Expression of ZnT and ZIP zinc transporters in the human RPE and their regulation by neurotrophic factors.

Expression of ZnT and ZIP Zinc Transporters in the Human RPE and Their Regulation by Neurotrophic Factors Kar Wah Leung, 1 Mugen Liu, 2 Xuming Xu, 2 Magdalene J. Seiler, 3 Colin J. Barnstable, 1,2 and Joyce Tombran-Tink 1,2 P URPOSE . Zinc is an essential cofactor for normal cell function. Altered expression and function of zinc transporters may con- tribute to the pathogenesis of neurodegenerative disorders including macular degeneration. The expression and regula- tion of zinc transporters in the RPE and the toxicity of zinc to these cells were examined. M ETHODS . Zinc transporters were identified in a human RPE cell line, ARPE19, using a 28K human array, and their expression was confirmed by PCR, immunocytochemistry, and Western blot analysis in primary human RPE cultures and ARPE19. Zinc toxicity to ARPE19 was determined using monotetrazolium, propidium iodide, and TUNEL assays, and Zn 2⫹ uptake was visualized with Zinquin ethyl ester. The effect of various growth factors on zinc transporter expression also was exam- ined. R ESULTS . Transcripts for 20 of 23 zinc transporters are ex- pressed in fetal human RPE, 16 of 23 in adult human RPE, and 21 of 23 in ARPE19. Zn transporter proteins were also detected in ARPE19. ZnT5 expression was not observed, whereas ZnT6, ZIP1, and ZIP13 were the most abundantly expressed in all RPE samples. The addition of low concentrations of Zn 2⫹ to cul- tures resulted in a dose-dependent increase in intracellular Zn 2⫹ content in ARPE19, and ⬎30 nM Zn 2⫹ induced necrosis with an LC 50 of 117.4 nM. Brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor (GDNF), and pigment epithelial-derived neurotrophic factor (PEDF) increased ZIP2 expression, GDNF and PEDF increased ZnT2 expression, and PEDF increased ZnT3 and ZnT8 expres- sion. These neurotrophic factors also promoted Zn 2⫹ uptake in the RPE. C ONCLUSIONS . The array of zinc transporters expressed by the RPE may play a key role in zinc homeostasis in the retina and in ocular health and diseases. (Invest Ophthalmol Vis Sci. 2008;49:1221–1231) DOI:10.1167/iovs.07-0781 From the 1 Department of Neural and Behavioral Sciences, Penn- sylvania State University College of Medicine, Hershey, Pennsylvania; Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut; and 3 Department of Ophthalmology, Doheny Retina Institute, Keck School of Medicine, University of Southern California, Los Angeles, California. Supported by The David Woods Kemper Foundation, by grants from the National Institutes of Health and the Macular Vision Research Foundation, and by the Ben Franklin Award from the Pennsylvania Department of Community and Economic Development. Submitted for publication June 26, 2007; revised September 7, 2007; accepted January 9, 2008. Disclosure: K.W. Leung, None; M. Liu, None; X. Xu, None; M.J. Seiler, None; C.J. Barnstable, None; J. Tombran-Tink, None The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Joyce Tombran-Tink, Department of Neu- ral and Behavioral Sciences, Pennsylvania State University College of Medicine, Hershey, PA 17033; jttink@aol.com. D isruption in zinc homeostasis is strongly implicated in the pathophysiology of many chronic neurodegenerative dis- eases and acute neural injuries. Excessive and inadequate levels of bioavailable zinc are detrimental to the health of neurons. 1 For example, increased concentration of Zn 2⫹ is associated with aggregation of ␤ -amyloid protein in patients with Alzhei- mer disease. 2–5 Zinc enrichment in the cerebrovasculature may be an underlying factor in the development of cerebral amyloid angiopathy, a condition characterized by ␤ -amyloid deposits in perivascular spaces of the brain. 6 Intense presynaptic activity in epilepsy, ischemia, or traumatic lesions can trigger the release of Zn 2⫹ to neurotoxic levels in surrounding tissues. This release may account for the unusually high Zn 2⫹ content in the somata of neurons degenerating after severe episodes of ischemia or seizure activity. 2,7–14 A deficiency in Zn 2⫹ , on the other hand, promotes brain malformations during development and has other adverse con- sequences in the nervous system. 15 Depleted pools of intracel- lular zinc in primary retinal cell cultures induce the caspase- dependent death of photoreceptors and other retinal neurons 16 in the eye, and decreased levels of zinc in the retina contribute to the pathogenesis of age-related macular degen- eration (AMD). The zinc–AMD connection is supported by the findings that retinas from monkeys with early-onset macular degeneration contain fourfold less Zn 2⫹ than monkeys with normal vision 17,18 and that levels of zinc in drusen and sub-RPE deposits 19 are increased, suggesting that the risk for or severity of AMD increases with the depletion of available intracellular zinc pools in the retina. Newsome et al. 20 were the first to report that replenishing Zn 2⫹ by oral administration has a positive effect on AMD. The 2004 AREDS report and other studies confirm that replacing zinc with a dietary supplement has beneficial effects against AMD. 21–23 Normal ocular tissues contain relatively high levels of zinc, ranging from approximately 25 ␮ g/g wet weight in the RPE/ choroid to 100 ␮ g/g dry weight in the retina, 24,25 with a high percentage of this localized in the photoreceptors and RPE cells. Recent studies show that intracellular localization of Zn 2⫹ pools in photoreceptors changes with light exposure, with the greatest intensity of zinc staining observed in the perikarya of photoreceptors of dark-adapted retinas and in the inner segments of light-adapted retinas. 25 Zn 2⫹ movement be- tween RPE and photoreceptors is also light dependent, sug- gesting that Zn 2⫹ is critical to normal visual function. The importance of Zn 2⫹ in biological processes is not re- stricted to the nervous system. It is the second most abundant trace element in the human body and is critical to housekeep- ing roles in physiology, cellular metabolism, protein structure, and gene expression. It provides structural stability to the Zn 2⫹ finger domains of many DNA-binding proteins and is a cofactor for more than 300 metalloenzymes, in which it is an essential element for the catalytic site of the enzymes or serves in a structural capacity to facilitate enzymatic function. 26 For ex- ample, mutations in the copper-zinc superoxide dismutase (SOD1), an anti-oxidative Zn-binding enzyme, promote loss of Investigative Ophthalmology & Visual Science, March 2008, Vol. 49, No. 3 Copyright © Association for Research in Vision and Ophthalmology Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/Journals/IOVS/933445/ on 05/29/2016