Heparanase overexpression reduces hepcidin expression, affects iron homeostasis and alters the response to inflammation

Hepcidin is the key regulator of systemic iron availability that acts by controlling the degradation of the iron exporter ferroportin. It is expressed mainly in the liver and regulated by iron, inflammation, erythropoiesis and hypoxia. The various agents that control its expression act mainly via the BMP6/SMAD signaling pathway. Among them are exogenous heparins, which are strong hepcidin repressors with a mechanism of action not fully understood but that may involve the competition with the structurally similar endogenous Heparan Sulfates (HS). To verify this hypothesis, we analyzed how the overexpression of heparanase, the HS degrading enzyme, modified hepcidin expression and iron homeostasis in hepatic cell lines and in transgenic mice. The results showed that transient and stable overexpression of heparanase in HepG2 cells caused a reduction of hepcidin expression and of SMAD5 phosphorylation. Interestingly, the clones showed also altered level of TfR1 and ferritin, indices of a modified iron homeostasis. The heparanase transgenic mice showed a low level of liver hepcidin, an increase of serum and liver iron with a decrease in spleen iron content. The hepcidin expression remained surprisingly low even after treatment with the inflammatory LPS. The finding that modification of HS structure mediated by heparanase overexpression affects hepcidin expression and iron homeostasis supports the hypothesis that HS participate in the mechanisms controlling hepcidin expression

Mitochondrial ferritin deficiency reduces male fertility in mice

Mitochondrial ferritin (FtMt) is a functional ferritin targeted to mitochondria that is highly expressed in the testis. To investigate the role of FtMt in the testis we set up a series of controlled matings between FtMt gene-deletion mice (FtMt–/–) with FtMt+/+ mice. We found that the number of newborns per litter and the fertility rate were strongly reduced for the FtMt–/– males, but not for the females, indicating that FtMt has an important role for male fertility. The morphology of the testis and of the spermatozoa of FtMt–/– mice was normal and we did not detect alterations in sperm parameters or in oxidative stress indices. In contrast, we observed that the cauda epididymides of FtMt–/– mice were significantly lighter and contained a lower number of spermatozoa compared with the controls. Also, the ATP content of FtMt–/– spermatozoa was found to be lower than that of FtMt+/+ spermatozoa. These data show that FtMt contributes to sperm epididymis maturation and to male fertility.The work was partially supported by MIUR grant PRIN10–11 to P. A. and by Telethon grant GGP1099 to P. A

The Hyperferritinemic Syndrome:macrophage activation syndrome, Still's disease, septic shock and catastrophic antiphospholipid syndrome

BACKGROUND: Over the last few years, accumulating data have implicated a role for ferritin as a signaling molecule and direct mediator of the immune system. Hyperferritinemia is associated with a multitude of clinical conditions and with worse prognosis in critically ill patients. DISCUSSION: There are four uncommon medical conditions characterized by high levels of ferritin, namely the macrophage activation syndrome (MAS), adult onset Still’s disease (AOSD), catastrophic antiphospholipid syndrome (cAPS) and septic shock, that share a similar clinical and laboratory features, and also respond to similar treatments, suggesting a common pathogenic mechanism. Ferritin is known to be a pro-inflammatory mediator inducing expression of pro-inflammatory molecules, yet it has opposing actions as a pro-inflammatory and as an immunosuppressant. We propose that the exceptionally high ferritin levels observed in these uncommon clinical conditions are not just the product of the inflammation but rather may contribute to the development of a cytokine storm. SUMMARY: Here we review and compare four clinical conditions and the role of ferritin as an immunomodulator. We would like to propose including these four conditions under a common syndrome entity termed “Hyperferritinemic Syndrome”

Cardiac Protection by Preconditioning Is Generated via an Iron-Signal Created by Proteasomal Degradation of Iron Proteins

<div><p>Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient ‘iron-signal’ that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this ‘iron signal’ and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after <em>ex vivo</em> ischemia-and-reperfusion (I/R) were found in hearts from <em>in vivo</em> hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min (‘delay’) prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the ‘delay’. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the ‘iron-signal’ by IPC, ensuing <em>de-novo</em> apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the ‘delay’. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.</p> </div

Parameters for recovery of hearts subjected to 3 days of <i>in vivo</i> hypoxia followed by <i>ex vivo</i> I/R.

<p>Hemodynamic parameters; the heart’s hemodynamic parameters at the end of a 10 min stabilization period are considered 100%.</p><p>Ferritin levels given as µg/mg protein.</p><p>mRNA levels given as arbitrary units per unit β-actin.</p><p>Results are mean±SE. Numbers in parentheses represents the number of repetitious experiments.</p>*<p> = Significantly different from Normoxia (p<0.05).</p

Hemodynamic parameters measured throughout the <i>ex vivo</i> procedure following <i>in vivo</i> hypoxic-preconditioning.

<p>a. Heart rate (beats per minutes). b. Developed pressure (mmHg). c. Working index (A.U. = heart rate x developed presure). Results are means±SE of 5–6 experiments. Hypoxia- white square, Normoxia- black square. Stab = stabilization period.</p

Ferritin and ferritin-bound iron levels in hearts subjected to I/R.

<p>Ferritin and ferritin-bound iron levels with or without prior IPC and a ‘delay’ period. a. Relative ferritin protein levels (% of the post stabilization period). b. Ferritin-bound iron (number of iron atoms per molecule of ferritin). c. Relative ferritin protein levels (% of the post stabilization period) in hearts infused with a cocktail of protease inhibitors (MG132-3 µmol/L, leupeptin-11.7 µmol/L, pepstatin-A-4.4 µmol/L): perfusion -black diamond, IPC+‘delay’ +I/R - black circle, ‘delay’+I/R- white square. Results are means±SE of 6 experiments. SE for perfusion and ‘delay’+I/R groups are too small to be seen.</p

Representative EMSA using extracts of hearts subjected to I/R with and without prior IPC.

<p>a. Original phosphorimaging blots. Upper panel shows active IRPs. Lower panel displays total IRPs (+βME). b. Graphic presentation of active IRP; three scanned blots each (±SE). Perfusion only- black, I/R- gray, IPC+ I/R- light gray. No efforts were made to distinguish between IRP1 and IRP2.</p