Increased Expression Of NGF In Hepatocytes Is An Early Event In Iron Overloaded Mouse By Transcriptome Analysis

The liver plays a central role in iron metabolism by storing and sensing the amounts of iron in the body. The dietary iron from duodenum and recycled-iron by reticuloendothelial system are the main source of body iron. When excess iron enters the liver, hepatocytes secrete hepcidin, an anti-microbial peptide which negatively regulates iron excretion from enterocytes and macrophages, and stores the excess iron as ferritin-bound iron. A dysfunction of this regulatory system causes iron overload in the liver. Aberrant iron accumulation in the liver is found in hereditary hemochromatosis and chronic liver disease, and this is considered to be an exacerbating factor in liver cirrhosis and hepatocellular carcinoma. It is therefore important to understand the precise molecular events that take place as a result of iron accumulation during the early stages of iron overload.

In the present study, we performed transcriptome analysis on the liver of dietary iron overloaded mice. Transcriptome analysis using a high throughput sequencer is capable of comprehensive analysis with high sensitivity. We hypothesized that this method will be suitable in detecting the changes in gene expression induced by iron overload, even in slightly expressed genes. C57B1/6 mice were fed a normal diet, and a 2.5% iron diet for 8 weeks. Serum and liver tissue samples were then collected, and histological analysis showed the features of early stage iron overload without significant hepatic damage in the iron-fed mice.

From the results of the transcriptome analysis, we found that nerve growth factor (NGF) was significantly expressed in the slightly iron overloaded liver. This observation was also confirmed by real time RT-PCR, Western blotting and immunohistochemistry. Similarly, NGF upregulation was induced in mice primary hepatocytes cultured in conditioned iron overloaded medium (with high concentration of holo-transferrin or ferric ammonium citrate). Furthermore, immunohistochemical analysis showed that TrkA, a high affinity NGF receptor, was expressed in liver sinusoidal endothelial cells (LSECs). Using scanning electron microscopy, we sought to examine any morphological changes in the sinusoids of the iron overloaded liver and observed that although sieve plate structures (so-called ‘fenestrae’) were found in the LSECs of mice fed a normal diet, they were not visible in the iron-fed mice. The loss of fenestrae was also observed in the LSECs of mice that received intraperitoneal injections of NGF. In cultured isolated primary LSECs, treatment with NGF, or conditioned medium from iron overloaded primary hepatocytes reduced the fenestrae while the anti-NGF neutralization antibody or TrkA inhibitor K252a cancelled this effect. In addition, a fresh iron overloaded medium did not reduce the fenestrae in primary LSECs, indicating that iron itself has no direct effects on the fenestrae in LSECs.

LSECs constitute the sinusoidal wall in the liver and can be regarded as unique capillaries which differ from other capillaries in the body due to the presence of fenestrae which lack a diaphragm, and are therefore open connections between the lumen of the sinusoid and the space of Disse. The fenestrae in LSECs therefore play an important role in the exchange of solutes between the lumen of the sinusoid and hepatocytes.

The results of this study indicate that iron accumulation induces the expression of NGF in hepatoctyes, which in turn leads to the loss of fenestrae in LSECs via TrkA. This phenomenon may therefore contribute to the defensive machinery against iron accumulation in hepatocytes in the early stages of iron overload. These data further suggest a novel function of NGF in the regulation of iron transport.