RESUMEN
Crustacean molting is highly related to energy and lipid metabolism. This study was conducted to detect the changes of total lipids (TL), triacylglyceride (TAG), phospholipid (PL) and lipid droplets in hepatopancreas, and then to investigate the gene expression patterns related to hepatopancreatic lipid metabolism during the molting cycle of Chinese mitten crab Eriocheir sinensis. Hepatopancreatic TL and TAG increased significantly from post-molt stage to pre-molt stage, then decreased significantly from pre-molt stage to ecdysis stage, which is consistent to the changes of neutral lipid-rich adipocytes in hepatopancreas. By transcriptomic analysis, 65,325 transcripts were sequenced and assembled, and 28,033 transcripts were annotated. Most genes were related to energy metabolism, and the enriched genes were involved in carbohydrate and lipid metabolism and biosynthesis, especially in de novo synthesis of fatty acids and TAG, and ketone body production. Compared to the inter-molt stages, acetyl-CoA carboxylase, fatty acid synthase and other genes related to the synthesis of fatty acids were upregulated in the pre-molt stage. TAG synthesis related genes, including Glycerol-3-phosphate acyltransferase and 1-acylglycerol-3-phosphate acyltransferases, were upregulated in the post-molt stage compared to the inter-molt stage. The expression of ketone body-related genes had no significant changes during the molting cycle. Compared to the TAG synthetic pathway, ketone body biosynthesis may contribute less/secondarily to fatty acid metabolic processes, which could be involved in the other physiological processes or metabolism. In conclusion, these results showed that TAG is the major lipid deposition during inter- and pre-molt stages, and the most genes are related to the fatty acids and TAG metabolism in the hepatopancreas during the molting cycle of E. sinensis.
Asunto(s)
Braquiuros , Transcriptoma , Animales , Muda/genética , Metabolismo de los Lípidos/genética , Ácidos Grasos/metabolismo , Fosfatos/metabolismo , Cetonas/metabolismo , Braquiuros/genética , Hepatopáncreas/metabolismoRESUMEN
Secretory-pathway Ca2+-ATPases (SPCAs) play critical roles in maintaining Ca2+ homeostasis, but the exact mechanism of SPCAs-mediated Ca2+ transport remains unclear. Here, we determined six cryo-electron microscopy (cryo-EM) structures of human SPCA1 (hSPCA1) in a series of intermediate states, revealing a near-complete conformational cycle. With the aid of molecular dynamics simulations, these structures offer a clear structural basis for Ca2+ entry and release in hSPCA1. We found that hSPCA1 undergoes unique conformational changes during ATP binding and phosphorylation compared to other well-studied P-type II ATPases. In addition, we observed a conformational distortion of the Ca2+-binding site induced by the separation of transmembrane helices 4L and 6, unveiling a distinct Ca2+ release mechanism. Particularly, we determined a structure of the long-sought CaE2P state of P-type IIA ATPases, providing valuable insights into the Ca2+ transport cycle. Together, these findings enhance our understanding of Ca2+ transport by hSPCA1 and broaden our knowledge of P-type ATPases.
Asunto(s)
ATPasas Transportadoras de Calcio , Calcio , Humanos , Calcio/metabolismo , Microscopía por Crioelectrón , ATPasas Transportadoras de Calcio/metabolismo , Adenosina Trifosfatasas/metabolismoRESUMEN
Prolonged or excessive stimulation from inhaled toxins may cause oxidative stress and DNA damage that can lead to stress-induced senescence in epithelial cells, which can contribute to several airway diseases. Mounting evidence has shown carbon monoxide (CO) confers cytoprotective effects. We investigated the effects of CO on oxidative stress-induced senescence in human airway epithelium and elucidated the underlying molecular mechanisms. Here, CO pretreatment reduced H2O2-mediated increases in total reactive oxygen species (ROS) production and mitochondrial superoxide in a human bronchial epithelial cell line (BEAS-2B). H2O2 treatment triggered a premature senescence-like phenotype with enlarged and flattened cell morphology accompanied by increased SA-ß-gal activity, cell cycle arrest in G0/G1, reduced cell viability, and increased transcription of senescence-associated secretory phenotype (SASP) genes. Additionally, exposure to H2O2 increased protein levels of cellular senescence markers (p53 and p21), reduced Sirtuin 3 (SIRT3) and manganese superoxide dismutase (MnSOD) levels, and increased p53 K382 acetylation. These H2O2-mediated effects were attenuated by pretreatment with a CO-containing solution. SIRT3 silencing induced mitochondrial superoxide production and triggered a senescence-like phenotype, whereas overexpression decreased mitochondrial superoxide production and alleviated the senescence-like phenotype. Air-liquid interface (ALI) culture of primary human bronchial cells, which becomes a fully differentiated pseudostratified mucociliary epithelium, was used as a model. We found that apical and basolateral exposure to H2O2 induced a vacuolated structure that impaired the integrity of ALI cultures, increased goblet cell numbers, decreased SCGB1A1+ club cell numbers, increased p21 protein levels, and increased SASP gene transcription, consistent with our observations in BEAS-2B cells. These effects were attenuated in the apical presence of a CO-containing solution. In summary, we revealed that CO has a pivotal role in epithelial senescence by regulating ROS production via the SIRT3/MnSOD/p53/p21 pathway. This may have important implications in the prevention and treatment of age-associated respiratory pathologies.
Asunto(s)
Sirtuina 3 , Monóxido de Carbono/metabolismo , Senescencia Celular , Epitelio , Humanos , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 3/metabolismo , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVE: ß2-Adrenoceptor agonists are widely used to treat asthma because of their bronchial-dilation effects. We previously reported that isoprenaline, via the apical and basolateral ß2-adrenoceptor, induced Cl- secretion by activating cyclic AMP (cAMP)-dependent pathways in human bronchial epithelia. Despite these results, whether and how the ß2-adrenoceptor-mediated cAMP-dependent pathway contributes to pro-inflammatory cytokine release in human bronchial epithelia remains poorly understood. METHODS: We investigated ß2-adrenoceptor-mediated signaling pathways involved in the production of two pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, in 16HBE14o- human bronchial epithelia. The effects of isoprenaline or formoterol were assessed in the presence of protein kinase A (PKA), exchange protein directly activated by cAMP (EPAC), Src, and extracellular signal-regulated protein kinase (ERK)1/2 inhibitors. The involvement of ß-arrestin2 was examined using siRNA knockdown. RESULTS: Isoprenaline and formoterol (both ß2 agonists) induced IL-6, but not IL-8, release, which could be inhibited by ICI 118,551 (ß2 antagonist). The PKA-specific inhibitor, H89, partially inhibited IL-6 release. Another intracellular cAMP receptor, EPAC, was not involved in IL-6 release. Isoprenaline-mediated IL-6 secretion was attenuated by dasatinib, a Src inhibitor, and PD98059, an ERK1/2 inhibitor. Isoprenaline treatment also led to ERK1/2 phosphorylation. In addition, knockdown of ß-arrestin2 by siRNA specifically suppressed cytokine release when a high concentration of isoprenaline (1 mM) was used. CONCLUSION: Our results suggest that activation of the ß2-adrenoceptor in 16HBE14o- cells stimulated the PKA/Src/ERK1/2 and/or ß-arrestin2 signaling pathways, leading to IL-6 release. Therefore, our data reveal that ß2-adrenoceptor signaling plays a role in the immune regulation of human airway epithelia.
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Proteínas Quinasas Dependientes de AMP Cíclico , Interleucina-6 , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas , Transducción de Señal , Arrestina beta 2RESUMEN
We investigated the regulation of Cl- secretion by adrenoceptors in polarized 16HBE14o- human bronchial epithelial cells. Treatment with the nonselective ß adrenoceptor agonist isoprenaline stimulated an increase in short-circuit current (ISC ), which was inhibited by the ß adrenoceptor blocker propranolol. Treatment with procaterol, an agonist specific for the ß2 adrenoceptor subtype, stimulated a similar increase in ISC , which was inhibited by the ß2 adrenoceptor antagonist ICI 118551. Inhibitors of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated Cl- channel (CaCC), but not K+ channel blockers, were able to inhibit the increase in ISC . "Trimultaneous" recording of ISC and intracellular cyclic adenosine monophosphate (cAMP) and Ca2+ levels in 16HBE14o- epithelia confirmed that the ISC induced by isoprenaline or procaterol involved both cAMP and Ca2+ signaling. Our results demonstrate that ß2 adrenoceptors regulate Cl- secretion in the human airway epithelium by activating apical CFTRs and CaCCs via cAMP-dependent and intracellular Ca2+ -dependent mechanisms, respectively.
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Canales de Cloruro/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Mucosa Respiratoria/metabolismo , Transporte Biológico Activo , Bronquios/metabolismo , Línea Celular , Células Epiteliales/metabolismo , Humanos , Transporte Iónico/fisiología , Transducción de Señal/fisiologíaRESUMEN
Carbon monoxide (CO) is an anti-inflammatory gaseous molecule produced endogenously by heme oxygenases (HOs) HO-1 and HO-2. However, the mechanisms underlying the anti-inflammatory effects of CO in the human bronchial epithelium are still not fully understood. In this study, the cationic peptide poly-l-arginine (PLA) was utilized to induce bronchial epithelial damage and subsequent pro-inflammatory cytokine release in the human bronchial epithelial cell line 16HBE14o-. Expression of both HO-1 and HO-2 after PLA exposure was examined. The polarized secretion of two pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, was determined by ELISA. The anti-inflammatory effects of CO liberated from CO-releasing molecules (CORMs) were examined by both ELISA and western blot analysis. Our results indicate that PLA exposure leads to upregulation of HO-1 expression and p65 NF-κB phosphorylation, as well as IL-6 and IL-8 release. HO-1 induction by hemin or CORMs significantly suppressed IL-6 and IL-8 release. In addition, HO-1 knockdown further increased IL-6 and IL-8 release under basal and PLA-stimulated conditions. Our results thereby demonstrate that the HO-1/CO axis exerts significant anti-inflammatory activity during bronchial epithelial damage caused by cationic protein.
Asunto(s)
Antiinflamatorios/farmacología , Bronquios/inmunología , Monóxido de Carbono/farmacología , Hemo-Oxigenasa 1/inmunología , Péptidos/farmacología , Mucosa Respiratoria/inmunología , Línea Celular , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/inmunología , Hemo-Oxigenasa 1/genética , Humanos , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-8/genética , Interleucina-8/inmunologíaRESUMEN
The activity of the CDK inhibitor p21 is associated with diverse biological activities, including cell proliferation, senescence, and tumorigenesis. However, the mechanisms governing transcription of p21 need to be extensively studied. In this study, we demonstrate that the high-mobility group box-containing protein 1 (HBP1) transcription factor is a novel activator of p21 that works as part of a complex mechanism during senescence and tumorigenesis. We found that HBP1 activates the p21 gene through enhancing p53 stability by inhibiting Mdm2-mediated ubiquitination of p53, a well known positive regulator of p21. HBP1 was also found to enhance p21 transcription by inhibiting Wnt/ß-catenin signaling. We identified histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2, as a target of Wnt/ß-catenin signaling. HBP1-mediated repression of EZH2 through Wnt/ß-catenin signaling decreased the level of trimethylation of histone H3 at lysine 27 of overall and specific histone on the p21 promoter, resulting in p21 transactivation. Although intricate, the reciprocal partnership of HBP1 and p21 has exceptional importance. HBP1-mediated elevation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to both cellular senescence and tumor inhibition. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence and tumorigenesis with an impact on protein ubiquitination and overall histone methylation state.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteínas del Grupo de Alta Movilidad/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Células A549 , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Western Blotting , Carcinogénesis/genética , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Senescencia Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Regulación de la Expresión Génica , Células HEK293 , Células Hep G2 , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Ratones Desnudos , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Interferencia de ARN , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Factor de Transcripción 4 , Factores de Transcripción/metabolismo , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
AIMS/HYPOTHESIS: Growing evidence supports that dysregulation of adipose tissue-derived factors contributes to the pathogenesis of diabetes and its complications. Since our global gene profiling analysis has identified lipocalin-14 (LCN14)-a secretory protein with lipid-binding properties-as a potential adipokine highly expressed in white adipose tissue (WAT), this study aims to explore the metabolic roles of LCN14 in obese mice, and to investigate the functional mechanisms involved. METHODS: Immunoassays and western blotting were performed to determine the circulating level and tissue distribution of LCN14, respectively. Recombinant adeno-associated virus (rAAV)-mediated gene delivery was used to overexpress LCN14 in diet-induced obese (DIO) mice and the effects on glucose and lipid metabolism were examined. RESULTS: LCN14 is expressed predominantly in WAT. Both circulating levels of LCN14 and its expression in adipose tissues are repressed in DIO and genetically inherited diabetic (db/db) mice. Overexpression of LCN14 by rAAV-mediated gene delivery in DIO mice significantly increased insulin sensitivity in major metabolic tissues and ameliorated hyperglycaemia by inhibiting hepatic gluconeogenesis. The reduced hepatic glucose production is attributed to the suppressive effects of LCN14 on the expression of gluconeogenic genes and on glycerol efflux in adipocytes, possibly by reducing the expression of aquaporin-7. CONCLUSIONS/INTERPRETATION: Reduced LCN14 expression is involved in the pathogenesis of obesity-related metabolic dysregulation. LCN14 exerts its beneficial effects on glucose homeostasis and insulin sensitivity via its actions in both adipocytes and hepatocytes.
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Adipocitos/metabolismo , Glicerol/metabolismo , Hiperglucemia/metabolismo , Lipocalinas/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Gluconeogénesis/fisiología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
A processing is a tightly regulated and highly complex pathway which includes transcription, splicing, editing, transportation, translation and degradation. It has been well-documented that splicing of RNA polymerase II medicated nascent transcripts occurs co-transcriptionally and is functionally coupled to other RNA processing. Recently, increasing experimental evidence indicated that pre-mRNA splicing influences RNA degradation and vice versa. In this review, we summarized the recent findings demonstrating the coupling of these two processes. In addition, we highlighted the importance of splicing in the production of intronic miRNA and circular RNAs, and hence the discovery of the novel mechanisms in the regulation of gene expression.
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Precursores del ARN/metabolismo , Empalme del ARN , Exosomas/metabolismo , Humanos , MicroARNs/metabolismoRESUMEN
Ferredoxins are iron-sulfur proteins that play important roles in electron transport and redox homeostasis. Yeast Apd1p is a novel member of the family of thioredoxin-like ferredoxins. In this study, we characterized the hydroxyurea (HU)-hypersensitive phenotype of apd1Δ cells. HU is an inhibitor of DNA synthesis, a cellular stressor and an anticancer agent. Although the loss of APD1 did not influence cell proliferation or cell cycle progression, it resulted in HU sensitivity. This sensitivity was reverted in the presence of antioxidant N-acetyl-cysteine, implicating a role for intracellular redox. Mutation of the iron-binding motifs in Apd1p abrogated its ability to rescue HU sensitivity in apd1Δ cells. The iron-binding activity of Apd1p was verified by a color assay. By mass spectrometry two irons were found to be incorporated into one Apd1p protein molecule. Surprisingly, ribonucleotide reductase genes were not induced in apd1Δ cells and the HU sensitivity was unaffected when dNTP production was boosted. A suppressor screen was performed and the expression of stress-regulated transcription factor Yap1p was found to effectively rescue the HU sensitivity in apd1Δ cells. Taken together, our work identified Apd1p as a new ferredoxin which serves critical roles in cellular defense against HU.
Asunto(s)
Replicación del ADN/genética , Ferredoxinas/genética , Hidroxiurea/farmacología , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Acetilcisteína/química , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Replicación del ADN/efectos de los fármacos , Ferredoxinas/química , Hierro/química , Oxidación-Reducción , Fenotipo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Most unwanted RNA transcripts in the nucleus of eukaryotic cells, such as splicing-defective pre-mRNAs and spliced-out introns, are rapidly degraded by the nuclear exosome. In budding yeast, a number of these unwanted RNA transcripts, including spliced-out introns, are first recognized by the nuclear exosome cofactor Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex before subsequent nuclear-exosome-mediated degradation. However, it remains unclear when spliced-out introns are recognized by TRAMP, and whether TRAMP may have any potential roles in pre-mRNA splicing. Here, we demonstrated that TRAMP is cotranscriptionally recruited to nascent RNA transcripts, with particular enrichment at intronic sequences. Deletion of TRAMP components led to further accumulation of unspliced pre-mRNAs even in a yeast strain defective in nuclear exosome activity, suggesting a novel stimulatory role of TRAMP in splicing. We also uncovered new genetic and physical interactions between TRAMP and several splicing factors, and further showed that TRAMP is required for optimal recruitment of the splicing factor Msl5p. Our study provided the first evidence that TRAMP facilitates pre-mRNA splicing, and we interpreted this as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP before or during splicing to prepare for the subsequent targeting of spliced-out introns to rapid degradation by the nuclear exosome.
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Intrones , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , ARN Helicasas DEAD-box/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Eliminación de Gen , Genes Reporteros , Factores de Empalme de ARN , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Empalme U2AF , Elongación de la Transcripción Genética , Transcripción Genética , beta-Galactosidasa/genéticaRESUMEN
The activity of DNA methyltransferase 1 (DNMT1) is associated with diverse biological activities, including cell proliferation, senescence, and cancer development. In this study, we demonstrated that the HMG box-containing protein 1 (HBP1) transcription factor is a new repressor of DNMT1 in a complex mechanism during senescence. The DNMT1 gene contains an HBP1-binding site at bp -115 to -134 from the transcriptional start site. HBP1 repressed the endogenous DNMT1 gene through sequence-specific binding, resulting in both gene-specific (e.g., p16(INK4)) and global DNA hypomethylation changes. The HBP1-mediated repression by DNMT1 contributed to replicative and premature senescence, the latter of which could be induced by Ras and HBP1 itself. A detailed investigation unexpectedly revealed that HBP1 has dual and complex transcriptional functions, both of which contribute to premature senescence. HBP1 both repressed the DNMT1 gene and activated the p16 gene in premature senescence. The opposite transcriptional functions proceeded through different DNA sequences and differential protein acetylation. While intricate, the reciprocal partnership between HBP1 and DNMT1 has exceptional importance, since its abrogation compromises senescence and promotes tumorigenesis. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence, with an impact on overall DNA methylation state.
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Senescencia Celular , ADN (Citosina-5-)-Metiltransferasas/genética , Regulación hacia Abajo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas Represoras/metabolismo , Acetilación , Línea Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , ADN/genética , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Genes p16 , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Activación TranscripcionalRESUMEN
HBP1 is a sequence-specific DNA-binding transcription factor with many important biological roles. It activates or represses the expression of some specific genes during cell growth and differentiation. Previous studies have exhibited that HBP1 binds to p16(INK4A) promoter and activates p16(INK4A) expression. We found that trichostatin A (TSA), an inhibitor of HDAC (histone deacetylase), induces p16(INK4A) expression in an HBP1-dependent manner. This result was drawn from a transactivation experiment by measuring relative luciferase activities of p16(INK4A) promoter with HBP1-binding site in comparison with that of the wild-type p16(INK4A) promoter by transient cotransfection with HBP1 into HEK293T cells and 2BS cells. HBP1 acetylation after TSA treatment was confirmed by immunoprecipitation assay. Our data showed that HBP1 interacted with histone acetyltransferase p300 and CREB-binding protein (CBP) and also recruited p300/CBP to p16(INK4A) promoter. HBP1 was acetylated by p300/CBP in two regions: repression domain (K297/305/307) and P domain (K171/419). Acetylation of Repression domain was not required for HBP1 transactivation on p16(INK4A). However, luciferase assay and western blotting results indicate that acetylation of P domain, especially K419 acetylation is essential for HBP1 transactivation on p16(INK4A). As assayed by SA-beta-gal staining, the acetylation of HBP1 at K419 enhanced HBP1-induced premature senescence in 2BS cells. In addition, HDAC4 repressed HBP1-induced premature senescence through permanently deacetylating HBP1. We conclude that our data suggest that HBP1 acetylation at K419 plays an important role in HBP1-induced p16(INK4A) expression.