Molecular Vision 2007; 13:1469-1474 <http://www.molvis.org/molvis/v13/a163/> Received 14 May 2007 | Accepted 21 August 2007 | Published 27 August 2007

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Localization and H₂O₂-specific induction of PRDX3 in the eye lens

Wanda Lee, Tracy Wells, Marc Kantorow
 
 

Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL

Correspondence to: Wanda Lee, Florida Atlantic University, Department of Biomedical Science, 777 Glades Road, Boca Raton, FL, 33431; Phone: (561) 297-2918; FAX: (561) 297-2221; email: wlee10@fau.edu

Abstract

Purpose: Peroxiredoxin III (PRDX3) is a mitochondrial peroxidase that defends cells against oxidative damage and therefore could play a role in cataract formation. To establish a possible role for PRDX3 in lens function, PRDX3 was localized to specific human lens sub-regions and the levels of PRDX3 in human lens cells and rat lenses exposed to exogenously-added oxidative stress determined.

Methods: PRDX3 levels were monitored by RT-PCR, western analysis, and immunofluorescence. PRDX3 levels in human lens epithelial cells and whole rat lenses exposed to H₂O₂, TBHP, and heat-treatment were also examined relative to untreated controls by RT-PCR and western analysis.

Results: Significant levels of PRDX3 mRNA and protein were detected in human lens epithelia and fiber cells. PRDX3 was localized to the mitochondria in human lens epithelial cells. PRDX3 was highly induced in human lens epithelial cells by as little as 2 μM H₂O₂ and by 50 μM H₂O₂ in cultured rat lenses. Induction of PRDX3 was specific for H₂O₂ in cultured lens cells since sub-lethal levels of TBHP or heat-shock did not result in detectable increases in the level of PRDX3.

Conclusions: These data demonstrate that PRDX3 is present throughout the lens and localized to the mitochondria in lens epithelial cells. PRDX3 was specifically induced by low levels of H₂O₂ in human lens epithelial cells and rat lenses suggesting that induction of PRDX3 is an acute response of the lens to increased H₂O₂ levels. These data provide evidence for an important role for PRDX3 in lens H₂O₂-detoxification, mitochondrial maintenance, and possibly cataract formation.

Introduction

The eye lens has evolved a wide variety of protective and repair systems to combat oxidative stress. These include high levels of reduced glutathione (GSH) [1], abundant antioxidant enzymes [2], and the chaperone function of α-crystallin [3]. Aging of the lens is characterized by diminishing levels of these systems [1,2] and their loss can result in cataract formation [4,5].

Of the many systems believed to be important for lens defense, mitochondrial antioxidant enzymes have been shown to be essential for lens cell viability and resistance to oxidative stress damage [6-12]. For instance, deletion of the mitochondrial antioxidant/repair enzyme, methionine sulfoxide reductase A (MSRA), results in loss of human lens cell mitochondrial function, increased lens cell reactive oxygen species (ROS) levels, and loss of lens cell viability [6,8]. Similar results were also shown in human lens cells upon deletion of a second mitochondrial methionine sulfoxide reductase B2 (MSRB2) [7]. Artificial targeting of catalase to the mitochondria delayed cataract formation in mice [13] suggesting that scavenging of mitochondrial H₂O₂ is important for lens maintenance and delay of cataract formation.

In the present report, we have identified Peroxiredoxin III (AOP-1, Mer5, and SP-22), a member of the Perxiredoxin family of peroxidases, to be present at high levels in the human lens. Of the six known PRDXs, PRDX3 is specifically targeted to the mitochondria [14] and has been localized to the mitochondria of HeLa [15] and bovine aortic endothelial [16] cells. PRDX3 is categorized as 2-Cys Typical PRDX which refers to the two reactive cysteines involved in its peroxidase activity [14,17]. Upon peroxide attack, the redox-active peroxidatic cysteine oxidizes to a cysteine sulfenic acid (Cys-SOH) which is attacked by the resolving cysteine of a neighboring PRDX3 to form an intersubunit disulfide bond. This disulfide bond is then reduced by thioredoxin [14] regenerating the original cysteine and completing the catalytic cycle. In addition to its peroxidase activity, PRDX3 has been reported to be induced by several oxidants including H₂O₂ and TBHP in bovine aortic endothelial cells [16], to act as a free radical scavenger [18] and to participate in redox-related signaling transduction pathways [15,19,20] suggesting that multiple functions of PRDX3 could play important roles in the lens and other tissues.

In the present report, we have localized PRDX3 to the lens epithelia and fiber cells and we have localized PRDX3 to lens epithelial cell mitochondria. Moreover, we show that PRDX3 is specifically induced in cultured human lens epithelial (HLE) cells and cultured whole lenses by low (2 μM to 50 μM) levels of H₂O₂. These data suggest an important role for PRDX3 in the regulation of lens mitochondrial H₂O₂ levels which is likely important for lens defense against oxidative stress damage.

Methods

Cell culture

HLE cell cultures (SRA01/04 [21]) were grown and cultured in Dulbecco's modified minimum Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 15% fetal bovine serum (Invitrogen), gentamicin (50 units/ml; Invitrogen), penicillin-streptomycin antibiotic mix (50 units/ml; Invitrogen), and Fungizone (5 μg/ml; Invitrogen) at 36.5 °C in the presence of 5% CO₂.

Monitoring of PRDX3 mRNA and protein levels in microdissected human lens epithelia and human lens fibers

PRDX3 transcript levels were monitored in RNA obtained from microdissected human lens epithelium and fiber cells that were prepared from five clear human lenses obtained 24-36 h post-mortem. Lenses were microdissected into 6-9 mm central epithelium and the remainder of the lens saved as fiber cells. Lens epithelium was cleaned to remove contaminating lens fibers and the absence of lens fibers was confirmed by microscopy. The average age of the lenses used in this study was 60 years old. RNA was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA) and transcript levels were detected by semi-quantitative RT-PCR (Superscript One-Step RT-PCR kit, Invitrogen). GAPDH levels were analyzed in the same RNA sample for comparison. PRDX3 PCR primer sequences did not distinguish between the two known variants of the PRDX3 gene (GenBank Locus: Variant 1-NM_006793 and Variant 2-NM_014098). The primer sequences used in this study along with the expected product sizes and PCR annealing temperatures employed are listed in Table 1. All reactions were determined to be linear over the number of PCR cycles indicated and all PCR products were sequenced to ensure authenticity.

PRDX3 protein levels were monitored using protein extracts prepared from lens epithelium and fiber cells microdissected as described above. The ages of the lenses used in the protein study was 53 and 63 years old. Microdissected lens portions were combined and homogenized in lysis buffer containing 1% SDS in 1X PBS and 1 μl Protease Inhibitor Cocktail (Sigma, St. Louis, MO). The resulting homogenate was centrifuged at 10,000 RPM in a microcentrifuge and the resulting supernatant collected. Protein levels of the resultant extracts were measured by Bradford analysis. Protein levels were examined by coomassie staining and western analysis using 10 μg of the above protein extracts using 14% Tris-Glycine Gels (Invitrogen) and a 1:10,000 dilution of PRDX3 specific polyclonal antibody (Abcam, Cat Number: ab15573, Cambridge, MA). Immunoreactive PRDX3 was visualized using the ECL Western Blotting Analysis System (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's specifications using a 1:2500 dilution of secondary antibody.

PRDX3 mitochondrial colocalization in HLE cells

HLE cells were stained with 100 nM Mitotracker Red CMXRos (Invitrogen) for 45 min. After fixation and permeabilization, the cells were then treated with 1:100 dilution of PRDX3-specific polyclonal antibody (Abcam) and then labeled with 1:500 dilution of Alexa Flour 488 goat anti-rabbit IgG antibody (Invitrogen) for 45 min. Mitochondrial co-localization of PRDX3 was visualized using an Olympus Provis AX70 fluorescence microscope.

Analysis of PRDX3 transcript and protein levels in HLE cells exposed to H₂O₂

Cells were treated as previously described [22] with indicated concentrations of H₂O₂ in the absence of serum for one h and then returned to complete media for indicated times prior to RNA or protein isolation using methods described above. H₂O₂ concentrations lower than 500 μM were employed since concentrations above 500 μM are lethal to HLEs [6]. PRDX3 transcript and protein levels were examined by semi-quantitative RT-PCR and western analysis as described above. The levels of PRDX3-immunoreactive bands were quantified by scanning densitometry.

Induction of PRDX3 transcript in cultured whole lenses

Eight lenses were dissected from 53-day-old Sprague-Dawley male rats and immediately cultured in the presence or absence of 50 μM H₂O₂ for one h in phenol-free, serum-free DMEM. After one h, the media was changed and the lenses were incubated for an additional 12 h prior to RNA isolation. Rat PRDX3 transcript levels were evaluated between treated and control lenses by semi-quantitative RT-PCR relative to the levels of rat ribosomal 28 S RNA as a control. PCR primers, expected product sizes and PCR annealing temperatures are listed in Table 1. All PCR reactions were conducted in the linear PCR range and all products were sequenced to ensure authenticity.

Analysis of PRDX3 transcript in HLE cells exposed to heat-shock and TBHP

Cells were exposed to 45 °C for heat-shock treatment for one h in complete DMEM and cultured for 12 h. For TBHP exposure, cells were treated with indicated concentrations in the absence of serum for one h and then returned to complete media for 12 h. After the 12 h recovery period, RNA was isolated using the RNeasy Mini Kit (Qiagen). PRDX3 transcript was examined by semi-quantitative RT-PCR as described above.

Results

PRDX3 is present in human lens epithelia and fiber cells

To determine the potential levels of PRDX3 in the human lens, PRDX3 mRNA was examined in microdissected human lens epithelia and fiber cells by semi-quantitative RT-PCR. The analysis revealed that PRDX3 mRNA was expressed in both human lens epithelial and fiber cells (Figure 1A) with apparently higher levels of PRDX3 in the fibers relative to the lens epithelium. PRDX3 protein levels were also examined in protein extracts prepared from lens sub-regions by western analysis (Figure 1B). Significant levels of immunoreactive PRDX3 protein were detected in both lens epithelia and fiber cells. The protein levels of PRDX3 paralleled the levels of PRDX3 transcript (compare Figure 1A and Figure 1B).

PRDX3 is localized to the mitochondria in HLE cells

To examine the mitochondrial specificity for PRDX3 in HLEs, cells were stained with the mitochondrial-specific marker Mitotracker (red, Figure 2A) and PRDX3 levels detected using PRDX3 polyclonal antibody visualized with Alexa Flour 488 goat anti-rabbit antibody (green, Figure 2B). The analysis revealed that PRDX3 co-localized with the mitochondrial specific marker in HLE cells (Figure 2C).

Oxidative stress induces PRDX3 mRNA and protein in HLE cells

To examine PRDX3 induction in HLEs, cells were treated with increasing concentrations of H₂O₂ and the resulting levels of PRDX3 examined at the transcript (Figure 3A) and protein levels (Figure 3B). H₂O₂-treatment resulted in significantly increased levels of PRDX3 transcript and protein in the cells after exposure to H₂O₂ (Figure 3A and Figure 3B). PRDX3 transcript and protein levels were highest when cells were exposed to 10 μM H₂O₂ (Figure 3A and Figure 3B). Densitometric analysis performed on immunoreactive PRDX3 protein bands indicated that PRDX3 was induced to levels as much as 26 times above uninduced control levels by 2 μM H₂O₂ and 34 times above control levels by 10 μM H₂O₂ (Figure 3B). Beyond 10 μM H₂O₂, PRDX3 levels decreased possibly as a consequence of H₂O₂ damaging the cells.

PRDX3 is induced in cultured rat lenses exposed to H₂O₂

Since it is possible that the responses of the cultured lens cells might not reflect those responses of intact lenses, PRDX3 transcript levels were compared between rat lenses cultured in the absence of serum and exposed to 50 μM H₂O₂ relative to untreated control lenses. Exposure of the cultured rat lenses to 50 μM H₂O₂ caused some lens opacity in four out of eight lenses examined, compare Figure 4A (-) and (+). Consistent with the lens epithelial cell culture studies, PRDX3 transcript levels were significantly increased in the pooled cultured rat lenses exposed to H₂O₂ relative to untreated control lenses (Figure 4B).

PRDX3 induction in lens cells is specific for H₂O₂-oxidative stress

To evaluate the specificity for PRDX3 H₂O₂-induction, we chose to examine the induction of PRDX3 in response to TBHP and heat-shock. No PRDX3 induction upon heat-shock treatment was detected by semi-quantitative RT-PCR (Figure 5A) relative to HSP27, a known heat-shock protein, which was induced in the cells upon heat-shock (Figure 5A). Interestingly, PRDX3 levels decreased slightly upon heat-shock. No PRDX3 induction was detected in HLE cells exposed to increasing concentrations of TBHP (Figure 5B). PRDX3 transcript levels actually decreased in the TBHP-treated lens cells at concentrations above 100 μM TBHP (Figure 5B).

Discussion

The present report demonstrates that PRDX3 is localized to the mitochondria in HLEs and is present in human lens epithelium and fiber cells. We also demonstrate that PRDX3 is induced at both the transcript and protein levels by H₂O₂ in cultured HLE cells and at the mRNA level in whole rat lenses. It is likely that PRDX3 mRNA is made in both lens epithelial cells and elongating lens fiber cells since both contain mitochondria. Further sub-cellular localization studies will be needed to determine the possible presence of PRDX3 in the mitochondria-free nuclear lens fibers.

Since PRDX3 has been reported to be induced in bovine aortic endothelial cells by 500 μM H₂O₂ [16], we examined if the induction of PRDX3 could occur in HLEs. We detected PRDX3 induction in HLEs using as little as 2 μM H₂O₂ at both the mRNA and protein levels. The induction of PRDX3 was optimal at 10 μM H₂O₂ and then dropped off at 50 μM H₂O₂ potentially as a result of cell damage. Transcript levels peaked at approximately 12 h following H₂O₂ exposure, and protein levels from 24 to 48 h post- H₂O₂ exposure (data not shown). Densitometric analysis revealed that PRDX3 protein was induced to levels over 20 times above those detected in untreated control cells by 2 μM H₂O₂. PRDX3 induction was also examined in a separate HLE cell line (HLE-B3) and similar induction levels of PRDX3 were detected upon H₂O₂-exposure (data not shown) suggesting that PRDX3 induction is not confined to the SRA01/04 HLE cells. To further confirm that PRDX3 induction is a response of H₂O₂ and is not an artifact of lens cells grown in serum containing media, PRDX3 induction was also examined in whole rat lenses cultured in serum-free media and PRDX3 was highly induced in the cultured lenses upon H₂O₂ exposure. Though not definitive, this data provides strong evidence that PRDX3 is induced in the actual lens in response to H₂O₂ in vivo. TBHP and heat shock treatment were also examined to determine the specificity of PRDX3 induction in HLEs since several oxidants including 500 μM TBHP has been reported to induce PRDX3 in bovine aortic endothelial cells [16]. The induction of PRDX3 appears specific for H₂O₂ in the lens since low levels of TBHP or heat shock treatment did not induce PRDX3 in HLEs. It is possible that peroxinitrite and/or other unknown oxidants and/or factors could induce PRDX3 in lens cells.

Although the mechanism governing the induction of PRDX3 in the lens is not known, it has been demonstrated that the lens expresses a wide range of transcription factors that are regulated by changes in redox state including AP-1, NfκB, P53 and USF-1 [23]. In addition, JunD and the Nf-κB-p50 subunit have been found to be up-regulated as a direct response of human lens epithelial cells to 50 μM H₂O₂ [22]. Thus, induction of PRDX3 likely depends on the redox sensitive activation of these or other lens transcription factors responding to the presence of low H₂O₂ levels.

At present, the function of PRDX3 in the lens is not known. In previous studies, we have shown that the levels of PRDX3 are not reduced in the aging human lens [24]. Other studies have reported that targeted silencing of the PRDX3 gene in bovine aortic cells [16] and HeLa cells [15] resulted in ROS accumulation and loss of cell viability. Increased ROS levels were also observed in blood cells of PRDX3 knockout mice [25] suggesting that PRDX3 may defend and/or protect these cells against oxidative stress insult through its antioxidant functions. In preliminary studies, we have shown that the deletion of PRDX3 causes reduced sensitivity of lens cells to H₂O₂-treatment (unpublished data). In addition to its direct antioxidant properties [17], PRDX3 has been proposed to participate in redox-mediated signaling pathways [19,20] and it is possible that PRDX3 could have a signaling function in lens cells.

Regardless of its exact functions in the lens, localization of PRDX3 in HLE mitochondria suggest an important role for PRDX3 in lens mitochondrial function and its presence in lens fibers suggest a role for protection of lens proteins and prevention of the protein aggregation. Its induction by low levels of H₂O₂ suggests that increased levels of PRDX3 is an acute response of the lens to the presence of H₂O₂. Further studies will be needed to determine the exact mechanisms governing the expression of PRDX3 and the exact functions of PRDX3 in the lens. Other PRDXs are also present in the lens that likely play important roles in lens cell function including PRDX6 which is induced by LEDGF [26] as well as H₂O₂ [27]. It is possible that these PRDXs have coordinate functions in the lens and that they play important roles in lens maintenance, lens defense, and possibly cataract formation.

Acknowledgements

The authors would like to thank Dr. Venkat Reddy for providing the SRA01/04 cells used in this study and the West Virginia Eye Bank for human lenses. This report is in partial fulfillment of the PhD requirements for WL. This work was funded by NIH grant EY13022 (MK).

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