Project description: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) supple-mentation 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 initi-ated by iron-loaded Lcn-2.
Project description: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.
Project description:Hemolysis drives susceptibility to lung injury and predicts poor outcomes in diseases, such as malaria and sickle cell disease (SCD). However, the underlying pathological mechanism remains unknown. Here, we report that major facilitator superfamily domain containing 7C (MFSD7C) protects the lung from hemolytic-induced damage by preventing ferroptosis. Mechanistically, MFSD7C deficiency leads to mitochondrial dysfunction and lipid remodeling caused by increased fatty acid uptake and decreased consumption, the excess accumulation of lipids sensitizes cells to peroxidation and ferroptosis. Moreover, systematic delivery of MFSD7C mRNA-loaded nanoparticles to the lungs effectively prevented lung injury in mice with hemolysis. These findings present the detailed link between hemolytic complications and ferroptosis, providing potential therapeutic targets for patients with hemolytic disorders.
Project description:Hypertonicity in renal medulla is essential for urine concentration and water homeostasis. However, how renal medullary collecting duct cells (MCDs) survive and function under the harsh hypertonic stress remains incompletely understood. By using the RNA-seq technique, we identified SNAT2 (slc38a2) as a novel osmoresponsive neutral amino acid transporter in MCD cells. We found that hypertonic stress induced cell death of MCDs mainly via ferroptosis.
Project description: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 (M?) 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 M? 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 M?-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.
Project description:Ferroptosis is an iron-dependent form of cell death driven by biochemical and metabolic alterations resulting in oxidation within the lipid compartment. Calcium is a potent signaling molecule ascribed to diverse cellular processes including migration, neurotransmitter function, and cell death. Here we elucidate a crucial link between calcium homeostasis and ferroptotic cell death through the identification of the tetraspanin MS4A15. Ectopic MS4A15 expression specifically protects against ferroptosis by depleting endoplasmic reticulum stores. In an unexpected connection, prolonged calcium dysregulation stimulates fundamental remodeling to ferroptosis-resistant monounsaturated and plasmalogen lipid species. Application of this discovery revealed that augmenting luminal calcium sensitizes cancer cell lines previously refractory to ferroptosis. This finding provides a unique mechanistic basis for ferroptosis sensitivity and resolves a long-standing query into the role of calcium in oxidative cell death. Manipulating calcium homeostasis offers an unprecedented strategy for overcoming therapy resistance in cancer.
Project description:Sepsis is a prevalent health issue that can lead to central nervous system (CNS) inflammation with long-term behavioral and cognitive alterations. Using unbiased proteomic profiling of over 100 different cytokines, we found that Lipocalin-2 (LCN2) was the most substantially elevated protein in the CNS after peripheral administration of lipopolysaccharide (LPS). To determine whether the high level of LCN2 in the CNS is protective or deleterious, we challenged Lcn2-/- mice with peripheral LPS and determined effects on behavior and neuroinflammation. At a time corresponding to peak LCN2 induction in WT mice injected with LPS, Lcn2-/- mice challenged with LPS had exacerbated levels of pro-inflammatory cytokines and exhibited significantly worsened behavioral phenotypes. To determine the extent of global inflammatory changes dependent upon LCN2, we performed an RNAseq transcriptomic analysis. Compared to WT mice injected with LPS, Lcn2-/- mice injected with LPS had unique transcriptional profiles and significantly elevated levels of multiple pro-inflammatory molecules. Several LCN2-dependent pathways were revealed with this analysis including, cytokine and chemokine signaling, NOD-like receptor signaling, and Jak-STAT signaling. These findings demonstrate that LCN2 serves as a potent protective factor in the CNS in response to systemic inflammation and may be a potential candidate for limiting sepsis-related CNS sequelae.