Overview of the expression patterns and roles of Lipocalin 2 in the reproductive system.

The 25 kDa-sized protein Lipocalin 2 (LCN2) was originally isolated from human neutrophil granulocytes more than 30 years ago. LCN2 is an emerging player in innate immune defense, as it reduces bacterial growth due to its ability to sequester iron-containing bacterial siderophores. On the other hand, LCN2 also serves as a transporter for various hydrophobic substances due to its ß-barrel shaped structure. Over the years, LCN2 has been detected in many other cell types including epithelial cells, astrocytes, and hepatocytes. Studies have clearly shown that aberrant expression of LCN2 is associated with a variety of disorders and malignancies, including several diseases of the reproductive system. Furthermore, LCN2 was proposed as a non-invasive prognostic and/or diagnostic biomarker in this context. Although several studies have shed light on the role of LCN2 in various disorders of the female and male reproductive systems, including tumorigenesis, a comprehensive understanding of the physiological function of LCN2 in the reproductive tract is still lacking. However, there is evidence that LCN2 is directly related to fertility, as global depletion of Lcn2 in mice has a negative effect on their pregnancy rate. Since LCN2 expression can be regulated by steroid hormones, it is not surprising that its expression fluctuates greatly during remodeling processes in the female reproductive tract, especially in the uterus. Well-founded details about the expression and regulation of LCN2 in a healthy reproductive state and also about possible changes during reproductive aging could contribute to a better understanding of LCN2 as a target in various diseases. Therefore, the present review summarizes current knowledge about LCN2 in the reproductive system, including studies in rodents and humans, and discusses changes in LCN2 expression during pathological events. The limited data suggest that LCN2 is expressed and regulated differently in healthy male and female reproductive organs.

Exploring the ferroptosis-related gene lipocalin 2 as a potential biomarker for sepsis-induced acute respiratory distress syndrome based on machine learning.

BACKGROUND: Sepsis is a major cause of mortality in patients, and ARDS is one of the most common outcomes. The pathophysiology of acute respiratory distress syndrome (ARDS) caused by sepsis is significantly impacted by genes related to ferroptosis. METHODS: In this study, Weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) networks, functional enrichment analysis, and machine learning were employed to identify characterized genes and to construct receiver operating characteristic (ROC) curves. Additionally, DNA methylation levels were quantified and single-cell analysis was conducted. To validate the alterations in the expression of Lipocalin-2 (LCN2) and ferroptosis-related proteins in the in vitro model, Western blotting was carried out, and the changes in intracellular ROS and Fe2+ levels were detected. RESULTS: A combination of eight machine learning algorithms, including RFE, LASSO, RandomForest, SVM-RFE, GBDT, Bagging, XGBoost, and Boruta, were used with a machine learning model to highlight the significance of LCN2 as a key gene in sepsis-induced ARDS. Analysis of immune cell infiltration showed a positive correlation between neutrophils and LCN2. In a cell model induced by LPS, it was found that Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, was able to reverse the expression of LCN2. Knocking down LCN2 in BEAS-2B cells reversed the LPS-induced lipid peroxidation, Fe2+ levels, ACSL4, and GPX4 levels, indicating that LCN2, a ferroptosis-related gene (FRG), plays a crucial role in mediating ferroptosis. CONCLUSION: Upon establishing an FRG model for individuals with sepsis-induced ARDS, we determined that LCN2 could be a dependable marker for predicting survival in these patients. This finding provides a basis for more accurate ARDS diagnosis and the exploration of innovative treatment options.

LCN2 and ELANE overexpression induces sepsis.

Sepsis is a syndrome characterized by a systemic inflammatory response due to the invasion of pathogenic microorganisms. The relationship between Lipocalin-2 (LCN2), elastase, neutrophil expressed (ELANE) and sepsis remains unclear. The sepsis datasets GSE137340 and GSE154918 profiles were downloaded from gene expression omnibus generated from GPL10558. Batch normalization, differentially expressed Genes (DEGs) screening, weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, Gene Set Enrichment Analysis (GSEA), immune infiltration analysis, construction and analysis of protein-protein interaction (PPI) networks, Comparative Toxicogenomics Database (CTD) analysis were performed. Gene expression heatmaps were generated. TargetScan was used to screen miRNAs of DEGs. 328 DEGs were identified. According to Gene Ontology (GO), in the Biological Process analysis, they were mainly enriched in immune response, apoptosis, inflammatory response, and immune response regulation signaling pathways. In cellular component analysis, they were mainly enriched in vesicles, cytoplasmic vesicles, and secretory granules. In Molecular Function analysis, they were mainly concentrated in hemoglobin binding, Toll-like receptor binding, immunoglobulin binding, and RAGE receptor binding. In Kyoto Encyclopedia of Genes and Genomes (KEGG), they were mainly enriched in NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, P53 signaling pathway, and legionellosis. Seventeen modules were generated. The PPI network identified 4 core genes (MPO, ELANE, CTSG, LCN2). Gene expression heatmaps revealed that core genes (MPO, ELANE, CTSG, LCN2) were highly expressed in sepsis samples. CTD analysis found that MPO, ELANE, CTSG and LCN2 were associated with sepsis, peritonitis, meningitis, pneumonia, infection, and inflammation. LCN2 and ELANE are highly expressed in sepsis and may serve as molecular targets.

Lipocalin-2 expression identifies an intestinal regulatory neutrophil population during acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT), for which therapeutic options are limited. Strategies to promote intestinal tissue tolerance during aGVHD may improve patient outcomes. Using single-cell RNA sequencing, we identified a lipocalin-2 (LCN2)-expressing neutrophil population in mice with intestinal aGVHD. Transfer of LCN2-overexpressing neutrophils or treatment with recombinant LCN2 reduced aGVHD severity, whereas the lack of epithelial or hematopoietic LCN2 enhanced aGVHD severity and caused microbiome alterations. Mechanistically, LCN2 induced insulin-like growth factor 1 receptor (IGF-1R) signaling in macrophages through the LCN2 receptor SLC22A17, which increased interleukin-10 (IL-10) production and reduced major histocompatibility complex class II (MHCII) expression. Transfer of LCN2-pretreated macrophages reduced aGVHD severity but did not reduce graft-versus-leukemia effects. Furthermore, LCN2 expression correlated with IL-10 expression in intestinal biopsies in multiple cohorts of patients with aGVHD, and LCN2 induced IGF-1R signaling in human macrophages. Collectively, we identified a LCN2-expressing intestinal neutrophil population that reduced aGVHD severity by decreasing MHCII expression and increasing IL-10 production in macrophages. This work provides the foundation for administration of LCN2 as a therapeutic approach for aGVHD.

Lipocalin 2 (LCN2) confers acquired resistance to almonertinib in NSCLC through LCN2-MMP-9 signaling pathway.

Almonertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is highly selective for EGFR-activating mutations as well as the EGFR T790M mutation in patients with advanced non-small cell lung cancer (NSCLC). However, the development of resistance inevitably occurs and poses a major obstacle to the clinical efficacy of almonertinib. Therefore, a clear understanding of the mechanism is of great significance to overcome drug resistance to almonertinib in the future. In this study, NCI-H1975 cell lines resistant to almonertinib (NCI-H1975 AR) were developed by concentration-increasing induction and were employed for clarification of underlying mechanisms of acquired resistance. Through RNA-seq analysis, the HIF-1 and TGF-ß signaling pathways were significantly enriched by gene set enrichment analysis. Lipocalin-2 (LCN2), as the core node in these two signaling pathways, were found to be positively correlated to almonertinib-resistance in NSCLC cells. The function of LCN2 in the drug resistance of almonertinib was investigated through knockdown and overexpression assays in vitro and in vivo. Moreover, matrix metalloproteinases-9 (MMP-9) was further identified as a critical downstream effector of LCN2 signaling, which is regulated via the LCN2-MMP-9 axis. Pharmacological inhibition of MMP-9 could overcome resistance to almonertinib, as evidenced in both in vitro and in vivo models. Our findings suggest that LCN2 was a crucial regulator for conferring almonertinib-resistance in NSCLC and demonstrate the potential utility of targeting the LCN2-MMP-9 axis for clinical treatment of almonertinib-resistant lung adenocarcinoma.

Relationship between methylenetetrahydrofolate reductase C677T gene polymorphism and neutrophil gelatinase-associated lipocalin in early renal injury in H-type hypertension.

OBJECTIVE: To analyse the relationship between the polymorphisms of the H-type hypertensive methylenetetrahydrofolate reductase (MTHFR) C677T gene and neutrophil gelatinase-associated lipocalin (NGAL) in early kidney injury. METHOD: A total of 279 hospitalised patients with hypertension were selected and grouped according to their homocysteine (Hcy) level. If their blood Hcy level was ≥ 10 µmol/L they were assigned to the H-type hypertensive group, and if it was < 10 µmol/L they were assigned to the non-H-type hypertensive group. Blood lipid indexes, renal function indexes and blood glucose indexes were collected, and the differences between the two groups were compared. Furthermore, MTHFR C677T genotype distribution and allele frequency and Hcy level of MTHFR C677T genotype were compared, and logistic multiple regression analysis was conducted for the correlation of different genotypes of MTHFR C677T and the early kidney injury marker NGAL. RESULTS: In the non-H-type hypertensive group, the levels of Hcy and NGAL, cystatin, blood urea nitrogen, serum creatinine, uric acid, serum ß2-microglobulin and urinary microalbumin-to-creatinine ratio increased significantly, and the glomerular filtration rate level decreased significantly, when compared with the H-type hypertensive group, with statistical differences (p < 0.05). The H-type hypertensive group and the non-H-type hypertensive group had significant differences in the CC, CT and TT genotypes and allele frequencies at the MTHFR C677T locus. The MTHFR C677T gene mutation rate of the H-type hypertensive group was significantly higher than that of the non-H-type hypertensive group. The H-type hypertensive group had higher levels of the TT genotype and CT genotype Hcy. There was a statistical difference (p < 0.05). CONCLUSION: Methylenetetrahydrofolate reductase C677T polymorphism is correlated with the Hcy level, and its gene polymorphism will affect the Hcy level. Methylenetetrahydrofolate reductase C677T polymorphism has an interactive effect with NGAL. Screening NGAL and reducing Hcy levels are valuable methods for the prevention and treatment of early renal injury in patients with H-type hypertension and help improve the prognosis of patients and their quality of life.

Specific expression and blood kinetics for relaxin 2, lipocalin 2, and tissue factor pathway inhibitor 2 at the canine placenta and pregnant bloods.

In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

Inhibiting LCN2 can suppress the development of NSCLC by promoting ferroptosis.

BACKGROUND: Tumor progression is intricately linked to ferroptosis, a recently discovered form of regulated cell death. However, the specific causes of ferroptosis in non-small cell lung cancer (NSCLC) remain unclear. METHODS: In this study, we conducted transcriptome sequencing on NSCLC samples and identified Lipocalin-2 (LCN2) as a significantly differentially expressed gene associated with ferroptosis in NSCLC. Through the intersection of the set of significantly different genes with ferroptosis-related genes, we unveiled the relevance of LCN2 in NSCLC. To validate our findings, several cell lines (BEAS-2B, A549, H1299, PC-9, H1975) were utilized, and Western blot (WB) analysis was performed. We employed a variety of assays, including CCK8, EDU, scratch, Transwell, and specific assays targeting ferroptosis, to investigate the effects of LCN2 on NSCLC cell proliferation, migration, and ferroptosis. Additionally, LCN2 was evaluated in vivo using a mouse tumor xenograft model. RESULTS: In both NSCLC patients and cells, LCN2 exhibited upregulation and was associated with a poor prognosis. Inhibition of LCN2 promoted ferroptosis, resulting in the inhibition of NSCLC proliferation and migration. Conversely, the ferroptosis inhibitor Fer-1 promoted NSCLC cell proliferation and migration while inhibiting ferroptosis. Furthermore, down-regulating LCN2 reduced Fer-1's promotion of NSCLC cell migration and proliferation, as well as its prevention of ferroptosis. In vivo inhibition of LCN2 prevented NSCLC cell growth and enhanced ferroptosis. CONCLUSION: Based on our research, reducing LCN2 could effectively induce ferroptosis and hinder the growth of NSCLC.

Lipocalin-2 promotes neutrophilic inflammation in nasal polyps and its value as biomarker.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common chronic inflammatory disease of the nasal cavity and paranasal sinuses. The role of neutrophils in the pathogenesis of CRSwNP has attracted more attention in recent years, due to its association with more severe disease and reduced steroid responsiveness. Lipocalin-2 (LCN2) has been found to modulate neutrophils infiltration in other neutrophilic inflammation including inflammatory bowel disease, rheumatoid arthritis, and psoriasis. The aim was to evaluate the expression and regulator role of LCN2 in neutrophilic inflammation in CRSwNP, and its role as a potential biomarker predicting non-eosinophilic CRSwNP (neCRSwNP). METHODS: Bioinformatic analysis, immunostainings, real-time PCR and ELISA were used to analyze the expression and location of LCN2 in nasal tissues. The expression of proinflammatory mediators were assessed in nasal tissues and secretions. LCN2 production in human nasal epithelial cells (HNECs) and neutrophils, as well as its role in neutrophilic inflammation was evaluated by in vitro experiments. RESULTS: LCN2 was mainly located in neutrophils and HNECs of nasal polyps. LCN2 expression was also significantly higher in the polyp tissue and nasal secretions from patients with neCRSwNP. The LCN2 levels were positively correlated with type 3 inflammation markers, including G-CSF, IL-8, and IL-17. LCN2 expression could be upregulated by IL-17 A and TNF-α in HNECs, and LCN2 could also promote the expression of IL-8 in dispersed polyp cells and HNECs. CONCLUSIONS: LCN2 could serve as a novel biomarker predicting patients with neCRSwNP, and the increased expression of LCN2 may participate in the pathogenesis of neCRSwNP.

Tumor-associated astrocytes promote tumor progression of Sonic Hedgehog medulloblastoma by secreting lipocalin-2.

Sonic Hedgehog (SHH) subgroup of medulloblastoma (MB) accounts for about 25% of all subgroups of MB. Tumor microenvironment (TME) may play a key role in the tumor progression and therapeutic resistance. Tumor-associated astrocytes (TAAs) are reshaped to drive tumor progression through multiple paracrine signals. However, the mechanism by which TAAs modulate MB cells remains elusive. Here, we illuminated that TAAs showed a specific and dynamic pattern during SHH-MB development. Most TAAs gathered to the tumor margin during the tumor progression, rather than evenly distributed in the early-stage tumors. We further demonstrated that lipocalin-2 (LCN2) secreted by TAAs could promote the tumor growth and was correlated with the poor prognosis of MB patients. Knocking down LCN2 in TAAs in vitro impeded the proliferation and migration abilities of MB cells. In addition, we identified that TAAs accelerated the tumor growth by secreting LCN2 via STAT3 signaling pathway. Accordingly, blockade of STAT3 signaling by its inhibitor WP1066 and AAV-Lcn2 shRNA, respectively, in TAAs abrogated the effects of LCN2 on tumor progression in vitro and in vivo. In summary, we for the first time clarified that LCN2, secreted by TAAs, could promote MB tumor progression via STAT3 pathway and has potential prognostic value. Our findings unveiled a new sight in reprogramming the TME of SHH-MB and provided a potential therapeutic strategy targeting TAAs.

NR3C2 activates LCN2 transcription to promote endoplasmic reticulum stress and cell apoptosis in ischemic cerebral infarction.

Endoplasmic reticulum (ER) stress can lead to cell death and worsen tissue damage during ischemic events. Nuclear receptor subfamily 3 group C member 2 (NR3C2) and lipocalin 2 (LCN2) are known to be associated with ER stress. In this study, we obtained a potential interaction between NR3C2 and LCN2 through bioinformatics. The primary objective was to investigate their roles and interactions in the context of ER stress in ischemic cerebral infarction (ICI). A mouse model of ICI was generated by middle cerebral artery occlusion, resulting in elevated levels of NR3C2 and LCN2 in brain tissues. NR3C2 bound to the LCN2 promoter, thereby activating its transcription. Either knockdown of LCN2 or NR3C2 led to an improvement in neurologic deficits in mice, along with a reduction in infract size, tissue damage, ER stress, inflammation, and cell apoptosis in their brain tissues. Similar results were reproduced in HT22 cells, where LCN2 or NR3C2 knockdown alleviated oxygen-glucose deprivation-induced ER stress, inflammation, and cell apoptosis while improving cell viability. However, the protective effects of NR3C2 knockdown were counteracted when LCN2 was overexpressed, both in vitro and in vivo. Overall, this study demonstrates that NR3C2 activates LCN2 transcription, ultimately promoting ER stress and cell apoptosis in the context of ICI.

NGAL Mediates Anaplastic Thyroid Carcinoma Cells Survival Through FAS/CD95 Inhibition.

Neutrophil gelatinase-associated lipocalin (NGAL), a siderophore-mediated iron binding protein, is highly expressed in human anaplastic thyroid carcinomas (ATCs) where it plays pleiotropic protumorigenic roles including that of a prosurvival protein. Here we show that NGAL inhibits FAS/CD95 death receptor to control ATC cell survival. FAS/CD95 expression in human specimens from patients with ATC and in ATC-derived cell lines negatively correlate with NGAL expression. Silencing of NGAL in ATC cells leads to FAS/CD95 upregulation, whereas NGAL overexpression determines the opposite effect. As a result, an agonist anti-FAS/CD95 antibody induces cell death in NGAL-silenced cells while it is ineffective on NGAL-overexpressing cells. Interestingly, the inhibitory activity of NGAL on FAS/CD95 is due to its iron carrier property given that perturbing iron homeostasis of NGAL-proficient and -deficient ATC cells directly influences FAS/CD95 expression. Accordingly, conditioned media containing a mutant form of NGAL unable to bind siderophores cannot rescue cells from FAS/CD95-dependent death, whereas NGAL wild type-containing conditioned media abolish the effects of the agonist antibody. We also find that downregulation of FAS/CD95 expression is mediated by iron-dependent NGAL suppression of p53 transcriptional activity. Our results indicate that NGAL contributes to ATC cell survival by iron-mediated inhibition of p53-dependent FAS/CD95 expression and suggest that restoring FAS/CD95 by NGAL suppression could be a helpful strategy to kill ATC cells.

The Effects of Stress on Hippocampal Neurogenesis and Behavior in the Absence of Lipocalin-2.

Lipocalin-2 (LCN2) is an acute phase protein able to bind iron when complexed with bacterial siderophores. The recent identification of a mammalian siderophore also suggested a physiological role for LCN2 in the regulation of iron levels and redox state. In the central nervous system, the deletion of LCN2 induces deficits in neural stem cells proliferation and commitment, with an impact on the hippocampal-dependent contextual fear discriminative task. Additionally, stress is a well-known regulator of cell genesis and is known to decrease adult hippocampal cell proliferation and neurogenesis. Although voluntary running, another well-known regulator of neurogenesis, is sufficient to rescue the defective hippocampal neurogenesis and behavior in LCN2-null mice by promoting stem cells' cell cycle progression and maturation, the relevance of LCN2-regulated hippocampal neurogenesis in response to stress has never been explored. Here, we show a lack of response by LCN2-null mice to the effects of chronic stress exposure at the cellular and behavioral levels. Together, these findings implicate LCN2 as a relevant mediator of neuronal plasticity and brain function in the adult mammalian brain.

Mild Oxidative Stress Induced by Sodium Arsenite Reduces Lipocalin-2 Expression Levels in Cortical Glial Cells.

Astrocytes and microglia, the most abundant glial cells in the central nervous system, are involved in maintaining homeostasis in the brain microenvironment and in the progression of various neurological disorders. Lipocalin-2 (LCN2) is a small secretory protein that can be transcriptionally upregulated via nuclear factor kappa B (NF-κB) signaling. It is synthesized and secreted by glial cells, resulting in either the restoration of damaged neural tissues or the induction of neuronal apoptosis in a context-dependent manner. It has recently been reported that when glial cells are under lipopolysaccharide-induced inflammatory stress, either reduced production or accelerated degradation of LCN2 can alleviate neurotoxicity. However, the regulatory mechanisms of LCN2 in glial cells are not yet fully understood. In this study, we used primary astroglial-enriched cells which produce LCN2 and found that the production of LCN2 could be reduced by sodium arsenite treatment. Surprisingly, the reduced LCN2 production was not due to the suppression of NF-κB signaling. Mild oxidative stress induced by sodium arsenite treatment activated antioxidant responses and downregulated Lcn2 expression without reducing the viability of astroglial-enriched cells. Intriguingly, reduced LCN2 production could not be achieved by simple activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway in astroglial-enriched cells. Thus, it appears that mild oxidative stress, occurring in an Nrf2-independent manner, is required for the downregulation of Lcn2 expression. Taken together, our findings provide new insights into the regulatory mechanisms of LCN2 and suggest that mild oxidative stress may alter LCN2 homeostasis, even under neuroinflammatory conditions.

Lcn2 deficiency accelerates the infection of Escherichia coli O157:H7 by disrupting the intestinal barrier function.

Bacterial infections result in intestinal inflammation and injury, which affects gut health and nutrient absorption. Lipocalin 2 (Lcn2) is a protein that reacts to microbial invasion, inflammatory responses, and tissue damage. However, it remains unclear whether Lcn2 has a protective effect against bacterial induced intestinal inflammation. Therefore, this study endeavors to investigate the involvement of Lcn2 in the intestinal inflammation of mice infected with Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157:H7). Lcn2 knockout (Lcn2-/-) mice were used to evaluate the changes of inflammatory responses. Lcn2 deficiency significantly exacerbated clinical symptoms of E. coli O157:H7 infection by reducing body weight and encouraging bacterial colonization of. Compared to infected wild type mice, infected Lcn2-/- mice had significantly elevated levels of pro-inflammatory cytokines in serum and ileum, including interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α), as well as severe villi destruction in the jejunum. Furthermore, Lcn2 deficiency aggravated intestinal barrier degradation by significantly reducing the expression of tight junction proteins occludin and claudin 1, the content of myeloperoxidase (MPO) in the ileum, and the number of goblet cells in the colon. Our findings indicated that Lcn2 could alleviate inflammatory damage caused by E. coli O157:H7 infection in mice by enhancing intestinal barrier function.

Macrophage CARD9 mediates cardiac injury following myocardial infarction through regulation of lipocalin 2 expression.

Immune cell infiltration in response to myocyte death regulates extracellular matrix remodeling and scar formation after myocardial infarction (MI). Caspase-recruitment domain family member 9 (CARD9) acts as an adapter that mediates the transduction of pro-inflammatory signaling cascades in innate immunity; however, its role in cardiac injury and repair post-MI remains unclear. We found that Card9 was one of the most upregulated Card genes in the ischemic myocardium of mice. CARD9 expression increased considerably 1 day post-MI and declined by day 7 post-MI. Moreover, CARD9 was mainly expressed in F4/80-positive macrophages. Card9 knockout (KO) led to left ventricular function improvement and infarct scar size reduction in mice 28 days post-MI. Additionally, Card9 KO suppressed cardiomyocyte apoptosis in the border region and attenuated matrix metalloproteinase (MMP) expression. RNA sequencing revealed that Card9 KO significantly suppressed lipocalin 2 (Lcn2) expression post-MI. Both LCN2 and the receptor solute carrier family 22 member 17 (SL22A17) were detected in macrophages. Subsequently, we demonstrated that Card9 overexpression increased LCN2 expression, while Card9 KO inhibited necrotic cell-induced LCN2 upregulation in macrophages, likely through NF-κB. Lcn2 KO showed beneficial effects post-MI, and recombinant LCN2 diminished the protective effects of Card9 KO in vivo. Lcn2 KO reduced MMP9 post-MI, and Lcn2 overexpression increased Mmp9 expression in macrophages. Slc22a17 knockdown in macrophages reduced MMP9 release with recombinant LCN2 treatment. In conclusion, our results demonstrate that macrophage CARD9 mediates the deterioration of cardiac function and adverse remodeling post-MI via LCN2.

Role of LCN2 in a murine model of hindlimb ischemia and in peripheral artery disease patients, and its potential regulation by miR-138-5P.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is a leading cause of morbimortality worldwide. Lipocalin-2 (LCN2) has been associated with higher risk of amputation or mortality in PAD and might be involved in muscle regeneration. Our aim is to unravel the role of LCN2 in skeletal muscle repair and PAD. METHODS AND RESULTS: WT and Lcn2-/- mice underwent hindlimb ischemia. Blood and crural muscles were analyzed at the inflammatory and regenerative phases. At day 2, Lcn2-/- male mice, but not females, showed increased blood and soleus muscle neutrophils, and elevated circulating pro-inflammatory monocytes (p < 0.05), while locally, total infiltrating macrophages were reduced (p < 0.05). Moreover, Lcn2-/- soleus displayed an elevation of Cxcl1 (p < 0.001), and Cxcr2 (p < 0.01 in males), and a decrease in Ccl5 (p < 0.05). At day 15, Lcn2 deficiency delayed muscle recovery, with higher density of regenerating myocytes (p < 0.04) and arterioles (αSMA+, p < 0.025). Reverse target prediction analysis identified miR-138-5p as a potential regulator of LCN2, showing an inverse correlation with Lcn2 mRNA in skeletal muscles (rho = -0.58, p < 0.01). In vitro, miR-138-5p mimic reduced Lcn2 expression and luciferase activity in murine macrophages (p < 0.05). Finally, in human serum miR-138-5p was inversely correlated with LCN2 (p ≤ 0.001 adjusted, n = 318), and associated with PAD (Odds ratio 0.634, p = 0.02, adjusted, PAD n = 264, control n = 54). CONCLUSIONS: This study suggests a possible dual role of LCN2 in acute and chronic conditions, with a probable role in restraining inflammation early after skeletal muscle ischemia, while being associated with vascular damage in PAD, and identifies miR-138-5p as one potential post-transcriptional regulator of LCN2.

Hepatocytic lipocalin-2 controls HDL metabolism and atherosclerosis via Nedd4-1-SR-BI axis in mice.

High-density lipoprotein (HDL) metabolism is regulated by complex interplay between the scavenger receptor group B type 1 (SR-BI) and multiple signaling molecules in the liver. Here, we show that lipocalin-2 (Lcn2) is a key regulator of hepatic SR-BI, HDL metabolism, and atherosclerosis. Overexpression of human Lcn2 in hepatocytes attenuates the development of atherosclerosis via SR-BI in western-diet-fed Ldlr-/- mice, whereas hepatocyte-specific ablation of Lcn2 has the opposite effect. Mechanistically, hepatocyte Lcn2 improves HDL metabolism and alleviates atherogenesis by blocking Nedd4-1-mediated SR-BI ubiquitination at K500 and K508. The Lcn2-improved HDL metabolism is abolished in mice with hepatocyte-specific Nedd4-1 or SR-BI deletion and in SR-BI (K500A/K508A) mutation mice. This study identifies a regulatory axis from Lcn2 to HDL via blocking Nedd4-1-mediated SR-BI ubiquitination and demonstrates that hepatocyte Lcn2 may be a promising target to improve HDL metabolism to treat atherosclerotic cardiovascular diseases.

Lipocalin-2: a therapeutic target to overcome neurodegenerative diseases by regulating reactive astrogliosis.

Glial cell activation precedes neuronal cell death during brain aging and the progression of neurodegenerative diseases. Under neuroinflammatory stress conditions, lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin or 24p3, is produced and secreted by activated microglia and reactive astrocytes. Lcn2 expression levels are known to be increased in various cells, including reactive astrocytes, through the activation of the NF-κB signaling pathway. In the central nervous system, as LCN2 exerts neurotoxicity when secreted from reactive astrocytes, many researchers have attempted to identify various strategies to inhibit LCN2 production, secretion, and function to minimize neuroinflammation and neuronal cell death. These strategies include regulation at the transcriptional, posttranscriptional, and posttranslational levels, as well as blocking its functions using neutralizing antibodies or antagonists of its receptor. The suppression of NF-κB signaling is a strategy to inhibit LCN2 production, but it may also affect other cellular activities, raising questions about its effectiveness and feasibility. Recently, LCN2 was found to be a target of the autophagy‒lysosome pathway. Therefore, autophagy activation may be a promising therapeutic strategy to reduce the levels of secreted LCN2 and overcome neurodegenerative diseases. In this review, we focused on research progress on astrocyte-derived LCN2 in the central nervous system.

Lipocalin-2 Upregulation in Nasopharyngeal Carcinoma: A Novel Potential Diagnostic Biomarker.

Background: Lipocalin-2 (LCN2) deregulation has been reported in several types of cancer and is implicated in the proliferation, migration, angiogenesis, and progression of tumors. However, its aberrant expression has been rarely studied in nasopharyngeal carcinoma (NPC). In the present study, we investigated the expression of LCN2 in NPC patients. Methods: In this descriptive cross-sectional study, 29 NPC and 20 non-cancerous control paraffin pathology blocks were obtained from the seven-year (2011 to 2018) archive of Razi Laboratory in Rasht, Iran. LCN2 mRNA expression was evaluated through quantitative real-time PCR. In addition, immunohistochemistry was performed to evaluate LCN2 expression at the protein level. The fold change value and total immunostaining score (TIS) were applied for quantitative evaluation. The nonparametric Mann-Whitney U test and Fisher's exact test were used through GraphPad Prism 8.3.0 software. P<0.05 was considered statistically significant. Results: Our results revealed that LCN2 mRNA and protein levels in NPC tissues were significantly higher than control tissues (P=0.028 and P=0.002, respectively). At the protein level, 65.51% (19/29) of NPC patients were categorized as having high LCN2 expression (TIS>3) and 34.47% (10/29) as low expression (TIS≤3). While in the control group, 25% (5/20) of subjects represented a high expression of LCN2 (TIS>3), and 75% (15/20) showed no or weak expression (TIS≤3). No significant correlation was found between the overexpression of LCN2 at the protein level and the demographic features of the patients. Conclusion: Our findings suggest that LCN2 might be considered a potential new diagnostic marker for NPC. However, this warrants further studies.