TNF-α controls Lipocalin-2 expression in PC-3 prostate cancer cells.

Prostate cancer (PCa) is one of the most common and deadly cancers in men worldwide. The surrounding tumor microenvironment (TME) is important in tumor progression, as cytokines and soluble mediators including tumor necrosis factor (TNF-α) or lipocalin-2 (LCN2) can influence tumor growth and formation of metastasis. The exact mechanisms on how these pleiotropic factors affect PCa are still unknown. In this study, we showed for the first time that LCN2 mRNA and protein expression are strongly inducible by TNF-α in the highly metastatic human PCa cell line PC-3. In addition, we observed higher levels of secreted LCN2 in cell culture medium of TNF-α-treated PC-3 cells. We found that different signaling pathways such as p38, NF-κB or JNK were activated shortly after TNF-α treatment. Moreover, the mRNA levels of IL-1ß and IL-8 were also significantly increased after 24 h stimulation. Mechanistically, the NF-κB pathway and the JNK signaling axis are directly responsible for LCN2 upregulation. This was shown by the fact that pretreatment with the JNK inhibitors SP600125 or JNK-IN-8 strongly downregulated phosphorylation of c-Jun protein and markedly reduced TNF-α-mediated LCN2 upregulation in PC-3 cells. Likewise, the NF-κB inhibitor QNZ was able to repress TNF-α-induced LCN2 expression in PC-3 cells. Taking into consideration that LCN2 has been described as a tumor promoting factor in PCa, our results indicate that JNK regulates LCN2 expression and unmasks the JNK signaling axis as a possible therapeutic target for patients with PCa.

Targeting Activated Hepatic Stellate Cells Using Collagen-Binding Chitosan Nanoparticles for siRNA Delivery to Fibrotic Livers.

Activated hepatic stellate cells (aHSCs) are the main orchestrators of the fibrotic cascade in inflamed livers, with transforming growth factor-beta (TGF-ß) being the most potent pro-fibrotic cytokine. Hence, aHSCs serve as interesting therapeutic targets. However, drug delivery to aHSCs is hindered by excessive collagen deposition in the extracellular matrix (ECM) and capillarization of liver sinusoids. Chitosan-nanoparticles (CS-NPs) show intrinsic affinity for collagen, holding potential for drug delivery to fibrotic livers. Here, we employed CS-NPs for anti-TGF-ß siRNA delivery, promoting delivery into aHSCs via modification with platelet-derived growth factor receptor-beta binding peptides. In-vitro experiments using aHSCs demonstrated the association of unmodified CS-NPs to the collagen-rich ECM, with reduced intracellular accumulation. Peptide-modified CS-NPs showed a higher propensity to localize intracellularly; however, this was only the case upon ECM-collagen reduction via collagenase treatment. Peptide-modified CS-NPs were more potent than unmodified CS-NPs in reducing TGF-ß expression, implying that while collagen binding promotes liver accumulation, it hinders cell-specific siRNA delivery. In-vivo, CS-NPs successfully accumulated in fibrotic livers via collagen binding. Similar to in-vitro findings, when mice were pretreated with collagenase-loaded CS-NPs, the accumulation of peptide-modified NPs increased. Our findings demonstrate the usefulness of NPs modification with targeting ligands and collagenase treatment for aHSCs targeting and highlight the importance of chitosan-collagen binding in drug delivery to fibrotic diseases.

Deletion of Perilipin 5 Protects Against Hepatic Injury in Nonalcoholic Fatty Liver Disease via Missing Inflammasome Activation.

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver diseases with an increasing prevalence due to rising rates of obesity, metabolic syndrome and type II diabetes. Untreated NAFLD may progress to steatohepatitis (NASH) and ultimately liver cirrhosis. NAFLD is characterized by lipid accumulation, and when sufficient excess lipids are obtained, irreversible liver injury may follow. Perilipin 5 (PLIN5), a known lipid droplet coating protein and triglyceride metabolism regulator, is highly expressed in oxidatively modified tissues but it is still unclear how it affects NAFLD/NASH progress. We here studied how PLIN5 affects NAFLD development induced by a 30-week high-fat diet (HFD) administration in wild type and PLIN5 knock out (Plin5-/-) mice. The disruption of PLIN5 induced differences in lipid metabolism during HFD feeding and was associated with reduced hepatic fat accumulation. Surprisingly, Plin5-/- mice showed mitigated activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome, leading to minor hepatic damage. We conclude that PLIN5 is a pleiotropic regulator of hepatic homeostasis in NASH development. Targeting the PLIN5 expression appears critical for protecting the liver from inflammatory activation during chronic NAFLD.

Perilipin 5 and Lipocalin 2 Expression in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly cancers worldwide. Therefore, current global research focuses on molecular tools for early diagnosis of HCC, which can lead to effective treatment at an early stage. Perilipin 5 (PLIN5) has been studied as one of the main proteins of the perilipin family, whose role is to maintain lipid homeostasis by inhibiting lipolysis. In this study, we show for the first time that PLIN5 is strongly expressed in tumors of human patients with HCC as well as in mouse livers, in which HCC was genetically or experimentally induced by treatment with the genotoxic agent diethylnitrosamine. Moreover, the secreted acute phase glycoprotein Lipocalin 2 (LCN2) established as a biomarker of acute kidney injury, is also proven to indicate liver injury with upregulated expression in numerous cases of hepatic damage, including steatohepatitis. LCN2 has been studied in various cancers, and it has been assigned roles in multiple cellular processes such as the suppression of the invasion of HCC cells and their metastatic abilities. The presence of this protein in blood and urine, in combination with the presence of α -Fetoprotein (AFP), is hypothesized to serve as a biomarker of early stages of HCC. In the current study, we show in humans and mice that LCN2 is secreted into the serum from liver cancer tissue. We also show that AFP-positive hepatocytes represent the main source for the massive expression of LCN2 in tumoral tissue. Thus, the strong presence of PLIN5 and LCN2 in HCC and understanding their roles could establish them as markers for diagnosis or as treatment targets against HCC.

Prevention of hepatic stellate cell activation using JQ1- and atorvastatin-loaded chitosan nanoparticles as a promising approach in therapy of liver fibrosis.

Preventing hepatic stellate cell (HSC) activation represents a promising approach to resolve liver fibrosis. Several drugs have been reported to delay/prevent HSCs activation, however with limited clinical benefits. The latter may be in part attributed to the limited ability of such drugs in targeting more than one pathway of HSC activation. Added to that, is their inability of reaching their target cell in sufficient amounts to induce a therapeutic effect. In this work, chitosan NPs were loaded with JQ1 and atorvastatin, two drugs that have been reported to prevent HSCs activation, however via different mechanisms. NPs were then modified with different densities of retinol (Rt) for active targeting of HSCs. The NP HSCs targeting ability as a function of Rt density was assessed in vitro on primary HSCs and in vivo in carbon tetrachloride (CCl4) induced fibrotic mouse models. In vitro NPs modified with a low Rt density (LRt-NPs) showed ≈2 folds enhanced HSCs uptake in comparison to unmodified NPs, whereas NPs modified with a high Rt density (HRt-NPs) showed ≈0.8 folds change in uptake relative to unmodified NPs. Similarly, in vivo LRt-NPs showed higher accumulation in fibrotic livers in comparison to healthy livers whereas HRt-NPs and unmodified NPs showed lower accumulation in fibrotic livers relative to healthy controls respectively. Finally, the ability of drug-loaded NPs in preventing HSCs activation was assessed by monitoring the reduction in α-smooth muscle actin (α-SMA) expression by Western blot. NPs loaded with both JQ1 and atorvastatin showed reduction in α-SMA expression. In addition, a synergistic reduction in α-SMA was observed when cells were co-treated with JQ1 and atorvastatin loaded NPs.

Data on Lipocalin 2 and phosphatidylinositol 3-kinase signaling in a methionine- and choline-deficient model of non-alcoholic steatohepatitis.

The data presented in this brief report support the research article "Altered mitochondrial and peroxisomal integrity in lipocalin-2-deficient mice with hepatic steatosis" [1, doi: 10.1016/j.bbadis.2017.04.006]. We tested whether the absence of Lipocalin-2 (LCN2) could dysregulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K-PKB) pathway and hepatic homeostasis in Non-Alcoholic-Steatohepatitis (NASH). The article highlights the role of LCN2 in hepatic homeostasis.

Altered mitochondrial and peroxisomal integrity in lipocalin-2-deficient mice with hepatic steatosis.

Lipocalin-2 (LCN2) is a secreted adipokine that transports small hydrophobic molecules such as fatty acids and steroids. LCN2 limits bacterial growth by sequestering iron-containing siderophores and in mammalian liver protects against inflammation, infection, injury and other stressors. Because LCN2 modulates hepatic fat metabolism and homeostasis, we performed a comparative profiling of proteins and lipids of wild type (WT) and Lcn2-deficient mice fed either standard chow or a methionine- and choline-deficient (MCD) diet. Label-free proteomics and 2D-DIGE protein expression profiling revealed differential expression of BRIT1/MCPH1, FABP5, HMGB1, HBB2, and L-FABP, results confirmed by Western blotting. Gene ontology enrichment analysis identified enrichment for genes associated with mitochondrial membrane permeabilization and metabolic processes involving carboxylic acid. Measurements of mitochondrial membrane potential, mitochondrial chelatable iron pool, intracellular lipid peroxidation, and peroxisome numbers in primary hepatocytes confirmed that LCN2 regulates mitochondrial and peroxisomal integrity. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry imaging identified significant changes to sphingomyelins, triglycerides, and glycerophospholipids in livers of mice fed an MCD diet regardless of LCN2 status. However, two arachidonic acid-containing glycerophospholipids were increased in Lcn2-deficient livers. Thus, LCN2 influences peroxisomal and mitochondrial biology in the liver to maintain triglyceride balance, handle oxidative stress, and control apoptosis.

Lipocalin 2 (LCN2) Expression in Hepatic Malfunction and Therapy.

Lipocalin 2 (LCN2) is a secreted protein that belongs to the Lipocalins, a group of transporters of small lipophilic molecules such as steroids, lipopolysaccharides, iron, and fatty acids in circulation. Two decades after its discovery and after a high variety of published findings, LCN2's altered expression has been assigned to critical roles in several pathological organ conditions, including liver injury and steatosis, renal damage, brain injury, cardiomyopathies, muscle-skeletal disorders, lung infection, and cancer in several organs. The significance of this 25-kDa lipocalin molecule has been impressively increased during the last years. Data from several studies indicate the role of LCN2 in physiological conditions as well as in response to cellular stress and injury. LCN2 in the liver shows a protective role in acute and chronic injury models where its expression is highly elevated. Moreover, LCN2 expression is being considered as a potential strong biomarker for pathological conditions, including rheumatic diseases, cancer in human organs, hepatic steatosis, hepatic damage, and inflammation. In this review, we summarize experimental and clinical findings linking LCN2 to the pathogenesis of liver disease.

Lipocalin-2 (LCN2) regulates PLIN5 expression and intracellular lipid droplet formation in the liver.

Lipocalin-2 (LCN2) belongs to the superfamily of lipocalins and plays critical roles in the control of cellular homeostasis during inflammation and in responses to cellular stress or injury. In the liver, LCN2 triggers protective effects following acute or chronic injury, and its expression is a reliable indicator of liver damage. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis. In this study, we fed wild type (WT) and LCN2-deficient (Lcn2(-/-)) mice a methionine- and choline-deficient (MCD) diet as a nutritional model of non-alcoholic steatohepatitis, and compared intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins that regulate cellular lipid metabolism. We found that Lcn2(-/-) mice fed an MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5, also known as OXPAT) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. Restoration of LCN2 in Lcn2(-/-) primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. Our data indicate that LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with elevated fat accumulation and steatosis.

Microsatellite and Wolbachia analysis in Rhagoletis cerasi natural populations: population structuring and multiple infections.

Rhagoletis cerasi (Diptera: Tephritidae) is a major pest of sweet and sour cherries in Europe and parts of Asia. Despite its economic significance, there is a lack of studies on the genetic structure of R. cerasi populations. Elucidating the genetic structure of insects of economic importance is crucial for developing phenological-predictive models and environmental friendly control methods. All natural populations of R. cerasi have been found to harbor the endosymbiont Wolbachia pipientis, which widely affects multiple biological traits contributing to the evolution of its hosts, and has been suggested as a tool for the biological control of insect pests and disease vectors. In the current study, the analysis of 18 R. cerasi populations collected in Greece, Germany, and Russia using 13 microsatellite markers revealed structuring of R. cerasi natural populations, even at close geographic range. We also analyzed the Wolbachia infection status of these populations using 16S rRNA-, MLST- and wsp-based approaches. All 244 individuals screened were positive for Wolbachia. Our results suggest the fixation of the wCer1 strain in Greece while wCer2, wCer4, wCer5, and probably other uncharacterized strains were also detected in multiply infected individuals. The role of Wolbachia and its potential extended phenotypes needs a thorough investigation in R. cerasi. Our data suggest an involvement of this symbiont in the observed restriction in the gene flow in addition to a number of different ecological factors.