Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis

Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron-deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT-expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)-transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 microM treatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose-dependent manner, with no apparent change in K(m), in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration- and time-dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three-fivefold in both cell types exposed to 50 microM DFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting that in vitro iron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.     

The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines

The dopamine transporter (DAT) removes dopamine from the extracellular milieu and is potently inhibited by number of psychoactive drugs, including cocaine, amphetamines, and methylphenidate (Ritalin). Multiple lines of evidence demonstrate that protein kinase C (PKC) down-regulates dopamine transport, primarily by redistributing DAT from the plasma membrane to endosomal compartments, although the mechanisms facilitating transporter sequestration are not defined. Here, we demonstrate that DAT constitutively internalizes and recycles in rat pheochromocytoma (PC12) cells. Temperature blockades demonstrated basal internalization and reliance on recycling to maintain DAT cell surface levels. In contrast, recycling blockade with bafilomycin A1 significantly decreased transferrin receptor (TfR) surface expression but had no effect on DAT surface levels, suggesting that DAT and TfR traffic via distinct endosomal mechanisms. Kinetic analyses reveal robust constitutive DAT cycling to and from the plasma membrane, independent of transporter expression levels. In contrast, phorbol ester-mediated PKC activation accelerated DAT endocytosis and attenuated transporter recycling in a manner sensitive to DAT expression levels. These data demonstrate constitutive DAT trafficking and that PKC-mediated DAT sequestration is achieved by a combination of accelerated internalization and reduced recycling. Additionally, the differential sensitivity to expression level exhibited by constitutive and regulated DAT trafficking suggests that these two processes are mediated by independent cellular mechanisms.     

Divalent Metal Transporter 1 Expression and Regulation in Human Placenta

Divalent metal transporter 1 (DMT1) is likely responsible for the release of iron from endosomes to the cytoplasm in placental syncytiotrophoblasts (STB). To determine the localization and the regulation of DMT1 expression by iron directly in placenta, the expression of DMT1 in human term placental tissues and BeWo cells (human placental choriocarcinoma cell line) was detected and the change in expression in response to different iron treatments on BeWo cells was observed. DMT1 was shown to be most prominent near the maternal side in human term placenta and predominantly in the cytoplasm of BeWo cells. BeWo cells were treated with desferrioxamine (DFO) and human holotransferrin (hTf-2Fe) and it was found that both DMT1 mRNA and protein increased significantly with DFO treatment and decreased with hTf-2Fe treatment. Further, DMT1 mRNA responded more significantly to treatments if it possessed an iron-responsive element than mRNA without this element. This study indicated that DMT1 is likely involved in endosomal iron transport in placental STB and placental DMT1 + IRE expression was primarily regulated by the IRE/IRP mechanism.     

Regulation of divalent metal transporter expression in human intestinal epithelial cells following exposure to non-haem iron

A number of regulatory factors including dietary iron levels can dramatically alter the expression of the intestinal iron transporter DMT1. Here we show that Caco-2 cells exposed to iron for 4h exhibited a significant decrease in plasma membrane DMT1 protein, though total cellular DMT1 levels were unaltered. Following biotinylation of cell surface proteins, there was a significant increase in intracellular biotin-labelled DMT1 in iron-exposed cells. Furthermore, iron-treatment increased levels of DMT1 co-localised with LAMP1, suggesting that the initial response of intestinal epithelial cells to iron involves internalisation and targeting of DMT1 transporter protein towards a late endosomal/lysosomal compartment.     

Endosomal and lysosomal effects of desferrioxamine: protection of HeLa cells from hydrogen peroxide-induced DNA damage and induction of cell-cycle arrest

The role of endosomal/lysosomal redox-active iron in H2O2-induced nuclear DNA damage as well as in cell proliferation was examined using the iron chelator desferrioxamine (DFO). Transient transfections of HeLa cells with vectors encoding dominant proteins involved in the regulation of various routes of endocytosis (dynamin and Rab5) were used to show that DFO (a potent and rather specific iron chelator) enters cells by fluid-phase endocytosis and exerts its effects by chelating redox-active iron present in the endosomal/lysosomal compartment. Endocytosed DFO effectively protected cells against H2O2-induced DNA damage, indicating the importance of endosomal/lysosomal redox-active iron in these processes. Moreover, exposure of cells to DFO in a range of concentrations (0.1 to 100 microM) inhibited cell proliferation in a fluid-phase endocytosis-dependent manner. Flow cytometric analysis of cells exposed to 100 microM DFO for 24 h showed that the cell cycle was transiently interrupted at the G2/M phase, while treatment for 48 h led to permanent cell arrest. Collectively, the above results clearly indicate that DFO has to be endocytosed by the fluid-phase pathway to protect cells against H2O2-induced DNA damage. Moreover, chelation of iron in the endosomal/lysosomal cell compartment leads to cell cycle interruption, indicating that all cellular labile iron is propagated through this compartment before its anabolic use is possible.     

Differential regulation of the ascorbic acid transporter SVCT2 during development and in response to ascorbic acid depletion

The sodium-dependent vitamin C transporter-2 (SVCT2) is the only ascorbic acid (ASC) transporter significantly expressed in brain. It is required for life and is critical during brain development to supply adequate levels of ASC. To assess SVCT2 function in the developing brain, we studied time-dependent SVCT2 mRNA and protein expression in mouse brain, using liver as a comparison tissue because it is the site of ASC synthesis. We found that SVCT2 expression followed an inverse relationship with ASC levels in the developing brain. In cortex and cerebellum, ASC levels were high throughout late embryonic stages and early post-natal stages and decreased with age, whereas SVCT2 mRNA and protein levels were low in embryos and increased with age. A different response was observed for liver, in which ASC levels and SVCT2 expression were both low throughout embryogenesis and increased post-natally. To determine whether low intracellular ASC might be capable of driving SVCT2 expression, we depleted ASC by diet in adult mice unable to synthesize ASC. We observed that SVCT2 mRNA and protein were not affected by ASC depletion in brain cortex, but SVCT2 protein expression was increased by ASC depletion in the cerebellum and liver. The results suggest that expression of the SVCT2 is differentially regulated during embryonic development and in adulthood.     

Dopamine transporter-mediated cytotoxicity of 6-hydroxydopamine in vitro depends on expression of mutant alpha-synucleins related to Parkinson's disease

6-Hydroxydopamine (6-OHDA) is widely used to produce animal models of Parkinson's disease (PD) by selectively destroying the nigro-striatal dopaminergic systems, but selective toxicity of 6-OHDA towards dopaminergic cells in vitro remains controversial. Mutant (A30P and A53T) alpha-synuclein isoforms cause increased vulnerability of cells towards various toxic insults and enhance dopamine transporter (DAT)-mediated toxicity of the selective dopaminergic neurotoxin and mitochondrial complex I inhibitor MPP(+) in vitro. Here we extend our recent studies on DAT-mediated toxicity to elucidate the mechanisms involved in selective dopaminergic toxicity of 6-OHDA. We studied the cytotoxicity as well as the toxic mechanisms of 6-OHDA in human embryonic kidney HEK-293 cells ectopically co-expressing mutant alpha-synucleins and the human DAT protein. 6-OHDA showed half-maximal toxic concentration (TC(50)) of 88 microM in HEK-hDAT cells without alpha-synuclein expression after 24 h, whereas the TC(50) values significantly decreased to 58 and 39 microM by expression of A30P and A53T alpha-synuclein, respectively. alpha-Synuclein expression did not affect 6-OHDA toxicity in HEK-293 cells not expressing the DAT. Analysis of intracellular parameters of cellular energy metabolism revealed that the co-expression of mutant alpha-synucleins in HEK-hDAT cells accelerates the reduction of intracellular net ATP levels and ATP/ADP ratios induced by 6-OHDA. Uptake function of the DAT was not altered by expression of alpha-synuclein isoforms. Our data suggest a mechanism of 6-OHDA-induced dopaminergic toxicity involving an interaction of mutant alpha-synucleins with the DAT molecule and subsequent acceleration of cellular energy depletion that might be relevant for the pathogenesis of PD.     

Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells.

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I-Tf binding sites. Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short-term (1-2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non-heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.     

Iron chelator triggers inflammatory signals in human intestinal epithelial cells: involvement of p38 and extracellular signal-regulated kinase signaling pathways

Competition for cellular iron (Fe) is a vital component of the interaction between host and pathogen. Most bacteria have an obligate requirement for Fe to sustain infection, growth, and survival in host. To obtain iron required for growth, many bacteria secrete iron chelators (siderophores). This study was undertaken to test whether a bacterial siderophore, deferoxamine (DFO), could trigger inflammatory signals in human intestinal epithelial cells as a single stimulus. Incubation of human intestinal epithelial HT-29 cells with DFO increased the expression of IL-8 mRNA, as well as the release of IL-8 protein. The signal transduction study revealed that both p38 and extracellular signal-regulated kinase-1/2 were significantly activated in response to DFO. Accordingly, the selective inhibitors for both kinases, either alone or in combination, completely abolished DFO-induced IL-8 secretion, indicating an importance of mitogen-activated protein kinases pathway. These proinflammatory effects of DFO were, in large part, mediated by activation of Na(+)/H(+) exchangers, because selective blockade of Na(+)/H(+) exchangers prevented the DFO-induced IL-8 production. Interestingly, however, DFO neither induced NF-kappaB activation by itself nor affected IL-1beta- or TNF-alpha-mediated NF-kappaB activation, suggesting a NF-kappaB-independent mechanism in DFO-induced IL-8 production. Global gene expression profiling revealed that DFO significantly up-regulates inflammation-related genes including proinflammatory genes, and that many of those genes are down-modulated by the selective mitogen-activated protein kinase inhibitors. Collectively, these results demonstrate that, in addition to bacterial products or cell wall components, direct chelation of host Fe by infected bacteria may also contribute to the evocation of host inflammatory responses.     

Iron chelation study in a normal human hepatocyte cell line suggests that tumor necrosis factor receptor-associated protein 1 (TRAP1) regulates production of reactive oxygen species

Iron is an essential component of many proteins, and has crucial roles in the proper functioning of proteins involved in cellular respiration, proliferation, and differentiation. It has been recently reported that the deferoxamine (DFO), an iron chelator, induces mitochondrial dysfunction, characterized by an attenuation of oxidative phosphorylation, as well as senescence-like cellular morphology. However, the effects of DFO on mitochondrial heat shock proteins (HSPs) remain poorly understood. In this study, we examined the effect of DFO on tumor necrosis factor receptor-associated protein 1 (TRAP1), a representative mitochondrial HSP, in a normal human hepatocyte cell line, Chang cells. DFO specifically decreased TRAP1 levels, increasing reactive oxygen species (ROS) and caveolin-1 (Cav-1), a marker protein of senescence. To examine whether these effects of DFO are reversed, we established TRAP1-overexpressing Chang cells. DFO treatment to TRAP1-overexpressing cells resulted in decreases in levels of ROS, Cav-1, glutathione peroxidase (GPX), and manganese superoxide dismutase (MnSOD) levels as well as senescence-associated beta-galactosidase (SA beta-gal) activity. These results suggest that TRAP1 might play a role in protecting mitochondria against damaging stimuli via decrease of ROS generation.     

Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.     

Modulation of monoamine transporter expression and function by repetitive transcranial magnetic stimulation

Repetitive transcranial magnetic stimulation (rTMS) is a new tool for the treatment of neuropsychiatric disorders. However, the mechanisms underlying the effects of rTMS are still unclear. In this study, we analyzed mRNA expression changes of monoamine transporter (MAT) genes, which are targets for antidepressants and psychostimulants. Following a 20-day rTMS treatment, these genes were found to be differentially expressed in the mouse brain. Down-regulation of serotonin transporter (SERT) mRNA levels and the subsequent decrease in serotonin uptake and binding were observed after chronic rTMS. In contrast to the SERT changes, increased mRNA levels of dopamine transporter (DAT) and norepinephrine transporter (NET) were observed. For NET, but not DAT, there were accompanying changes in uptake and binding. Similar effect on NET was observed in PC12 cells stimulated by rTMS for 15 days. These results indicate that modulation of MATs by chronic rTMS may be one therapeutic mechanism for the treatment of neuropsychiatric disorders.