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.

Ovaries of estrogen receptor 1-deficient mice show iron overload and signs of aging.

Introduction: Estrogens are crucial regulators of ovarian function, mediating their signaling through binding to estrogen receptors. The disruption of the estrogen receptor 1 (Esr1) provokes infertility associated with a hemorrhagic, cystic phenotype similar to that seen in diseased or aged ovaries. Our previous study indicated the possibility of altered iron metabolism in Esr1-deficient ovaries showing massive expression of lipocalin 2, a regulator of iron homeostasis. Methods: Therefore, we examined the consequences of depleting Esr1 in mouse ovaries, focusing on iron metabolism. For that reason, we compared ovaries of adult Esr1-deficient animals and age-matched wild type littermates. Results and discussion: We found increased iron accumulation in Esr1-deficient animals by using laser ablation inductively coupled plasma mass spectrometry. Western blot analysis and RT-qPCR confirmed that iron overload alters iron transport, storage and regulation. In addition, trivalent iron deposits in form of hemosiderin were detected in Esr1-deficient ovarian stroma. The depletion of Esr1 was further associated with an aberrant immune cell landscape characterized by the appearance of macrophage-derived multinucleated giant cells (MNGCs) and increased quantities of macrophages, particularly M2-like macrophages. Similar to reproductively aged animals, MNGCs in Esr1-deficient ovaries were characterized by iron accumulation and strong autofluorescence. Finally, deletion of Esr1 led to a significant increase in ovarian mast cells, involved in iron-mediated foam cell formation. Given that these findings are characteristics of ovarian aging, our data suggest that Esr1 deficiency triggers mechanisms similar to those associated with aging.

Isolation of Bovine and Human Milk Extracellular Vesicles.

Extracellular vesicles such as exosomes are small-sized, bilayered extracellular biovesicles generated by almost every cell and released into the surrounding body fluids upon the fusion of multivesicular bodies and the plasma membrane. Based on their origin, they are enriched with a variety of biologically active components including proteins, lipids, nucleic acids, cellular metabolites, and many other constituents. They can either attach or fuse with the membrane of a target cell, or alternatively be taking up via endocytosis by a recipient cell. In particular, milk exosomes have been recently shown to be a fundamental factor supporting infant growth, health, and development. In addition, exosomes derived from different cell types have been shown to possess regenerative, immunomodulatory, and anti-inflammatory properties, suggesting that they are a potential therapeutic tool in modulating the pathogenesis of diverse diseases. Therefore, efficient protocols for the isolation of milk exosomes in a high quantity and purity are the basis for establishing clinical applications. Here, we present an easy-to-follow protocol for exosome isolation from bovine and human milk. Electron microscopic analysis and nanoparticle tracking analysis reveal that the protocols allow the isolation of highly enriched fractions of exosomes. The purified exosomes express the typical exosomal protein markers, CD81 and ALIX.

Lipocalin 2 receptors: facts, fictions, and myths.

The human 25-kDa Lipocalin 2 (LCN2) was first identified and purified as a protein that in part is associated with gelatinase from neutrophils. This protein shows a high degree of sequence similarity with the deduced sequences of rat α2-microglobulin-related protein and the mouse protein 24p3. Based on its typical lipocalin fold, which consists of an eight-stranded, anti-parallel, symmetrical ß-barrel fold structure it was initially thought that LCN2 is a circulating protein functioning as a transporter of small lipophilic molecules. However, studies in Lcn2 null mice have shown that LCN2 has bacteriostatic properties and plays a key role in innate immunity by sequestering bacterial iron siderophores. Numerous reports have further shown that LCN2 is involved in the control of cell differentiation, energy expenditure, cell death, chemotaxis, cell migration, and many other biological processes. In addition, important roles for LCN2 in health and disease have been identified in Lcn2 null mice and multiple molecular pathways required for regulation of Lcn2 expression have been identified. Nevertheless, although six putative receptors for LCN2 have been proposed, there is a fundamental lack in understanding of how these cell-surface receptors transmit and amplify LCN2 to the cell. In the present review we summarize the current knowledge on LCN2 receptors and discuss inconsistencies, misinterpretations and false assumptions in the understanding of these potential LCN2 receptors.

Expression Analysis of Lipocalin 2 (LCN2) in Reproductive and Non-Reproductive Tissues of Esr1-Deficient Mice.

Estrogen receptor alpha (ERα) is widely expressed in reproductive organs, but also in non-reproductive tissues of females and males. There is evidence that lipocalin 2 (LCN2), which has diverse immunological and metabolic functions, is regulated by ERα in adipose tissue. However, in many other tissues, the impact of ERα on LCN2 expression has not been studied yet. Therefore, we used an Esr1-deficient mouse strain and analyzed LCN2 expression in reproductive (ovary, testes) and non-reproductive tissues (kidney, spleen, liver, lung) of both sexes. Tissues collected from adult wild-type (WT) and Esr1-deficient animals were analyzed by immunohistochemistry, Western blot analysis, and RT-qPCR for Lcn2 expression. In non-reproductive tissues, only minor genotype- or sex-specific differences in LCN2 expression were detected. In contrast, significant differences in LCN2 expression were observed in reproductive tissues. Particularly, there was a strong increase in LCN2 in Esr1-deficient ovaries when compared to WTs. In summary, we found an inverse correlation between the presence of ERα and the expression of LCN2 in testes and ovaries. Our results provide an important basis to better understand LCN2 regulation in the context of hormones and in health and disease.

Lipocalin-2 regulates the expression of interferon-stimulated genes and the susceptibility of prostate cancer cells to oncolytic virus infection.

Lipocalin-2 (LCN2) performs pleiotropic and tumor context-dependent functions in cancers of diverse etiologies. In prostate cancer (PCa) cells, LCN2 regulates distinct phenotypic features, including cytoskeleton organization and expression of inflammation mediators. Oncolytic virotherapy uses oncolytic viruses (OVs) to kill cancer cells and induce anti-tumor immunity. A main source of specificity of OVs towards tumor cells stems from cancer-induced defects in interferon (IFN)-based cell autonomous immune responses. However, the molecular underpinnings of such defects in PCa cells are only partially understood. Moreover, LCN2 effects on IFN responses of PCa cells and their susceptibility to OVs are unknown. To examine these issues, we queried gene expression databases for genes coexpressed with LCN2, revealing co-expression of IFN-stimulated genes (ISGs) and LCN2. Analysis of human PCa cells revealed correlated expression of LCN2 and subsets of IFNs and ISGs. CRISPR/Cas9-mediated stable knockout of LCN2 in PC3 cells or transient overexpression of LCN2 in LNCaP cells revealed LCN2-mediated regulation of IFNE (and IFNL1) expression, activation of JAK/STAT pathway, and expression of selected ISGs. Accordingly, and dependent on a functional JAK/STAT pathway, LCN2 reduced the susceptibility of PCa cells to infection with the IFN-sensitive OV, EHDV-TAU. In PC3 cells, LCN2 knockout increased phosphorylation of eukaryotic initiation factor 2α (p-eIF2α). Inhibition of PKR-like ER kinase (PERK) in PC3-LCN2-KO cells reduced p-eIF2α while increasing constitutive IFNE expression, phosphorylation of STAT1, and ISG expression; and decreasing EHDV-TAU infection. Together, these data propose that LCN2 regulates PCa susceptibility to OVs through attenuation of PERK activity and increased IFN and ISG expression.

Genetic Characterization of Rat Hepatic Stellate Cell Line PAV-1.

The rat hepatic stellate cell line PAV-1 was established two decades ago and proposed as a cellular model to study aspects of hepatic retinoic acid metabolism. This cell line exhibits a myofibroblast-like phenotype but also has the ability to store retinyl esters and synthesize retinoic acid from its precursor retinol. Importantly, when cultured with palmitic acid alone or in combination with retinol, the cells switch to a deactivated phenotype in which the proliferation and expression of profibrogenic marker genes are suppressed. Despite these interesting characteristics, the cell line has somehow fallen into oblivion. However, based on the fact that working with in vivo models is becoming increasingly complicated, genetically characterized established cell lines that mimic aspects of hepatic stellate cell biology are of fundamental value for biomedical research. To genetically characterize PAV-1 cells, we performed karyotype analysis using conventional chromosome analysis and multicolor spectral karyotyping (SKY), which allowed us to identify numerical and specific chromosomal alteration in PAV-1 cells. In addition, we used a panel of 31 species-specific allelic variant sites to define a unique short tandem repeat (STR) profile for this cell line and performed bulk mRNA-sequencing, showing that PAV-1 cells express an abundance of genes specific for the proposed myofibroblastic phenotype. Finally, we used Rhodamine-Phalloidin staining and electron microscopy analysis, which showed that PAV-1 cells contain a robust intracellular network of filamentous actin and process typical ultrastructural features of hepatic stellate cells.

Phalloidin Staining for F-Actin in Hepatic Stellate Cells.

During the development of liver fibrosis, hepatic stellate cells undergo a transition from a quiescent phenotype into a proliferative, fibrogenic, and contractile, α-smooth muscle actin-positive myofibroblast. These cells acquire properties that are strongly associated with the reorganization of the actin cytoskeleton. Actin possesses a unique ability to polymerize into filamentous actin (F-actin) form its monomeric globular state (G-actin). F-actin can form robust actin bundles and cytoskeletal networks by interacting with a number of actin-binding proteins that provide important mechanical and structural support for a multitude of cellular processes including intracellular transport, cell motility, polarity, cell shape, gene regulation, and signal transduction. Therefore, stains with actin-specific antibodies and phalloidin conjugates for actin staining are widely used to visualize actin structures in myofibroblasts. Here we present an optimized protocol for F-actin staining for hepatic stellate cells using a fluorescent phalloidin.

Lipid-Independent Regulation of PLIN5 via IL-6 through the JAK/STAT3 Axis in Hep3B Cells.

Perilipin 5 (PLIN5) is a lipid droplet coat protein that is highly expressed in oxidative tissues such as those of muscles, the heart and the liver. PLIN5 expression is regulated by a family of peroxisome proliferator-activated receptors (PPARs) and modulated by the cellular lipid status. So far, research has focused on the role of PLIN5 in the context of non-alcoholic fatty liver disease (NAFLD) and specifically in lipid droplet formation and lipolysis, where PLIN5 serves as a regulator of lipid metabolism. In addition, there are only limited studies connecting PLIN5 to hepatocellular carcinoma (HCC), where PLIN5 expression is proven to be upregulated in hepatic tissue. Considering that HCC development is highly driven by cytokines present throughout NAFLD development and in the tumor microenvironment, we here explore the possible regulation of PLIN5 by cytokines known to be involved in HCC and NAFLD progression. We demonstrate that PLIN5 expression is strongly induced by interleukin-6 (IL-6) in a dose- and time-dependent manner in Hep3B cells. Moreover, IL-6-dependent PLIN5 upregulation is mediated by the JAK/STAT3 signaling pathway, which can be blocked by transforming growth factor-ß (TGF-ß) and tumor necrosis factor-α (TNF-α). Furthermore, IL-6-mediated PLIN5 upregulation changes when IL-6 trans-signaling is stimulated through the addition of soluble IL-6R. In sum, this study sheds light on lipid-independent regulation of PLIN5 expression in the liver, making PLIN5 a crucial target for NAFLD-induced HCC.

A Beginner's Guide to Cell Culture: Practical Advice for Preventing Needless Problems.

The cultivation of cells in a favorable artificial environment has become a versatile tool in cellular and molecular biology. Cultured primary cells and continuous cell lines are indispensable in investigations of basic, biomedical, and translation research. However, despite their important role, cell lines are frequently misidentified or contaminated by other cells, bacteria, fungi, yeast, viruses, or chemicals. In addition, handling and manipulating of cells is associated with specific biological and chemical hazards requiring special safeguards such as biosafety cabinets, enclosed containers, and other specialized protective equipment to minimize the risk of exposure to hazardous materials and to guarantee aseptic work conditions. This review provides a brief introduction about the most common problems encountered in cell culture laboratories and some guidelines on preventing or tackling respective problems.

Immunohistochemical Detection of Estrogen Receptor-Beta (ERß) with PPZ0506 Antibody in Murine Tissue: From Pitfalls to Optimization.

The estrogen receptor beta (ERß) is physiologically essential for reproductive biology and is implicated in various diseases. However, despite more than 20 years of intensive research on ERß, there are still uncertainties about its distribution in tissues and cellular expression. Several studies show contrasts between mRNA and protein levels, and the use of knockout strategies revealed that many commercially available antibodies gave false-positive expression results. Recently, a specific monoclonal antibody against human ERß (PPZ0506) showed cross-reactivity with rodents and was optimized for the detection of rat ERß. Herein, we established an immunohistochemical detection protocol for ERß protein in mouse tissue. Staining was optimized on murine ovaries, as granulosa cells are known to strongly express ERß. The staining results were confirmed by western blot analysis and RT-PCR. To obtain accurate and reliable staining results, different staining conditions were tested in paraffin-embedded tissues. Different pitfalls were encountered in immunohistochemical detection. Strong heat-induced epitope retrieval (HIER) and appropriate antibody dilution were required to visualize specific nuclear expression of ERß. Finally, the specificity of the antibody was confirmed by using ovaries from Esr2-depleted mice. However, in some animals, strong (non-specific) background staining appeared. These signals could not be significantly alleviated with commercially available additional blocking solutions and are most likely due to estrus-dependent expression of endogenous immunoglobulins. In summary, our study showed that the antibody PPZ0506, originally directed against human ERß, is also suitable for reliable detection of murine ERß. An established staining protocol mitigated ambiguities regarding the expression and distribution of ERß in different tissues and will contribute to an improved understanding of its role and functions in murine tissues in the future.

Rat Hepatic Stellate Cell Line CFSC-2G: Genetic Markers and Short Tandem Repeat Profile Useful for Cell Line Authentication.

Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS).

Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology.

Primary hepatocytes are a major tool in biomedical research. However, obtaining high yields of variable hepatocytes is technically challenging. Most protocols rely on the two-step collagenase perfusion protocol introduced by Per Ottar Seglen in 1976. In this procedure, the liver is perfused in situ with a recirculating, constant volume of calcium-free buffer, which is maintained at 37 °C and continuously oxygenated. In a second step, the liver is removed from the carcass and perfused with a collagenase solution in order to dissociate the extracellular matrix of the liver and liberate individual cells. Finally, the dissected hepatocytes are further purified and concentrated by density-based centrifugation. However, failure in proper cannulation, incomplete enzymatic digestion or over-digestion can result in low cell yield and viability. Here we present a novel semi-automated perfusion device, which allows gentle, rapid and efficient generation of a single-cell suspension from rodent livers. In combination with prefabricated buffers, the system allows reliable and highly reproducible isolation of primary hepatocytes.

Genetic Characterization of Rat Hepatic Stellate Cell Line HSC-T6 for In Vitro Cell Line Authentication.

Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS).

Genetic and Molecular Characterization of the Immortalized Murine Hepatic Stellate Cell Line GRX.

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells.

Lipocalin-2 (LCN2) Deficiency Leads to Cellular Changes in Highly Metastatic Human Prostate Cancer Cell Line PC-3.

The transporter protein lipocalin-2 (LCN2) also termed neutrophil-gelatinase-associated lipocalin (NGAL) has pleiotropic effects in tumorigenesis in various cancers. Since the precise role of LCN2 in prostate cancer (PCa) is poorly understood, we aimed to elucidate its functions in PCa in vitro. For this purpose, LCN2 was transiently suppressed or permanently depleted in human PC-3 cells using siRNA or CRISPR/Cas9-mediated knockout. Effects of LCN2 suppression on expression of different tumorigenic markers were investigated by Western blot analysis and RT-qPCR. LCN2 knockout cells were analyzed for cellular changes and their ability to cope endoplasmic stress compared to parenteral PC-3 cells. Reduced LCN2 was accompanied by decreased expression of IL-1ß and Cx43. In PC-3 cells, LCN2 deficiency leads to reduced proliferation, diminished expression of pro-inflammatory cytokines, lower adhesion, and disrupted F-actin distribution. In addition, IL-1ß expression strongly correlated with LCN2 levels. LCN2 knockout cells showed enhanced and sustained activation of unfolded protein response proteins when treated with tunicamycin or cultured under glucose deprivation. Interestingly, an inverse correlation between phosphorylation of eukaryotic initiation factor 2 α subunit (p-eIF2α) and LCN2 expression was observed suggesting that LCN2 triggers protein synthesis under stress conditions. The finding that LCN2 depletion leads to significant phenotypic and cellular changes in PC-3 cells adds LCN2 as a valuable target for the treatment of PCa.

The Unfolded Protein Response Is a Major Driver of LCN2 Expression in BCR-ABL- and JAK2V617F-Positive MPN.

Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression >100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N-terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1-JNK-NF-κB-C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.

In Vitro Compression Model for Orthodontic Tooth Movement Modulates Human Periodontal Ligament Fibroblast Proliferation, Apoptosis and Cell Cycle.

Human Periodontal Ligament Fibroblasts (hPDLF), as part of the periodontal apparatus, modulate inflammation, regeneration and bone remodeling. Interferences are clinically manifested as attachment loss, tooth loosening and root resorption. During orthodontic tooth movement (OTM), remodeling and adaptation of the periodontium is required in order to enable tooth movement. hPDLF involvement in the early phase-OTM compression side was investigated for a 72-h period through a well-studied in vitro model. Changes in the morphology, cell proliferation and cell death were analyzed. Specific markers of the cell cycle were investigated by RT-qPCR and Western blot. The study showed that the morphology of hPDLF changes towards more unstructured, unsorted filaments under mechanical compression. The total cell numbers were significantly reduced with a higher cell death rate over the whole observation period. hPDLF started to recover to pretreatment conditions after 48 h. Furthermore, key molecules involved in the cell cycle were significantly reduced under compressive force at the gene expression and protein levels. These findings revealed important information for a better understanding of the preservation and remodeling processes within the periodontium through Periodontal Ligament Fibroblasts during orthodontic tooth movement. OTM initially decelerates the hPDLF cell cycle and proliferation. After adapting to environmental changes, human Periodontal Ligament Fibroblasts can regain homeostasis of the periodontium, affecting its reorganization.

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.