Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the subtilases that promotes the internalization and degradation of LDL receptor in liver and thereby controls the level of LDL cholesterol in plasma. Here, we show that the expression of PCSK9 in HepG2 cells is completely dependent on the absence or presence of sterols. The minimal promoter region of the PCSK9 gene contains a sterol-regulatory element (SRE), which makes the transcription of PCSK9 dependent on sterols. Expression of nuclear forms of sterol-regulatory element binding protein-1 (SREBP-1) and SREBP-2 dramatically increased the promoter activity of PCSK9. In vitro-translated nuclear forms of SREBPs showed interactions with SRE, whereas mutations in SRE abolished their binding. In vivo studies in mice showed that Pcsk9 protein and mRNA were decreased significantly by fasting and increased by refeeding. However, supplementation with 2% cholesterol in the diet prevented the increase in Pcsk9. The amounts of Pcsk9 mRNA in livers of refed mice showed correlated regulation by the changes in the nuclear form of Srebp-2. In summary, it is suggested that the expression of PCSK9 is regulated by sterol at the transcriptional level in HepG2 cells and that both SREBP-1 and SREBP-2 can transcriptionally activate PCSK9 via SRE in its proximal promoter region in vitro. However, in vivo, it is suggested that the sterol-dependent regulation of PCSK9 is mediated predominantly by SREBP-2. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilisin serine proteases of which the gain-of-function mutations cause hypercholesterolemia (1-3). PCSK9 was independently identified as one of the genes that are regulated by sterol-regulatory element binding proteins (SREBPs) (4, 5). The SREBPs are members of the basic helix-loop-helix leucine zipper family of transcription factors that regulate the expression of the target genes by binding to the sterolregulatory elements (SREs) in their promoter regions (6). Using microarrays hybridized with RNA from livers of mice that either overexpressed nuclear forms of human SREBPs (transgenic model) or lacked SREBP-activating protein (knockout model), PCSK9 was identified as a SREBP target gene. Soon after the first cloning of this gene, with its relationship to neural apoptosis and liver regeneration, studies focused on its relationship with the regulation of cholesterol in plasma. Subsequently, the loss-of-function mutations of PCSK9 have been reported to decrease LDL cholesterol level (7-9) and reduce the risk of coronary heart disease (10). The definite evidence for a role of PCSK9 in LDL metabolism was revealed by a set of in vivo animal experiments. Adenovirus-mediated overexpression of PCSK9 reduced the amount of low density lipoprotein receptor (LDLR) in livers posttranscriptionally (11,12), whereas the amount of LDLR increased significantly in livers of Pcsk9 knockout mice (13). The mechanism by which PCSK9 reduces LDLR suggests that secreted PCSK9 in plas...

SummaryAccumulating evidence shows that extracellular vesicles (EVs) produced by mesenchymal stem/stromal cells (MSCs) exert their therapeutic effects in several disease models. We previously demonstrated that MSCs suppress autoimmunity in models of type 1 diabetes (T1D) and experimental autoimmune uveoretinitis (EAU). Therefore, here, we investigated the therapeutic potential of MSC-derived EVs using our established mouse models for autoimmune diseases affecting the pancreas and the eye: T1D and EAU. The data demonstrate that MSC-derived EVs effectively prevent the onset of disease in both T1D and EAU. In addition, the mixed lymphocyte reaction assay with MSC-derived EVs indicated that EVs inhibit activation of antigen-presenting cells and suppress development of T helper 1 (Th1) and Th17 cells. These results raise the possibility that MSC-derived EVs may be an alternative to cell therapy for autoimmune disease prevention.

Intravenously administered mesenchymal stem/stromal cells (MSCs) engraft only transiently in recipients, but confer long-term therapeutic benefits in patients with immune disorders. This suggests that MSCs induce immune tolerance by long-lasting effects on the recipient immune regulatory system. Here, we demonstrate that i.v. infusion of MSCs preconditioned lung monocytes/macrophages toward an immune regulatory phenotype in a TNF-α-stimulated gene/protein (TSG)-6-dependent manner. As a result, mice were protected against subsequent immune challenge in two models of alloand autoimmune ocular inflammation: corneal allotransplantation and experimental autoimmune uveitis (EAU). The monocytes/macrophages primed by MSCs expressed high levels of MHC class II, B220, CD11b, and IL-10, and exhibited T-cell-suppressive activities independently of FoxP3 + regulatory T cells. Adoptive transfer of MSCinduced B220 + CD11b + monocytes/macrophages prevented corneal allograft rejection and EAU. Deletion of monocytes/macrophages abrogated the MSC-induced tolerance. However, MSCs with TSG-6 knockdown did not induce MHC II + B220 + CD11b + cells, and failed to attenuate EAU. Therefore, the results demonstrate a mechanism of the MSC-mediated immune modulation through induction of innate immune tolerance that involves monocytes/macrophages. corneal allotransplantation | experimental autoimmune uveitis | immune tolerance | mesenchymal stem/stromal cell | monocyte/macrophage

Post-translational modifications of one or more central ''clock'' proteins, most notably time-of-day-dependent changes in phosphorylation, are critical for setting the pace of circadian (y24 h) clocks. In animals, PERIOD (PER) proteins are the key state variable regulating circadian clock speed and undergo daily changes in abundance and cytoplasmic-nuclear distribution that are partly driven by a complex phosphorylation program. Here, we identify O-GlcNAcylation (O-GlcNAc) as a critical post-translational modification in circadian regulation that also contributes to setting clock speed. Knockdown or overexpression of Drosophila O-GlcNAc transferase (ogt) in clock cells either shortens or lengthens circadian behavioral rhythms, respectively. The Drosophila PERIOD protein (dPER) is a direct target of OGT and undergoes daily changes in O-GlcNAcylation, a modification that is mainly observed during the first half of the night, when dPER is predominantly located in the cytoplasm. Intriguingly, the timing of when dPER translocates from the cytoplasm to the nucleus is advanced or delayed in flies, wherein ogt expression is reduced or increased, respectively. Our results suggest that O-GlcNAcylation of dPER contributes to setting the correct pace of the clock by delaying the timing of dPER nuclear entry. In addition, OGT stabilizes dPER, suggesting that O-GlcNAcylation has multiple roles in circadian timing systems.

Highlights d MSCs enhance macrophage phagocytosis through MSC mitochondria-containing vesicles d MSCs elicit secretion and transcription of amphiregulin in macrophages d Amphiregulin secretion depends on macrophage phagocytosis and MSC vesicle uptake d Macrophage amphiregulin increases Treg cells and preserves epithelial stem cells