Although Prx proteins have been found to be ubiquitously expressed in human tissues, including skin, neuronal tissue and blood cells,18, 20, 21
this is the first study reporting the expression of Prx proteins in human cornea. Results of the proteomic analysis indicate that Prx-2, -3, -5, and -6 are expressed in normal corneal endothelium. Western blot studies confirmed the expression of Prx-2, -3, and -5 in these cells. The 2-D gel methodology showed that Prx-2 was the most abundantly expressed Prx protein in the endothelium, even though Prx-5 appeared as a more intense band in the Western blot analysis. Since the Western blot data is dependent on many factors, one of which is the sensitivity of the primary antibody, the spot intensity of the 2-D gels is a more accurate representation of the relative Prx levels in the sample. As indicated previously, expression of Prx-6 could not be verified by Western blot due to the lack of an antibody that worked sufficiently well. In the eye, Prx-6 expression has been noted in bovine iris stroma, lens, ciliary epithelium and its blood vessels, and retina.22, 23
In bovine cornea, Prx-6 was present in the epithelial layer, but not found in corneal endothelium.23
The proteomic identification of Prx-6 in the current studies requires verification; however, the fact that Prx-6 was not detected in bovine corneal endothelium, but was detected in human, may be due to species-related differences in protein expression or to differences in detection methods. It is also possible that Prx-1 or -4 is expressed in human corneal endothelium, but that they were not identified using our 2-D gel-based method.
In the current study, corneal stroma and epithelium showed lower levels of Prx expression when compared to endothelium from the same donors. This is the first study indicating that Prx-2 and -3 expression is significantly higher in human corneal endothelium than in the stroma and epithelium. Prx-5 was expressed in HCEC and to a lesser extent in epithelium/stroma; however, the difference in levels of this Prx isoform were not statistically significant. Selective expression of certain Prx isoforms in HCEC-DM, but not in epithelium/stroma, indicates distinct functional roles of antioxidant enzymes that reflect different physiologic activities of corneal cell types. It is possible that significantly higher expression of Prx-2 and -3 in corneal endothelium suggests selective vulnerability of specific corneal cell types to oxidative stress.
On the proteomic level we detected a significant reduction in Prx-2, -3, and -5 in FED endothelium. Tissues were used from age-matched normal donors to eliminate any age-related variation. The average differences in age between normal and FED pooled samples were within a decade of each other. The normal donors were matched by gender to FED patients. For studies comparing protein expression in normal and FED endothelium, extracts from FED and normal donors were pooled to eliminate individual variations that might skew the results. One of the limitations of our study is that some samples contained unequal number of pooled corneas between FED and normals. Since the overall endothelial cell count was much lower in FED samples, for the most part, more tissue was required to be pooled for FED samples. Two different semi-quantitative methods were used to compare the relative expression of Prx isoforms. Both the software-based 2D gel analysis and the Western blot analysis demonstrated a significant reduction in the expression of Prx-2, -3, and -5 in FED corneal endothelium compared with age and gender-matched normals. Proteomic analysis comparing the normal and FED endothelial extracts revealed a number of differences in relative protein expression patterns, the significance of which should be further investigated. The fact that the relative expression of SOD-1, another anti-oxidant enzyme, as well as the intermediate filament protein, vimentin, did not differ significantly between the FED and normal tissue strongly indicates that the reduction in expression of the Prx isoforms is specific and not the result of a general reduction in anti-oxidant or total protein expression.
Proteomic analysis of normal endothelium demonstrated that Prx-2 was the most abundant Prx isoform and thus of potential greatest significance at the functional level. To corroborate the decrease of Prx-2 levels in FED, its expression at the gene level was compared between normal and FED samples. The finding that levels of Prx-2 mRNA were significantly decreased in FED samples, further substantiates the proteomic data and indicates that the source of the differences detected by the proteomic analysis, at least for Prx-2, stem from decreased gene transcription.
Prx proteins have varying subcellular locations reflecting their multifunctional isoform diversity. Prx-2 is mainly a cytosolic protein that inhibits release of cytochrome c from mitochondria to cytosol and blocks hydrogen peroxide-induced apoptosis upstream of the site of Bcl-2 action. 24
In addition, activation of NF-kB induced by hydrogen peroxide is blocked by Prx-2, indicating its role in gene transcription regulation in response to reduction/oxidation status.25, 26
Since NF-kB has been implicated in regulation of corneal endothelial cell apoptosis in response to ROS, the potential role of Prx-2 as an endogenous inhibitor of NF-kB is particularly pertinent to HCEC physiology.27
Prx-3 (MER 5, SP-22, and AOP-1) is a mitochondrion-specific peroxidase that uses mitochondrial thioredoxin-2 as an electron donor and provides primary antioxidant defense of the mitochondrial respiratory chain.26
Under-expression of Prx-3 has been shown to exacerbate mitochondrial macromolecule damage via membrane potential collapse, cytochrome c release, and caspase activation.28
Similarly to our findings, Prx-3 was also noted to be under-expressed in Alzheimer’s disease (AD) and Down’s syndrome (DS), disorders in which ROS-induced apoptosis accounts for the neuronal cell loss.29, 30
It was postulated that the instability of Prx-3 in the neurodegenerative disorders accounts for the cell susceptibility to oxidative stress. Prx-5 has been localized to the mitochondria, peroxisomes and cell nucleus. Prx-5 also has a strong anti-apoptotic function by preventing intracellular ROS production via a p53-dependent pathway.17, 31, 32
For future studies, it would be important to correlate the down-regulation of Prx’s with apoptosis-related proteins, such NF-kB, caspase, and proteins involved in the p53-dependant pathways, as well as to substantiate further the role of Prx under-expression in the apoptotic cell death seen in FED endothelium. Since FED has a notable female preponderance, it would be important to further explore Prx expression differences between the genders. Also, because FED is for the most part a disease of the older population (>50 years of age), the changes in antioxidant expression between young and old donors might be of significance. Whether there is a variation in Prx expression between male and female gender and young and older donors requires further investigation. Furthermore, the down-regulation of antioxidants in FED endothelium needs to be correlated with their upstream regulators, since Prx-2 under-expression is seen on both RNA and protein level.
Oxidative damage via generation of ROS can lead to corneal endothelial cell apoptosis and has been implicated in the pathogenesis of Fuchs’ endothelial dystrophy. 12, 13
Antioxidant enzymes are critical for regulating intracellular levels of ROS and averting the deleterious effects associated with oxidative damage. Prx proteins constitute a potent antioxidant defense system by neutralizing ROS. The significant decrease in Prx levels in FED-affected cells may represent an alteration in the functioning of mechanisms required to combat oxidative stress related to the pathogenesis of this disease.