| Author: | Ke, Ya |
| Title: | Expression of iron transport proteins in the brain |
| Degree: | Ph.D. |
| Year: | 2003 |
| Subject: | Hong Kong Polytechnic University -- Dissertations Iron -- Metabolism Carrier proteins |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | 281 leaves : ill. ; 30 cm |
| Language: | English |
| Abstract: | The studies decribed in this thesis were performed to gain more knowledge of brain iron metabolism. The regional and/or cellular distribution of the newly identified iron transport proteins, including divalent metal transporter 1 (DMT1), ferroportini (FP1), hephaestin (HP), and duodenal cytochrome b (Dcytb), in the brain were investigated, and the effects of iron status and age on expression of these important proteins involved in brain iron metabolism were determined. The regulation of DMT1 expression in the heart was also investigated for comparison with that in the brain. In addition, the study on effect of intracellular Ca2+ on the transferrin receptor-mediated cellular iron uptake was conducted. This thesis consists of 8 chapters, beginning with a general introduction, followed by a general methods section, then 5 chapters (chapter 3-7) describing the experimental work involved, and finally ending with a general discussion. CHAPTER 1 Chapter 1 presents a general introduction to this thesis. The current knowledge on iron and its functions and hazards in biological system, mechanisms involved in body and cellular iron balance and brain iron metabolism, and iron transport proteins that might play important roles in brain iron homeostasis, including some newly identified iron transporters such as DMT1, FP1, HP, Dcytb, are reviewed. The relationship betweenabnormal regulation of brain iron metabolism and CNS diseases is discussed. In the end of this chapter, the objectives of this project are addressed. CHAPTER 2 Chapter 2 describes the materials and methods used in this study. Other methods used specially in certain experiments are described in the Methods section of the relevant chapter. Apparatuses used in the investigation are described in this chapter as well. CHAPTER 3 In this chapter, the effects of iron status and age on DMT1 mRNA expression and protein synthesis in different brain regions, including the cortex, hippocampus, striatum, and substantia nigra, were investigated. By RT-PCR and western blot analysis, it was found that two isoforms of DMT1 are expressed in different brain regions and presented a high ratio of DMT1 (IRE- iron response element) to DMT1 (no-IRE) in each brain region. The expression of DMT1 (IRE) in the brain was not regulated by iron but influenced by age. DMT1 (IRE) protein synthesis during the brain development almost corresponded to its mRNA expression in each examined region, while this consistent regulation of mR.NA and protein was not found in DMT1 (no-IRE) expression in the cortex and striatum. The result also demonstrated that DMT1 (IRE) and DMT1 (no-IRE) mRNAs present a similar regulation pattern by age in the cortex and hippocampus. In addition, the results revealed that the level of DMT 1 (IRE) expression in the substantia nigra is significantly correlated with the change of iron content at different ages. Based on these results, we suggest that DMT1 (IRE) response to the iron is tissue, cell or status specific and two isoforms ofDMT1 show age-dependent and regional-dependent expression. DMT1 (IRE) and DMT (no-IRE) may have the same regulation mechanism in certain brain regions. CHAPTER 4 The purpose of this study was to evaluate the expression of DMT1 in rat hearts and compare its expression regulation inside and outside the brain. By RT-PCR, the two isoforms of DMT1 mRNA with IRE or without IRE were both expressed in the heart. The relative amount of DMT1 (IRE) to DMT1 (non-IRE) in the heart is the same as that found in the brain. Both of the levels of two DMT1 transcripts expression increased from 1, 3, 9, 28 weeks as did the iron content of the heart. The low dietary iron, which induced the iron deficiency in the heart, increased the level of DMT 1 protein in the heart. In contrast, high dietary iron caused a decline in the level of DMT1 protein in the heart, and the two isoforms of DMT1 proteins showed the same trend. However, the amount of DMT1 transcript expression did not change. Combining this data with the result from the brain, we suggest that the expression of both isoforms of DMT1 are age-dependent and the regulation of DMT1 (IRE) expression by iron is organ and tissue specific. The IRE motif of DMT1 is not involved in the process of iron-regulating protein expression through IRE-IRP interaction in all cases. CHAPTER 5 The experiments of this chapter were desigued to investigate expression of ferroportini (FPI) protein in different brain regions, including the cortex, hippocampus, striatum and substantia nigra, in developing male SD rats and different iron status rats. The results provided direct evidence for the existence of FP1 proteinin the rat brain. All brain areas examined were found to have the ability to synthesize FP1 protein. The findings addressed a possibility that FP1 might play a role in iron release in the brain. It also showed that age has significant effect on the expression of FP1 protein in the cortex, hippocampus, striatum and substantia nigra of the rat brain, but iron has no effect on the expression of FP1 in the brain although FP1 has a 5' RE motif. This suggested that the FP1 IRE response to iron is also organ and tissue specific. Based on the findings of this study and other reports, it appears likely that IRE-IRP dependent and unknown IRE-IRP independent regulatory mechanisms both are involved in the regulation of FP1 expression. CHAPTER 6 Hephaestin (HP), a newly discovered ferroxidase, which plays a role in iron transport across basolateral membrane of enterocytes in the gut. It has been suggested that this enzyme might have the same role in iron transport across the abluminal membrane of the blood-brain barrier as it has in the enterocytes. In this study, I therefore investigated expression of HP mRNA in different brain regions, including the cortex, hippocampus, striatum and substantia nigra, in male SD rats, using RT-PCR and sequence analysis. The expression of mRNA of duodenal cytocbrome b (Dcytb), another newly discovered enzyme (a ferric reductase) also involved in intestinal iron transport, was measured as well. The results provided direct evidence for the existence of the mRNAs of two enzymes in the rat brain. All brain areas examined have the ability to express rnRNAs of these two enzymes, but the levels vary in different regions. The existence of HP and Dcytb mRNAs in the brain implies that these proteins might have a role in brain iron metabolism. The results also showedthat age and iron could affect the expression of HP and Dcytb mRNAs with different response in different brain regions. CHAPTER 7 In this chapter, fluorescence quenching method was used to study kinetics of cellular iron uptake in K562 cells loaded with the fluorescence probe calcein in both cell suspension and single cells. It was found that the iron uptake process followed a first order kinetics. Artificial elevation of the intracellular free Ca2+ speeds up the initial rate of iron uptake and increases the overall capacity of the cells in taking up iron. Depletion of the intracellular free Ca2+ or complete chelation of extracellular Ca2+ results in complete inhibition of the iron uptake in cells. Both of them strongly suggest that the TfR-mediated signal transduction is calcium-dependent. Increase of the TfR expressed on cell surface and accelaration of endocytosis and recycling of the Tf-TfR complex are found to be involved in the up-regulation of the cellular uptake of iron by elevating intracellular Ca2+. CHAPTER 8 This chapter presents a general discussion of the methods and results of the experiments described in this thesis. Some relevant aspects for future research are suggested. |
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