Control of division in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex.

In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ('CDK') CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1, confirming and extending previous results. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle, but is degraded after all divisions have ceased. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1 signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by 'comets' of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC modulate microtubule function and assembly while regulating mitotic progression. Genetic results suggest an independent additional role for the APC in regulating sister chromatid cohesion; this role is likely conserved across eukaryotes.

Insulin Suppresses Ubiquitination via the Deubiquitinating Enzyme Ubiquitin-Specific Protease 14, Independent of Proteasome Activity in H4IIEC3 Hepatocytes.

Ubiquitin-proteasome dysfunction contributes to obesity-related metabolic disorders, such as diabetes and fatty liver disease. However, the regulation of ubiquitin-proteasome activity by insulin remains to be elucidated. Here, we show that prolonged insulin stimulation activates proteasome function even though it reduces the ubiquitinated proteins in H4IIEC3 hepatocytes. Looking for a pathway by which insulin inhibits ubiquitination, we found that hepatic expression of ubiquitin-specific protease 14 (USP14) was upregulated in the liver of patients with insulin resistance. Indeed, the USP14-specific inhibitor IU1 canceled the insulin-mediated reduction of ubiquitinated proteins. Furthermore, insulin-induced endoplasmic reticulum (ER) stress, which was canceled by IU1, suggesting that USP14 activity is involved in insulin-induced ER stress. Co-stimulation with insulin and IU1 for 2 hours upregulated the nuclear translocation of the lipogenic transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), upregulated the expression of the lipogenic gene, fatty acid synthase (Fasn), and repressed the gluconeogenic genes. In conclusion, insulin activates proteasome function even though it inhibits protein ubiquitination by activating USP14 in hepatocytes. USP14 activation by insulin inhibits mature SREBP-1c while upregulating ER stress and the expression of genes involved in gluconeogenesis. Further understanding mechanisms underlying the USP14 activation and its pleiotropic effects may lead to therapeutic development for obesity-associated metabolic disorders, such as diabetes and fatty liver disease. SIGNIFICANCE STATEMENT: This study shows that insulin stimulation inhibits ubiquitination by activating USP14, independent of its effect on proteasome activity in hepatocytes. USP14 also downregulates the nuclear translocation of the lipogenic transcription factor SREBP-1c and upregulates the expression of genes involved in gluconeogenesis. Since USP14 is upregulated in the liver of insulin-resistant patients, understanding mechanisms underlying the USP14 activation and its pleiotropic effects will help develop treatments for metabolic disorders such as diabetes and fatty liver.

Forkhead box protein O1 (FoxO1) knockdown accelerates the eicosapentaenoic acid (EPA)-mediated Selenop downregulation independently of sterol regulatory element-binding protein-1c (SREBP-1c) in H4IIEC3 hepatocytes.

Selenoprotein P is upregulated in type 2 diabetes, causing insulin and exercise resistance. We have previously reported that eicosapentaenoic acid (EPA) negatively regulates Selenop expression by suppressing Srebf1 in H4IIEC3 hepatocytes. However, EPA downregulated Srebf1 long before downregulating Selenop. Here, we report additional novel mechanisms for the Selenop gene regulation by EPA. EPA upregulated Foxo1 mRNA expression, which was canceled with the ERK1/2 inhibitor, but not with the PKA inhibitor. Foxo1 knockdown by siRNA initiated early suppression of Selenop, but not Srebf1, by EPA. However, EPA did not affect the nuclear translocation of the FoxO1 protein. Neither ERK1/2 nor PKA inhibitor affected FoxO1 nuclear translocation. In summary, FoxO1 knockdown accelerates the EPA-mediated Selenop downregulation independent of SREBP-1c in hepatocytes. EPA upregulates Foxo1 mRNA via the ERK1/2 pathway without altering its protein and nuclear translocation. These findings suggest redundant and conflicting transcriptional networks in the lipid-induced redox regulation.

Eicosapentaenoic acid down-regulates expression of the selenoprotein P gene by inhibiting SREBP-1c protein independently of the AMP-activated protein kinase pathway in H4IIEC3 hepatocytes.

Selenoprotein P (encoded by SELENOP in humans, Selenop in rat), a liver-derived secretory protein, induces resistance to insulin and vascular endothelial growth factor (VEGF) in type 2 diabetes. Suppression of selenoprotein P may provide a novel therapeutic approach to treating type 2 diabetes; however, few drugs inhibiting SELENOP expression in hepatocytes have been identified. The present findings demonstrate that eicosapentaenoic acid (EPA) suppresses SELENOP expression by inactivating sterol regulatory element-binding protein-1c (SREBP-1c, encoded by Srebf1 in rat) in H4IIEC3 hepatocytes. Treatment with EPA caused concentration- and time-dependent reduction in SELENOP promoter activity. EPA activated AMP-activated protein kinase (AMPK); however, the inhibitory effect of EPA on SELENOP promoter activity was not canceled with an AMPK inhibitor compound C and dominant-negative AMPK transfection. Deletion mutant promoter assays and computational analysis of transcription factor-binding sites conserved among the species resulted in identification of a sterol regulatory element (SRE)-like site in the SELENOP promoter. A chromatin immunoprecipitation (ChIP) assay revealed that EPA decreases binding of SREBP-1c to the SELENOP promoter. Knockdown of Srebf1 resulted in a significant down-regulation of Selenop expression. Conversely, SREBP-1c overexpression inhibited the suppressive effect of EPA. These data provide a novel mechanism of action for EPA involving improvement of systemic insulin sensitivity through the regulation of selenoprotein P production independently of the AMPK pathway and suggest an additional approach to developing anti-diabetic drugs.

Peretinoin, an Acyclic Retinoid, Inhibits Hepatitis B Virus Replication by Suppressing Sphingosine Metabolic Pathway In Vitro.

Hepatocellular carcinoma (HCC) frequently develops from hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. We previously reported that peretinoin, an acyclic retinoid, inhibits HCV replication. This study aimed to examine the influence of peretinoin on the HBV lifecycle. HBV-DNA and covalently closed circular DNA (cccDNA) were evaluated by a qPCR method in HepG2.2.15 cells. Peretinoin significantly reduced the levels of intracellular HBV-DNA, nuclear cccDNA, and HBV transcript at a concentration that did not induce cytotoxicity. Conversely, other retinoids, such as 9-cis, 13-cis retinoic acid (RA), and all-trans-retinoic acid (ATRA), had no effect or rather increased HBV replication. Mechanistically, although peretinoin increased the expression of HBV-related transcription factors, as observed for other retinoids, peretinoin enhanced the binding of histone deacetylase 1 (HDAC1) to cccDNA in the nucleus and negatively regulated HBV transcription. Moreover, peretinoin significantly inhibited the expression of SPHK1, a potential inhibitor of HDAC activity, and might be involved in hepatic inflammation, fibrosis, and HCC. SPHK1 overexpression in cells cancelled the inhibition of HBV replication induced by peretinoin. This indicates that peretinoin activates HDAC1 and thereby suppresses HBV replication by inhibiting the sphingosine metabolic pathway. Therefore, peretinoin may be a novel therapeutic agent for HBV replication and chemoprevention against HCC.

Rapid response of the steatosis-sensing hepatokine LECT2 during diet-induced weight cycling in mice.

Dieting often leads to body weight cycling involving repeated weight loss and regain. However, little information is available regarding rapid-response serum markers of overnutrition that predict body weight alterations during weight cycling. Here, we report the rapid response of serum leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine that induces insulin resistance in skeletal muscle, during diet-induced weight cycling in mice. A switch from a high-fat diet (HFD) to a regular diet (RD) in obese mice gradually decreased body weight but rapidly decreased serum LECT2 levels within 10 days. In contrast, a switch from a RD to a HFD rapidly elevated serum LECT2 levels. Serum LECT2 levels showed a positive correlation with liver triglyceride contents but not with adipose tissue weight. This study demonstrates the rapid response of LECT2 preceding body weight alterations during weight cycling in mice and suggests that measurement of serum LECT2 may be clinically useful in the management of obesity.

Leukocyte cell-derived chemotaxin 2 is an antiviral regulator acting through the proto-oncogene MET.

Retinoic acid-inducible gene (RIG)-I is an essential innate immune sensor that recognises pathogen RNAs and induces interferon (IFN) production. However, little is known about how host proteins regulate RIG-I activation. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine and ligand of the MET receptor tyrosine kinase is an antiviral regulator that promotes the RIG-I-mediated innate immune response. Upon binding to MET, LECT2 induces the recruitment of the phosphatase PTP4A1 to MET and facilitates the dissociation and dephosphorylation of phosphorylated SHP2 from MET, thereby protecting RIG-I from SHP2/c-Cbl-mediated degradation. In vivo, LECT2 overexpression enhances RIG-I-dependent IFN production and inhibits lymphocytic choriomeningitis virus (LCMV) replication in the liver, whereas these changes are reversed in LECT2 knockout mice. Forced suppression of MET abolishes IFN production and antiviral activity in vitro and in vivo. Interestingly, hepatocyte growth factor (HGF), an original MET ligand, inhibits LECT2-mediated anti-viral signalling; conversely, LECT2-MET signalling competes with HGF-MET signalling. Our findings reveal previously unrecognized crosstalk between MET-mediated proliferation and innate immunity and suggest that targeting LECT2 may have therapeutic value in infectious diseases and cancer.

The osteopontin-CD44 axis in hepatic cancer stem cells regulates IFN signaling and HCV replication.

Osteopontin (OPN) is involved in cell proliferation, migration, inflammation, and tumor progression in various tissues. OPN induces stemness by interacting with CD44, but the functional relevance of OPN-mediated interferon (IFN) signaling and hepatitis C virus (HCV) replication in stem cell populations remains unclear. In this study, we investigated the effect of OPN on HCV replication and IFN signaling in cancer stem cells (CSCs) positive for epithelial cell adhesion molecule (EpCAM) and CD44. We show that the EpCAM+/CD44+ CSCs show marked HCV replication when compared to EpCAM-/CD44- cells. In addition, OPN significantly enhances this HCV replication in EpCAM+/CD44+ CSCs and markedly suppresses IFN-stimulated gene expression. The GSK-3ß inhibitor BIO increases the EpCAM+/CD44+ CSC population and OPN expression and impairs IFN signaling via STAT1 degradation. Taken together, our data suggest that OPN enhances HCV replication in the EpCAM+/CD44+ CSCs, while it also negatively regulates the IFN signaling pathway via inhibition of STAT1 phosphorylation and degradation. Therefore, OPN may represent a novel therapeutic target for treating HCV-related hepatocellular carcinoma.