Lipocalin-2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron-laden bacterial siderophores, chemo-attracts neutrophils and has immunomodulatory and apoptosis-regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella-infected macrophages, which reduces cellular iron content and enhances the generation of pro-inflammatory cytokines. Lcn2 represses IL-10 production while augmenting Nos2, TNF-α, and IL-6 expression. Lcn2(-/-) macrophages have elevated IL-10 levels as a consequence of increased iron content. The crucial role of Lcn-2/IL-10 interactions was further demonstrated by the greater ability of Lcn2(-/-) IL-10(-/-) macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2(-/-) counterparts. Overexpression of the iron exporter ferroportin-1 in Lcn2(-/-) macrophages represses IL-10 and restores TNF-α and IL-6 production to the levels found in wild-type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL-10 production, and that both functions are linked to its ability to shuttle iron from macrophages.

Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion injury. The level of lipocalin-2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose-dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce stroke-reperfusion injury.

Lipocalin-2 (LCN2) is a member of the highly heterogeneous secretory protein family of lipocalins and increases in its levels can contribute to neurodegeneration in the adult brain. However, there are no reports on the role of LCN2 in Parkinson's disease (PD). Here, we report for the first time that LCN2 expression is increased in the substantia nigra (SN) of patients with PD. In mouse brains, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment for a neurotoxin model of PD significantly upregulated LCN2 expression, mainly in reactive astrocytes in both the SN and striatum. The increased LCN2 levels contributed to neurotoxicity and neuroinflammation, resulting in disruption of the nigrostriatal dopaminergic (DA) projection and abnormal locomotor behaviors, which were ameliorated in LCN2-deficient mice. Similar to the effects of MPTP treatment, LCN2-induced neurotoxicity was also observed in the 6-hydroxydopamine (6-OHDA)-treated animal model of PD. Moreover, treatment with the iron donor ferric citrate (FC) and the iron chelator deferoxamine mesylate (DFO) increased and decreased, respectively, the LCN2-induced neurotoxicity in vivo In addition to the in vivo results, 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in cocultures of mesencephalic neurons and astrocytes was reduced by LCN2 gene deficiency in the astrocytes and conditioned media derived from MPP(+)-treated SH-SY5Y neuronal enhanced glial expression of LCN2 in vitro Therefore, our results demonstrate that astrocytic LCN2 upregulation in the lesioned DA system may play a role as a potential pathogenic factor in PD and suggest that inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal DA system in the adult brain.

Lipocalin-2 (LCN2), a member of the highly heterogeneous secretory protein family of lipocalins, may contribute to neuroinflammation and neurotoxicity in the brain. However, LCN2 expression and its role in Parkinson's disease (PD) are largely unknown. Here, we report that LCN2 is upregulated in the substantia nigra of patients with PD and neurotoxin-treated animal models of PD. Our results suggest that LCN2 upregulation might be a potential pathogenic mechanism of PD, which would result in disruption of the nigrostriatal dopaminergic system through neurotoxic iron accumulation and neuroinflammation. Therefore, inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in PD.

Anaemia is a frequent complication of chronic infectious diseases but the exact mechanisms by which it develops remain to be clarified. In the present work, we used a mouse model of mycobacterial infection to study molecular alterations of iron metabolism induced by infection. We show that four weeks after infection with Mycobacterium avium BALB/c mice exhibited a moderate anaemia, which was not accompanied by an increase on hepatic hepcidin mRNA expression. Instead, infected mice presented increased mRNA expression of ferroportin (Slc40a1), ceruloplasmin (Cp), hemopexin (Hpx), heme-oxygenase-1 (Hmox1) and lipocalin-2 (Lcn2). Both the anaemia and the mRNA expression changes of iron-related genes were largely absent in C.D2 mice which bear a functional allele of the Nramp1 gene. Data presented in this work suggest that anaemia due to a chronic mycobacterial infection may develop in the absence of elevated hepcidin expression, is influenced by Nramp1 and may involve lipocalin-2.

The kidney is one of the main targets of drug toxicity, but early detection of renal damage is often difficult. As part of the InnoMed PredTox project, a collaborative effort aimed at assessing the value of combining omics technologies with conventional toxicology methods for improved preclinical safety assessment, we evaluated the performance of a panel of novel kidney biomarkers in preclinical toxicity studies. Rats were treated with a reference nephrotoxin or one of several proprietary compounds that were dropped from drug development in part due to renal toxicity. Animals were dosed at two dose levels for 1, 3, and 14 days. Putative kidney markers, including kidney injury molecule-1 (Kim-1), lipocalin-2 (Lcn2), clusterin, and tissue inhibitor of metalloproteinases-1, were analyzed in kidney and urine using quantitative real-time PCR, ELISA, and immunohistochemistry. Changes in gene/protein expression generally correlated well with renal histopathological alterations and were frequently detected at earlier time points or at lower doses than the traditional clinical parameters blood urea nitrogen and serum creatinine. Urinary Kim-1 and clusterin reflected changes in gene/protein expression and histopathological alterations in the target organ in the absence of functional changes. This confirms clusterin and Kim-1 as early and sensitive, noninvasive markers of renal injury. Although Lcn2 did not appear to be specific for kidney toxicity, its rapid response to inflammation and tissue damage in general may suggest its utility in routine toxicity testing.

BACKGROUND

The kidney functions in key physiological processes to filter blood and regulate blood pressure via key molecular transporters and ion channels. Sex-specific differences have been observed in renal disease incidence and progression, as well as acute kidney injury in response to certain drugs. Although advances have been made in characterizing the molecular components involved in various kidney functions, the molecular mechanisms responsible for sex differences are not well understood. We hypothesized that the basal expression levels of genes involved in various kidney functions throughout the life cycle will influence sex-specific susceptibilities to adverse renal events.

METHODS

Whole genome microarray gene expression analysis was performed on kidney samples collected from untreated male and female Fischer 344 (F344) rats at eight age groups between 2 and 104 weeks of age.

RESULTS

A combined filtering approach using statistical (ANOVA or pairwise t test, FDR 0.05) and fold-change criteria (>1.5 relative fold change) was used to identify 7,447 unique differentially expressed genes (DEGs). Principal component analysis (PCA) of the 7,447 DEGs revealed sex-related differences in mRNA expression at early (2 weeks), middle (8, 15, and 21 weeks), and late (104 weeks) ages in the rat life cycle. Functional analysis (Ingenuity Pathway Analysis) of these sex-different genes indicated over-representation of specific pathways and networks including renal tubule injury, drug metabolism, and immune cell and inflammatory responses. The mRNAs that code for the qualified urinary protein kidney biomarkers KIM-1, Clu, Tff3, and Lcn2 were also observed to show sex differences.

CONCLUSIONS

These data represent one of the most comprehensive in-life time course studies to be published, assessing sex differences in global gene expression in the F344 rat kidney. PCA and Venn analyses reveal specific periods of sexually dimorphic gene expression which are associated with functional categories (xenobiotic metabolism and immune cell and inflammatory responses) of key relevance to acute kidney injury and chronic kidney disease, which may underlie sex-specific susceptibility. Analysis of the basal gene expression patterns of renal genes throughout the life cycle of the rat will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

Lipocalin 2 (Lcn2), a mammalian protein that is expressed and secreted in various pathologic states, binds siderophores, which are high-affinity iron chelators. Besides its role in limiting iron availability to pathogens in the setting of bacterial infection, Lcn2:siderophore complexes can also deliver iron to cells. In this study, we examined Lcn2 regulation in the liver of mice in situations of increased iron utilization, namely, during anemia. Anemia induced by phlebotomy, iron deprivation, or phenylhydrazine treatment was associated with upregulation of Lcn2 gene expression in the liver and elevation of serum Lcn2 protein levels. We further explored the participation of several factors known to co-occur during anemia, including hypoxia, changes in iron levels, and erythropoietic drive, in the regulation of Lcn2 by anemia. We found that hypoxia, but not iron or erythropoietin, caused an induction of Lcn2 expression. The upregulation of Lcn2 levels by anemia and hypoxia, which is not directly mediated by iron or erythropoietin, suggests a possible physiological role for Lcn2 during increased iron utilization and mobilization from stores.

Microarray analysis of paired cultures of normal and cancerous urothelial cells revealed differences in cytokeratin and adhesion gene expression. Normal cells expressed autocrine growth factor genes more strongly whereas carcinoma cells were distinguished by concomitant expression of urothelial and epidermal differentiation markers. Expression of SNCG, S100A9 and LCN2 was also enhanced. In other cancers, overexpression of SNCG, LCN2 and S100A4 has been ascribed to DNA hypomethylation. We therefore investigated expression and methylation of SNCG, S100A4, S100A9 and LCN2 in urothelial cancer cell lines and tissues. SNCG and S100A4 were overexpressed in some cancer tissues and cell lines, but downregulated in others, whereas LCN2 and S100A9 were upregulated in few cancer cell lines, but regularly in tissues. Normal and cancerous urothelial cells expressing SNCG lacked promoter methylation. SNCG downregulation was associated with hypermethylation and could be reversed by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. S100A4 methylation at regulatory intronic sites and in the promoter region was lowest in leukocytes and fibroblasts, and denser in urothelial cells. Gene expression responded to 5-aza-2'-deoxycytidine. LCN2 promoter methylation was variable and even less consistently related to expression. The S100A9 promoter was partially methylated in nonexpressing cells, but 5-aza-2'-deoxycytidine had no effect. Our data indicate that SNCG methylation is cell type-specific and the gene is hypermethylated in some urothelial cancers. S100A4, S100A9 and LCN2 are genes with moderate CpG-density that show a less stringent relationship between DNA methylation and gene expression. Therefore, changes in methylation of these genes in cancer should be interpreted cautiously.

BACKGROUND

Structural remodeling is associated with the fibroinflammatory process in the atrial extracellular matrix. In the present study we aimed to investigate whether serum levels of new circulating remodeling markers differ in patients with atrial fibrillation (AF) compared to patients with sinus rhythm.

METHODS

The study population included 52 patients diagnosed with non-valvular AF and 33 age-matched patients with sinus rhythm. Serum levels of Galectin-3, matrix metalloproteinase-9 (MMP-9), lipocalin-2 (Lcn2/NGAL), N-terminal propeptide of type III procollagen (PIIINP), Hs-Crp, and neutrophil-to-lymphocyte ratio (NLR) were measured. The left atrial volume (LAV) was calculated by echocardiographic method and LAV index was calculated.

RESULTS

Galectin-3, MMP-9, and PIIINP levels were significantly higher in AF patients except NGAL levels (1166 pg/ml (1126-1204) and 1204 pg/ml (1166-1362) p=0.001, 104 (81-179) pg/ml and 404 (162-564) pg/ml p<0.0001, and 1101 (500-1960) pg/ml and 6710 (2370-9950) pg/ml p<0.0001, respectively). The NLR and Hs-CRP levels were also higher in AF (2.1 ± 1.0 and 2.7 ± 1.1 p=0.02 and 4.2 ± 1.9 mg/L and 6.0 ± 4.7 mg/L p=0.04, respectively). In correlation analyses, NLR showed a strongly significant correlation with LAVi, but Hs-CRP did not (p=0.007 r=0.247, Pearson test and p=0.808 r=0.025, Pearson test, respectively). Moreover, Galectin-3, MMP-9, and PIIINP had a strong positive correlation with LAVi (p=0.021 r=640, Spearman test and p=0.004 r=0.319 Pearson test, and p=0.004 r=0.325 Pearson test, respectively).

CONCLUSIONS

Novel fibrosis and inflammation markers in AF are correlated with atrial remodeling. Several unexplained mechanisms of atrial remodeling remain, but the present study has taken the first step in elucidating the mechanisms involving fibrosis and inflammation markers.

OBJECTIVE

A global gene expression profile of non-inflamed colonic mucosal cells from patients with Crohn's disease (CD) and of colonic mucosal cells from controls was performed.

METHODS

Tissue specimens from macroscopically non-inflamed descending colon were obtained colonoscopically from 33 CD patients and from 17 control subjects. All controls and 10 CD patients were medication-free at the time of colonoscopy. The Human Genome U133 Plus 2.0 GeneChip Array was used for gene profiling. Hybridization data were analysed with dChip software. Results were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Protein product expression of selected genes was assessed by immunohistochemistry using the Envision+ visualization technique.

RESULTS

The expression profile was not homogeneous with the statistical cut-point settings applied. In comparison with controls, it was found that 19 CD patients had three differentially expressed genes, two of them related to the innate immune system: dual oxidase 2 on chromosome 15 (DUOX2, fold change 4.1) and lipocalin 2 on chromosome 9 (LCN2, fold change 3.1). The third gene, regenerating islet-derived 1 alpha (REG1A, fold change 3.9), codes for a mitogenic protein; this could not be confirmed by RT-PCR. Medication-free patients had no differentially expressed genes as compared with controls. Immunohistochemistry indicated that these proteins were produced by epithelial cells (REG1A, LCN2) and leucocytes (DUOX2 and LCN2).

CONCLUSIONS

As compared with controls, non-inflamed colonic mucosal cells contain two up-regulated genes related to the innate immune system. Up-regulation of these genes, known to be induced by microorganisms, suggests either increased microflora antigenicity or an altered function in mucosal barrier defence.

Functional neuroimaging studies have implicated the prefrontal cortex (PFCTX) in descending modulation of pain and the placebo effect. This study was performed to elucidate comprehensive PFCTX gene expression in an animal model of persistent trigeminal pain. Adult male C57BL/6J mice received facial carrageenan injection and showed sustained increase in nociceptive responses. Microarray analyses of differentially expressed genes in the PFCTX at 3 d after injection showed "immune system process" as the dominant ontology term and increased mRNA expression of S100a8, S100a9, Lcn2, Il2rg, Fcgr1, Fcgr2b, C1qb, Ptprc, Ccl12, and Cd52 were verified by RT-PCR. Upregulation of S100A8, S100A9, and lipocalin 2 (LCN2) were confirmed by Western blots, and cells in the PFCTX were double immunolabeled with MPO, indicating they were neutrophils. Analyses of blood of facial carrageenan-injected mice also showed increased mRNA expression of these markers, suggesting transmigration of activated neutrophils into the brain. Other immune-related genes, Il2rg, Fcgr2b, C1qb, Ptprc, and Ccl12 were upregulated in the PFCTX but not blood. Approximately 70% of S100A9-positive cells in the PFCTX of carrageenan-injected mice were located in capillaries adherent to endothelial cells, whereas 30% were within the brain parenchyma. Carrageenan-injected mice showed significantly reduced nociceptive responses after injection of C terminus of murine S100A9 protein in the lateral ventricles and PFCTX but not somatosensory barrel cortex. Together, these findings demonstrate activation of immune-related genes in the PFCTX during inflammatory pain and highlight an exciting role of neutrophils in linking peripheral inflammation with immune activation of the PFCTX and antinociception.

Lipocalin-2 (LCN2) is a protein largely expressed in many tissues, associated with different biological phenomena such as cellular differentiation, inflammation and cancer acting as a survival/apoptotic signal. We found that LCN2 was expressed during osteoblast differentiation and we generated transgenic (Tg) mice over-expressing LCN2 in bone. Tg mice were smaller and presented bone microarchitectural changes in both endochondral and intramembranous bones. In particular, Tg bones displayed a thinner layer of cortical bone and a decreased trabecular number. Osteoblast bone matrix deposition was reduced and osteoblast differentiation was slowed-down. Differences were also observed in the growth plate of young transgenic mice where chondrocyte displayed a more immature phenotype and a lower proliferation rate. In bone marrow cell cultures from transgenic mice, the number of osteoclast progenitors was increased whereas in vivo it was increased the number of mature osteoclasts expressing tartrate-resistant acid phosphatase (TRAP). Finally, while osteoprotegerin (OPG) levels remained unchanged, the expression of the conventional receptor activator of nuclear factor-κB ligand (RANKL) and of the IL-6 was enhanced in Tg mice. In conclusion, we found that LCN2 plays a role in bone development and turnover having both a negative effect on bone formation, by affecting growth plate development and interfering with osteoblast differentiation, and a positive effect on bone resorption by enhancing osteoclast compartment.

Lipocalin 2 (Lcn2) is a promising therapeutic target as well as a potential diagnostic biomarker for breast cancer. It has been previously shown to promote breast cancer progression by inducing the epithelial to mesenchymal transition in breast cancer cells as well as by enhancing angiogenesis. Lcn2 levels in urine and tissue samples of breast cancer patients has also been correlated with breast cancer status and poor patient prognosis. In this study, we have engineered a novel liposomal small interfering RNA (siRNA) delivery system to target triple negative breast cancer (TNBC) via a recently identified molecular target, intercellular adhesion molecule-1 (ICAM-1). This ICAM-1-targeted, Lcn2 siRNA- encapsulating liposome (ICAM-Lcn2-LP) binds human TNBC MDA-MB-231cells significantly stronger than non-neoplastic MCF-10A cells. Efficient Lcn2 knockdown by ICAM-Lcn2-LPs led to a significant reduction in the production of vascular endothelial growth factor (VEGF) from MDA-MB-231 cells, which, in turn, led to reduced angiogenesis both in vitro and in vivo. Angiogenesis (neovascularization) is a requirement for solid tumor growth and progression, and its inhibition is an important therapeutic strategy for human cancers. Our results indicate that a tumor-specific strategy such as the TNBC-targeted, anti-angiogenic therapeutic approach developed here, may be clinically useful in inhibiting TNBC progression.

Acute-on-chronic liver failure (ACLF) is a syndrome that occurs in cirrhosis characterized by organ failure(s) and high mortality rate. There are no biomarkers of ACLF. The LCN2 gene and its product, neutrophil gelatinase-associated lipocalin (NGAL), are upregulated in experimental models of liver injury and cultured hepatocytes as a result of injury by toxins or proinflammatory cytokines, particularly Interleukin-6. The aim of this study was to investigate whether NGAL could be a biomarker of ACLF and whether LCN2 gene may be upregulated in the liver in ACLF.

We analyzed urine and plasma NGAL levels in 716 patients hospitalized for complications of cirrhosis, 148 with ACLF. LCN2 expression was assessed in liver biopsies from 29 additional patients with decompensated cirrhosis with and without ACLF.

Urine NGAL was markedly increased in ACLF vs. no ACLF patients (108(35-400) vs. 29(12-73)μg/g creatinine; p<0.001) and was an independent predictive factor of ACLF; the independent association persisted after adjustment for kidney function or exclusion of variables present in ACLF definition. Urine NGAL was also an independent predictive factor of 28day transplant-free mortality together with MELD score and leukocyte count (AUROC 0.88(0.83-0.92)). Urine NGAL improved significantly the accuracy of MELD in predicting prognosis. The LCN2 gene was markedly upregulated in the liver of patients with ACLF. Gene expression correlated directly with serum bilirubin and INR (r=0.79; p<0.001 and r=0.67; p<0.001), MELD (r=0.68; p<0.001) and Interleukin-6 (r=0.65; p<0.001).

NGAL is a biomarker of ACLF and prognosis and correlates with liver failure and systemic inflammation. There is remarkable overexpression of LCN2 gene in the liver in ACLF syndrome.

Urine NGAL is a biomarker of acute-on-chronic liver failure (ACLF). NGAL is a protein that may be expressed in several tissues in response to injury. The protein is filtered by the kidneys due to its small size and can be measured in the urine. Ariza, Graupera and colleagues found in a series of 716 patients with cirrhosis that urine NGAL was markedly increased in patients with ACLF and correlated with prognosis. Moreover, gene coding NGAL was markedly overexpressed in the liver tissue in ACLF.

The thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), regulates cell growth, development and differentiation via interactions with thyroid hormone receptors (TR), but the mechanisms underlying T3-mediated modulation of cancer progression are currently unclear. Lipocalin 2 (LCN2), a tumor-associated protein, is overexpressed in a variety of cancer types. Oligonucleotide microarray, coupled with proteomic analysis, has revealed that LCN2 is positively regulated by T3/TR. However, the physiological role and pathway of T3-mediated regulation of LCN2 in hepatocellular carcinogenesis remain to be characterized. Upregulation of LCN2 after T3 stimulation was observed in a time- and dose-dependent manner. Additionally, TRE on the LCN2 promoter was identified at positions -1444/-1427. Overexpression of LCN2 enhanced tumor cell migration and invasion, and conversely, its knockdown suppressed migration and invasion, both in vitro and in vivo. LCN2-induced migration occurred through activation of the Met/FAK cascade. LCN2 was overexpressed in clinical hepatocellular carcinoma (HCC) patients, compared with normal subjects, and positively correlated with TRα levels. Both TRα and LCN2 showed similar expression patterns in relation to survival rate, tumor grade, tumor stage and vascular invasion. Our findings collectively support a potential role of T3/TR in cancer progression through regulation of LCN2 via the Met/FAK cascade. LCN2 may thus be effectively utilized as a novel marker and therapeutic target in HCC.

Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knock-out (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Because adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned medium from ERα-null adipocytes and medium containing pure Lcn2 increased proliferation and migration of a subset of BrCA cells in culture. The proliferative and promigratory effects of ERα-deficient adipocyte-conditioned medium on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. SLC22A17 and 3-hydroxybutyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity.

BACKGROUND

Tumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production.

METHODS

We interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways.

RESULTS

Induction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation.

CONCLUSIONS

We conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression.

Inflammation is a key part of central nervous system pathophysiology. However, inflammatory factors are now thought to have both beneficial and deleterious effects. Here, we examine the hypothesis that lipocalin-2 (LCN2), an inflammatory molecule that can be up-regulated in the distressed central nervous system, may enhance angiogenesis in brain endothelial cells. Adding LCN2 (0.5-2.0 μg/mL) to RBE (Rat brain endothelial cells). 4 rat brain endothelial cells significantly increased matrigel tube formation and scratch migration, and also elevated levels of iron and reactive oxygen species. Co-treatment with a radical scavenger (U83836E), a Nox inhibitor (apocynin) and an iron chelating agent (deferiprone) significantly dampened the ability of LCN2 to enhance tube formation and scratch migration in brain endothelial cells. These findings provide in vitro proof of the concept that LCN2 can promote angiogenesis via iron- and reactive oxygen species-related pathways, and support the idea that LCN2 may contribute to the neurovascular recovery aspects of inflammation. Angiogenesis is an important part of stroke recovery. In the present study, we examined the hypothesis that lipocalin-2 (LCN2) may enhance angiogenesis in brain endothelial cells. LCN2 promoted tube formation and migration via iron and ROS-related pathways in rat brain endothelial cells. ROS scavengers, Nox inhibitors and iron chelators all dampened the ability of LCN2 to enhance in vitro angiogenesis. These findings support the idea that LCN2 that is released by damaged neurons may act as a 'help me' signal that promotes neurovascular recovery after stroke and brain injury.

Male adult germ cell tumors (GCTs) comprise two major histologic groups: seminomas and nonseminomas. Nonseminomatous GCTs (NSGCTs) can be further divided into embryonal carcinoma (EC), teratoma (T), yolk sac tumor (YS), and choriocarcinoma (CC) on the basis of the lineage differentiation that they exhibit. NSGCTs frequently present as mixed tumors consisting of two or more histological subtypes, often limiting correlative studies of clinical and molecular features to histology. We sought to develop a molecular classifier that could predict the predominant histologic subtype within mixed NSGCT tumor samples. The expression profiles of 84 NSGCTs (42 pure and 42 mixed) and normal age-matched testes were obtained using Affymetrix microarrays. Using prediction analysis for microarrays, we identified 146 transcripts that classified the histology of pure NSGCTs samples with 93% accuracy. When applied to mixed NSGCTs, the classifier predicted a histology that was consistent with one of the reported components in 93% of cases. Among the predictive transcripts were CGB (high in CC), LCN2 (high in T), BMP2 (high in YS), and POU5F1 (high in EC). Thus, the expression-based classifier accurately assigned a single predominant histology to mixed NSGCTs, and identified transcripts differentially expressed between histologic components with relevance to NSGCT differentiation.

Lipocalin-2 (LCN2), a secretory protein, regulates diverse cellular processes such as cell death/survival, cell migration/invasion, cell differentiation, iron delivery, inflammation, insulin resistance, and tissue regeneration. Recently, we reported that LCN2 is secreted by brain astrocytes under inflammatory conditions and that it promotes apoptosis, morphological changes, and migration in astrocytes both in vitro and in vivo. Activated astrocytes release LCN2 not only to induce the morphological transformation associated with reactive astrocytosis, but also to promote their own death. Under inflammatory conditions, activated astrocytes also show functional dichotomy similar to the M1/M2 phenotypes of microglia and macrophages. LCN2 is thought to be a chemokine inducer and an autocrine promoter of the classical proinflammatory activation of astrocytes. This article summarizes the current knowledge regarding the role of astrocyte-derived LCN2 as a proinflammatory mediator in the central nervous system and discusses LCN2’s role in neuroinflammatory disorders.

The production, accumulation and aggregation of amyloid beta (Aβ) peptides in Alzheimer's disease (AD) are influenced by different modulators. Among these are iron and iron-related proteins, given their ability to modulate the expression of the amyloid precursor protein and to drive Aβ aggregation. Herein, we describe that lipocalin 2 (LCN2), a mammalian acute-phase protein involved in iron homeostasis, is highly produced in response to Aβ1-42 by choroid plexus epithelial cells and astrocytes, but not by microglia or neurons. Although Aβ1-42 stimulation decreases the dehydrogenase activity and survival of wild-type astrocytes, astrocytes lacking the expression of Lcn2 are not affected. This protection results from a lower expression of the proapoptotic gene Bim and a decreased inflammatory response. Altogether, these findings show that Aβ toxicity to astrocytes requires LCN2, which represents a novel mechanism to target when addressing AD.

OBJECTIVE

Preeclampsia (PE) can be sub-divided into early- and late-onset phenotypes. The pathogenesis of these two phenotypes has not been elucidated. To gain insight into the mechanisms of disease, the transcriptional profiles of whole blood from women with early- and late-onset PE were examined.

METHODS

A cross-sectional study was conducted to include women with: i) early-onset PE (diagnosed prior to 34 weeks, n=25); ii) late-onset PE (after 34 weeks, n=47); and iii) uncomplicated pregnancy (n=61). Microarray analysis of mRNA expression in peripheral whole blood was undertaken using Affymetrix microarrays. Differential gene expression was evaluated using a moderated t-test (false discovery rate <0.1 and fold change >1.5), adjusting for maternal white blood cell count and gestational age. Validation by real-time qRT-PCR was performed in a larger sample size [early PE (n=31), late PE (n=72) and controls (n=99)] in all differentially expressed genes. Gene ontology analysis and pathway analysis were performed.

RESULTS

i) 43 and 28 genes were differentially expressed in early- and late-onset PE compared to the control group, respectively; ii) qRT-PCR confirmed the microarray results for early and late-onset PE in 77% (33/43) and 71% (20/28) of genes, respectively; iii) 20 genes that are involved in coagulation (SERPINI2), immune regulation (VSIG4, CD24), developmental process (H19) and inflammation (S100A10) were differentially expressed in early-onset PE alone. In contrast, only seven genes that encoded proteins involved in innate immunity (LTF, ELANE) and cell-to-cell recognition in the nervous system (CNTNAP3) were differentially expressed in late-onset PE alone. Thirteen genes that encode proteins involved in host defense (DEFA4, BPI, CTSG, LCN2), tight junctions in blood-brain barrier (EMP1) and liver regeneration (ECT2) were differentially expressed in both early- and late-onset PE.

CONCLUSIONS

Early- and late-onset PE are characterized by a common signature in the transcriptional profile of whole blood. A small set of genes were differentially regulated in early- and late-onset PE. Future studies of the biological function, expression timetable and protein expression of these genes may provide insight into the pathophysiology of PE.

Although chronic inflammation is believed to contribute to the pathology of age-related macular degeneration (AMD), knowledge regarding the events that elicit the change from para-inflammation to chronic inflammation in the pathogenesis of AMD is lacking. We propose here that lipocalin-2 (LCN2), a mammalian innate immunity protein that is trafficked to the lysosomes, may contribute to this process. It accumulates significantly with age in retinal pigment epithelial (RPE) cells of Cryba1 conditional knockout (cKO) mice, but not in control mice. We have recently shown that these mice, which lack βA3/A1-crystallin specifically in RPE, have defective lysosomal clearance. The age-related increase in LCN2 in the cKO mice is accompanied by increases in chemokine (C-C motif) ligand 2 (CCL2), reactive gliosis, and immune cell infiltration. LCN2 may contribute to induction of a chronic inflammatory response in this mouse model with AMD-like pathology.

The cystic fibrosis (CF) locus has been localized to the long arm of chromosome 7 by linkage analysis, and the genetic relationship between CF and the probes J3.11, met, and 7C22 has been extensively studied. To extend this genetic analysis to higher resolution, to provide information on physical distances underlying the genetic relationships, and to set limits to the position of the cystic fibrosis mutation, we have constructed a partial restriction map covering approximately 5 Mb that defines the physical relationship between these and the more recently isolated markers CS.7, XV-2c, Lcn2, and C2/5. Allelic association indicates that CS.7 and XV-2c are close to the CF locus, and an expressed sequence from this region has been described as a candidate gene for this mutation (X. Estivill et al., 1987, Nature (London) 326: 840-845). Using pulsed-field gel electrophoresis we have determined the physical order of these markers to be cen-7C22-Lcn2-met-C2/5-XV-2c-CS.7-J3.11-tel and have localized the CF mutation to an interval of less than 1500 kb. A (not unexpected) disproportionality was observed between the currently best estimates of genetic and physical distances, with the interval J3.11-met showing an approximately fourfold higher frequency of recombination than the met-7C22 interval.