Abstract
Purpose: :
Metallothioneins (MTs) are cytosolic zinc ion-binding proteins involved in neuroprotection, metal detoxification, protection against oxidative damage and inflammatory stress. The purpose of this work was: i) to determine the expression and tissue distribution of MT isoforms in glaucoma eyes, and ii) to assay the effect of metals and inflammatory stress on MT expression and metabolism.
Methods: :
Tissues (cornea, TM, iris, lens, CB, retina and RPE) were excised from glaucoma and normal eye donors. Total RNA was isolated from each tissue with TRIzol, purified with Qiagen RNeasy and processed on Illumina BeadChip array platform. MT isoform-specific antibodies were applied to paraffin-embedded human eye sections by indirect immunofluorescence. The effect of ZnSO4 (10µM-100µM) and IL1α (10-100ng/ml) on MTs was carried out on a cultured cornea HCEsv cell line, in a dose- and time-dependent manner. Total zinc bound to MTs was determined in the presence of 68ZnSO4 and 67Zn as tracers. Zinc levels were detected and quantified by the ICP-MS technique and the Isotope Pattern Deconvolution (IPD) method. MTs were separated by size exclusion (SEC) anionic exchange (MonoQ) chromatography and identified by MALDI-TOF.
Results: :
Profiling of MTs in normal eyes indicated that MT-1A and MT-2A are abundant in all the ocular tissues examined, and MT-3 is restricted to the retina and iris. In glaucoma eyes, MT-1A and MT-2A expression is significantly downregulated in most of the ocular tissues, except in TM where they are up-regulated. MT-1/2 antibodies labeled the corneal epithelium and endothelium, lens epithelium and the INL/IPL of the retina; whereas MT-3 antibodies labeled the retina INL, RGC and NFL. Cultured HCEsv cells, when exposed to ZnSO4 or IL-1α did not alter MT patterns of expression, but they elicited a synergistic effect when added together on MT-1A and MT-2A up to 1.6-fold. This effect coincided with increased levels of zinc bound to purified MTs.
Conclusions: :
These results provide new evidence of the coordinate effect of zinc and interleukin IL-1α on MT expression in eye cells under conditions mimicking pro-inflammatory stress. The ability of MTs in sequestering metals (i.e., zinc) and being responsive to pro-inflammatory cytokines, supports their potential role as molecular sensors of oxidative stress and inflammation in eye disease.
Keywords: gene microarray • protective mechanisms • proteomics