RESUMEN
Plant growth occurs via the interconnection of cell growth and proliferation in each organ following specific developmental and environmental cues. Therefore, different photoperiods result in distinct growth patterns due to the integration of light and circadian perception with specific Carbon (C) partitioning strategies. In addition, the TARGET OF RAPAMYCIN (TOR) kinase pathway is an ancestral signaling pathway that integrates nutrient information with translational control and growth regulation. Recent findings in Arabidopsis (Arabidopsis thaliana) have shown a mutual connection between the TOR pathway and the circadian clock. However, the mechanistical network underlying this interaction is mostly unknown. Here, we show that the conserved TOR target, the 40S ribosomal protein S6 kinase (S6K) is under circadian and photoperiod regulation both at the transcriptional and post-translational level. Total S6K (S6K1 and S6K2) and TOR-dependent phosphorylated-S6K protein levels were higher during the light period and decreased at dusk especially under short day conditions. Using chemical and genetic approaches, we found that the diel pattern of S6K accumulation results from 26S proteasome-dependent degradation and is altered in mutants lacking the circadian F-box protein ZEITLUPE (ZTL), further strengthening our hypothesis that S6K could incorporate metabolic signals via TOR, which are also under circadian regulation. Moreover, under short days when C/energy levels are limiting, changes in S6K1 protein levels affected starch, sucrose and glucose accumulation and consequently impacted root and rosette growth responses. In summary, we propose that S6K1 constitutes a missing molecular link where day-length perception, nutrient availability and TOR pathway activity converge to coordinate growth responses with environmental conditions.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Fotoperiodo , Proteínas Quinasas S6 Ribosómicas , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas Quinasas S6 Ribosómicas/genética , Fosforilación , Transducción de Señal , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Relojes Circadianos/genética , Mutación/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Fosfatidilinositol 3-QuinasasRESUMEN
Fish growth and health are predominantly governed by dietary nutrient supply. Although the beneficial effects of omega-3 polyunsaturated fatty acids supplementation have been shown in a number of fish species, the underlying mechanisms are still mostly unknown. In this study, we conducted an investigation into the effects of EPA and DHA on cell proliferation, nutrient sensing signaling, and branched-chain amino acids (BCAA) transporting in primary turbot muscle cells. The findings revealed that EPA and DHA could stimulate cell proliferation, promote protein synthesis and inhibit protein degradation through activation of target of rapamycin (TOR) signaling pathway, a pivotal nutrient-sensing signaling cascade. While downregulating the expression of myogenin and myostatin, EPA and DHA increased the level of myogenic regulatory factors, such as myoD and follistatin. Furthermore, we observed a significant increase in the concentrations of intracellular BCAAs following treatment with EPA or DHA, accompanied by an upregulation of the associated amino acid transporters. Our study providing valuable insights into the mechanisms underlying the growth-promoting effects of omega-3 fatty acids in fish.
Asunto(s)
Proliferación Celular , Ácidos Docosahexaenoicos , Ácido Eicosapentaenoico , Peces Planos , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR , Animales , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Proliferación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas de Peces/metabolismoRESUMEN
BACKGROUND: We previously demonstrated that insulin-like growth factor-1 (IGF-1) regulates sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) in vascular smooth muscle cells (VSMC) via phosphatidylinositol-3 kinase (PI3K). Taking into account that others' work show that IGF-1 activates the PI3K/protein kinase B (Akt) signaling pathway in many different cells, we here further questioned if the Akt/mammalian target of rapamycin (mTOR)/ribosomal protein p70 S6 kinase (S6K) pathway stimulates Na+/K+-ATPase, an essential protein for maintaining normal heart function. METHODS AND RESULTS: There were 14 adult male Wistar rats, half of whom received bolus injections of IGF-1 (50 µg/kg) for 24 h. We evaluated cardiac Na+/K+-ATPase expression, activity, and serum IGF-1 levels. Additionally, we examined the phosphorylated forms of the following proteins: insulin receptor substrate (IRS), phosphoinositide-dependent kinase-1 (PDK-1), Akt, mTOR, S6K, and α subunit of Na+/K+-ATPase. Additionally, the mRNA expression of the Na+/K+-ATPase α1 subunit was evaluated. Treatment with IGF-1 increases levels of serum IGF-1 and stimulates Na+/K+-ATPase activity, phosphorylation of α subunit of Na+/K+-ATPase on Ser23, and protein expression of α2 subunit. Furthermore, IGF-1 treatment increased phosphorylation of IRS-1 on Tyr1222, Akt on Ser473, PDK-1 on Ser241, mTOR on Ser2481 and Ser2448, and S6K on Thr421/Ser424. The concentration of IGF-1 in serum positively correlates with Na+/K+-ATPase activity and the phosphorylated form of mTOR (Ser2448), while Na+/K+-ATPase activity positively correlates with the phosphorylated form of IRS-1 (Tyr1222) and mTOR (Ser2448). CONCLUSION: These results indicate that the Akt/mTOR/S6K signalling pathway may be involved in the IGF-1 regulating cardiac Na+/K+-ATPase expression and activity.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina , Proteínas Proto-Oncogénicas c-akt , Animales , Masculino , Ratas , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas S6 RibosómicasRESUMEN
Suppression of target of rapamycin complex 1 (TORC1) by rapamycin ameliorates aging in diverse species. S6 kinase (S6K) is an essential mediator, but the mechanisms involved are unclear. Here we show that activation of S6K specifically in Drosophila fat-body blocked extension of lifespan by rapamycin, induced accumulation of multilamellar lysosomes and blocked age-associated hyperactivation of the NF-κB-like immune deficiency (IMD) pathway, indicative of reduced inflammaging. Syntaxin 13 mediated the effects of TORC1-S6K signaling on lysosome morphology and inflammaging, suggesting they may be linked. Inflammaging depended on the IMD receptor regulatory isoform PGRP-LC, and repression of the IMD pathway from midlife extended lifespan. Age-related inflammaging was higher in females than in males and was not lowered in males by rapamycin treatment or lowered S6K. Rapamycin treatment also elevated Syntaxin 12/13 levels in mouse liver and prevented age-related increase in noncanonical NF-κB signaling, suggesting that the effect of TORC1 on inflammaging is conserved from flies to mammals.
Asunto(s)
Longevidad , FN-kappa B , Animales , Femenino , Masculino , Ratones , Drosophila , Inflamación/tratamiento farmacológico , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , FN-kappa B/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Sirolimus/farmacologíaRESUMEN
The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.
Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Gefitinib , Neoplasias Pulmonares , Proteína Elk-1 con Dominio ets , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos/genética , Receptores ErbB/metabolismo , Gefitinib/farmacología , Gefitinib/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones Desnudos , Proteínas Quinasas S6 Ribosómicas , ARN Interferente Pequeño/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , /uso terapéuticoRESUMEN
Mitochondrial function is essential for plant growth, but the mechanisms involved in adjusting growth and metabolism to changes in mitochondrial energy production are not fully understood. We studied plants with reduced expression of CYTC-1, one of two genes encoding the respiratory chain component cytochrome c (CYTc) in Arabidopsis, to understand how mitochondria communicate their status to coordinate metabolism and growth. Plants with CYTc deficiency show decreased mitochondrial membrane potential and lower ATP content, even when carbon sources are present. They also exhibit higher free amino acid content, induced autophagy, and increased resistance to nutritional stress caused by prolonged darkness, similar to plants with triggered starvation signals. CYTc deficiency affects target of rapamycin (TOR)-pathway activation, reducing S6 kinase (S6K) and RPS6A phosphorylation, as well as total S6K protein levels due to increased protein degradation via proteasome and autophagy. TOR overexpression restores growth and other parameters affected in cytc-1 mutants, even if mitochondrial membrane potential and ATP levels remain low. We propose that CYTc-deficient plants coordinate their metabolism and energy availability by reducing TOR-pathway activation as a preventive signal to adjust growth in anticipation of energy exhaustion, thus providing a mechanism by which changes in mitochondrial activity are transduced to the rest of the cell.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Citocromos c/genética , Citocromos c/metabolismo , Sirolimus/farmacología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Adenosina Trifosfato/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Immunity-related GTPase M (IRGM), an Interferon-inducible protein, functions as a pivotal immunoregulator in multiple autoimmune diseases and infection. However, the role of IRGM in hepatocellular carcinoma (HCC) development remains unveiled. Here, we found interferon-γ (IFN-γ) treatment in HCC drastically triggered the expression of IRGM, and the high level of IRGM indicated poor prognosis in HCC patients. Functionally, IRGM promoted the malignant progression of HCC. Single-cell sequencing revealed that IRGM inhibition promoted the infiltration of CD8+ cytotoxic T lymphocytes (CTLs) with significant downregulation of PD-L1 expression in HCC. Furthermore, Immunoprecipitation-Mass Spectrometry assay revealed that IRGM interacted with transcription factor YBX1, which facilitated PD-L1 transcription. Mechanistically, IRGM promoted the interaction of YBX1 and phosphokinase S6K1, increasing phosphorylation and nuclear localization of YBX1, transcription of PD-L1. Additionally, the combination of IRGM inhibition with α-PD1 demonstrated a stronger anti-tumor effect compared to the single application of α-PD1. In summary, IRGM is a novel regulator of PD-L1, which suppresses CD8+ CTLs infiltration and function in HCC, resulting in cancer progression. This study may raise a novel therapeutic strategy combined with immune checkpoint inhibitors (ICIs) against HCC.
Asunto(s)
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Antineoplásicos/uso terapéutico , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Linfocitos T CD8-positivos , Proteínas de Unión al GTP/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Fosforilación , Microambiente Tumoral , Proteína 1 de Unión a la Caja Y/genética , Proteína 1 de Unión a la Caja Y/metabolismo , Proteínas Quinasas S6 RibosómicasRESUMEN
Objective: To investigate the effect and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats (hereinafter referred to as burn serum). Methods: Experimental research methods were adopted. Thirty gender equally balanced Wistar rats aged 7 to 8 weeks were collected, 10 of which were used to prepare normal rat serum (hereinafter referred to as normal serum), and the other 20 were inflicted with full-thickness burn of 30% total body surface area to prepare burn serum. Primary cardiomyocytes were isolated and cultured from the apical tissue of 180 Wistar rats aged 1 to 3 days by either gender for follow-up experiments. Cells were divided into normal serum group and burn serum group treated with corresponding serum according to the random number table (the same grouping method below). Trypanosoma blue staining was performed at post treatment hour (PTH) 1, 3, 6, 9, and 12 to detect the cell survival rate. Cells were divided into burn serum alone group treated with burn serum for 6 h followed by routine culture of 30 min and 0.4 mmol/L glycine group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, 1.6 mmol/L glycine group, and 2.0 mmol/L glycine group treated with burn serum for 6 h followed by culture of 30 min with corresponding final molarity of glycine, i.e., at post intervention hour (PIH) 6.5, the cell survival rate was detected as before. Cells were divided into normal serum group, burn serum alone group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, and 1.6 mmol/L glycine group, with the same intervention of 6.5 h as before, respectively. The content of adenosine monophosphate (AMP) and adenosine triphosphate (ATP) was detected by high performance liquid chromatography, and the AMP/ATP ratio was calculated. The protein expressions of phosphorylated mammalian target of rapamycin complex 1 (p-mTORC1), phosphorylated p70 ribosomal protein S6 kinase (p-p70 S6K), phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4E-BP1), and phosphorylated AMP-activated protein kinase (p-AMPK) were detected by Western blotting. Cells were divided into normal serum group, burn serum alone group, 0.8 mmol/L glycine group intervened as before and 0.8 mmol/L glycine+25 ng/mL rapamycin group treated with burn serum followed by culture with two reagents. The expressions of heat shock protein 70 (HSP70), metallothionein (MT), and tubulin were detected by immunofluorescence method after 30 min of corresponding culture at PTH 1, 3, and 6, i.e., at PIH 1.5, 3.5, and 6.5, and the microtubule morphology was observed at PIH 6.5. The sample number at each time point was 10. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference (LSD)-t test, LSD test, and Bonferroni correction. Results: At PTH 1, 3, 6, 9, and 12, the cell survival rates in burn serum group were significantly lower than those in normal serum group (with t values of 4.96, 16.83, 35.51, 34.33, and 27.88, P<0.05). In burn serum group, the cell survival rate at PTH 3, 6, 9, or 12 was significantly lower than that at PTH 1 (P<0.05), the cell survival rate at PTH 6, 9, or 12 was significantly lower than that at PTH 3 (P<0.05), and the cell survival rate at PTH 6 was similar to that at PTH 9 (P>0.05) but significantly higher than that at PTH 12 (P<0.05). Treatment of 6 h was selected as the follow-up intervention time of burn serum. At PIH 6.5, compared with that in burn serum alone group, the cell survival rate in each glycine group was significantly increased (P<0.05). The cell survival rate in 0.8 mmol/L glycine group was the highest, and 0.8, 1.2, and 1.6 mmol/L were selected as subsequent glycine intervention concentrations. At PIH 6.5, the AMP/ATP ratio of cells in burn serum alone group was significantly higher than that in normal serum group, 1.2 mmol/L glycine group, or 1.6 mmol/L glycine group (P values all <0.05), and the AMP/ATP ratio of cells in 1.6 mmol/L glycine group was significantly lower than that in 0.8 mmol/L glycine group (P<0.05). At PIH 6.5, the protein expressions of p-mTORC1, p-p70 S6K, and p-4E-BP1 of cells in normal serum group, burn serum alone group, 0.8 mmol/L glycine group, 1.2 mmol/L glycine group, and 1.6 mmol/L glycine group were 1.001±0.037, 0.368±0.020, 1.153±0.019, 1.128±0.062, 1.028±0.037, 0.96±0.07, 0.63±0.12, 1.17±0.13, 1.13±0.16, 1.11±0.11, and 0.98±0.06, 0.45±0.08, 1.13±0.05, 0.77±0.12, 0.51±0.13. Compared with those in burn serum alone group, the protein expressions of p-mTORC1, p-p70 S6K, and p-4E-BP1 of cells in normal serum group and each glycine group were significantly increased (P<0.05), while the protein expressions of p-AMPK were significantly decreased (P<0.05). Compared with those in 0.8 mmol/L glycine group, the protein expression of p-4E-BP1 of cells in 1.2 mmol/L glycine group and the protein expressions of p-mTORC1 and p-4E-BP1 of cells in 1.6 mmol/L glycine group were significantly decreased (P<0.05). Compared with those in 1.2 mmol/L glycine group, the protein expressions of p-mTORC1 and p-4E-BP1 of cells in 1.6 mmol/L glycine group were significantly decreased (P<0.05), while the protein expression of p-AMPK was significantly increased (P<0.05). Compared with those in normal serum group, the expression of tubulin of cells in burn serum alone group was significantly decreased at PIH 1.5, 3.5, and 6.5 (P<0.05), while the expression of HSP70 of cells at PIH 1.5 and 3.5 and the expression of MT at PIH 3.5 and 6.5 were significantly increased (P<0.05). The expressions of HSP70 and MT of cells at PIH 1.5, 3.5, and 6.5 and the expression of tubulin at PIH 1.5 and 3.5 in burn serum alone group and 0.8 mmol/L glycine+25 ng/mL rapamycin group were significantly lower than those in 0.8 mmol/L glycine group (P<0.05). At PIH 6.5, compared with that in normal serum group, the cell microtubule structure in burn serum alone group was disordered; the cell boundary in 0.8 mmol/L glycine group was clearer than that in burn serum alone group, and the microtubule structure arranged neatly near the nucleus. Compared with that in 0.8 mmol/L glycine group, 0.8 mmol/L glycine+25 ng/mL rapamycin group had unclear cell boundaries and disordered microtubule structure. Conclusions: Burn serum can cause cardiomyocytes damage in rats. Glycine can significantly up-regulate mammalian target of rapamycin/p70 ribosomal protein S6 kinase/eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway through AMP-activated protein kinase, promote the synthesis of protective proteins HSP70, MT, and tubulin, stabilize the microtubule structure, and exert cardiomyocytes protection function.
Asunto(s)
Quemaduras , Miocitos Cardíacos , Ratas , Animales , Ratas Sprague-Dawley , Miocitos Cardíacos/metabolismo , Ratas Wistar , Proteínas Quinasas Activadas por AMP , Ápice del Diente/metabolismo , Tubulina (Proteína) , Quemaduras/metabolismo , Adenosina Trifosfato , Diana Mecanicista del Complejo 1 de la Rapamicina , Sirolimus , Serina-Treonina Quinasas TOR , Proteínas Quinasas S6 Ribosómicas , Factores de Iniciación de Péptidos , Adenosina Monofosfato , MamíferosRESUMEN
OBJECTIVE: The prognosis of acute myeloid leukemia (AML) remains poor although the basic and translational research has been highly productive in understanding the genetics and pathopoiesis of AML and a plethora of targeted therapies have been developed. Consequently, it is crucial to deepen the knowledge of molecular pathogenesis underlying AML for the advancement of new treatment options. METHOD: A RSK gene family-related signature was constructed to investigate whether RSK gene family members were useful in predicting the prognosis of AML patients. The relationship between the RSK gene family-related signature and the infiltration of immune cells was further assessed using the CIBERSORT algorithm. The 'oncoPredict' package was used to analyze relationships between the RSK gene family-related signature and the sensitivity to drugs or small molecules. RESULTS: Patients were classified into two groups using the RSK gene family-related signature following the median risk score. Overall survival (OS) was significantly longer in patients with low-risk scores than that in patients with high-risk scores as showed by both training and validation datasets. Moreover, the signature was helpful in predicting 1-year, 3-year, and 5-year OS in training and validation datasets. In addition, it was identified that low-risk patients exhibited greater sensitivity to 20 drugs or small molecules and that high-risk patients had higher sensitivity to 38 drugs or small molecules. CONCLUSION: RSK gene family members, particularly RPS6KA1 and RPS6KA4, may help to predict prognosis for AML patients. Furthermore, RPS6KA1 may serve as a novel drug target for AML.
Asunto(s)
Familia , Leucemia Mieloide Aguda , Proteínas Quinasas S6 Ribosómicas , Humanos , Algoritmos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Pronóstico , Proteínas Quinasas S6 Ribosómicas/genéticaRESUMEN
Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.
Asunto(s)
Azacitidina , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Azacitidina/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas Quinasas S6 Ribosómicas , Proteínas Quinasas S6 Ribosómicas 90-kDa , ARN Guía de Sistemas CRISPR-CasRESUMEN
The 5' untranslated regions (UTRs) in messenger RNAs (mRNAs) play an important role in the regulation of protein synthesis. We had previously identified a group of mRNAs that includes human semaphorin 7A (SEMA7A) whose translation is upregulated by the Erk/p90S6K pathway in human eosinophils, with a potential negative impact in asthma and airway inflammation. In the current study, we aimed to find a common 5'UTR regulatory cis-element, and determine its impact on protein synthesis. We identified a common and conserved 5'UTR motif GGCTG-[(C/G)T(C/G)]n-GCC that was present in this group of mRNAs. Mutations of the first two GG bases in this motif in SEMA7A 5'UTR led to a complete loss of S6K activity dependence for maximal translation. In conclusion, the newly identified 5'UTR motif present in SEMA7A has a critical role in regulating S6K-dependent protein synthesis.
Asunto(s)
Biosíntesis de Proteínas , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 5' , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , MutaciónRESUMEN
Hepatocellular carcinoma (HCC) is a symptomatic disease involed multi-stage program. Here, we elucidated the molecular mechanism of LncTUG1 in the regulation of HCC evolvement. And that may in all likelyhood supply a innovative latent target for HCC's diagnoses and prognosis. LncRNA TUG1, miR-144-3p, RRAGD and mTOR signaling pathway were screened as target genes in the database, and their expression levels at the cytological level were verified utilized qRT-PCR, Western Blot and immunohistochemistry. Then, we adopted CCK-8, Transwell and flow cytometry assays to estimate cell proliferation, invasion and apoptosis. By use of luciferase reporter assay, the relationships of LncRNA TUG1, miR-144-3p and RRAGD was confirmed. In addition, the LncRNA TUG1-miR-144-3p-RRAGD-mTOR signaling pathway in HCC cells was verified adopted rescue experiment and confirmed by xenotransplantation animal experiment. LncTUG1 in HCC tissues from three databases were identified and further verified through qRT-PCR in HCC cells (Huh7, Hep3B). Knockdown the LncTUG1 could increase apoptosis and inhibite invasion and proliferation in HCC cells. Using inhibitors and activators of the mTOR/S6K pathway, LncTUG1 was confirmed to regulate HCC progression by the mTOR/S6K pathway. Luciferase reporter assay demonstrated that TUG1 negatively regulates miR-144-3p. Furthermore, miR-144-3p negativly regulates RRAGD by way of interacting with the 3'UTR of the RRAGD mRNA in HCC utilized luciferase reporter assay. In vivo, we also discovered that neoplasm weight and tumor volume reduced significantly in subcutaneous xenograft nude mouse models derived from sh-LncTUG1-expressing Huh7 cells. And the expressions of p-mTOR, p-S6K and RRAGD were decreased obviously while the miR144-3p increased in subcutaneous xenograft nude mouse models. In a word, the research suggests that LncTUG1 targets miR-144-3p while miR-144-3p binds to RRAGD mRNA, which induces mTOR/S6K pathway activation and promotes the progression of HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Transducción de Señal , Línea Celular Tumoral , Humanos , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas Quinasas S6 Ribosómicas/metabolismo , MicroARNs/genética , Animales , Ratones , Ratones Endogámicos BALB C , Progresión de la Enfermedad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ribosomal S6 kinases (S6Ks) are critical regulators of cell growth, homeostasis, and survival, with dysregulation of these kinases found to be associated with various malignancies. While S6K1 has been extensively studied, S6K2 has been neglected despite its clear involvement in cancer progression. Protein arginine methylation is a widespread post-translational modification regulating many biological processes in mammalian cells. Here, we report that p54-S6K2 is asymmetrically dimethylated at Arg-475 and Arg-477, two residues conserved amongst mammalian S6K2s and several AT-hook-containing proteins. We demonstrate that this methylation event results from the association of S6K2 with the methyltransferases PRMT1, PRMT3, and PRMT6 in vitro and in vivo and leads to nuclear the localisation of S6K2 that is essential to the pro-survival effects of this kinase to starvation-induced cell death. Taken together, our findings highlight a novel post-translational modification regulating the function of p54-S6K2 that may be particularly relevant to cancer progression where general Arg-methylation is often elevated.
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Fenómenos Biológicos , Proteínas Quinasas S6 Ribosómicas 90-kDa , Animales , Fosforilación , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Mamíferos/metabolismoRESUMEN
Why do adult mammalian central nervous system axons not regenerate, when peripheral axons do? Two studies in PLOS Biology point to the role of 2 related ribosomal S6 kinase family members in the differences in regeneration capacity between central and peripheral axons.
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Sistema Nervioso Central , Regeneración Nerviosa , Animales , Proteínas Quinasas S6 Ribosómicas , Regeneración Nerviosa/fisiología , Axones/fisiología , MamíferosRESUMEN
This study aimed to investigate whether the developmental changes in glucose metabolism were associated with insulin signaling in the middle and later stages of goose embryos. Serum and liver were sampled on embryonic day 19, 22, 25, 28, and day of hatchment, with 30 eggs at each sampling time point, and 6 replicates of 5 embryos. The embryonic growth traits, serum glucose, hormone levels, and the hepatic mRNA expressions of target genes related to glucose metabolism and insulin signaling were measured at each time point. Relative body weight, relative liver weight, and relative body length decreased linearly and quadratically from embryonic day 19 to day of hatchment, while relative yolk weight decreased linearly from embryonic day 19 to day of hatchment. Serum glucose, insulin, and free triiodothyronine levels increased linearly with increasing incubation time, while no differences were observed in serum glucagon and free thyroxine levels. The hepatic mRNA expression related to glucose catabolism (hexokinase, phosphofructokinase, and pyruvate kinase) and insulin signaling (insulin receptor, insulin receptor substrate protein, Src homology collagen protein, extracellular signal-regulated kinase, and ribosomal protein S6 kinase, 70 ku) increased quadratically from embryonic day 19 to day of hatchment. The expression of citrate synthase and isocitrate dehydrogenase mRNA decreased linearly and quadratically respectively from embryonic day 19 to day of hatchment. Serum glucose levels were positively related to serum insulin (r = 1.00) and free triiodothyronine (r = 0.90) levels, as well as the hepatic mRNA expression of insulin receptor (r = 1.00), insulin receptor substrate protein (r = 0.64), extracellular signal-regulated kinase (r = 0.81), and ribosomal protein S6 kinase, 70 ku (r = 0.81) related to insulin signaling. In conclusion, glucose catabolism was enhanced and had positive correlations with the insulin signaling in the middle and later stages of geese embryogenesis.
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Gansos , Insulina , Animales , Insulina/metabolismo , Gansos/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Triyodotironina , Proteínas Sustrato del Receptor de Insulina/metabolismo , Pollos/genética , Óvulo/metabolismo , Hígado/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Glucosa/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , ARN Mensajero/genéticaRESUMEN
OBJECTIVE: Intrauterine growth restriction followed by postnatal catch-up growth (CG-IUGR) increases the risk of insulin resistance-related diseases. Low-density lipoprotein receptor-related protein 6 (LRP6) plays a substantial role in glucose metabolism. However, whether LRP6 is involved in the insulin resistance of CG-IUGR is unclear. This study aimed to explore the role of LRP6 in insulin signaling in response to CG-IUGR. METHODS: The CG-IUGR rat model was established via a maternal gestational nutritional restriction followed by postnatal litter size reduction. The mRNA and protein expression of the components in the insulin pathway, LRP6/ß-catenin and mammalian target of rapamycin (mTOR)/S6 kinase (S6K) signaling, was determined. Liver tissues were immunostained for the expression of LRP6 and ß-catenin. LRP6 was overexpressed or silenced in primary hepatocytes to explore its role in insulin signaling. RESULTS: Compared with the control rats, CG-IUGR rats showed higher homeostasis model assessment for insulin resistance (HOMA-IR) index and fasting insulin level, decreased insulin signaling, reduced mTOR/S6K/ insulin receptor substrate-1 (IRS-1) serine307 activity, and decreased LRP6/ß-catenin in the liver tissue. The knockdown of LRP6 in hepatocytes from appropriate-for-gestational-age (AGA) rats led to reductions in insulin receptor (IR) signaling and mTOR/S6K/IRS-1 serine307 activity. In contrast, LRP6 overexpression in hepatocytes of CG-IUGR rats resulted in elevated IR signaling and mTOR/S6K/IRS-1 serine307 activity. CONCLUSION: LRP6 regulated the insulin signaling in the CG-IUGR rats via two distinct pathways, IR and mTOR-S6K signaling. LRP6 may be a potential therapeutic target for insulin resistance in CG-IUGR individuals.
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Retardo del Crecimiento Fetal , Resistencia a la Insulina , Insulina , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Proteínas Quinasas S6 Ribosómicas , Animales , Femenino , Humanos , Ratas , beta Catenina/genética , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Receptor de Insulina/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The principal mechanism underlying the reduced rate of protein synthesis in atrophied skeletal muscle is largely unknown. Eukaryotic elongation factor 2 kinase (eEF2k) impairs the ability of eukaryotic translation elongation factor 2 (eEF2) to bind to the ribosome via T56 phosphorylation. Perturbations in the eEF2k/eEF2 pathway during various stages of disuse muscle atrophy have been investigated utilizing a rat hind limb suspension (HS) model. Two distinct components of eEF2k/eEF2 pathway misregulation were demonstrated, observing a significant (P < 0.01) increase in eEF2k mRNA expression as early as 1-day HS and in eEF2k protein level after 3-day HS. We set out to determine whether eEF2k activation is a Ca2+-dependent process with involvement of Cav1.1. The ratio of T56-phosphorylated/total eEF2 was robustly elevated after 3-day HS, which was completely reversed by 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and decreased by 1.7-fold (P < 0.05) by nifedipine. Transfection of C2C12 with cytomegalovirus promoter (pCMV)-eEF2k and administration with small molecules were used to modulate eEF2k and eEF2 activity. More importantly, pharmacologic enhancement of eEF2 phosphorylation induced phosphorylated ribosomal protein S6 kinase (T389) up-regulation and restoration of global protein synthesis in the HS rats. Taken together, the eEF2k/eEF2 pathway was up-regulated during disuse muscle atrophy involving calcium-dependent activation of eEF2k partly via Cav1.1. The study provides evidence, in vitro and in vivo, of the eEF2k/eEF2 pathway impact on ribosomal protein S6 kinase activity as well as protein expression of key atrophy biomarkers, muscle atrophy F-box/atrogin-1 and muscle RING finger-1.
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Quinasa del Factor 2 de Elongación , Músculo Esquelético , Ratas , Animales , Quinasa del Factor 2 de Elongación/genética , Quinasa del Factor 2 de Elongación/metabolismo , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Fosforilación , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismoRESUMEN
BACKGROUND: Oncostatin M produced by osteal macrophages, a cytokine that belongs to the interleukin-6 family, is implicated in bone fracture healing. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts plays an important role in osteoclastogenesis. We have previously reported that tumor necrosis factor-α (TNF-α), a potent bone resorptive agent, stimulates the activation of p44/p42 mitogen-activated protein (MAP) kinase, Akt, and p70 S6 kinase in osteoblast-like MC3T3-E1 cells, and induces the synthesis of M-CSF at least in part via Akt. OBJECTIVE: In the present study, we investigated whether oncostatin M affects the TNF-α-induced M-CSF synthesis in MC3T3-E1 cells and the underlying mechanisms. METHODS: Clonal osteoblast-like MC3T3-E1 cells were treated with oncostatin M or rapamycin and then stimulated with TNF-α. M-CSF release was assessed by ELISA. M-CSF mRNA expression level was assessed by real-time RT-PCR. Phosphorylation of Akt, p44/p42 MAP kinase, and p70 S6 kinase was detected by Western blot analysis. RESULTS: Oncostatin M dose-dependently reduced the TNF-α-stimulated M-CSF release. The expression of M-CSF mRNA induced by TNF-α was significantly suppressed by oncostatin M. Rapamycin, an inhibitor of mTOR/p70 S6 kinase, had little effect on the M-CSF release by TNF-α. Oncostatin M significantly reduced the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. However, the p70 S6 kinase phosphorylation by TNF-α was not affected by oncostatin M. CONCLUSION: These results strongly suggest that oncostatin M attenuates TNF-α-stimulated synthesis of M-CSF in osteoblasts, and the inhibitory effect is exerted at a point upstream of Akt and p44/p42 MAP kinase but not p70 S6 kinase.
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Factor Estimulante de Colonias de Macrófagos , Factor de Necrosis Tumoral alfa , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/farmacología , Oncostatina M/farmacología , Oncostatina M/metabolismo , Fosforilación , Sirolimus/farmacología , Osteoblastos/metabolismo , ARN Mensajero/metabolismo , Macrófagos/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas Quinasas S6 Ribosómicas/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Leucine (Leu), an essential amino acid, is known to stimulate protein synthesis in the skeletal muscle via mTOR complex 1 (mTORC1) activation. However, the intrinsic contribution of other amino acids to Leu-mediated activation of mTORC1 signaling remains unexplored. This study aimed to identify amino acids that can promote mTORC1 activity in combination with Leu and to assess the effectiveness of these combinations in vitro and in vivo. We found that tyrosine (Tyr) enhanced Leu-induced phosphorylation of S6 kinase (S6K), an indicator of mTORC1 activity, although it exerted no such effect individually. This booster effect was observed in C2C12 cells, isolated murine muscle, and the skeletal muscles of mice orally administered the amino acids. To explore the molecular mechanisms underlying this Tyr-mediated booster effect, the expression of the intracellular Leu sensors, Sestrin1 and 2, was suppressed, and the cells were treated with Leu and Tyr. This suppression enabled Tyr alone to induce S6K phosphorylation and enhanced the booster effect, suggesting that Tyr possibly contributes to mTORC1 activation when Sestrin-GAP activity toward Rags 2 (GATOR2) is dissociated through Sestrin knockdown or the binding of Sestrins to Leu. Collectively, these results indicate that Tyr is a key regulator of Leu-mediated protein synthesis.
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Aminoácidos , Tirosina , Animales , Ratones , Leucina/farmacología , Músculo Esquelético , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas Quinasas S6 RibosómicasRESUMEN
Ribosomal protein S6 kinase 1 (S6K1), a key downstream effector of the mammalian target of rapamycin (mTOR), regulates diverse functions, such as cell proliferation, cell growth, and protein synthesis. Because S6K1 was previously known to be localized in the cytoplasm, its function has been mainly studied in the cytoplasm. However, the nuclear localization and function of S6K1 have recently been elucidated and other nuclear functions are expected to exist but remain elusive. Here, we show a novel nuclear role of S6K1 in regulating the expression of the Wnt target genes. Upon activation of the Wnt signaling, S6K1 translocated from the cytosol into the nucleus and subsequently bound to ß-catenin and the cofactors of the Wnt/ß-catenin transcriptional complex, leading to the upregulation of the Wnt target genes. The depletion or repression of S6K1 downregulated the Wnt target gene expression by inhibiting the formation of the Wnt/ß-catenin transcriptional complex. The S6K1-depleted colon cancer cell lines showed lower transcription levels of the Wnt/ß-catenin target genes and a decrease in the cell proliferation and invasion compared to the control cell lines. Taken together, these results indicate that nuclear S6K1 positively regulates the expression of the Wnt target genes by inducing the reciprocal interaction of the subunits of the transcriptional complex.