RESUMEN
Beyond the antimicrobial activity, doxycycline (DOX) exhibits longevity-promoting effect in nematodes, while its effect on mammals is unclear. Here, we applied a mouse model of Hutchinson-Gilford progeria syndrome (HGPS), Zmpste24 knockout (KO) mice, and analyzed the antiaging effect of DOX. We found that the DOX treatment prolongs lifespan and ameliorates progeroid features of Zmpste24 KO mice, including the decline of body and tissue weight, exercise capacity and cortical bone density, and the shortened colon length. DOX treatment alleviates the abnormal nuclear envelope in multiple tissues, and attenuates cellular senescence and cell death of Zmpste24 KO and HGPS fibroblasts. DOX downregulates the level of proinflammatory IL6 in both serum and tissues. Moreover, the elevated α-tubulin (K40) acetylation mediated by NAT10 in progeria, is rescued by DOX treatment in the aorta tissues in Zmpste24 KO mice and fibroblasts. Collectively, our study uncovers that DOX can decelerate aging in progeria mice via counteracting IL6 expression and NAT10-mediated acetylation of α-tubulin.
Asunto(s)
Envejecimiento , Doxiciclina , Ratones Noqueados , Progeria , Animales , Progeria/tratamiento farmacológico , Progeria/metabolismo , Progeria/patología , Ratones , Envejecimiento/efectos de los fármacos , Doxiciclina/farmacología , Metaloendopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Senescencia Celular/efectos de los fármacosRESUMEN
AIMS: Pulmonary hypertension (PH) is a pulmonary vascular disease characterized by a high mortality rate. Pulmonary arterial endothelium cells (PAECs) serve as a primary sensor of various environmental cues, such as shear stress and hypoxia, but PAEC dysfunction may trigger vascular remodelling during the onset of PH. This study aimed to illustrate the role of Sirtuin 7 (SIRT7) in endothelial dysfunction during PH and explore the potential therapeutic strategy for PH. METHODS AND RESULTS: SIRT7 levels were measured in human and murine experimental PH samples. Bioinformatic analysis, immunoprecipitation, and deacetylation assay were used to identify the association between SIRT7 and Krüpple-like factor 4 (KLF4), a key transcription factor essential for endothelial cell (EC) homeostasis. Sugen5416 + hypoxia (SuHx)-induced PH mouse models and cell cultures were used for the study of the therapeutic effect of SIRT7 for PH. SIRT7 level was significantly reduced in lung tissues and PAECs from PH patients and the SuHx-induced PH mouse model as compared with healthy controls. Pulmonary endothelium-specific depletion of Sirt7 increased right ventricular systolic pressure and exacerbated right ventricular hypertrophy in the SuHx-induced PH model. At the molecular level, we identified KLF4 as a downstream target of SIRT7, which deacetylated KLF4 at K228 and inhibited the ubiquitination-proteasome degradation. Thus, the SIRT7/KLF4 axis maintained PAEC homeostasis by regulating proliferation, migration, and tube formation. PAEC dysfunction was reversed by adeno-associated virus type 1 vector-mediated endothelial overexpression of Sirt7 or supplementation with nicotinamide adenine dinucleotide (NAD)+ intermediate nicotinamide riboside which activated Sirt7; both approaches successfully reversed PH phenotypes. CONCLUSION: The SIRT7/KLF4 axis ensures PAEC homeostasis, and pulmonary endothelium-specific SIRT7 targeting might constitute a PH therapeutic strategy.
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Hipertensión Pulmonar , Sirtuinas , Animales , Humanos , Ratones , Endotelio Vascular/metabolismo , Hipoxia/metabolismo , Pulmón/metabolismo , Arteria Pulmonar , Sirtuinas/genética , Sirtuinas/metabolismoRESUMEN
Age-associated hypercoagulability is accompanied by the increase of plasma levels of some coagulation factors including fibrinogen which may contribute to the increased risk of cardiovascular, cerebrovascular, and thrombotic diseases in elderly people. However, the underlying mechanism of increased plasma fibrinogen concentration during aging is still elusive. GRSF1 belongs to the heterogeneous nuclear ribonucleoproteins F/H (hnRNP F/H) subfamily. Here, we report that GRSF1 attenuates hypercoagulability via negative modulation of fibrinogen expression. We demonstrated that GRSF1 negatively regulated fibrinogen expression at both mRNA and protein levels. GRSF1 directly interacted with the coding region (CDS) of FGA, FGB, and FGG mRNAs, and decreased their stability thus mitigating fibrinogen expression. We further identified that only a few G-tracts within the Fib C domain of FGA, FGB, and FGG CDS and the qRRM2 domain of GRSF1 were required for their interaction. Moreover, we confirmed hypercoagulability and the decrease of GRSF1 expression level during mice aging. Functionally, GRSF1 overexpression in old mice liver decreased fibrinogen plasma level, reduced hypercoagulability, and mitigated blood coagulation activity, whereas GRSF1 knockdown in young mice liver increased fibrinogen plasma level and promoted blood coagulation activity. Collectively, our findings unveil a novel posttranscriptional regulation of fibrinogen by GRSF1 and uncover a critical role of GRSF1 in regulating blood coagulation activity.
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Fibrinógeno , Trombofilia , Anciano , Animales , Humanos , Ratones , Fibrinógeno/genética , Fibrinógeno/metabolismo , Regulación de la Expresión Génica , Proteínas de Unión a Poli(A)/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Cutaneous wound healing, an integral part for protection of skin barrier, is a complex biological process and intimately associated with keratinocyte migration. However, mechanisms regulating keratinocyte migration in the process of cutaneous wound repair remain largely unknown. Here, we found that N-acetyltransferase 10 (NAT10) is essential for cutaneous wound repair in an in vivo skin wound healing model-a significant delay of wound repair in Nat10 haploinsufficient mice and a remarkable inhibition of keratinocyte migration by NAT10 knockdown in an in vitro keratinocyte migration model. We further demonstrate that loss of NAT10 expression attenuates the wound-induced IL-6/IL-8 expression through inhibiting NF-κB/p65 activity in keratinocytes. By deeply digging, silencing NAT10 compromises the level of nuclear p65 by facilitating its poly-ubiquitination, thus accelerates its degradation in the nucleus. Notably, we detected a strong positive correlation between the expression of NAT10 and relevant NF-kB/p65-IL6 signaling activity in mouse wound skin tissues. Overall, our study reveals an important role of NAT10 on cutaneous wound repair by potentiating NF-κB/p65-IL-6/8-STAT3 signaling. Targeting NAT10 might be a potential strategy for the treatment of skin wound dysfunctions and related diseases.
RESUMEN
The progressive decline of physiological function and the increased risk of age-related diseases challenge healthy aging. Multiple anti-aging manipulations, such as senolytics, have proven beneficial for health; however, the biomarkers that label in vivo senescence at systemic levels are lacking, thus hindering anti-aging applications. In this study, we generate a Glb1+/mâGlb1-2A-mCherry (GAC) reporter allele at the Glb1 gene locus, which encodes lysosomal ß-galactosidase-an enzyme elevated in tissues of old mice. A linear correlation between GAC signal and chronological age is established in a cohort of middle-aged (9 to 13 months) Glb1+/m mice. The high GAC signal is closely associated with cardiac hypertrophy and a shortened lifespan. Moreover, the GAC signal is exponentially increased in pathological senescence induced by bleomycin in the lung. Senolytic dasatinib and quercetin (D + Q) reduce GAC signal in bleomycin treated mice. Thus, the Glb1-2A-mCherry reporter mice monitors systemic aging and function decline, predicts lifespan, and may facilitate the understanding of aging mechanisms and help in the development of anti-aging interventions.
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Senescencia Celular , Longevidad , Animales , Ratones , Envejecimiento/genética , Bleomicina , Dasatinib/farmacología , Longevidad/genética , Genes Reporteros , Glicósido HidrolasasRESUMEN
The histone acetyltransferase (HAT) KAT7/HBO1/MYST2 plays a crucial role in the pre-replication complex (pre-RC) formation, DNA replication and cell proliferation via acetylation of histone H4 and H3. In a search for protein kinase D1 (PKD1)-interacting proteins, we have identified KAT7 as a potential PKD1 substrate. We show that PKD1 directly interacts and phosphorylates KAT7 at Thr97 and Thr331 in vitro and in vivo. PKD1-mediated phosphorylation of KAT7 enhances its expression levels and stability by reducing its ubiquitination-mediated degradation. Significantly, the phospho-defective mutant KAT7-Thr97/331A attenuates histone H4 acetylation levels, MCM2/6 loading on the chromatin, DNA replication and cell proliferation. Similarly, PKD1 knockdown decreases, whereas the constitutive active mutant PKD1-CA increases histone H4 acetylation levels and MCM2/6 loading on the chromatin. Overall, these results suggest that PKD1-mediated phosphorylation of KAT7 may be required for pre-RC formation and DNA replication.
RESUMEN
Human telomerase RNA component hTERC comprises multiple motifs that contribute to hTERC biogenesis, holoenzyme activity, and enzyme recruitment to telomeres. hTERC contains several guanine tracts (G-tracts) at its 5'-end, but its associated proteins and potential roles in telomerase function are still poorly understood. The heterogeneous nuclear ribonucleoproteins F, H1, and H2 (hnRNP F/H) are splicing factors that preferentially bind to poly(G)-rich sequences RNA. Here, we demonstrate that hnRNP F/H associate with both hTERC and telomerase holoenzyme to regulate telomerase activity. We reveal hnRNP F/H bind to the 5'-end region of hTERC in vitro and in vivo, and identify the first three G-tracts of hTERC and qRRM1 domain of hnRNP F/H are required for their interaction. Furthermore, hnRNP F/H also directly interact with telomerase holoenzyme. Functionally, we show that hnRNP F/H plays important roles in modulating telomerase activity and telomere length. Moreover, hnRNP F/H deletion greatly impair cancer and stem cell proliferation, and induce stem cell senescence, while hnRNP F/H overexpression delay stem cell senescence. Collectively, our findings unveil a novel role of hnRNP F/H as the binding partners of hTERC and telomerase holoenzyme to regulate telomerase function.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , ARN/metabolismo , Telomerasa/metabolismo , Sitios de Unión , Proliferación Celular , Células HEK293 , Células HeLa , Humanos , Unión ProteicaRESUMEN
The activation of transcription factor NF-κB is currently identified as one of the driving forces to the aging process. Genetic impairment of NF-κB signaling pathway or pharmacological inhibition of NF-κB activity has been shown to extend healthspan and lifespan in animal models, and delay or reduce many age-related symptoms. However, the aging intervention strategies based on NF-κB inhibition by the suitable small molecular compound is currently still lacking. The water-soluble dimethylaminomicheliolide (DMAMCL), can inhibit NF-κB activity and is currently undergoing clinical trials. In this study, we showed that 15 months of DMAMCL administration started in 1-year old male mice was well-tolerated and safe, and improved or had little effect on some age-associated symptoms, such as neurobehavioral phenotypes, physical performance, cardiac function, hematological parameters, immune aging phenotypes, clinical chemistry parameters, and glucose homeostasis. At the molecular level, DMAMCL administration mitigated serum levels of several age-associated inflammatory cytokines, including IL-6, IL-1α, IL-1ß, TNF-α, IFN-γ, and CXCL2, and inhibited NF-κB activity in several aged tissues. Collectively, our results indicate that current strategy of DMAMCL administration may has little effect on aging process in mice, and provide basic clues to further exploit the possibility of DMAMCL-based aging intervention to promote healthy aging.
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Envejecimiento/efectos de los fármacos , Sesquiterpenos de Guayano/farmacología , Animales , Citocinas/genética , Citocinas/metabolismo , Esquema de Medicación , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Fenómenos Fisiológicos Musculoesqueléticos , Sesquiterpenos de Guayano/administración & dosificaciónRESUMEN
Post-translational modifications of the histone H2A represent an important mechanism by which cells modulate the structure and function of chromatin. Ubiquitination at K119 of histone H2A is associated transcriptional repression, which is shown to be regulated by deubiquitinases (DUBs). Here, we performed a screen to identify novel DUBs for histone H2A. Although RNAi-mediated knockdown of USP28, USP32 and USP36 showed that their depletion resulted in the increase of ub-K119-H2A, only USP28-depleted cells showed increased cell proliferation. Notably, USP28 knockdown cells had decreased expression of p53, p21 and p16INK4a, suggesting that the effect of USP28 on cell proliferation was mediated by regulating the expression of p53, p21 and p16INK4a. In summary, we have shown that USP28 is a deubiquitinase for histone H2A and is involved in regulation of cell proliferation. Thus, USP28 represents a potentially novel therapeutic target for cancer.
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Proliferación Celular/genética , Histonas/genética , Ubiquitina Tiolesterasa/genética , Ubiquitinación/genética , Línea Celular , Cromatina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Células HEK293 , Humanos , Procesamiento Proteico-Postraduccional/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Senescent cells display the senescence-associated secretory phenotype (SASP) which plays important roles in cancer, aging, etc. Cell surface-bound IL-1α is a crucial SASP factor and acts as an upstream regulator to induce NF-κB activity and subsequent SASP genes transcription. IL-1α exports to cell surface via S100A13 protein-dependent non-classical secretory pathway. However, the status of this secretory pathway during cellular senescence and its role in cellular senescence remain unknown. Here, we show that S100A13 is up-regulated in various types of cellular senescence. S100A13 overexpression increases cell surface-associated IL-1α level, NF-κB activity and subsequent multiple SASP genes induction, whereas S100A13 knockdown has an opposite role. We also exhibit that Cu2+ level is elevated during cellular senescence. Lowering Cu2+ level decreases cell surface-bound IL-1α level, NF-κB activity and SASP production. Moreover, S100A13 overexpression promotes oncogene Ras-induced cell senescence (Ras OIS), Doxorubicin-induced cancer cell senescence (TIS) and replicative senescence, while impairment of non-classical secretory pathway of IL-1α delays cellular senescence. In addition, intervention of S100A13 affects multiple SASP and cellular senescence mediators including p38, γ-H2AX, and mTORC1. Taken together, our findings unveil a critical role of the non-classical secretory pathway of IL-1α in cellular senescence and SASP regulation.
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Senescencia Celular , Regulación de la Expresión Génica/fisiología , Interleucina-1alfa/metabolismo , Proteínas S100/metabolismo , Cobre/metabolismo , Células HCT116 , Humanos , Interleucina-1alfa/genética , FN-kappa B/metabolismo , Proteínas S100/genética , TransfecciónRESUMEN
The nucleolus is an important organelle that is responsible for the biogenesis of ribosome RNA (rRNA) and ribosomal subunits assembly. It is also deemed to be the center of metabolic control, considering the critical role of ribosomes in protein translation. Perturbations of rRNA synthesis are closely related to cell proliferation and tumor progression. Telomeric repeat-binding factor 2 (TRF2) is a member of shelterin complex that is responsible for telomere DNA protection. Interestingly, it was recently reported to localize in the nucleolus of human cells in a cell-cycle-dependent manner, while the underlying mechanism and its role on the nucleolus remained unclear. In this study, we found that nucleolar and coiled-body phosphoprotein 1 (NOLC1), a nucleolar protein that is responsible for the nucleolus construction and rRNA synthesis, interacted with TRF2 and mediated the shuttle of TRF2 between the nucleolus and nucleus. Abating the expression of NOLC1 decreased the nucleolar-resident TRF2. Besides, the nucleolar TRF2 could bind rDNA and promoted rRNA transcription. Furthermore, in hepatocellular carcinoma (HCC) cell lines HepG2 and SMMC7721, TRF2 overexpression participated in the nucleolus stress-induced rRNA inhibition and cell-cycle arrest.
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Antineoplásicos/farmacología , Nucléolo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/genética , Fosfoproteínas/genética , ARN Ribosómico/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Secuencia de Aminoácidos , Camptotecina/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestructura , Proliferación Celular/efectos de los fármacos , Dactinomicina/farmacología , Etopósido/farmacología , Células HEK293 , Células Hep G2 , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Unión Proteica , Transporte de Proteínas , ARN Ribosómico/biosíntesis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/genética , Ribosomas/metabolismo , Transducción de Señal , Proteína 2 de Unión a Repeticiones Teloméricas/antagonistas & inhibidores , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
Senescent cells develop a senescence-associated secretory phenotype (SASP). The factors secreted by cells with a SASP have multiple biological functions that are mediated in an autocrine or paracrine manner. However, the status of the protein kinase D1 (PKD1; also known as PRKD1)-mediated classical protein secretory pathway, from the trans-Golgi network (TGN) to the cell surface, during cellular senescence and its role in the cellular senescence response remain unknown. Here, we show that the activities or quantities of critical components of this pathway, including PKD1, ADP-ribosylation factor 1 (ARF1) and phosphatidylinositol 4-kinase IIIß (PI4KIIIß), at the TGN are increased in senescent cells. Blocking of this pathway decreases IL-6 and IL-8 (hereafter IL-6/IL-8) secretion and results in IL-6/IL-8 accumulation in SASP-competent senescent cells. Inhibition of this pathway reduces IL-6/IL-8 secretion during Ras oncogene-induced senescence (OIS), retards Ras OIS and alleviates its associated ER stress and autophagy. Finally, targeting of this pathway triggers cell death in SASP factor-producing senescent cells due to the intracellular accumulation of massive amounts of IL-6/IL-8. Taken together, our results unveil the hyperactive state of the protein secretory pathway in SASP-competent senescent cells and its critical functions in mediating SASP factor secretion and the Ras OIS process, as well as in determining the fate of senescent cells.
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Senescencia Celular , Fibroblastos/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Proteína Quinasa C/metabolismo , Vías Secretoras , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Fibroblastos/citología , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Proteína Oncogénica p21(ras)/genética , Proteína Quinasa C/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
Sirtuin6(SIRT6) has been implicated as a key factor in aging and aging-related diseases. However, the role of SIRT6 in cellular senescence has not been fully understood. Here, we show that SIRT6 repressed the expression of p27Kip1 (p27) in cellular senescence. The expression of SIRT6 was reduced during cellular senescence, whereas enforced SIRT6 expression promoted cell proliferation and antagonized cellular senescence. In addition, we demonstrated that SIRT6 promoted p27 degradation by proteasome and SIRT6 decreased the acetylation level and protein half-life of p27. p27 acetylation increased its protein stability. Furthermore, SIRT6 directly interacted with p27. Importantly, p27 was strongly acetylated and had a prolonged protein half-life with the reduction of SIRT6 when cells were senescent, compared with those young cells. Finally, SIRT6 markedly rescued senescence induced by p27. Our findings indicate that SIRT6 decreases p27 acetylation, leading to its degradation via ubiquitin-proteasome pathway and then delays cellular senescence.
Asunto(s)
Proliferación Celular/fisiología , Senescencia Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Sirtuinas/metabolismo , Acetilación , Línea Celular , Humanos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Senescent cells display a senescence-associated secretory phenotype (SASP) which contributes to tumor suppression, aging, and cancer. However, the underlying mechanisms for SASP regulation are not fully elucidated. SIRT1, a nicotinamide adenosine dinucleotide-dependent deacetylase, plays multiple roles in metabolism, inflammatory response, and longevity, etc. However, its posttranscriptional regulation and its roles in cellular senescence and SASP regulation are still elusive. Here, we identify the RNA-binding protein hnRNP A1 as a posttranscriptional regulator of SIRT1, as well as cell senescence and SASP regulator. hnRNP A1 directly interacts with the 3' untranslated region of SIRT1 mRNA, promotes its stability, and increases SIRT1 expression. hnRNP A1 delays replicative cellular senescence and prevents from Ras OIS via upregulation of SIRT1 expression to deacetylate NF-κB, thus blunting its transcriptional activity and subsequent IL-6/IL-8 induction. hnRNP A1 overexpression promotes cell transformation and tumorigenesis in a SIRT1-dependent manner. Together, our findings unveil a novel posttranscriptional regulation of SIRT1 by hnRNP A1 and uncover a critical role of hnRNP A1-SIRT1-NF-κB pathway in regulating cellular senescence and SASP expression.