ABSTRACT
Parietal epithelial cells (PECs) are kidney progenitor cells with similarities to a bone marrow stem cell niche. In focal segmental glomerulosclerosis (FSGS) PECs become activated and contribute to extracellular matrix deposition. Colony stimulating factor-1 (CSF-1), a hematopoietic growth factor, acts via its specific receptor, CSF-1R, and has been implicated in several glomerular diseases, although its role on PEC activation is unknown. Here, we found that CSF-1R was upregulated in PECs and podocytes in biopsies from patients with FSGS. Through in vitro studies, PECs were found to constitutively express CSF-1R. Incubation with CSF-1 induced CSF-1R upregulation and significant transcriptional regulation of genes involved in pathways associated with PEC activation. Specifically, CSF-1/CSF-1R activated the ERK1/2 signaling pathway and upregulated CD44 in PECs, while both ERK and CSF-1R inhibitors reduced CD44 expression. Functional studies showed that CSF-1 induced PEC proliferation and migration, while reducing the differentiation of PECs into podocytes. These results were validated in the Adriamycin-induced FSGS experimental mouse model. Importantly, treatment with either the CSF-1R-specific inhibitor GW2580 or Ki20227 provided a robust therapeutic effect. Thus, we provide evidence of the role of the CSF-1/CSF-1R pathway in PEC activation in FSGS, paving the way for future clinical studies investigating the therapeutic effect of CSF-1R inhibitors on patients with FSGS.
Subject(s)
Glomerulosclerosis, Focal Segmental , Hyaluronan Receptors , Macrophage Colony-Stimulating Factor , Podocytes , Glomerulosclerosis, Focal Segmental/pathology , Glomerulosclerosis, Focal Segmental/metabolism , Glomerulosclerosis, Focal Segmental/genetics , Animals , Humans , Podocytes/metabolism , Podocytes/pathology , Macrophage Colony-Stimulating Factor/metabolism , Macrophage Colony-Stimulating Factor/genetics , Hyaluronan Receptors/metabolism , Hyaluronan Receptors/genetics , Mice , Cell Proliferation/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial Cells/drug effects , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Receptor, Macrophage Colony-Stimulating Factor/genetics , Kidney Glomerulus/pathology , Kidney Glomerulus/metabolism , Male , Disease Models, Animal , Cells, Cultured , Female , Up-Regulation , Cell Movement/drug effects , MAP Kinase Signaling System/drug effects , Signal Transduction , Mice, Inbred C57BL , Receptors, Granulocyte-Macrophage Colony-Stimulating FactorABSTRACT
Throughout life, macrophages are located in every tissue of the body, where their main roles are to phagocytose cellular debris and recycle aging red blood cells. In the tissue niche, they promote homeostasis through trophic, regulatory, and repair functions by responding to internal and external stimuli. This in turn polarizes macrophages into a broad spectrum of functional activation states, also reflected in their iron-regulated gene profile. The fast adaptation to the environment in which they are located helps to maintain tissue homeostasis under physiological conditions.
Subject(s)
Iron/immunology , Macrophage Activation , Macrophages/immunology , Animals , Homeostasis , Humans , Inflammation/immunology , Inflammation/metabolism , Iron/metabolism , Macrophages/metabolism , PhagocytosisABSTRACT
BACKGROUND: We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function. METHODS: LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression. RESULTS: LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects. CONCLUSIONS: We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options.
Subject(s)
Carcinoma, Renal Cell/metabolism , Iron/metabolism , Kidney Neoplasms/metabolism , Lipocalin-2/metabolism , RNA, Messenger/metabolism , Adult , Aged , Aged, 80 and over , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Carcinoma, Renal Cell/surgery , Cell Adhesion/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Disease Progression , Female , Humans , In Vitro Techniques , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Kidney Neoplasms/surgery , Lipocalin-2/genetics , Male , Middle Aged , Prognosis , Real-Time Polymerase Chain Reaction , Spectrophotometry, Atomic , Spheroids, Cellular , Tumor Cells, CulturedABSTRACT
During the course of sepsis in critically ill patients, kidney dysfunction and damage are among the first events of a complex scenario toward multi-organ failure and patient death. Acute kidney injury triggers the release of lipocalin-2 (Lcn-2), which is involved in both renal injury and recovery. Taking into account that Lcn-2 binds and transports iron with high affinity, we aimed at clarifying if Lcn-2 fulfills different biological functions according to its iron-loading status and its cellular source during sepsis-induced kidney failure. We assessed Lcn-2 levels both in serum and in the supernatant of short-term cultured renal macrophages (MΦ) as well as renal tubular epithelial cells (TEC) isolated from either Sham-operated or cecal ligation and puncture (CLP)-treated septic mice. Total kidney iron content was analyzed by Perls' staining, while Lcn-2-bound iron in the supernatants of short-term cultured cells was determined by atomic absorption spectroscopy. Lcn-2 protein in serum was rapidly up-regulated at 6 h after sepsis induction and subsequently increased up to 48 h. Lcn-2-levels in the supernatant of TEC peaked at 24 h and were low at 48 h with no change in its iron-loading. In contrast, in renal MΦ Lcn-2 was low at 24 h, but increased at 48 h, where it mainly appeared in its iron-bound form. Whereas TEC-secreted, iron-free Lcn-2 was associated with renal injury, increased MΦ-released iron-bound Lcn-2 was linked to renal recovery. Therefore, we hypothesized that both the cellular source of Lcn-2 as well as its iron-load crucially adds to its biological function during sepsis-induced renal injury.
Subject(s)
Iron/metabolism , Lipocalin-2/metabolism , Renal Insufficiency/metabolism , Sepsis/complications , Animals , Biomarkers/metabolism , Cells, Cultured , Epithelial Cells/metabolism , Kidney Tubules/cytology , Kidney Tubules/metabolism , Lipocalin-2/genetics , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Protein Binding , Renal Insufficiency/etiology , Renal Insufficiency/pathologyABSTRACT
Lipocalin-2 (Lcn-2) is rapidly upregulated in macrophages after renal tubular injury and acts as renoprotective and pro-regenerative agent. Lcn-2 possesses the ability to bind and transport iron with high affinity. Therefore, the present study focuses on the decisive role of the Lcn-2 iron-load for its pro-regenerative function. Primary mouse tubular epithelial cells were isolated from kidney tissue of wildtype mice and incubated with 5µM Cisplatin for 24h to induce injury. Bone marrow-derived macrophages of wildtype and Lcn-2-/- mice were isolated and polarized with IL-10 towards an anti-inflammatory, iron-release phenotype. Their supernatants as well as recombinant iron-loaded holo-Lcn-2 was used for stimulation of Cisplatin-injured tubular epithelial cells. Incubation of tubular epithelial cells with wildtype supernatants resulted in less damage and induced cellular proliferation, whereas in absence of Lcn-2 no protective effect was observed. Epithelial integrity as well as cellular proliferation showed a clear protection upon rescue experiments applying holo-Lcn-2. Notably, we detected a positive correlation between total iron amounts in tubular epithelial cells and cellular proliferation, which, in turn, reinforced the assumed link between availability of Lcn-2-bound iron and recovery. We hypothesize that macrophage-released Lcn-2-bound iron is provided to tubular epithelial cells during toxic cell damage, whereby injury is limited and recovery is favored.
Subject(s)
Epithelial Cells/metabolism , Kidney/metabolism , Lipocalin-2/metabolism , Macrophages/metabolism , Regeneration , Animals , Cell Proliferation , Cisplatin/adverse effects , Iron/metabolism , Kidney/drug effects , Kidney/injuries , Lipocalin-2/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Recombinant Proteins , Up-RegulationABSTRACT
Iron is an essential element for virtually all organisms. On the one hand, it facilitates cell proliferation and growth. On the other hand, iron may be detrimental due to its redox abilities, thereby contributing to free radical formation, which in turn may provoke oxidative stress and DNA damage. Iron also plays a crucial role in tumor progression and metastasis due to its major function in tumor cell survival and reprogramming of the tumor microenvironment. Therefore, pathways of iron acquisition, export, and storage are often perturbed in cancers, suggesting that targeting iron metabolic pathways might represent opportunities towards innovative approaches in cancer treatment. Recent evidence points to a crucial role of tumor-associated macrophages (TAMs) as a source of iron within the tumor microenvironment, implying that specifically targeting the TAM iron pool might add to the efficacy of tumor therapy. Here, we provide a brief summary of tumor cell iron metabolism and updated molecular mechanisms that regulate cellular and systemic iron homeostasis with regard to the development of cancer. Since iron adds to shaping major hallmarks of cancer, we emphasize innovative therapeutic strategies to address the iron pool of tumor cells or cells of the tumor microenvironment for the treatment of cancer.
Subject(s)
Iron/metabolism , Neoplasms/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/metabolism , DNA Damage , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Metabolic Networks and Pathways/drug effects , Neoplasms/drug therapy , Neoplasms/genetics , Oxidative Stress , Tumor MicroenvironmentABSTRACT
Macrophage-epithelial cross-talk regulates cell cycle progression and represents an important factor in rescuing epithelial cells from cell cycle arrest in order to maintain a healthy epithelial phenotype. However, the underlying mechanisms are still not well defined. We provide evidence that macrophage-secreted lipocalin-2 (Lcn-2) plays a key role during this process. In a co-culture setup using cell cycle arrested NRK52e renal epithelial cells and primary bone marrow-derived macrophages, Lcn-2 restores proliferation through inhibition of peroxisome proliferator-activated receptor (PPAR)-γ. Lcn-2 overexpression in macrophages overcomes epithelial cell cycle arrest and enhances epithelial markers via megalin and the downstream activation of PI3K/Akt signalling pathway, whereas a knockdown of Lcn-2 in macrophages prevented this effect. Our results show that macrophage-secreting Lcn-2 is crucial in rescuing epithelial cells from cell cycle arrest and in promoting epithelial proliferation.
Subject(s)
Cell Proliferation , Epithelial Cells/physiology , Lipocalin-2/physiology , PPAR gamma/metabolism , Animals , Cell Cycle , Cell Line , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Macrophages/metabolism , Phenotype , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats, Sprague-DawleyABSTRACT
Iron is an essential element for virtually all organisms. It facilitates cell proliferation and growth but also contributes to major hallmarks of cancer such as tumor initiation, growth, and metastasis. Often, iron handling of tumor cells is disturbed, with altered iron acquisition, efflux, and storage. Targeting perturbed iron metabolic pathways might open opportunities towards novel approaches in cancer treatment. It is becoming clear that cells of the tumor microenvironment such as macrophages contribute to tumor progression. Since macrophages evolved a multitude of mechanisms to sequester, transport, store, and release iron it can be speculated that tumor cells educate them to supply iron to support tumor growth. Recent evidence supports the existence of transferrin-independent iron transport mechanisms in the tumor microenvironment, which points to local iron transport proteins such as lipocalin-2 and/or low molecular weight iron-trafficking substances such as siderophores. We hypothesize that tumor cells educate immune cells, i.e. macrophages in their neighborhood to make them delivering iron for the benefit of cancer progression. In particular, we pay attention to recent developments, pointing to lipocalin-2 and siderophores as alternative iron transport molecules in the tumor microenvironment.
Subject(s)
Iron/metabolism , Lipocalin-2/metabolism , Neoplasms/metabolism , Tumor Microenvironment , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biological Transport/drug effects , Humans , Iron Chelating Agents/pharmacology , Iron Chelating Agents/therapeutic use , Macrophages/metabolism , Neoplasms/drug therapy , Neoplasms/pathology , Tumor Microenvironment/drug effectsABSTRACT
Tumour cell-secreted factors skew infiltrating immune cells towards a tumour-supporting phenotype, expressing pro-tumourigenic mediators. However, the influence of lipocalin-2 (Lcn2) on the metastatic cascade in the tumour micro-environment is still not clearly defined. Here, we explored the role of stroma-derived, especially macrophage-released, Lcn2 in breast cancer progression. Knockdown studies and neutralizing antibody approaches showed that Lcn2 contributes to the early events of metastasis in vitro. The release of Lcn2 from macrophages induced an epithelial-mesenchymal transition programme in MCF-7 breast cancer cells and enhanced local migration as well as invasion into the extracellular matrix, using a three-dimensioanl (3D) spheroid model. Moreover, a global Lcn2 deficiency attenuated breast cancer metastasis in both the MMTV-PyMT breast cancer model and a xenograft model inoculating MCF-7 cells pretreated with supernatants from wild-type and Lcn2-knockdown macrophages. To dissect the role of stroma-derived Lcn2, we employed an orthotopic mammary tumour mouse model. Implantation of wild-type PyMT tumour cells into Lcn2-deficient mice left primary mammary tumour formation unaltered, but specifically reduced tumour cell dissemination into the lung. We conclude that stroma-secreted Lcn2 promotes metastasis in vitro and in vivo, thereby contributing to tumour progression. Our study highlights the tumourigenic potential of stroma-released Lcn2 and suggests Lcn2 as a putative therapeutic target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Subject(s)
Breast Neoplasms/genetics , Lipocalin-2/metabolism , Lung Neoplasms/secondary , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Cell Transformation, Neoplastic , Disease Progression , Epithelial-Mesenchymal Transition , Female , Humans , Lipocalin-2/genetics , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , RNA, Small Interfering , Stromal Cells/metabolism , Tumor Microenvironment , Xenograft Model Antitumor AssaysABSTRACT
A pleiotropic signaling lipid, sphingosine-1-phosphate (S1P), has been implicated in various pathophysiological processes supporting tumor growth and metastasis. However, there are only a few descriptive studies suggesting a role of S1P in tumor lymphangiogenesis, which is critical for tumor growth and dissemination. Corroborating own data, the literature suggests that apoptotic tumor cell-derived S1P alters the phenotype of tumor-associated macrophages (TAMs) to gain protumor functions. However, mechanistically, the role of TAM-induced lymphangiogenesis has only been poorly described, mostly linked to the production of lymphangiogenic factors such as vascular endothelial growth factor C (VEGF-C) and VEGF-D, or transdifferentiation into lymphatic endothelial cells. Recent findings highlight a rather underappreciated role of S1P in tumor lymphangiogenesis, referring to the production of interleukin-1ß (IL-1ß) and lipocalin-2 (LCN2) by a tumor-promoting macrophage phenotype. In this review, we aim to provide to the readers with the current understanding of the molecular mechanism how apoptotic cell-derived S1P triggers TAMs to promote lymphangiogenesis.
Subject(s)
Interleukin-1beta/metabolism , Lipocalin-2/metabolism , Lymphangiogenesis/physiology , Lysophospholipids/metabolism , Sphingosine/analogs & derivatives , Animals , Humans , Lipocalin-2/genetics , Lymphangiogenesis/genetics , Receptors, Lysosphingolipid/genetics , Receptors, Lysosphingolipid/metabolism , Sphingosine/metabolism , Vascular Endothelial Growth Factor C/genetics , Vascular Endothelial Growth Factor C/metabolismABSTRACT
Great diversity of shape, size, and skin color is observed among the fruits of different apple genotypes. These traits are critical for consumers and therefore interesting targets for breeding new apple varieties. However, they are difficult to phenotype and their genetic basis, especially for fruit shape and ground color, is largely unknown. We used the FruitPhenoBox to digitally phenotype 525 genotypes of the apple reference population (apple REFPOP) genotyped for 303,148 single nucleotide polymorphism (SNP) markers. From the apple images, 573 highly heritable features describing fruit shape and size as well as 17 highly heritable features for fruit skin color were extracted to explore genotype-phenotype relationships. Out of these features, seven principal components (PCs) and 16 features with the Pearson's correlation r < 0.75 (selected features) were chosen to carry out genome-wide association studies (GWAS) for fruit shape and size. Four PCs and eight selected features were used in GWAS for fruit skin color. In total, 69 SNPs scattered over all 17 apple chromosomes were significantly associated with round, conical, cylindrical, or symmetric fruit shapes and fruit size. Novel associations with major effect on round or conical fruit shapes and fruit size were identified on chromosomes 1 and 2. Additionally, 16 SNPs associated with PCs and selected features related to red overcolor as well as green and yellow ground color were found on eight chromosomes. The identified associations can be used to advance marker-assisted selection in apple fruit breeding to systematically select for desired fruit appearance.
Subject(s)
Fruit , Genome-Wide Association Study , Genotype , Malus , Phenotype , Polymorphism, Single Nucleotide , Malus/genetics , Fruit/genetics , Fruit/anatomy & histology , Quantitative Trait Loci , Quantitative Trait, HeritableABSTRACT
Betulinic acid (BA) exhibits antitumoral activity by blocking proliferation, invasion, and angiogenesis. However, the impact of BA on epithelial-to-mesenchymal transition (EMT), a hallmark of cancer metastasis induced among others by neutrophil gelatinase-associated lipocalin (NGAL), remains unknown. The present study aimed at determining the effect of BA on NGAL-induced EMT. In A375 melanoma cells, BA downregulated mesenchymal markers, increased epithelial markers, and inhibited cytoskeletal reorganization. In addition, BA limited endogenous NGAL production and further suppressed EMT induced by exogenously added NGAL and the corresponding invasive cellular phenotype. In conclusion, BA interferes with EMT-associated changes, a mechanism to antagonize invasive melanoma cells.
Subject(s)
Acute-Phase Proteins/metabolism , Epithelial-Mesenchymal Transition/drug effects , Lipocalins/metabolism , Melanoma/pathology , Proto-Oncogene Proteins/metabolism , Triterpenes/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Lipocalin-2 , Neoplasm Invasiveness , Pentacyclic Triterpenes , Phenotype , Triterpenes/chemistry , Betulinic AcidABSTRACT
Ischemia/reperfusion injury is a leading cause of acute renal failure triggering an inflammatory response associated with infiltrating macrophages, which determine disease outcome. To repair the inflammation we designed a procedure whereby macrophages that overexpress the anti-inflammatory agent interleukin (IL)-10 were adoptively transferred. These bone marrow-derived macrophages were able to increase their intracellular iron pool that, in turn, augmented the expression of lipocalin-2 and its receptors. Infusion of these macrophages into rats after 1 h of reperfusion resulted in localization of the cells to injured kidney tissue, caused increases in regenerative markers, and a notable reduction in both blood urea nitrogen and creatinine. Furthermore, IL-10 therapy decreased the local inflammatory profile and upregulated the expression of pro-regenerative lipocalin-2 and its receptors. IL-10-mediated protection and subsequent renal repair were dependent on the presence of iron and lipocalin-2, since the administration of a neutralizing antibody for lipocalin-2 or administration of IL-10 macrophages pretreated with the iron chelating agent deferoxamine abrogated IL-10-mediated protective effects. Thus, adoptive transfer of IL-10 macrophages to ischemic kidneys blunts acute kidney injury. These effects are mediated through the action of intracellular iron to induce lipocalin-2.
Subject(s)
Acute Kidney Injury/prevention & control , Adoptive Transfer , Interleukin-10/biosynthesis , Ischemia/therapy , Kidney/metabolism , Lipocalins/metabolism , Macrophages/transplantation , Reperfusion Injury/prevention & control , Acute Kidney Injury/genetics , Acute Kidney Injury/immunology , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Animals , Blood Urea Nitrogen , Cell Survival , Deferoxamine/pharmacology , Disease Models, Animal , HEK293 Cells , Humans , Inflammation Mediators/metabolism , Interleukin-10/genetics , Iron/metabolism , Iron Chelating Agents/pharmacology , Ischemia/genetics , Ischemia/immunology , Ischemia/metabolism , Ischemia/pathology , Kidney/blood supply , Kidney/immunology , Kidney/pathology , Lipocalin-2 , Lipocalins/genetics , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Male , Rats , Rats, Sprague-Dawley , Reperfusion Injury/genetics , Reperfusion Injury/immunology , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Time Factors , Transfection , Up-RegulationABSTRACT
Inflammatory reactions are initiated to eliminate pathogens, but also to promote repair of damaged tissue after acute inflammation is terminated. In this regard, macrophages play a prominent role during induction as well as resolution of inflammation and injury in various organs including the kidney. The present study describes a mechanism for renal tissue regeneration after ischaemia/reperfusion injury. Following injury, apoptotic cell-derived sphingosine-1-phosphate (S1P) or exogenously administered sphingosine analogue FTY720 activates macrophages to support the proliferation and healing of renal epithelium, once inflammatory conditions are terminated. Both suppression of inflammation and renal regeneration might require S1P receptor 3 (S1P3) signalling and downstream release of neutrophil gelatinase-associated lipocalin (NGAL/Lcn-2) from macrophages. Overall, our data point to a macrophage-dependent S1P-S1P3-Lcn-2 axis that might be beneficial for restoration of kidney function after an ischaemic insult.
Subject(s)
Acute-Phase Proteins/metabolism , Kidney/physiology , Lipocalins/metabolism , Lysophospholipids/metabolism , Macrophages/metabolism , Oncogene Proteins/metabolism , Regeneration/physiology , Sphingosine/analogs & derivatives , Animals , Disease Models, Animal , Kidney/pathology , Lipocalin-2 , Macrophages/pathology , Male , Mice , Receptors, Lysosphingolipid/metabolism , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Signal Transduction , Sphingosine/metabolism , Sphingosine-1-Phosphate ReceptorsABSTRACT
Implementation of genomic tools is desirable to increase the efficiency of apple breeding. Recently, the multi-environment apple reference population (apple REFPOP) proved useful for rediscovering loci, estimating genomic predictive ability, and studying genotype by environment interactions (G × E). So far, only two phenological traits were investigated using the apple REFPOP, although the population may be valuable when dissecting genetic architecture and reporting predictive abilities for additional key traits in apple breeding. Here we show contrasting genetic architecture and genomic predictive abilities for 30 quantitative traits across up to six European locations using the apple REFPOP. A total of 59 stable and 277 location-specific associations were found using GWAS, 69.2% of which are novel when compared with 41 reviewed publications. Average genomic predictive abilities of 0.18-0.88 were estimated using main-effect univariate, main-effect multivariate, multi-environment univariate, and multi-environment multivariate models. The G × E accounted for up to 24% of the phenotypic variability. This most comprehensive genomic study in apple in terms of trait-environment combinations provided knowledge of trait biology and prediction models that can be readily applied for marker-assisted or genomic selection, thus facilitating increased breeding efficiency.
ABSTRACT
Macrophages supply iron to the breast tumor microenvironment by enforced secretion of lipocalin-2 (Lcn-2)-bound iron as well as the increased expression of the iron exporter ferroportin (FPN). We aimed at identifying the contribution of each pathway in supplying iron for the growing tumor, thereby fostering tumor progression. Analyzing the expression profiles of Lcn-2 and FPN using the spontaneous polyoma-middle-T oncogene (PyMT) breast cancer model as well as mining publicly available TCGA (The Cancer Genome Atlas) and GEO Series(GSE) datasets from the Gene Expression Omnibus database (GEO), we found no association between tumor parameters and Lcn-2 or FPN. However, stromal/macrophage-expression of Lcn-2 correlated with tumor onset, lung metastases, and recurrence, whereas FPN did not. While the total iron amount in wildtype and Lcn-2-/- PyMT tumors showed no difference, we observed that tumor-associated macrophages from Lcn-2-/- compared to wildtype tumors stored more iron. In contrast, Lcn-2-/- tumor cells accumulated less iron than their wildtype counterparts, translating into a low migratory and proliferative capacity of Lcn-2-/- tumor cells in a 3D tumor spheroid model in vitro. Our data suggest a pivotal role of Lcn-2 in tumor iron-management, affecting tumor growth. This study underscores the role of iron for tumor progression and the need for a better understanding of iron-targeted therapy approaches.
ABSTRACT
While the importance of the iron-load of lipocalin-2 (Lcn-2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an iron-transporter, we aimed at clarifying iron-loaded, holo-Lcn-2 (hLcn-2)-dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn-2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn-2-stimulated cells are protected from erastin-induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. In contrast, preventing oxidative stress through N-acetyl-l-cysteine (NAC) supplementation was still able to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF-2α and induction of ATF4 after hLcn-2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn-2-treated renal tumor cells to ferroptosis, thus linking the ISR to pro-tumor characteristics of hLcn-2. Our study provides mechanistic details to better understand tumor pro-survival pathways initiated by iron-loaded Lcn-2.
ABSTRACT
Iron is an essential co-factor for cellular processes. In the immune system, it can activate macrophages and represents a potential therapeutic for various diseases. To specifically deliver iron to macrophages, iron oxide nanoparticles are embedded in polymeric micelles of reactive polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine). Upon surface functionalization via dihydrolipoic acid, iron oxide cores act as crosslinker themselves and undergo chemoselective disulfide bond formation with the surrounding poly(S-ethylsulfonyl-l-cysteine) block, yielding glutathione-responsive core cross-linked polymeric micelles (CCPMs). When applied to primary murine and human macrophages, these nanoparticles display preferential uptake, sustained intracellular iron release, and induce a strong inflammatory response. This response is also demonstrated in vivo when nanoparticles are intratracheally administered to wild-type C57Bl/6N mice. Most importantly, the controlled release concept to deliver iron oxide in redox-responsive CCPMs induces significantly stronger macrophage activation than any other iron source at identical iron levels (e.g., Feraheme), directing to a new class of immune therapeutics.
Subject(s)
Iron , Micelles , Animals , Inflammation/drug therapy , Macrophages , Mice , PolymersABSTRACT
Certain determinants of ischemic resistance in the Brown Norway rat strain have been proposed, but no studies to date have focused on the role of the Wnt pathway in the ischemic resistance mechanism. We performed a comparative genomic study in Brown Norway vs. Sprague-Dawley rats. Selective manipulations of the Wnt pathway in vivo and in vitro allowed us to study whether the action of the Wnt pathway on apoptosis through the regulation of osteopontin was critical to the maintenance of inherent ischemic resistance mechanisms. The results revealed a major gene upregulation of the Wnt family in Brown Norway rats after renal ischemia-reperfusion. Manipulation of the Wnt signaling cascade by selective antibodies increased mitochondrial cytochrome c release and caspase 3 activity. The antiapoptotic role of Wnt was mediated by osteopontin, a direct Wnt target gene. Osteopontin was reduced by Wnt antibody administration in vivo, and osteopontin gene silencing in vitro significantly increased mitochondrial cytochrome c release. The overexpression of Wnt pathway genes detected in Brown Norway rats is critical in the maintenance of their inherent ischemic resistance. Activation of the Wnt signaling cascade reduces mitochondrial cytochrome c release and caspase 3 activity through the action of osteopontin.
Subject(s)
Apoptosis , Cytochromes c/metabolism , Kidney/blood supply , Kidney/enzymology , Mitochondria/enzymology , Osteopontin/metabolism , Reperfusion Injury/prevention & control , Wnt Proteins/metabolism , Animals , Antibodies , Apoptosis/genetics , Caspase 3/metabolism , Cell Line , Disease Models, Animal , Gene Expression Profiling/methods , Kidney/pathology , Male , Oligonucleotide Array Sequence Analysis , Osteopontin/genetics , RNA Interference , Rats , Rats, Inbred BN , Rats, Sprague-Dawley , Reperfusion Injury/enzymology , Reperfusion Injury/genetics , Reperfusion Injury/pathology , Species Specificity , Transfection , Wnt Proteins/genetics , Wnt Proteins/immunologyABSTRACT
Cellular iron, at the physiological level, is essential to maintain several metabolic pathways, while an excess of free iron may cause oxidative damage and/or provoke cell death. Consequently, iron homeostasis has to be tightly controlled. Under hypoxia these regulatory mechanisms for human macrophages are not well understood. Hypoxic primary human macrophages reduced intracellular free iron and increased ferritin expression, including mitochondrial ferritin (FTMT), to store iron. In parallel, nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy, decreased and was proven to directly regulate FTMT expression. Reduced NCOA4 expression resulted from a lower rate of hypoxic NCOA4 transcription combined with a micro RNA 6862-5p-dependent degradation of NCOA4 mRNA, the latter being regulated by c-jun N-terminal kinase (JNK). Pharmacological inhibition of JNK under hypoxia increased NCOA4 and prevented FTMT induction. FTMT and ferritin heavy chain (FTH) cooperated to protect macrophages from RSL-3-induced ferroptosis under hypoxia as this form of cell death is linked to iron metabolism. In contrast, in HT1080 fibrosarcome cells, which are sensitive to ferroptosis, NCOA4 and FTMT are not regulated. Our study helps to understand mechanisms of hypoxic FTMT regulation and to link ferritinophagy and macrophage sensitivity to ferroptosis.