[en] Highlights: • We established a high-throughput screening system for induction of chondrocytic characters. • We screened 5822 compounds and found that A-674563 enhanced transcription of chondrocyte markers. • A-674563 inhibited the degradation of Sox9 protein. • A-674563 increased expression of ubiquitin-specific peptidase 29, which is known to induce deubiquitination. The implantation of autologous chondrocytes is a therapeutic treatment for articular cartilage damage. However, the benefits are limited due to the expansion of chondrocytes in monolayer culture, which causes loss of chondrocytic characters. Therefore, culture conditions that enhance chondrocytic characters are needed. We screened 5822 compounds and found that A-674563 enhanced the transcription of several chondrocyte marker genes, including Col2a1, Acan and Col11a2, in mouse primary chondrocytes. Experiments using cycloheximide, MG132 and bafilomycin A1 have revealed that Sox9 is degraded through the ubiquitin-proteasome pathway and that A-674563 inhibits this degradation, resulting in larger amount of Sox9 protein. RNA sequencing transcriptome analysis showed that A-674563 increases the expression of the gene that encodes ubiquitin-specific peptidase 29, which is known to induce the deubiquitination of proteins. Although the precise mechanism remains to be determined, our findings indicated that A-674563 could contribute to culture conditions that expand chondrocytes without losing chondrocytic characters.

[en] Highlights: • Neurons predominantly express KATNAL1 compared with KATNA1. • When introduced in cells, KATNAL1 showed higher microtubule-severing activity. • Cycloheximide chase analysis revealed that KATNAL1 is more stable in cells. • Knockdown of KATNAL1 but not KATANA1 resulted in enhancement of process elongation. Microtubule severing is essential for reorganization of microtubules during neuronal migration and process elongation. Katanin is a microtubule-severing enzyme, of which the major catalytic subunits are katanin A1 (KATNA1) and katanin A-like 1 (KATNAL1). The domain organization of the two subunits are almost the same; however, little is known about their functional difference. Here, we compared the expression pattern, microtubule-severing activity, intracellular degradation and knockdown phenotype in cultured cells of the two subunits. While KATNA1 was expressed ubiquitously among tissues of young adult mice, KATNAL1 was highly expressed in the brain and the testis. Neurons expressed almost only KATNAL1. When introduced into Neuro2a cells, KATNAL1 showed higher microtubule-severing activity. Cycloheximide chase analysis revealed that KATNAL1 is more stable in cells. To elucidate which part of the molecules are responsible for these characteristics, we generated chimeric molecules by swapping the amino-terminal and carboxyl-terminal halves between the two subunits. Experiments using these chimeras revealed that the amino-terminal half region is the determinant for their characteristics. Furthermore, KATNAL1 knockdown in Neuro2a cells resulted in enhancement of process elongation, while KATNA1 knockdown showed no effect. These data suggest that more active and more stable katanin subunit, KATNAL1, plays more important role in process elongation.

[en] Highlights: • GCPII interacts specifically with HDAC1 which is present in the non-nuclear fraction. • HDAC1 regulates the stability of GCPII protein. • Mutation at lysine sites in human GCPII (K479R and K491R) reduces the ubiquitination. • Mutation at 479 lysine residue of human GCPII blocks HDAC1-mediated decrease in the level of GCPII protein. Our previous study showed that the level of glutamate carboxypeptidase II (GCPII) protein is regulated by valproic acid, a histone deacetylase (HDAC) inhibitor, through acetylation of lysine residue in the GCPII protein in human astrocytes, U-87MG. The present study further investigated which HDAC subtype is involved in the acetylation of GCPII. The results revealed that GCPII interacted with HDAC1 but not with HDAC2, HDAC3, HDAC4, HDAC5, and HDAC6. Overexpression of catalytic domain (1–56 aa)-deleted HDAC1, which poorly binds to GCPII, enhanced lysine acetylation in GCPII and increased the level of GCPII protein when compared with that of the wild-type HDAC1. Further experiments showed that HDAC1 regulated the stability of GCPII protein. These data suggest that acetylation of GCPII is facilitated by HDAC1, and the acetylated GCPII is more stable than the non-acetylated GCPII. Additional experiments using siRNA HDAC1 and by HDAC1 overexpression confirmed the role of HDAC1 in regulating the stability of GCPII protein. Further, database search of acetylation and ubiquitination sites showed four candidate lysine sites in human GCPII protein that can be both acetylated and ubiquitinylated (K207, K479, K491, and K699). Mutation (lysine residues to arginine (R)) analysis showed that in the presence of cycloheximide K479R- and K491R-hGCPII mutants were less ubiquitinylated and degraded, and decrease in the level of GCPII protein by HDAC1 was significantly blocked by K479R mutants. These data suggest that K479 is a possible site of acetylation or ubiquitination. Furthermore, the results also demonstrate that the stability of GCPII protein is regulated by HDAC1 through acetylation at the lysine 479 residue.

[en] Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34^cdc2-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11^p58 induces apoptosis is not clear. Some evidences suggested β1,4-galactosyltransferase 1 (β1,4-GT 1) might participate in apoptosis induced by CDK11^p58. In this study, we demonstrated that ectopically expressed β1,4-GT 1 increased CDK11^p58-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of β1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11^p58-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of β1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11^p58 by caspase-3 was reduced. We proposed that β1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11^p58. This may represent a new mechanism of β1,4-GT 1 in CHX-induced apoptosis of CDK11^p58-overexpressing cells

[en] We found that a specific isoform of hepatocyte nuclear factor 4α (HNF-4α), HNF-4α8, was expressed in mouse mammary epithelial NMuMG cells, and that its expression was repressed by TGF-β. The repression was interfered by dominant negative forms of activin receptor-like kinase 5 (ALK5) and Smad3, and sensitive to cycloheximide, suggesting the involvement of additional protein(s) as well as ALK5 and Smad3 in the repression. Further study showed that high mobility group A2 (HMGA2), which is reported to be directly upregulated by Smads, repressed HNF-4α8 expression. Therefore, it is likely that HMGA2 mediates the downregulation of HNF-4α8 downstream of ALK5 and Smads To determine the significance of the downregulation of HNF-4α8 in TGF-β signaling, we performed DNA microarray analysis and extracted a subgroup of TGF-β1-regulated genes, including tenascin C and tissue inhibitor of metalloproteinase 3 (TIMP-3), whose regulation by TGF-β1 was attenuated by forced expression of HNF-4α8. HMGA2 has recently emerged as a transcriptional organizer of TGF-β signaling, regulating several key factors involved in epithelial-mesenchymal transition (EMT). In this study, we identified an isoform of HNF-4α as a new target downstream of HMGA2 and assigned a new role to HNF-4α in the TGF-β signaling/transcriptional cascade driven by ALK5/Smad/HMGA2 and associated with the malignant transformation of cells

[en] Highlights: • Antioxidant-independent effects N-acetylcysteine afford tubular cytoprotection. • N-acetylcysteine binds intracellular cisplatin and reduces its reactivity. • N-acetylcysteine transforms cisplatin-induced necrosis into apoptosis. • N-acetylcysteine is a potential nephroprotectant of cisplatin nephrotoxicity. Nephrotoxicity is the main limitation to the dosage and anticancer efficacy of cisplatin. Cisplatin produces tubular epithelial cell apoptosis and necrosis depending on the concentration of the drug. Protection from cisplatin nephrotoxicity must therefore tackle both cell death modes. For its ability to reduce cisplatin reactivity, in addition to its antioxidant effect, we tested and found that N-acetylcysteine (NAC) was most effective at inhibiting cisplatin cytotoxicity. NAC has no significant effect on cell death induced by either cycloheximide or Fas activation, indicating a rather selective action. Pt-DNA-binding experiments suggest that the differential effectiveness of NAC is due to its capacity to quench cisplatin reactivity inside the cell. NAC abolishes cisplatin-induced apoptosis, and transforms the necrosis induced by high concentrations of cisplatin into apoptosis. In fact, NAC allows the anti-apoptotic molecule Bcl-2 to reduce the cell death caused by pro-necrotic concentrations of cisplatin, to a significantly greater extent than in the absence of NAC. In rats, a dosage of NAC that significantly ameliorates cisplatin nephrotoxicity, has little effect on gentamicin nephrotoxicity. These characteristics provide NAC with a rationale as a potential nephroprotectant specifically tailored to and especially effective for therapeutic courses with platinated antineoplastics, which prompts to deepening into further preclinical knowledge, and to initiate clinical studies with NAC and mixed therapies composed of NAC and antiapoptotic drugs.

[en] Highlights: • AMPK increases FOXO3 protein stability in skeletal muscle primary cells. • AMPK appears as a critical factor for increasing FOXO3 expression during nutrient privation. • The role of AMPK in starvation-induced mitophagic program appears not exclusive. • AICAR treatment and starvation enhance Mul1-related mitophagic axis. AMP-activated protein kinase (AMPK) is a critical enzyme in conditions of cellular energy deficit such as exercise, hypoxia or nutritional stress. AMPK is well known to regulate protein degradation pathways notably through FOXO-related axis. In this study, we investigated the implication of AMPK activation in FOXO3 expression and stability in skeletal muscle primary myotubes. First, time course and dose response studies revealed optimal AICAR treatment duration and dose in skeletal muscle cells. Then, experiments with cycloheximide treatment of primary myotubes highlighted that AICAR infusion extends FOXO3 protein half-life. Our results also showed that AICAR treatment or nutrient depletion increases FOXO3 expression in primary myotubes and the expression of the mitochondrial E3 ligase Mul1 involved in mitochondrial turnover (mitophagy). In AMPK KO cells, nutrient depletion failed to alter the level of some FOXO3-dependent atrophic genes, including LC3B, BNIP3, and the mitochondrial E3 ligase Mul1, but not the expression of other genes (i.e. FOXO1, Gabarapl1, MAFbx, MuRF1). In summary, our data highlight that AMPK stabilizes FOXO3 and suggest a role in the first initiation step of mitochondrial segregation in muscle cells.

[en] The radiation sensibility of different fungi was determined and the influence of cycloheximide on radioresistance was tested. The radiation sensitive Byssochlamys fulva and Penicillium terrestre were treated with cycloheximide before γ-irradiation, but no sensitization could be found. The radioresistent Pullularia pullulans, however, pre-treated with 20 μg/ml cycloheximide, showed an increase of the radiation effect; this effect was much lower the organisms were treater after γ-irradiation. (author)

[en] Virus-mediated apoptosis is well documented in various systems, including herpes simplex virus 1 (HSV-1). HSV-2 is closely related to HSV-1 but its apoptotic potential during infection has not been extensively scrutinized. We report that (i) HEp-2 cells infected with HSV-2(G) triggered apoptosis, assessed by apoptotic cellular morphologies, oligosomal DNA laddering, chromatin condensation, and death factor processing when a translational inhibitor (CHX) was added at 3 hpi. Thus, HSV-2 induced apoptosis but was unable to prevent the process from killing cells. (ii) Results from a time course of CHX addition experiment indicated that infected cell protein produced between 3 and 5 hpi, termed the apoptosis prevention window, are required for blocking virus-induced apoptosis. This corresponds to the same prevention time frame as reported for HSV-1. (iii) Importantly, CHX addition prior to 3 hpi led to less apoptosis than that at 3 hpi. This suggests that proteins produced immediately upon infection are needed for efficient apoptosis induction by HSV-2. This finding is different from that observed previously with HSV-1. (iv) Infected cell factors produced during the HSV-2(G) prevention window inhibited apoptosis induced by external TNFα plus cycloheximide treatment. (v) NF-κB translocated to nuclei and its presence in nuclei correlated with apoptosis prevention during HSV-2(G) infection. (vi) Finally, clinical HSV-2 isolates induced and prevented apoptosis in HEp-2 cells in a manner similar to that of laboratory strains. Thus, while laboratory and clinical HSV-2 strains are capable of modulating apoptosis in human HEp-2 cells, the mechanism of HSV-2 induction of apoptosis differs from that of HSV-1

[en] The cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) envelope (Env) transmembrane protein gp41 interacts with the viral matrix MA protein, which facilitates incorporation of the trimeric Env complex into the virus. It is thus feasible to design an anti-HIV strategy targeting this interaction. We herein describe that Gag expression can be downregulated by a cytoplasmic domain fusion protein of the Env transmembrane protein, β-galactosidase (β-gal)/706-856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli β-gal. This mediator depleted intracellular Gag molecules in a dose-dependent manner. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag and β-gal/706-856 had stable interactions and formed aggregated complexes in perinuclear, intracellular sites. Pulse-chase and cycloheximide chase analyses demonstrated that this mediator enhanced unmyristylated Gag degradation. The results demonstrate a novel mode of HIV-1 Gag downregulation by directing Gag to an intracellular site via the interaction of Gag with a gp41 cytoplasmic domain fusion protein