributes to the impaired alveolarization and septal thickening observed in BPD. Precise targeting of Wnt5A may represent a potential therapeutic strategy for the treatment of BPD.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Fibroblastos/metabolismo , Hiperoxia/genética , Pulmón/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteína Wnt-5a/genética , Animales , Displasia Broncopulmonar , Técnicas de Cocultivo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Hiperoxia/metabolismo , Hibridación in Situ , Pulmón/crecimiento & desarrollo , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Microscopía Confocal , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Técnicas de Cultivo de Órganos , Reacción en Cadena en Tiempo Real de la Polimerasa , Sulfonas/farmacología , Proteína Wnt-5a/efectos de los fármacos , Proteína Wnt-5a/metabolismoRESUMEN
X-linked inhibitor of apoptosis (XIAP) plays an important role in preventing apoptotic cell death. XIAP has been shown to participate in signaling pathways, including Wnt signaling. XIAP regulates Wnt signaling by promoting the monoubiquitylation of the co-repressor Groucho/TLE family proteins, decreasing its affinity for the TCF/Lef family of transcription factors and allowing assembly of transcriptionally active ß-catenin-TCF/Lef complexes. We now demonstrate that XIAP is phosphorylated by GSK3 at threonine 180, and that an alanine mutant (XIAPT180A) exhibits decreased Wnt activity compared to wild-type XIAP in cultured human cells and in Xenopus embryos. Although XIAPT180A ubiquitylates TLE3 at wild-type levels in vitro, it exhibits a reduced capacity to ubiquitylate and bind TLE3 in human cells. XIAPT180A binds Smac (also known as DIABLO) and inhibits Fas-induced apoptosis to a similar degree to wild-type XIAP. Our studies uncover a new mechanism by which XIAP is specifically directed towards a Wnt signaling function versus its anti-apoptotic function. These findings have implications for development of anti-XIAP therapeutics for human cancers.
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Treonina/metabolismo , Proteína Wnt3A/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación , Unión Proteica , Vía de Señalización Wnt , Proteína Wnt3A/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genética , XenopusRESUMEN
A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing ß-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program.
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Proteínas Co-Represoras/metabolismo , Drosophila/metabolismo , Embrión no Mamífero/metabolismo , Ubiquitinación , Vía de Señalización Wnt , Proteína Inhibidora de la Apoptosis Ligada a X/fisiología , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Células HEK293 , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Inhibidoras de la Apoptosis/fisiología , Modelos Genéticos , Interferencia de ARN , Proteínas Wnt/metabolismo , Proteína Wnt1/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Xenopus , Proteínas de Xenopus/metabolismoRESUMEN
Wnt signaling regulates numerous cellular processes during embryonic development and adult tissue homeostasis. Underscoring this physiological importance, deregulation of the Wnt signaling pathway is associated with many disease states, including cancer. Here, we review pivotal regulatory events in the Wnt signaling pathway that drive cancer growth. We then discuss the roles of the established negative Wnt regulator, casein kinase 1α (CK1α), in Wnt signaling. Although the study of CK1α has been ongoing for several decades, the bulk of such research has focused on how it phosphorylates and regulates its various substrates. We focus here on what is known about the mechanisms controlling CK1α, including its putative regulatory proteins and alternative splicing variants. Finally, we describe the discovery and validation of a family of pharmacological CK1α activators capable of inhibiting Wnt pathway activity. One of the important advantages of CK1α activators, relative to other classes of Wnt inhibitors, is their reduced on-target toxicity, overcoming one of the major impediments to developing a clinically relevant Wnt inhibitor. Therefore, we also discuss mechanisms that regulate CK1α steady-state homeostasis, which may contribute to the deregulation of Wnt pathway activity in cancer and underlie the enhanced therapeutic index of CK1α activators.
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Caseína Quinasa Ialfa/metabolismo , Neoplasias/metabolismo , Vía de Señalización Wnt , Animales , Antineoplásicos/uso terapéutico , Caseína Quinasa Ialfa/genética , Activadores de Enzimas/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
The serine/threonine protein kinase casein kinase 1α (CK1α) functions as a negative regulator of Wnt signaling, phosphorylating ß-catenin at serine 45 (P-S45) to initiate its eventual ubiquitin-mediated degradation. We previously showed that the repurposed, FDA-approved anthelminthic drug pyrvinium potently inhibits Wnt signaling in vitro and in vivo. Moreover, we proposed that pyrvinium's Wnt inhibitory activity was the result of its function as an activator of CK1α. An understanding of the mechanism by which pyrvinium activates CK1α is important because pyrvinium was given an orphan drug designation by the FDA to treat familial adenomatous polyposis, a precancerous condition driven by constitutive Wnt signaling. In the current study, we show that pyrvinium stimulates the phosphorylation of S45 ß-catenin, a known CK1α substrate, in a cell-based assay, and does so in a dose- and time-dependent manner. Alternative splicing of CK1α results in four forms of the protein with distinct biological properties. We evaluated these splice products and identified the CK1α splice variant, CK1αS, as the form that exhibits the most robust response to pyrvinium in cells. Kinetic studies indicate that pyrvinium also stimulates the kinase activity of purified, recombinant CK1αS in vitro, increasing its catalytic efficiency (kcat/Km) toward substrates. These studies provide strong and clear mechanistic evidence that pyrvinium enhances CK1α kinase activity.
Asunto(s)
Biocatálisis/efectos de los fármacos , Caseína Quinasa Ialfa/metabolismo , Compuestos de Pirvinio/farmacología , Activación Enzimática/efectos de los fármacos , Células HEK293 , Humanos , CinéticaRESUMEN
Blood vessel epicardial substance (BVES, otherwise known as POPDC1) is an integral membrane protein known to regulate tight junction formation and epithelial-mesenchymal transition. BVES is underexpressed in a number of malignancies, including colorectal cancer. BVES loss leads to activation of the Wnt pathway, suggesting that decreased BVES expression functionally contributes to tumorigenesis. However, the mechanism by which BVES modulates Wnt signaling is unknown. Here, we confirm that BVES loss increases ß-catenin protein levels, leads to Wnt pathway activation in a ligand-independent fashion and coordinates with Wnt ligand to further increase Wnt signaling. We show that BVES loss increases levels and activation of the Wnt co-receptor, LRP6, in cell lines, murine adenoma tumoroids and human-derived colonoids. We also demonstrate that BVES interacts with LRP6. Finally, murine tumor modeling using a Wnt-driven genetic model and a chemically induced model of colorectal carcinogenesis demonstrate that BVES loss increases tumor multiplicity and dysplasia. Together, these results implicate BVES as an inhibitor of Wnt signaling, provide one of the first examples of a tight junction-associated protein regulating Wnt receptor levels, and expand the number of putative molecular targets for therapeutic intervention in colorectal cancer.
RESUMEN
The TGF-ß and Wnt/ß-catenin pathways have important roles in modulating CKD, but how these growth factors affect the epithelial response to CKD is not well studied. TGF-ß has strong profibrotic effects, but this pleiotropic factor has many different cellular effects depending on the target cell type. To investigate how TGF-ß signaling in the proximal tubule, a key target and mediator of CKD, alters the response to CKD, we injured mice lacking the TGF-ß type 2 receptor specifically in this epithelial segment. Compared with littermate controls, mice lacking the proximal tubular TGF-ß receptor had significantly increased tubular injury and tubulointerstitial fibrosis in two different models of CKD. RNA sequencing indicated that deleting the TGF-ß receptor in proximal tubule cells modulated many growth factor pathways, but Wnt/ß-catenin signaling was the pathway most affected. We validated that deleting the proximal tubular TGF-ß receptor impaired ß-catenin activity in vitro and in vivo Genetically restoring ß-catenin activity in proximal tubules lacking the TGF-ß receptor dramatically improved the tubular response to CKD in mice. Deleting the TGF-ß receptor alters many growth factors, and therefore, this ameliorated response may be a direct effect of ß-catenin activity or an indirect effect of ß-catenin interacting with other growth factors. In conclusion, blocking TGF-ß and ß-catenin crosstalk in proximal tubules exacerbates tubular injury in two models of CKD.
Asunto(s)
Fallo Renal Crónico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , beta Catenina/metabolismo , Animales , Ácidos Aristolóquicos/química , Núcleo Celular/metabolismo , Colágeno/química , Cruzamientos Genéticos , Epitelio/metabolismo , Femenino , Eliminación de Gen , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Receptor Tipo II de Factor de Crecimiento Transformador beta , Transducción de Señal , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inhibidoresRESUMEN
We previously identified a Drosophila maternal effect-lethal mutant named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid cycles of syncytial embryogenesis because of activation of a Chk2-mediated DNA checkpoint. NOPO is the Drosophila homolog of human TNF receptor associated factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains closely resembling those of known E3 ubiquitin ligases. We herein sought to elucidate the mechanism by which TRIP/NOPO promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We show that TRIP and NOPO co-immunoprecipitate with human and Drosophila Polη, respectively, from cultured cells. We generated a null mutation in Drosophila Polη (dPolη) and found that dPolη-derived embryos have increased sensitivity to ultraviolet irradiation and exhibit nopo-like mitotic spindle defects. dPolη and nopo interact genetically in that overexpression of dPolη in hypomorphic nopo-derived embryos suppresses nopo phenotypes. We observed enhanced ubiquitylation of Polη by TRIP and NOPO E3 ligases in human cells and Drosophila embryos, respectively, and show that TRIP promotes hPolη localization to nuclear foci in human cells. We present a model in which TRIP/NOPO ubiquitylates Polη to positively regulate its activity in translesion synthesis.
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ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Animales Modificados Genéticamente , Daño del ADN , ADN Polimerasa Dirigida por ADN/deficiencia , ADN Polimerasa Dirigida por ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Inestabilidad Genómica , Células HeLa , Humanos , Modelos Biológicos , Mutación , Transducción de Señal , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Wound healing in mammals is a fibrotic process. The mechanisms driving fibrotic (as opposed to regenerative) repair are poorly understood. Herein we report that therapeutic Wnt inhibition with topical application of small-molecule Wnt inhibitors can reduce fibrosis and promote regenerative cutaneous wound repair. In the naturally stented model of ear punch injury, we found that Wnt/ß-catenin pathway is activated most notably in the dermis of the wound bed early (d 2) after injury and subsides to baseline levels by d10. Topical application of either of 2 mechanistically distinct small-molecule Wnt pathway inhibitors (a tankyrase inhibitor, XAV-939, and the U.S. Food and Drug Administration-approved casein kinase activator, pyrvinium) in C57Bl/6J mice resulted in significantly increased rates of wound closure (72.3 ± 14.7% with XAV-939; and 52.1 ± 20.9% with pyrvinium) compared with contralateral controls (38.1 ± 23.0 and 40.4.± 16.7%, respectively). Histologically, Wnt inhibition reduced fibrosis as measured by α-smooth muscle actin positive myofibroblasts and collagen type I α1 synthesis. Wnt inhibition also restored skin architecture including adnexal structures in ear wounds and dermal-epidermal junction with rete pegs in excisional wounds. Additionally, in ear punch injury Wnt inhibitor treatment enabled regeneration of auricular cartilage. Our study shows that pharmacologic Wnt inhibition holds therapeutic utility for regenerative repair of cutaneous wounds.
Asunto(s)
Cartílago/lesiones , Regeneración , Piel/lesiones , Proteínas Wnt/antagonistas & inhibidores , Cicatrización de Heridas , beta Catenina/antagonistas & inhibidores , Animales , Folículo Piloso , Ratones , Ratones Endogámicos C57BL , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
We previously showed that asunder (asun) is a critical regulator of dynein localization during Drosophila spermatogenesis. Because the expression of asun is much higher in Drosophila ovaries and early embryos than in testes, we herein sought to determine whether ASUN plays roles in oogenesis and/or embryogenesis. We characterized the female germline phenotypes of flies homozygous for a null allele of asun (asun(d93)). We find that asun(d93) females lay very few eggs and contain smaller ovaries with a highly disorganized arrangement of ovarioles in comparison to wild-type females. asun(d93) ovaries also contain a significant number of egg chambers with structural defects. A majority of the eggs laid by asun(d93) females are ventralized to varying degrees, from mild to severe; this ventralization phenotype may be secondary to defective localization of gurken transcripts, a dynein-regulated step, within asun(d93) oocytes. We find that dynein localization is aberrant in asun(d93) oocytes, indicating that ASUN is required for this process in both male and female germ cells. In addition to the loss of gurken mRNA localization, asun(d93) ovaries exhibit defects in other dynein-mediated processes such as migration of nurse cell centrosomes into the oocyte during the early mitotic divisions, maintenance of the oocyte nucleus in the anterior-dorsal region of the oocyte in late-stage egg chambers, and coupling between the oocyte nucleus and centrosomes. Taken together, our data indicate that asun is a critical regulator of dynein localization and dynein-mediated processes during Drosophila oogenesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , Oogénesis/genética , Alelos , Animales , Tipificación del Cuerpo , Linaje de la Célula , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Dineínas/metabolismo , Femenino , Genotipo , Homocigoto , Hibridación in Situ , Masculino , Oocitos/metabolismo , Ovario/metabolismo , Fenotipo , Factores Sexuales , Testículo/metabolismo , TransgenesRESUMEN
Dynein, a microtubule motor complex, plays crucial roles in cell-cycle progression in many systems. The LIS1 accessory protein directly binds dynein, although its precise role in regulating dynein remains unclear. Mutation of human LIS1 causes lissencephaly, a developmental brain disorder. To gain insight into the in vivo functions of LIS1, we characterized a male-sterile allele of the Drosophila homolog of human LIS1. We found that centrosomes do not properly detach from the cell cortex at the onset of meiosis in most Lis-1 spermatocytes; centrosomes that do break cortical associations fail to attach to the nucleus. In Lis-1 spermatids, we observed loss of attachments between the nucleus, basal body and mitochondria. The localization pattern of LIS-1 protein throughout Drosophila spermatogenesis mirrors that of dynein. We show that dynein recruitment to the nuclear surface and spindle poles is severely reduced in Lis-1 male germ cells. We propose that Lis-1 spermatogenesis phenotypes are due to loss of dynein regulation, as we observed similar phenotypes in flies null for Tctex-1, a dynein light chain. We have previously identified asunder (asun) as another regulator of dynein localization and centrosome positioning during Drosophila spermatogenesis. We now report that Lis-1 is a strong dominant enhancer of asun and that localization of LIS-1 in male germ cells is ASUN dependent. We found that Drosophila LIS-1 and ASUN colocalize and coimmunoprecipitate from transfected cells, suggesting that they function within a common complex. We present a model in which Lis-1 and asun cooperate to regulate dynein localization and centrosome positioning during Drosophila spermatogenesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Espermatogénesis/fisiología , Animales , Animales Modificados Genéticamente , Proteínas de Ciclo Celular/genética , Centrosoma/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Complejo Dinactina , Dineínas/genética , Humanos , Masculino , Proteínas Asociadas a Microtúbulos/genética , Modelos Biológicos , Fenotipo , Espermátides/metabolismo , Espermátides/ultraestructura , Espermatocitos/metabolismo , Espermatocitos/ultraestructura , Espermatogénesis/genéticaRESUMEN
The precise orchestration of two opposing protein complexes - one in the cytoplasm (ß-catenin destruction complex) and the other at the plasma membrane (LRP6 signaling complex) - is critical for controlling levels of the transcriptional co-factor ß-catenin, and subsequent activation of the Wnt/ß-catenin signal transduction pathway. The Wnt pathway component Axin acts as an essential scaffold for the assembly of both complexes. How the ß-catenin destruction and LRP6 signaling complexes are modulated following Wnt stimulation remains controversial. A recent study in Science by He and coworkers reveals an underlying logic for Wnt pathway control in which Axin phosphorylation toggles a switch between the active and inactive states. This mini-review focuses on this and two other recent studies that provide insight into the initial signaling events triggered by Wnt exposure. We emphasize regulation of the ß-catenin destruction and LRP6 signaling complexes and propose a framework for future work in this area.
Asunto(s)
Proteína Axina/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Humanos , Fosforilación , Unión Proteica , Transducción de Señal , Vía de Señalización Wnt/genética , beta Catenina/genéticaRESUMEN
BACKGROUND: Although perforator flaps have advanced the field of reconstructive microsurgery, these flaps increase operative time and difficulty of dissection. A prospective experimental animal study was performed to study the use of high-volume hydrodissection to simultaneously decrease the operative time while increasing the safety of perforator dissection. METHODS: Sixteen Sprague-Dawley rats underwent bilateral "deep inferior epigastric perforator" flap dissections with hydrodissection used on the study side and a traditional dissection performed on the control side. Primary outcome measurements included dissection time and dissection difficulty score (1-5 in order of increasing difficulty). RESULTS: The mean (SD) dissection time for the hydrodissected perforators was 9.29 (2.50) minutes versus 13.22 (2.44) minutes for the control perforators (P < 0.001). The mean (SD) dissection difficulty score was 4.44 (0.73) for the dissection of the control side compared with 1.69 (0.87) for the hydrodissected side (P < 0.001). CONCLUSION: The mechanical benefits of hydrodissection of perforators were evident in reduction of perforator dissection time and difficulty.