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To investigate the mechanism of B cell receptor (BCR)-mediated apoptosis, we utilized immature B cell lines, DT40 and WEHI-231. In both cell lines, BCR-crosslinking caused the increase in lysosomal pH with early apoptotic changes characterized by chromatin condensation and phosphatidylserine exposure. This increase was detected in c-Abl-deficient DT40 cells but not in Syk-deficient cells, which corresponded to the fact that the former cells but not the latter revealed BCR-induced apoptosis. In contrast, BCR-crosslinking caused no apparent change in mitochondrial transmembrane potential. Therefore, the lysosomal change might be a primary event in BCR-induced apoptosis in DT40 cells. The increased activity of cathepsin B and apoptosis-preventing effect of a cathepsin inhibitor suggested a significant role of lysosomal enzymes in this apoptosis. By microscopic studies, lysosomes of wild-type DT40 cells fused to BCR-carrying endosomes became enlarged and accumulated one another. In contrast, these changes of lysosomal dynamics did not occur in Syk-deficient cells but transfer of wild-type Syk restored the lysosomal changes and apoptosis. These results demonstrated that the lysosomal change accompanied with the activation of lysosomal enzymes is a primary step in BCR-crosslinking-mediated apoptosis and Syk is responsible for this step through the fusion of BCR-carrying endosomes to lysosomes.

Serine protease inhibitors (SPIs, serpins) are a protein superfamily involved in almost all physiological processes in all organisms. In this study, a novel serpin was identified from Apostichopus japonicus (Ajserpin) by using high-throughput sequencing and RACE approaches. The full-length cDNA of Ajserpin was 1893 bp with a 5′-untranslated region (UTR) of 130 bp, a 3′-UTR of 587 bp, and an open reading frame of 1176 bp encoding a polypeptide of 391 amino acids with a deduced molecular weight of 43.8 kDa. Ajserpin shares the standard structure of SPI, including three β-sheets and eight α-helices. The deduced amino acid sequences of Ajserpin had no nuclear location signal and signal peptide structure. The phylogenetic tree and immunofluorescence showed that Ajserpin belonged to the clade B subfamily and was mainly located in the cytoplasm and nucleus. Sequence comparison and protein inhibition experiments showed that the active site (P1–P1' site) of Ajserpin was Arginine and Serine, which displayed inhibitory activity toward trypsin in a dose-dependent manner. Tissue distribution analysis showed that Ajserpin transcripts were constitutively expressed in all examined tissues with the peak in the body wall. Ajserpin mRNA transcripts could be induced in Vibrio splendidus -challenged sea cucumber or lipopolysaccharide-exposed coelomocytes. Furthermore, Ajserpin knockdown by small interfering RNAs could inhibit coelomocytes apoptosis. All our results revealed that Ajserpin might serve as an immune regulator in sea cucumber. • Complete cDNA sequence of serine protease inhibitors was identified in Apostichopus japonicus. • Immunofluorescence showed Ajserpin was an intracellular protein belonging to the Clade B sub-family. • Ajserpin mRNA transcripts could be induced by Vibrio splendidus challenge or LPS exposure. • RAjserpin displayed inhibitory activity towards trypsin in a dose-dependent manner. • Ajserpin knockdown by siRNA could accelerate the coelomocytes apoptosis. [ABSTRACT FROM AUTHOR]

AU-rich element RNA-binding factor 1 (AUF1) is a classical RNA-binding protein. AUF1 influences the process of development, apoptosis and tumorigenesis by interacting with adenylate-uridylate rich element-bearing mRNAs. Human skin is the largest organ of the body and acts as a protective barrier against pathogens and injuries. The aim of the present study was to explore the function and potential molecular pathways of AUF1 in human skin cells. AUF1 was overexpressed in human keratinocyte HaCaT cells and human skin fibroblast WS1 cells using adenoviruses and silenced using lentiviruses. AUF1 overexpression facilitated cell proliferation, whereas AUF1 knockdown induced the opposite effect. AUF1 reduced apoptosis but did not affect cell cycle progression. Forced AUF1 expression promoted the migration of human skin cells, as demonstrated by a scratch wound healing assay. Cell senescence was alleviated in AUF1-overexpressing skin cells, while AUF1 knockdown increased cell senescence. WS1 cells with AUF1 overexpression and silencing were used for RNA-sequencing and Kyoto Encyclopedia of Genes and Genomes-based pathway analysis to identify AUF1-affected mRNAs. A total of 18 mRNAs (eight mRNAs with positive associations and 10 mRNAs with negative associations) revealed consistent associations with both AUF1 overexpression and silencing. Enriched pathways associated with AUF1 expression included 'MAPK', 'cell adhesion molecules', 'proteasome', 'cellular senescence' and 'TGF-β signaling', indicating a complex regulatory network. Overall, the results of the present study revealed that AUF1 is involved in the proliferation, migration and senescence of skin cells in vitro and may be a potential target for cosmetic and disease treatment of skin. [ABSTRACT FROM AUTHOR]

• Dysregulated transcripts of irradiated human skin keratinocytes were identified by Microarray-based expression profiling. • These dysregulated transcripts were implicated in multiple pathways including type I interferon-mediated pathways. • Interferon α inducible protein 6 (IFI6) is induced by radiation in human skin cells and confers radio protection. • IFI6 is translocated into nucleus and regulating the transcriptional activity of HSF1. 5. A certain amount of potential HSF1-targeted genes upregulated by IFI6 overexpression post radiation is involved. Radiation-induced skin injury is one of the main adverse effects and a dose-limiting factor of radiotherapy without feasible treatment. The underlying mechanism of this disease is still limited. To investigate the potential molecular pathways and mechanisms of radiation-induced skin injury. mRNA expression profiles were determined by Affymetrix Human HTA2.0 microarray. IFI6 overexpression and knockdown were mediated by lentivirus. The functional changes of skin cells were measured by flow cytometry, ROS probe and Edu probe. Protein distribution was detected by immunofluorescence experiment, and IFI6-interacting proteins were detected by immunoprecipitation (IP) combined with mass spectrometry. The global gene changes in IFI6-overexpressed skin cells after irradiation were detected by RNA-seq. mRNA expression profiling showed 50 upregulated and 13 down regulated genes and interferon alpha inducible protein 6 (IFI6) was top upregulated. Overexpression of IFI6 promoted cell proliferation and reduced cell apoptosis as well as ROS production following radiation, and conversely, increased the radiosensitivity of HaCaT and human skin fibroblast (WS1). IFI6 was translocated into nucleus in irradiated skin cells and the interacting relationship with mitochondrial single-stranded DNA-binding protein 1 (SSBP1), which could enhance the transcriptional activity of heat shock transcription factor 1 (HSF1). IFI6 augmented HSF1 activity following radiation in HaCaT and WS1 cells. RNA-seq analysis showed IFI6 modulated virus infection and cellular response to stress pathways, which may help to further explore how IFI6 regulate the transcriptional activity of HSF1. This study reveals that IFI6 is induced by ionizing radiation and confers radioprotection in skin cells. [ABSTRACT FROM AUTHOR]