Transferrin receptor induction by hypoxia. HIF-1-mediated transcriptional activation and cell-specific post-transcriptional regulation.

The tight relationship between oxygen and iron prompted us to investigate whether the expression of transferrin receptor (TfR), which mediates cellular iron uptake, is regulated by hypoxia. In Hep3B human hepatoma cells incubated in 1% O2 or treated with CoCl2, which mimics hypoxia, we detected a 3-fold increase of TfR mRNA despite a decrease of iron regulatory proteins activity. Increased expression resulted from a 4-fold stimulation of the nuclear transcription rate of the TfR gene by both hypoxia and CoCl2. A role for hypoxia-inducible factor (HIF-1), which activates transcription by binding to hypoxia-responsive elements in the activation of TfR, stems from the following observations. (a) Hypoxia and CoCl2-dependent expression of luciferase reporter gene in transiently transfected Hep3B cells was mediated by a fragment of the human TfR promoter containing a putative hypoxia-responsive element sequence, (b) mutation of this sequence prevented hypoxic stimulation of luciferase activity, (c) binding to this sequence of HIF-1α, identified by competition experiments and supershift assays, was induced in Hep3B cells by hypoxia and CoCl2. In erythroid K562 cells, the same treatments did not affect iron regulatory proteins activity, thus resulting in a stimulation of TfR gene expression higher than in hepatoma cells

Effect of extracellular matrix components on the expression of epithelial-to-mesenchymal transition markers in cultured human pancreatic ductal adenocarcinoma cells

Epithelial-to-mesenchymal transition (EMT) is a step-wise process leading to the phenotypic switch of epithelial to mesenchymal cells, providing these cells with a metastatic phenotype. During EMT epithelial cells loose adhesion by down regulation of E-cadherin and express N-cadherin, display cytoskeleton reorganization by expressing vimentin and α-smooth muscle actin (αSMA), acquire motile properties and become invasive by secretion of matrix metalloproteinases (MMPs). Cancer cell phenotype is influenced by the tumor microenvironment in relation to tumor progression, as well as to cell proliferation and invasion. The role of the extracellular matrix (ECM) in the microenvironment is particularly relevant in pancreatic ductal adenocarcinoma (PDAC) since this carcinoma is characterized by an intense desmoplastic reaction, representing the environment where the complex interplay between tumor cells, stromal fibroblasts and ECM components occurs. We aimed at analyzing in vitro the effect of the crosstalk between PDAC cells and their microenvironment by characterizing PDAC cell phenotype in cells cultured on different ECM proteins used as a substrate, in order to better understand the relationship between cancer cell behaviour and the proteins occurring in the desmoplastic tissue. We analyzed by immunofluorescence the expression of the main EMT markers such as E-cadherin, N-cadherin, β-catenin, αSMA, vimentin and collagen type I (COL-I) in PDAC cells cultured on laminin, fibronectin, COL-I and without coating (NC). Moreover, we investigated cell proliferation and MMPs activity in cell culture supernatants by SDS-zymography. Cell morphology was similar in PDAC cells cultured on laminin, fibronectin, COL-I, and in NC, as well as the E-cadherin/β-catenin complex, αSMA and COLI expression; by contrast, vimentin was undetectable in all the experimental conditions. N-cadherin was slightly detectable in cells cultured on fibronectin, COL-I, and laminin, and at lower extent in NC cells. Cell proliferation resulted similar in NC and in cells cultured on fibronectin, decreased on laminin and increased on COL-I. MMP-9 activity exhibited a similar trend, resulting similar on fibronectin, decreased on laminin and stimulated on COL-I. These preliminary results provide new insights in the characterization of the mutual effects elicited by the tumor-stroma interplay on the cancer cell, and will contribute to better understand the influence of the stroma on PDAC cancer cell phenotype, in order to develop new therapeutic strategies

Role of HIF-1 and NF-κB Transcription Factors in the Modulation of Transferrin Receptor by Inflammatory and Anti-inflammatory Signals*

Inflammation generates various changes in body iron homeostasis, including iron sequestration in the reticuloendothelial system with ensuing hypoferremia and anemia of chronic disease. Increased iron accumulation is caused by hepcidin-mediated down-regulation of the iron export protein ferroportin and higher iron uptake. However, enhanced iron acquisition by macrophages cannot be accounted for by the previously reported transferrin receptor (TfR1) down-regulation in macrophages exposed to lipopolysaccharide (LPS)/interferon γ (IFNγ) because it impairs a major iron uptake mechanism. Because TfR1 is up-regulated by the hypoxia-inducible factor (HIF-1), we investigated the effect of inflammatory and anti-inflammatory signals on HIF-1-mediated TfR1 gene expression. Exposure of mouse macrophages (RAW 264.7 and J774A.1 cells or peritoneal macrophages) to LPS/IFNγ up-regulated NF-κB, which in turn rapidly and transiently activated HIF-1-dependent TfR1 expression and iron uptake. Activation of an anti-inflammatory pathway by pre-exposure to the adenosine A2A receptor agonist CGS21680 prevented the inducing effect of LPS/IFNγ on HIF-1 and TfR1 expression by inhibiting NF-κB activity, whereas treatment with CGS21680 alone increased HIF-1-mediated TfR1 expression by means of an NF-κB-independent signaling pathway. In conclusion, an interplay of the HIF-1 and NF-κB pathways controls TfR1 transcription in inflammation. The consequent changes in TfR1 expression may be involved in modulating iron retention in inflammatory macrophages, thus possibly contributing to the development of hypoferremia in the early phases preceding the down-regulation of macrophage ferroportin by hepcidin

Osteopontin: The Molecular Bridge between Fat and Cardiac–Renal Disorders

Osteopontin (OPN) is a multifaceted matricellular protein, with well-recognized roles in both the physiological and pathological processes in the body. OPN is expressed in the main organs and cell types, in which it induces different biological actions. During physiological conditioning, OPN acts as both an intracellular protein and soluble excreted cytokine, regulating tissue remodeling and immune-infiltrate in adipose tissue the heart and the kidney. In contrast, the increased expression of OPN has been correlated with the severity of the cardiovascular and renal outcomes associated with obesity. Indeed, OPN expression is at the “cross roads” of visceral fat extension, cardiovascular diseases (CVDs) and renal disorders, in which OPN orchestrates the molecular interactions, leading to chronic low-grade inflammation. The common factor associated with OPN overexpression in adipose, cardiac and renal tissues seems attributable to the concomitant increase in visceral fat size and the increase in infiltrated OPN+ macrophages. This review underlines the current knowledge on the molecular interactions between obesity and the cardiac–renal disorders ruled by OPN

Up regulation of IL-6 by ischemic preconditioning in normal and fatty rat livers: Association with reduction of oxidative stress

We analyzed the role of IL-6 in the protection that ischemic preconditioning (IP) exerts against hepatic ischemia reperfusion-mediated (I/R) oxidative damage, particularly in fatty livers. IP-related IL-6 up-regulation during reperfusion in steatotic and non-steatotic livers was correlated with reduced indices of liver damage, as also demonstrated by pharmacological modulation of IL-6. IP activated NF-κB and HSF during ischemia (Isc), whereas AP-1 activity was unaffected. IP blunted the activation of STAT3 and stress-responsive genes, such as NF-κB, AP-1 and heme oxygenase (HO-1) during reperfusion. The role of reduced oxidative stress in hepatoprotection of fatty livers was further demonstrated by the fact that: (i) IP prevented the decrease of glutathione levels and the increase of lipid peroxidation; (ii) the anti-oxidant GSH-ester prevented lipid peroxidation and necrosis. In conclusion, IP modulates the activity of transcription factors and triggers IL-6 production; this may prevent hepatic I/R damage in a oxidative stress-dependent way, particularly in fatty livers.Peer Reviewe

Expression of the Receptor for Advanced Glycation End Products in Epicardial Fat: Link with Tissue Thickness and Local Insulin Resistance in Coronary Artery Disease

Increased expression of receptor for advanced glycation end products (RAGE) in adipose tissue has been associated with inflammation, adipocyte hypertrophy, and impaired insulin signal. Epicardial adipose tissue (EAT), a visceral fat surrounding the myocardium, is potentially involved in the onset/progression of coronary artery disease (CAD). To date, the role of RAGE in EAT has not been explored much. We examined whether the RAGE expression in EAT was associated with EAT adiposity and metabolic dysfunctions normally found in CAD patients. EAT samples were obtained from 33 patients undergoing open-heart surgery. EAT expression of RAGE, GLUT4, adiponenctin, GLO1, HMGB1, TLR-4, and MyD88 was analyzed by microarray. EAT thickness was quantified by echocardiography. Anthropometric measures and clinical parameters were taken. BMI, HOMA-IR, and LAP indices were calculated. With increasing RAGE expression in EAT we observed increases in EAT thickness, reduced expression of GLUT4, adiponectin, and GLO1, and elevations of HMGB1, TLR-4, and MyD88. There were significant correlations between RAGE and EAT thickness and between RAGE and the genes. LAP was higher in patients with increased RAGE expression. Our data suggest that in CAD patients RAGE may be involved in promoting EAT adiposity and metabolic dysfunction, such as impaired insulin signaling