Effects of hypoxic preconditioning on the expression of iron influx and efflux proteins in primary neuron culture

https://doi.org/10.1016/j.neuint.2012.01.008Get rights and content

Abstract

The mechanisms of neuroprotection induced by hypoxic preconditioning (HP) and the effects of HP on iron metabolism proteins in the brain have not been fully elucidated. Based on the accumulated information, we hypothesized that HP would be able to affect the expression of iron metabolism proteins in the brain and that the changes in the expression of these proteins induced by HP might be partly associated with the HP-induced neuroprotection. Here, we demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (Fpn1) proteins and thus increase transferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release, and also a progressive increase in cellular iron content in the cultured neurons. We concluded that HP has the ability to speed iron transport rate and proposed that the increase in iron transport rate and cellular iron in neurons might be one of the mechanisms involved in neuroprotection in the HP neurons. We also demonstrated that Fpn1 expression was significantly affected by HIF-1alpha, implying that the gene encoding this iron efflux protein is hypoxia-inducible.

Highlights

► Hypoxic preconditioning (HP) increased significantly HIF-1alpha expression. ► HP increased significantly the expression of TfR1, DMT1 and Fpn1. ► HP increased Tf-Fe and NTBI uptake and iron release. ► HP induced a progressive increase in cellular iron content in neurons. ► HP speeds up iron transport rate.

Introduction

Preconditioning was first described in a dog model of myocardial injury in which sub-lethal ischemia enabled cells to better tolerate the usually lethal subsequent ischemia (Murray et al., 1986). It is now recognized that this phenomenon can be induced not only by ischemia but also by hypoxia in the central nervous system (Gustavsson et al., 2005, Ran et al., 2005, He et al., 2007). In the past few years, the phenomenon has received much attention because of its potential therapeutic importance (Stone, 2003). A number of studies have demonstrated that preconditioning induced by hypoxia can produce a significant effect to protect neurons in cells, animals and humans (Matsushima and Hakim., 1995, Vannucci et al., 1998, Gidday et al., 1999, Wu et al., 2005, Wagner et al., 2002). However, the mechanisms of neuroprotection induced by hypoxic preconditioning (HP) have not yet been fully elucidated although the involvement of hypoxia-inducible factor-1alpha (HIF-1alpha) in this process is confirmed.

Adaptation to hypoxia in cells and tissues leads to the transcriptional induction of a series of genes, including several with important functions in iron metabolism (Ke and Costa, 2006). It has been known for a long time that transferrin (Tf) and transferrin receptor (TfR1) are two key proteins involved in iron metabolism under physiological conditions (Qian et al., 1997). Studies have demonstrated that hypoxia can increase iron uptake by cells as well as the expression of Tf and TfR1, both of which have been identified as hypoxia-inducible genes (Rolfs et al., 1997, Bianchi et al., 1999, Lok and Ponka, 1999). It has also been reported that several other proteins that are involved in iron transport or regulation, such as ceruloplasmin (Cp) (Mukhopadhyay et al., 2000), iron regulatory protein 1 (IRP1) (Hanson et al., 1999) and 2 (IRP2) (Hanson and Leibold, 1998), are regulated by HIF-1 in response to hypoxic conditions. Furthermore, it has been demonstrated that the gene encoding hepcidin, a newly discovered iron regulatory hormone, is regulated by hypoxia (Nicolas et al., 2002). In our recent studies (Zhu et al., 2008, Qian et al., 2011), we demonstrated that divalent metal transporter 1 (DMT1), another key iron uptake protein under physiological conditions, is a hypoxia-inducible gene.

Currently, we know very little about the effects of HP on the expression of iron metabolism proteins in the brain. Based on the above findings, however, we speculated that the responses of the expression of iron metabolism proteins in neurons or other brain cells to HP might also be associated with the HP-induced neuroprotective role or the increased hypoxic tolerance. We also proposed that the expression of ferroportin1 (Fpn1), a key iron efflux protein, might also be regulated by HIF-1alpha. In this study, we therefore investigated systematically the effects of HP on the expression of iron transport proteins in primary cultured neurons. Our findings showed for the first time that HP could induce a significant increase in the expression of iron uptake (TfR1 and DMT1) and release (Fpn1) proteins and thus speed iron transport rate in neurons. Iron is a transition metal that is essential for oxygen transport, cell growth and survival (Zhu et al., 2008). Therefore, we believed that the increase in iron transport rate in neurons might be one of the mechanisms involved in the neuroprotection role induced by HP.

Section snippets

Materials

Unless otherwise stated, all chemicals were obtained from Sigma Chemical Co., St. Louis, MO, USA. The scintillation cocktail and tubes were purchased from Beckman Coulter Company, Fullerton, CA, USA and 55FeCl3 from Perkin–Elmer Company, Wellesley, MA, USA. The mouse anti-rat TfR1 monoclonal antibody was obtained from Zymed Laboratories, South San Francisco, CA, USA and antibodies against DMT1 with (DMT1 + IRE) or without (DMT1−IRE) iron response element and Fpn1 were purchased from Alpha

Effects of hypoxic preconditioning on cell viabilities

We first investigated the effects of different periods of HP (0, 0.5, 1, 2, 4 or 12 h) or HP (0, 0.5, 1, 2, 4 or 12 h)/R (24 h) on cell viability. The MTT assay results, as shown in Fig. 1, indicated that the cell viability was generally lower in HP/R cells than in HP cells at all time points. Treatments of the neurons with HP-0.5 h, HP-1 h or HP-2 h and HP-0.5 h/R-24 h or HP-1 h/R-24 h did not induce a significant effect on cell viability, no significant differences being found between these groups and

Discussion

The regulation and management of iron at the cellular level is primarily controlled by iron uptake, release and storage (ferritin) proteins found in most cells throughout the body (Qian et al., 1997, Qian and Wang, 1998). TfR1 and DMT1 are the most important iron uptake proteins (Qian and Wang, 1998, Qian and Shen, 2001), while Fpn1 plays a key role in iron release from cells (Abboud and Haile, 2000, Donovan et al., 2000, McKie et al., 2000). By controlling the level of expression of these

Author contributions

Y.K. and M.F. conceived, organized and supervised the study and obtained funding; F.D., G.Q., L.L.Z., Z.J.Z., and L.L. performed experiments; M.F. and Y.K. did statistical analysis, and Y.K. wrote the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

The studies in our laboratories were supported by The Competitive Earmarked Grants of NSFC-RGC Joint Research Grant (N-CUHK433/08; NSFC 30831160514), National 973 Grant (2011CB510004), and Grants from Shenzhen-Hong Kong Innovation Circle Program (2008, 2009).

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