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Hepcidin is an iron-regulatory protein that is upregulated in response to increased iron or inflammatory stimuli. Hepcidin reduces serum iron and induces iron sequestration in the reticuloendothelial macrophages – the hallmark of anaemia of inflammation. Iron deprivation is used as a defense mechanism against infection, and it also has a beneficial effect on the control of cancer. The tumour-suppressor p53 transcriptionally regulates genes involved in growth arrest, apoptosis and DNA repair, and perturbation of p53 pathways is a hallmark of the majority of human cancers. This study inspected a role of p53 in the transcriptional regulation of hepcidin. Based on preliminary bioinformatics analysis, we identified a putative p53 response-element (p53RE) contained in the hepcidin gene (HAMP) promoter. Chromatin immunoprecipitation (ChIP), reporter assays and a temperature sensitive p53 cell-line system were used to demonstrate p53 binding and activation of the hepcidin promoter. p53 bound to hepcidin p53RE in vivo, andthis p53RE could confer p53-dependent transcriptional activation. Activation of p53 increased hepcidin expression, while silencing of p53 resulted in decreased hepcidin expression in human hepatoma cells. Taken together, these results define HAMP as a novel transcriptional target of p53. We hypothesise that hepcidin upregulation by p53 is part of a defence mechanism against cancer, through iron deprivation. Hepcidin induction by p53 might be involved in the pathogenesis of anaemia accompanying cancer.
Hepcidin regulates iron efflux from enterocytes and other cells, such as hepatocytes and reticuloendothelial macrophages, by internalisation and degradation of ferroportin, the iron exporter of these cells (Nemeth et al, 2004a). Therefore, hepcidin serves as a regulator of iron absorption and distribution. Hepcidin is upregulated in response to increased body iron levels (Gehrke et al, 2003) or to inflammation (Nemeth et al, 2004b), and is downregulated in response to hypoxia, anaemia (Nicolas et al, 2002; Adamsky et al, 2004) and oxidative stress (Choi et al, 2007). An increase in hepcidin expression during infection or inflammation results in a decrease in iron availability (Means, 2000) through retention of iron by reticuloendothelial macrophages and decreased intestinal iron absorption. Recently, hepcidin has been shown to directly bind iron during expression in Escherichia. coli, revealing yet another potential role of hepcidin as an intracellular iron sequestering molecule (Gerardi et al, 2005).
Decreased plasma iron by its sequestration in macrophages and decreased intestinal iron absorption is thought to be the mechanism of anaemia of inflammation. As bacteriae depend on iron for their growth, dietary iron deprivation has been shown to have a beneficial effect in infectious diseases such as malaria, tuberculosis and candidiasis (Weinberg, 1999a). Restriction of dietary iron also showed beneficial effects in non-infectious inflammatory conditions, such as ulcerative colitis (Barollo et al, 2004), joint inflammation (Andrews et al, 1987) and bleomycin-induced pulmonary fibrosis (Chandler et al, 1988). Cancer cells also depend on iron for their proliferation. Several studies have shown that iron deprivation inhibits in-vitro tumour growth (Weinberg, 1999b; Gao & Richardson, 2001). Iron chelators inhibit the growth and induce the apoptosis of human Kaposi sarcoma-derived cells (Simonart et al, 2000) and treatment by iron chelation has been shown to have an anti proliferative effect in leukaemia, neuroblastoma (Richardson, 2002), lymphoma (Kemp et al, 1995) and breast cancer (Yang et al, 2001). Iron chelators have also been shown to inhibit the expression of vascular cell adhesion molecule-1 (VCAM-1), which is known to support angiogenesis and accounts for inflammation-augmented tumour development (Vidal-Vanaclocha et al, 2000; Nakao et al, 2003).
The molecular mechanisms controlling hepicidin gene (HAMP) expression are only starting to unfold. The HAMP promoter contains binding motifs for CCAAT/enhancer-binding protein (C/EBP), hepatocyte nuclear factor 4 (HNF4) (Courselaud et al, 2002), signal transducer and activator of transcription 3 (STAT3) (Pietrangelo et al, 2007) and SMAD4 (Milward et al, 2007), suggesting their role in controlling hepcidin synthesis.
P53 is a key tumour-suppressor gene. It is activated in response to a variety of cellular and genotoxic stress conditions, leading to the induction of growth arrest, apoptosis, DNA repair, senescence and differentiation (Vousden & Lu, 2002). p53 also regulates genes that participate in cell-to-cell communication (Komarova et al, 1998), and in the regulation of angiogenic signals to endothelial cells (Nishizaki et al, 1999). Many studies have shown that p53 exerts its various functions mainly as a transcription factor that regulates the expression of its target genes via a consensus DNA binding site (el-Deiry et al, 1992). P53 is mutated or lost in approximately half of human cancer cases worldwide (Levine et al, 1991). However, many human cancers are characterised by P53 that is only rarely mutated (Preudhomme et al, 1992; Neri et al, 1993; Corradini et al, 1994; Borsellino et al, 1995; Yasuga et al, 1995; Hangaishi et al, 1996), and about half of all human cancers are characterised by increased P53 expression, in many instances of the wild-type P53 allele (Shaulsky et al, 1991a,b; Moll et al, 1995; Ostermeyer et al, 1996; Levine, 1997). Increased levels of wild-type P53 have been found in subsets of human cancers, including neuroblastomas, mesotheliomas, and breast, colon and pancreatic cancer (Niedobitek et al, 1993; Bosari et al, 1995; Theobald et al, 1995; Gudas et al, 1996; Ropke et al, 1996; Chen & Carbone, 1997; Moretta, 1997).
We show here that the human HAMP promoter contains a p53-response element (p53RE). The p53 protein binds to this RE in vivo. Luciferase reporter assays revealed that the human HAMP promoter can confer p53-dependent transcriptional activation through the p53RE. HAMP mRNA levels increased in response to activation of P53 in human hepatoma cells. We suggest another mechanism of tumour suppression for p53 protein: iron deprivation from cancer cells by upregulation of HAMP expression. This affect may also tie p53 to the pathogenesis of anaemia of malignancy.
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- Materials and methods
Hepcidin is an anti-microbial peptide and a central regulator of iron homeostasis. Iron balance is achieved by the control of iron cycling through the reticuloendothelial system and dietary absorption. Disruption of the p53 pathway is crucial to the pathogenesis of malignancies. The present study showed that hepcidin is regulated by p53. However, the limitations of our study should be taken into account; HAMP regulation by p53 was examined only at the level of mRNA expression, which may not be equivalent to protein expression.
In a significant portion of tumours, either the wild type or a mutated form of p53 is upregulated. In addition, cancer patients are treated with chemotherapy and radiotherapy, which induce expression of the wild-type p53 protein present in normal cells and also in a significant portion of malignant cells. This induction causes the known side effects of cancer therapy, such as mucositis, alopecia and bone marrow suppression leading also to anaemia. In view of our findings here, that HAMP is a novel target of the P53 tumour suppressor gene, we hypothesise that HAMP expression will be induced in normal and malignant cells possessing a functional p53. HAMP induction is expected to decrease intestinal iron absorption, enhance iron sequestration in the reticuloendothelial system and, along with the recent finding proposing hepcidin itself as an iron sequestering molecule (Gerardi et al, 2005), diminish the availability of systemic free iron.
Anaemia is the most common haematological abnormality in cancer patients and is observed in about half of the patients at some time during their disease. Several unique causes for anaemia exist in patients with malignancy, such as the side effects of therapy, marrow replacement by tumour, nutritional deficiency, increased destruction of red blood cells and blood loss. Nevertheless, there is a significant overlap with the underlying mechanisms of anaemia of infection and chronic inflammation, where hepcidin was shown to play a central role (Deicher & Horl, 2004; Means, 2004). The elevated expression of P53 in many cancer patients and the p53-hepcidin link revealed here may therefore be relevant to the understanding of anaemia of malignancy.
The decrease in available iron mediated by increased hepcidin may also be a new pathway by which p53 exerts its tumour suppressor activity, by depriving the cancer cells of iron. Iron deprivation leads to cell growth arrest and apoptosis as iron is essential for cellular metabolism, oxygen transport and sensing, DNA synthesis, energy generation and other biological processes (Chaston & Richardson, 2003; Hentze et al, 2004).
Our hypothesis, that upregulation of hepcidin is part of the anti-tumour defence mechanism, is supported by recently published work that demonstrated an increased HAMP expression in a rat model of liver ischaemia/reperfusion, independent of IL-6 and C/EBPa regulatory pathways (Goss et al, 2005). An increase in HAMP mRNA expression was also demonstrated in a mouse model for lung ischaemia (Srisuma et al, 2003). Ischaemia also occurs within solid malignant tumours, due to their impaired neovascularisation that is unable to keep pace with the rapidly growing tumour cell mass and fails to meet its nutritional needs (Siemann et al, 2004). Ischaemia and ischaemia/reperfusion are known to increase P53 expression and p53-mediated apoptotic pathways (Cummings, 1996; Hatoko et al, 2001). Taken together with our findings, we suggest an ischaemia-p53-hepcidin tumour suppressor pathway.
On the other hand, we hypothesise that, in malignant cells which acquired mutations in P53 or other aberrations resulting in disruption of the p53 pathway, the consequent downregulation of HAMP expression will, in turn, result in increased iron-availability, which will enable rapid proliferation of the malignant cells.
The p53-hepcidin pathway may also take part in the inflammatory process. Overexpression and mutations of P53 are observed not only in cancer, but also in inflammatory conditions, such as rheumatoid arthritis and ulcerative colitis (Tak et al, 2000), thus p53 is hypothesised to have anti-inflammatory roles in addition to its role as a tumour suppressor and cell cycle regulator. During inflammation, p53 response pathway is activated. The free radical NO, for example, is produced during inflammation and induces p53 post-translational modifications, leading to an increase in the expression of p53 transcriptional targets (Hofseth et al, 2003). Iron has a role in the pathogenesis of inflammation mainly through generation of reactive oxygen species (Morris et al, 1995). Therefore iron deprivation or sequestration might decrease the generation of reactive oxygen species. Thus the p53-hepcidin axis may play a role in the reduction of inflammatory tissue damage as well.
As inflammation was shown to predispose and be involved in the pathogenesis of cancer, the anti-inflammatory hepcidin-mediated iron restrictive role of p53 may be yet another route of p53 tumour suppressive activity.
This study showed that incubating the hepatoma cell line with IL-6 activated the HAMP promoter. IL-6 is known as a stimulator of HAMP production via a STAT3-mediated pathway (Wrighting & Andrews, 2006; Verga Falzacappa et al, 2007). We found that p53 diminished the IL-6 effect on the HAMP promoter by approximately half (Fig 5). These results correspond with the known ability of p53 to repress the IL6 promoter (Santhanam et al, 1991). Moreover, p53 regulates the Janus kinase (JAK)-STAT signal transduction pathway of IL-6 by masking STAT3 and STAT5 accessibility to the genome (Rayanade et al, 1998). Constitutively active IL-6 is observed in many tumour cell lines (Margulies & Sehgal, 1993) and its action is essential to the development of some cancers (Hilbert et al, 1995). On the other hand, IL-6 has been shown to have a direct and indirect anti-tumour activity, and has been suggested as a potential immunotherapy for cancer (Chen et al, 1988; Mule et al, 1990; Givon et al, 1992). IL-6 has been linked to anaemia in cancer (Atkins et al, 1995), and this activity may be mediated through HAMP induction. Therefore both p53 and IL-6 may play a similar role in the defence mechanism against cancer by deprivation of iron from the tumour cells, while each of these factors possesses additional specific activities.
In conclusion, our results add p53 to the factors involved in the transcriptional control of HAMP and thereby link HAMP to cancer. These findings also show that iron deprivation is a new p53-mediated anti-tumoural and anti-inflammatory mechanism.