RESUMEN
5-Fluorouracil (5-FU) is widely used in the treatment of gastric cancer, and the emergence of drug resistance and toxic effects has limited its application. Therefore, there is an urgent need for safe and effective novel drugs or new therapies. ß-Ionone (BI) is found in vegetables and fruits and possesses an inhibitory proliferation of tumor cells in vitro and in vivo. In this study, we investigated whether BI could enhance the inhibitory effects of 5-FU on the proliferation of gastric adenocarcinoma cells and the growth of gastric cancer cell xenografts in a mouse model. The effects of BI and 5-FU alone or their combination on the cell viability, apoptosis, and mitochondrial membrane potential, the cell cycle, and its related proteins-Cyclin D1, and CDK4 as well as PCNA and GSK-3ß were evaluated in SGC-7901 cells and MKN45 cells by MTT, MB, flow cytometry and Western blot. In addition, the effects of BI and 5-FU alone or their combination on the growth of SGC-7901 cell xenografts in nude mice were investigated. The results showed that BI significantly enhanced the sensitivity of gastric adenocarcinoma cells to 5-FU in vitro and in vivo, i.e. proliferation inhibited, apoptosis induced and GSK-3ß protein activated. Therefore, our results suggest that BI increases the antitumor effect of 5-FU on gastric adenocarcinoma cells, at least partly from an activated GSK-3ß signaling pathway.
Asunto(s)
Adenocarcinoma , Apoptosis , Proliferación Celular , Fluorouracilo , Glucógeno Sintasa Quinasa 3 beta , Ratones Desnudos , Norisoprenoides , Transducción de Señal , Neoplasias Gástricas , Animales , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Fluorouracilo/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Proliferación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Norisoprenoides/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adenocarcinoma/metabolismo , Ratones , Apoptosis/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Sinergismo Farmacológico , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos BALB C , Glucógeno Sintasa Quinasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismoRESUMEN
Nowadays, GSK3 is accepted as an enzyme strongly involved in the regulation of inflammation by balancing the pro- and anti-inflammatory responses of cells and organisms, thus influencing the initiation, progression, and resolution of inflammatory processes at multiple levels. Disturbances within its broad functional scope, either intrinsically or extrinsically induced, harbor the risk of profound disruptions to the regular course of the immune response, including the formation of severe inflammation-related diseases. Therefore, this review aims at summarizing and contextualizing the current knowledge derived from animal models to further shape our understanding of GSK3α and ß and their roles in the inflammatory process and the occurrence of tissue/organ damage. Following a short recapitulation of structure, function, and regulation of GSK3, we will focus on the lessons learned from GSK3α/ß knock-out and knock-in/overexpression models, both conventional and conditional, as well as a variety of (predominantly rodent) disease models reflecting defined pathologic conditions with a significant proportion of inflammation and inflammation-related tissue injury. In summary, the literature suggests that GSK3 acts as a crucial switch driving pro-inflammatory and destructive processes and thus contributes significantly to the pathogenesis of inflammation-associated diseases.
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Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3 , Inflamación , Animales , Inflamación/metabolismo , Inflamación/patología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Humanos , Glucógeno Sintasa Quinasa 3 beta/metabolismoRESUMEN
BACKGROUND: Immune escape is an important feature of hepatocellular carcinoma (HCC). The overall response rate of immune checkpoint inhibitors (ICIs) in HCC is still limited. Revealing the immune regulation mechanisms and finding new immune targets are expected to further improve the efficacy of immunotherapy. Our study aims to use CRISPR screening mice models to identify potential targets that play a critical role in HCC immune evasion and further explore their value in improving immunotherapy. METHODS: We performed CRISPR screening in two mice models with different immune backgrounds (C57BL/6 and NPG mice) and identified the immunosuppressive gene Gsk3a as a candidate for further investigation. Flow cytometry was used to analyze the impact of Gsk3a on immune cell infiltration and T-cell function. RNA sequencing was used to identify the changes in neutrophil gene expression induced by Gsk3a and alterations in downstream molecules. The therapeutic value of the combination of Gsk3a inhibitors and anti-programmed cell death protein-1 (PD-1) antibody was also explored. RESULTS: Gsk3a, as an immune inhibitory target, significantly promoted tumor growth in immunocompetent mice rather than immune-deficient mice. Gsk3a inhibited cytotoxic T lymphocytes (CTLs) function by inducing neutrophil chemotaxis. Gsk3a promoted self-chemotaxis of neutrophil expression profiles and neutrophil extracellular traps (NETs) formation to block T-cell activity through leucine-rich α-2-glycoprotein 1 (LRG1). A significant synergistic effect was observed when Gsk3a inhibitor was in combination with anti-PD-1 antibody. CONCLUSIONS: We identified a potential HCC immune evasion target, Gsk3a, through CRISPR screening. Gsk3a induces neutrophil recruitment and NETs formation through the intermediate molecule LRG1, leading to the inhibition of CTLs function. Targeting Gsk3a can enhance CTLs function and improve the efficacy of ICIs.
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Carcinoma Hepatocelular , Inmunoterapia , Neoplasias Hepáticas , Animales , Humanos , Ratones , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/terapia , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Evasión Inmune , Inmunoterapia/métodos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/terapia , Ratones Endogámicos C57BL , Escape del Tumor/efectos de los fármacosRESUMEN
The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.
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Proteínas de Ciclo Celular , Glucógeno Sintasa Quinasa 3 , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas c-akt , Humanos , Células HEK293 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Ciclo Celular/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/genética , Proteínas Tirosina Quinasas/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.
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Proteínas de la Matriz Extracelular , Matriz Extracelular , Degeneración Macular , Epitelio Pigmentado de la Retina , Animales , Ratones , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Degeneración Macular/patología , Degeneración Macular/genética , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Humanos , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Modelos Animales de Enfermedad , Distrofias Retinianas/metabolismo , Distrofias Retinianas/patología , Distrofias Retinianas/genética , Drusas del Disco Óptico/congénitoRESUMEN
Non-invasive imaging of GSK-3 expression in the brain will help to understand the role of GSK-3 in disease pathology and progression. Herein, we report the radiosynthesis and evaluation of two novel isonicotinamide based 18F labeled PET probes, [18F]2 and [18F]6 for noninvasive imaging of GSK3. Among the developed PET probes, the in vitro blood-brain permeability coefficient of 2 (38 ± 20 × 10-6 cm/s, n = 3) was found to be better than 6 (8.75 ± 3.90 × 10-6 cm/s, n = 5). The reference compounds 2 and 6 showed nanomolar affinity towards GSK-3α and GSK-3ß. PET probe [18F]2 showed higher stability (100%) in mouse and human serums compared to [18F]6 (67.01 ± 4.93%, n = 3) in mouse serum and 66.20 ± 6.38%, n = 3) in human serum at 120 min post incubation. The in vivo imaging and blocking studies were performed in wild-type mice only with [18F]2 due to its observed stability. [18F]2 showed a SUV of 0.92 ± 0.28 (n = 6) in mice brain as early as 5 min post-injection followed by gradual clearance over time.
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Encéfalo , Radioisótopos de Flúor , Glucógeno Sintasa Quinasa 3 , Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones/métodos , Animales , Humanos , Ratones , Radioisótopos de Flúor/química , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Radiofármacos/química , Radiofármacos/síntesis química , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/diagnóstico por imagen , Distribución TisularRESUMEN
BACKGROUND: Physical exercise has been shown to be beneficial for individuals with Alzheimer's disease (AD), although the underlying mechanisms are not fully understood. METHODS: Six-month-old Amyloid precursor protein/Presenilin 1 (APP/PS1) transgenic (Tg) mice and wild-type (Wt) mice were randomly assigned to either a sedentary group (Tg-Sed, Wt-Sed) or an exercise group (Tg-Ex, Wt-Ex) undertaking a 12-week, moderate-intensity treadmill running program. Consequently, all mice were tested for memory function and amyloid ß (Aß) levels and phosphorylation of tau and protein kinase B (Akt)/glycogen synthase kinase-3 (GSK3) were examined in tissues of both the cortex and hippocampus. RESULTS: Tg-Sed mice had severely impaired memory, higher levels of Aß, and increased phosphorylation of tau, GSK3α tyrosine279, and GSK3ß tyrosine216, but less phosphorylation of GSK3α serine21, GSK3ß serine9, and Akt serine473 in both tissues than Wt-Sed mice in respective tissues. Tg-Ex mice showed significant improvement in memory function along with lower levels of Aß and less phosphorylation of tau (both tissues), GSK3α tyrosine279 (both tissues), and GSK3ß tyrosine216 (hippocampus only), but increased phosphorylation of GSK3α serine21 (both tissues), GSK3ß serine9 (hippocampus only), and Akt serine473 (both tissues) compared with Tg-Sed mice in respective tissues. CONCLUSIONS: Moderate-intensity aerobic exercise is highly effective in improving memory function in 9-month-old APP/PS1 mice, most likely through differential modulation of GSK3α/ß phosphorylation in the cortex and hippocampus.
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Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Corteza Cerebral , Glucógeno Sintasa Quinasa 3 beta , Glucógeno Sintasa Quinasa 3 , Hipocampo , Condicionamiento Físico Animal , Presenilina-1 , Animales , Masculino , Ratones , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hipocampo/metabolismo , Ratones Transgénicos , Fosforilación , Condicionamiento Físico Animal/fisiología , Presenilina-1/genética , Presenilina-1/metabolismo , Proteínas tau/metabolismoRESUMEN
Clarifying the cellular origin and regulatory mechanisms of intramuscular fat (IMF) deposition is crucial for improving beef quality. Here, we used single-nucleus RNA sequencing to analyze the structure and heterogeneity of skeletal muscle cell populations in different developmental stages of Yanbian cattle and identified eight cell types in two developmental stages of calves and adults. Among them, fibro/adipogenic progenitors (FAPs) expressing CD29 (ITGA7)pos and CD56 (NCAM1)neg surface markers were committed to IMF deposition in beef cattle and expressed major Wnt ligands and receptors. LY2090314/XAV-939 was used to activate/inhibit Wnt/ß-catenin signal. The results showed that the blockade of Glycogen Synthase Kinase 3 (GSK3) by LY2090314 promoted the stabilization of ß-catenin and reduced the expression of genes related adipogenic differentiation (e.g., PPARγ and C/EBPα) in bovine FAPs, confirming the anti-adipogenic effect of GSK3. XAV-939 inhibition of the Wnt/ß-catenin pathway promoted the lipid accumulation capacity of FAPs. Furthermore, we found that blocking GSK3 enhanced the paracrine effects of FAPs-MuSCs and increased myotube formation in muscle satellite cells (MuSCs). Overall, our results outline a single-cell atlas of skeletal muscle development in Yanbian cattle, revealed the role of Wnt/GSK3/ß-catenin signaling in FAPs adipogenesis, and provide a theoretical basis for further regulation of bovine IMF deposition.
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Adipogénesis , Glucógeno Sintasa Quinasa 3 , Músculo Esquelético , Vía de Señalización Wnt , Animales , Bovinos , beta Catenina/metabolismo , beta Catenina/genética , Diferenciación Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Células Madre/metabolismo , Células Madre/citologíaRESUMEN
Glycogen synthase kinase-3 (GSK3), consisting of GSK3α and GSK3ß subtypes, is a complex protein kinase that regulates numerous substrates. Research has observed increased GSK3 expression in the brains of Alzheimer's disease (AD) patients and models. AD is a neurodegenerative disorder with diverse pathogenesis and notable cognitive impairments, characterized by Aß aggregation and excessive tau phosphorylation. This article provides an overview of GSK3's structure and regulation, extensively analyzing its relationship with AD factors. GSK3 overactivation disrupts neural growth, development, and function. It directly promotes tau phosphorylation, regulates amyloid precursor protein (APP) cleavage, leading to Aß formation, and directly or indirectly triggers neuroinflammation and oxidative damage. We also summarize preclinical research highlighting the inhibition of GSK3 activity as a primary therapeutic approach for AD. Finally, pending issues like the lack of highly specific and affinity-driven GSK3 inhibitors, are raised and expected to be addressed in future research. In conclusion, GSK3 represents a target in AD treatment, filled with hope, challenges, opportunities, and obstacles.
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Enfermedad de Alzheimer , Glucógeno Sintasa Quinasa 3 , Animales , Humanos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/enzimología , Precursor de Proteína beta-Amiloide/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas tau/metabolismo , Proteínas tau/antagonistas & inhibidoresRESUMEN
The circadian system is a conserved time-keeping machinery that regulates a wide range of processes such as sleep/wake, feeding/fasting, and activity/rest cycles to coordinate behavior and physiology. Circadian disruption can be a contributing factor in the development of metabolic diseases, inflammatory disorders, and higher risk of cancer. Glioblastoma (GBM) is a highly aggressive grade 4 brain tumor that is resistant to conventional therapies and has a poor prognosis after diagnosis, with a median survival of only 12-15 months. GBM cells kept in culture were shown to contain a functional circadian oscillator. In seeking more efficient therapies with lower side effects, we evaluated the pharmacological modulation of the circadian clock by targeting the cytosolic kinases glycogen synthase kinase-3 (GSK-3) and casein kinase 1 ε/δ (CK1ε/δ) with specific inhibitors (CHIR99021 and PF670462, respectively), the cryptochrome protein stabilizer (KL001), or circadian disruption after Per2 knockdown expression in GBM-derived cells. CHIR99021-treated cells had a significant effect on cell viability, clock protein expression, migration, and cell cycle distribution. Moreover, cultures exhibited higher levels of reactive oxygen species and alterations in lipid droplet content after GSK-3 inhibition compared to control cells. The combined treatment of CHIR99021 with temozolomide was found to improve the effect on cell viability compared to temozolomide therapy alone. Per2 disruption affected both GBM migration and cell cycle progression. Overall, our results suggest that pharmacological modulation or molecular clock disruption severely affects GBM cell biology.
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Neoplasias Encefálicas , Glioblastoma , Glioblastoma/patología , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológico , Humanos , Línea Celular Tumoral , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Piridinas/farmacología , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , Citosol/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Pirimidinas/farmacología , Movimiento Celular/efectos de los fármacos , Relojes Circadianos/efectos de los fármacos , Relojes Circadianos/fisiología , Proteínas CLOCK/metabolismo , Proteínas CLOCK/genética , Proteínas Circadianas Period/metabolismo , Proteínas Circadianas Period/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Reactive astrocytes are important pathophysiologically and synthesize neurosteroids. We observed that LPS increased immunoreactive TLR4 and key steroidogenic enzymes in cortical astrocytes of rats and investigated whether corticosteroids are produced and mediate astrocytic TLR4-dependent innate immune responses. We found that LPS increased steroidogenic acute regulatory protein (StAR) and StAR-dependent aldosterone production in purified astrocytes. Both increases were blocked by the TLR4 antagonist TAK242. LPS also increased 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and corticosterone production, and both were prevented by TAK242 and by siRNAs against 11ß-HSD1, StAR, or aldosterone synthase (CYP11B2). Knockdown of 11ß-HSD1, StAR, or CYP11B2 or blocking either mineralocorticoid receptors (MR) or glucocorticoid receptors (GR) prevented dephosphorylation of p-Ser9GSK-3ß, activation of NF-κB, and the GSK-3ß-dependent increases of C3, IL-1ß, and TNF-α caused by LPS. Exogenous aldosterone mimicked the MR- and GSK-3ß-dependent pro-inflammatory effects of LPS in astrocytes, but corticosterone did not. Supernatants from astrocytes treated with LPS reduced MAP2 and viability of cultured neurons except when astrocytic StAR or MR was inhibited. In adrenalectomized rats, intracerebroventricular injection of LPS increased astrocytic TLR4, StAR, CYP11B2, and 11ß-HSD1, NF-κB, C3 and IL-1ß, decreased astrocytic p-Ser9GSK-3ß in the cortex and was neurotoxic, except when spironolactone was co-injected, consistent with the in vitro results. LPS also activated NF-κB in some NeuN+ and CD11b+ cells in the cortex, and these effects were prevented by spironolactone. We conclude that intracrine aldosterone may be involved in the TLR4-dependent innate immune responses of astrocytes and can trigger paracrine effects by activating astrocytic MR/GSK-3ß/NF-κB signaling.
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Astrocitos , Glucógeno Sintasa Quinasa 3 beta , Inmunidad Innata , Lipopolisacáridos , Receptor Toll-Like 4 , Animales , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Inmunidad Innata/efectos de los fármacos , Ratas , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Lipopolisacáridos/farmacología , Corticoesteroides/farmacología , Ratas Sprague-Dawley , Células Cultivadas , Receptores de Mineralocorticoides/metabolismo , Aldosterona/metabolismo , Aldosterona/farmacología , Masculino , FN-kappa B/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Corticosterona/farmacologíaRESUMEN
Alzheimer's disease (AD) is a major cause of dementia and one of the most common chronic diseases affecting the aging population. Because AD is considered a public health priority, there is a critical need to discover novel and effective agents for the treatment of this condition. In view of the known contribution of up-regulated glutaminyl cyclase (QC) and glycogen synthase kinase-3ß (GSK-3ß) to the initiation of AD, we previously evaluated a series of dual inhibitors containing maleimide and imidazole motifs as potential anti-AD agents. Here, we assessed another series of hybrids containing maleimide and imidazole motifs to gain an in-depth understanding of the structure-activity relationship (SAR). Based on the primary screening, the introduction of 5-methyl imidazole at one side of the molecule did not enhance the QC-specific inhibitory activity of these hybrids (2, IC50 = 1.22 µM), although the potency was increased by 2' substitution on the maleimide motif at the other side of the molecule. Interestingly, compounds containing 5-methyl imidazole exhibited stronger GSK-3ß-specific inhibitory activity (2, IC50 = 0.0021 µM), and the electron-withdrawing group and 2' and 3' substitution were favorable. Further investigation of substitutions on the maleimide motif in compounds 14-35 revealed that QC-specific inhibition in the presence of piperidine was improved by introduction of a methoxy group (R2). Increasing the linker length and introduction of a methoxy group (R2) also increased the GSK-3ß-specific inhibitory potency. These findings were further confirmed by molecular docking analysis of 33 and 24 with QC and GSK-3ß. Overall, these hybrids exhibited enhanced inhibitory potency against both QC and GSK-3ß, highlighting an important strategy for improving the potency of hybrids as dual-targeting anti-AD agents.
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Aminoaciltransferasas , Glucógeno Sintasa Quinasa 3 beta , Imidazoles , Maleimidas , Relación Estructura-Actividad , Maleimidas/química , Maleimidas/farmacología , Maleimidas/síntesis química , Imidazoles/química , Imidazoles/farmacología , Imidazoles/síntesis química , Humanos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Estructura Molecular , Enfermedad de Alzheimer/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Relación Dosis-Respuesta a DrogaRESUMEN
The amount of dietary sugars and the administration of lithium both impact the lifespan of the fruit fly Drosophila melanogaster. It is noteworthy that lithium is attributed with insulin-like activity as it stimulates protein kinase B/Akt and suppresses the activity of glycogen synthase kinase-3 (GSK-3). However, its interaction with dietary sugar has largely remained unexplored. Therefore, we investigated the effects of lithium supplementation on known lithium-sensitive parameters in fruit flies, such as lifespan, body composition, GSK-3 phosphorylation, and the transcriptome, while varying the dietary sugar concentration. For all these parameters, we observed that the efficacy of lithium was significantly influenced by the sucrose content in the diet. Overall, we found that lithium was most effective in enhancing longevity and altering body composition when added to a low-sucrose diet. Whole-body RNA sequencing revealed a remarkably similar transcriptional response when either increasing dietary sucrose from 1% to 10% or adding 1 mM LiCl to a 1% sucrose diet, characterized by a substantial overlap of nearly 500 differentially expressed genes. Hence, dietary sugar supply is suggested as a key factor in understanding lithium bioactivity, which could hold relevance for its therapeutic applications.
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Sacarosa en la Dieta , Drosophila melanogaster , Longevidad , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/efectos de los fármacos , Longevidad/efectos de los fármacos , Longevidad/genética , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Litio/farmacología , Cloruro de Litio/farmacología , Fosforilación/efectos de los fármacos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMEN
Glycogen synthase kinase 3 (GSK3) plays a pivotal role in signaling pathways involved in insulin metabolism and the pathogenesis of neurodegenerative disorders. In particular, the GSK3ß isoform is implicated in Alzheimer's disease (AD) as one of the key kinases involved in the hyperphosphorylation of tau protein, one of the neuropathological hallmarks of AD. As a constitutively active serine/threonine kinase, GSK3 is inactivated by Akt/PKB-mediated phosphorylation of Ser9 in the N-terminal disordered domain, and for most of its substrates, requires priming (prephosphorylation) by another kinase that targets the substrate to a phosphate-specific pocket near the active site. GSK3 has also been shown to be post-translationally modified by O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation), with still unknown functions. Here, we have found that binding of Akt inhibits GSK3ß kinase activity on both primed and unprimed tau substrates. Akt-mediated Ser9 phosphorylation restores the GSK3ß kinase activity only on primed tau, thereby selectively inactivating GSK3ß toward unprimed tau protein. Additionally, we have shown that GSK3ß is highly O-GlcNAcylated at multiple sites within the kinase domain and the disordered N- and C-terminal domains, including Ser9. In contrast to Akt-mediated regulation, neither the O-GlcNAc transferase nor O-GlcNAcylation significantly alters GSK3ß kinase activity, but high O-GlcNAc levels reduce Ser9 phosphorylation by Akt. Reciprocally, Akt phosphorylation downregulates the overall O-GlcNAcylation of GSK3ß, indicating a crosstalk between both post-translational modifications. Our results indicate that specific O-GlcNAc profiles may be involved in the phosphorylation-dependent Akt-mediated regulation of GSK3ß kinase activity.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta , Proteínas Proto-Oncogénicas c-akt , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/química , Fosforilación , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Procesamiento Proteico-Postraduccional , Glucógeno Sintasa Quinasa 3/metabolismo , Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilglucosaminiltransferasas/química , Glicosilación , AnimalesRESUMEN
Dysregulated autophagy/mitophagy is one of the major causes of cardiac injury in ischemic conditions. Glycogen synthase kinase-3alpha (GSK-3α) has been shown to play a crucial role in the pathophysiology of cardiac diseases. However, the precise role of GSK-3α in cardiac mitophagy remains unknown. Herein, we investigated the role of GSK-3α in cardiac mitophagy by employing AC16 human cardiomyocytes under the condition of acute hypoxia. We observed that the gain-of-GSK-3α function profoundly induced mitophagy in the AC16 cardiomyocytes post-hypoxia. Moreover, GSK-3α overexpression led to increased ROS generation and mitochondrial dysfunction in cardiomyocytes, accompanied by enhanced mitophagy displayed by increased mt-mKeima intensity under hypoxia. Mechanistically, we identified that GSK-3α promotes mitophagy through upregulation of BNIP3, caused by GSK-3α-mediated increase in expression of HIF-1α and FOXO3a in cardiomyocytes post-hypoxia. Moreover, GSK-3α displayed a physical interaction with BNIP3 and, inhibited PINK1 and Parkin recruitment to mitochondria was observed specifically under hypoxia. Taken together, we identified a novel mechanism of mitophagy in human cardiomyocytes. GSK-3α promotes mitochondrial dysfunction and regulates FOXO3a -mediated BNIP3 overexpression in cardiomyocytes to facilitate mitophagy following hypoxia. An interaction between GSK-3α and BNIP3 suggests a role of GSK-3α in BNIP3 recruitment to the mitochondrial membrane where it enhances mitophagy in stressed cardiomyocytes independent of the PINK1/Parkin.
Asunto(s)
Hipoxia de la Célula , Proteína Forkhead Box O3 , Glucógeno Sintasa Quinasa 3 , Proteínas de la Membrana , Mitofagia , Miocitos Cardíacos , Proteínas Quinasas , Proteínas Proto-Oncogénicas , Ubiquitina-Proteína Ligasas , Humanos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Mitofagia/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteína Forkhead Box O3/metabolismo , Proteína Forkhead Box O3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Proteínas Quinasas/metabolismo , Proteínas Quinasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Especies Reactivas de Oxígeno/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Transducción de Señal , Mitocondrias/metabolismo , Mitocondrias/patología , Mitocondrias/genética , Línea CelularRESUMEN
c-Myc is a proto-oncoprotein that regulates various cellular processes and whose abnormal expression leads to tumorigenesis. c-Myc protein stability has been shown to be predominantly controlled by the ubiquitin ligase (E3) CRL1Fbxw7 in a manner dependent on glycogen synthase kinase 3 (GSK3)-mediated phosphorylation. Here we show that, in some types of cancer cells, c-Myc degradation is largely insensitive to the GSK3 inhibitor (GSK3i) CHIR99021, suggesting the existence of an E3 other than CRL1Fbxw7 for c-Myc degradation. Mass spectrometry identified CRL2KLHDC3 as such an E3. In GSK3i-insensitive cancer cells, combined depletion of Fbxw7 and KLHDC3 resulted in marked stabilization of c-Myc, suggestive of a cooperative action of Fbxw7 and KLHDC3. Furthermore, transplantation of such cells deficient in both Fbxw7 and KLHDC3 into immunodeficient mice gave rise to larger tumors compared with those formed by cells lacking only Fbxw7. GSK3i-insensitive pancreatic cancer cells expressed lower levels of SHISA2, a negative regulator of the Wnt signaling pathway, than did GSK3i-sensitive cells. KLHDC3 mRNA abundance was associated with prognosis in pancreatic cancer patients with a low level of SHISA2 gene expression. These results suggest that KLHDC3 cooperates with Fbxw7 to promote c-Myc degradation in a subset of cancer cells with low GSK3 activity.
Asunto(s)
Proteína 7 que Contiene Repeticiones F-Box-WD , Proteolisis , Proteínas Proto-Oncogénicas c-myc , Ubiquitina-Proteína Ligasas , Humanos , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Ratones , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Línea Celular Tumoral , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMEN
Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying the progression from prehypertension to hypertension require further investigation. Recent proteomic studies have shown promising results in uncovering potential biomarkers related to disease development. In this study, serum proteomic data collected from Qatar Biobank were analyzed to identify altered protein expression between individuals with normal blood pressure, prehypertension, and hypertension and to elucidate the biological pathways contributing to this disease. The results revealed a cluster of proteins, including the SRC family, CAMK2B, CAMK2D, TEC, GSK3, VAV, and RAC, which were markedly upregulated in patients with hypertension compared to those with prehypertension (fold change ≥ 1.6 or ≤-1.6, area under the curve ≥ 0.8, and q-value < 0.05). Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling. Actin cytoskeleton reorganization affects various biological processes that contribute to the maintenance of blood pressure, including vascular tone, endothelial function, cellular signaling, inflammation, fibrosis, and mechanosensing. Therefore, the findings of this study suggest a potential novel role of actin cytoskeleton-related proteins in the progression from prehypertension to hypertension. The present study sheds light on the underlying pathological mechanisms involved in hypertension and could pave the way for new diagnostic and therapeutic approaches for the treatment of this disease.
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Citoesqueleto de Actina , Hipertensión , Proteómica , Femenino , Humanos , Masculino , Citoesqueleto de Actina/metabolismo , Biomarcadores , Presión Sanguínea , Hipertensión/metabolismo , Prehipertensión/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/ß-catenin pathway, which promotes the degradation of ß-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/ß-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of ß-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/ß-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate ß-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/ß KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-ß-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/ß-catenin pathway at the level of ß-catenin and Axin.
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Caseína Quinasa Ialfa , Vía de Señalización Wnt , beta Catenina , Humanos , Empalme Alternativo , beta Catenina/metabolismo , beta Catenina/genética , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Células HEK293 , Fosforilación , Proteína Wnt3A/metabolismo , Proteína Wnt3A/genéticaRESUMEN
Over the last six decades, lithium has been considered the gold standard treatment for the long-term management of bipolar disorder due to its efficacy in preventing both manic and depressive episodes as well as suicidal behaviors. Nevertheless, despite numerous observed effects on various cellular pathways and biologic systems, the precise mechanism through which lithium stabilizes mood remains elusive. Furthermore, there is recent support for the therapeutic potential of lithium in other brain diseases. This review offers a comprehensive examination of contemporary understanding and predominant theories concerning the diverse mechanisms underlying lithium's effects. These findings are based on investigations utilizing cellular and animal models of neurodegenerative and psychiatric disorders. Recent studies have provided additional support for the significance of glycogen synthase kinase-3 (GSK3) inhibition as a crucial mechanism. Furthermore, research has shed more light on the interconnections between GSK3-mediated neuroprotective, antioxidant, and neuroplasticity processes. Moreover, recent advancements in animal and human models have provided valuable insights into how lithium-induced modifications at the homeostatic synaptic plasticity level may play a pivotal role in its clinical effectiveness. We focused on findings from translational studies suggesting that lithium may interface with microRNA expression. Finally, we are exploring the repurposing potential of lithium beyond bipolar disorder. These recent findings on the therapeutic mechanisms of lithium have provided important clues toward developing predictive models of response to lithium treatment and identifying new biologic targets. SIGNIFICANCE STATEMENT: Lithium is the drug of choice for the treatment of bipolar disorder, but its mechanism of action in stabilizing mood remains elusive. This review presents the latest evidence on lithium's various mechanisms of action. Recent evidence has strengthened glycogen synthase kinase-3 (GSK3) inhibition, changes at the level of homeostatic synaptic plasticity, and regulation of microRNA expression as key mechanisms, providing an intriguing perspective that may help bridge the mechanistic gap between molecular functions and its clinical efficacy as a mood stabilizer.
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Compuestos de Litio , Humanos , Animales , Compuestos de Litio/farmacología , Compuestos de Litio/uso terapéutico , Antimaníacos/farmacología , Antimaníacos/uso terapéutico , Trastorno Bipolar/tratamiento farmacológico , Plasticidad Neuronal/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidoresRESUMEN
Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.